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Lipopolysaccharides of Vibrio cholerae. I. Physical and chemical characterization
Abstract
Vibrio cholerae is the causative organism of the disease cholera. The lipopolysaccharide (LPS) of V. cholerae plays an important role in eliciting the antibacterial immune response of the host and in classifying the vibrios into some 200 or more serogroups. This review presents an account of our up-to-date knowledge of the physical and chemical characteristics of the three constituents, lipid-A, core-polysaccharide (core-PS) and O-antigen polysaccharide (O-PS), of the LPS of V. cholerae of different serogroups including the disease-causing ones, O1 and O139. The structure and occurrence of the capsular polysaccharide (CPS) on V. cholerae O139 have been discussed as a relevant topic. Similarity and dissimilarity between the structures of LPS of different serogroups, and particularly between O22 and O139, have been analysed with a view to learning their role in the causation of the epidemic form of the disease by avoiding the host defence mechanism and in the evolution of the newer pathogenic strains in future. An idea of the emerging trends of research involving the use of immunogens prepared from synthetic oligosaccharides that mimic terminal epitopes of the O-PS of V. cholerae O1 in the development of a conjugate anti cholera vaccine is also discussed.
... Several virulence factors, e.g., enterotoxin, hemolysin, and LPS contribute considerably to the pathogenicity of Vibrio, thus being studied intensively (Zhang and Austin 2005). In V. cholerae, the LPS inner core is usually composed of at least three l-glycero-α-d-manno-heptoses, which are loaded by inverting glycosyltransferases employing ADP-l,d-manno-heptose as a sugar donor (Chatterjee and Chaudhuri 2003). However, little is known about the biosynthesis of ADP-activated β-d-mannoheptoses in Vibrio, let alone the influences of those ADP-β-dmanno-heptoses on Vibrio pathogenesis. ...
... To our knowledge, three l,d-manno-heptoses are contained in the LPS inner core of V. cholerae O1 strains as in E. coli, while the LPS structure of V. parahaemolyticus CGMCC 1.1997 remains unknown (Chatterjee and Chaudhuri 2003). In E. coli, ADP-d,d-manno-heptose is converted to ADPl,d-manno-heptose by an NAD + -dependent C-6″ epimerase HldD EC (Fig. 2a). ...
... Many people around the world are suffering waterborne and foodborne vibriosis with symptomatic entities such as watery diarrhea, stomach cramping, nausea, vomiting, fever, and chills (Dutta et al. 2021). ADP-β-d-mannoheptoses participate in the assembly of LPS, a virulence factor of Vibrio infections (Qadri et al. 2003;Chatterjee and Chaudhuri 2003), and are also powerful agonists that could trigger NF-κB-mediated innate immune responses (Janeway Jr. and Medzhitov 2002;Zhou et al. 2018). However, little is known about the formation mechanism of ADP-β-d-mannoheptoses in Vibrio species or the influences of those ADPsugars on their pathogenesis. ...
ADP-activated β-d-manno-heptoses (ADP-β-d-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-d-glycero-β-d-manno-heptose (ADP-d,d-manno-heptose) and its C-6′′ epimer, ADP-l-glycero-β-d-manno-heptose (ADP-l,d-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-d,d-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-β-d-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-d,d-manno-heptose and the epimerase that converts it to ADP-l,d-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that β-d-mannose 1-phosphate, but not α-d-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldEVC (from V. cholerae) and HldEVP (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldEVC and HldEVP together with the E. coli–derived HldEEC were thus carried out using β-d-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-β-d-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains.
Key points
• Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-β-d-manno-heptoses.
• HldEs from two Vibrio strains and E. coli could activate β-d-mannose 1-phosphate to ADP-β-d-mannose.
• Comparable nucleotidyltransfer efficiencies were observed in the kinetic studies of HldEs.
... Ogawa serotype strain expresses A and B antigens as well as a small amount of C antigens, while Inaba only expresses A and C antigens. A third serotype, Hikojima expressing both the B and C antigens, is also reported but is rare and unstable (Chatterjee and Chaudhuri, 2003). Ogawa and Inaba serotypes differ only by a single 2-O-methyl group that is present in the upstream (nonreducing) terminal perosamine unit of the Ogawa O-antigen polysaccharide (O-PS) but is absent in Inaba (Chatterjee and Chaudhuri, 2003). ...
... A third serotype, Hikojima expressing both the B and C antigens, is also reported but is rare and unstable (Chatterjee and Chaudhuri, 2003). Ogawa and Inaba serotypes differ only by a single 2-O-methyl group that is present in the upstream (nonreducing) terminal perosamine unit of the Ogawa O-antigen polysaccharide (O-PS) but is absent in Inaba (Chatterjee and Chaudhuri, 2003). An isolate is serotyped as Ogawa if its O1 serogroup O-PS is methylated and as Inaba, if its O-PS is not methylated (Ito et al., 1993). ...
Vibrio cholerae serogroup O1 (V. cholerae O1) is closely associated with cholera epidemics and has two main immunologically distinguishable serotypes, Ogawa and Inaba. Isolates serotype as Ogawa if the O-antigen polysaccharide (O-PS) is methylated or as Inaba if the O-PS is not methylated. This methylation is mediated by a methyltransferase encoded by the rfbT gene, and the mutation and low expression of rfbT results in serotype switch from Ogawa to Inaba. Previously, we have shown that cAMP receptor protein (CRP) activates rfbT. In this study, we demonstrated that histone-like nucleoid structuring protein (H-NS) is directly involved in the transcriptional repression of rfbT. This finding is supported by the analyses of rfbT mRNA level, rfbT-lux reporter fusions, electrophoretic mobility shift assay (EMSA), and DNase I footprinting assay. The rfbT mRNA abundances were significantly increased by deleting hns rather than fis which also preferentially associates with AT-rich sequences. A single-copy chromosomal complement of hns partly restored the down-regulation of rfbT. Analysis of rfbT-lux reporter fusions validated the transcriptional repression of hns. Subsequent EMSA and DNase I footprinting assay confirmed the direct binding of H-NS to rfbT promoter and mapped the exact binding site which was further verified by site-directed mutagenesis and promoter functional analysis. Furthermore, we found that in hns deletion mutant, CRP is no longer required for transcriptionally activating rfbT, suggesting that CRP functions as a dedicated transcription factor to relieve H-NS repression at rfbT. Together, this study expanded our understanding of the genetic regulatory mechanism of serotype conversion by global regulators in V. cholerae O1.
... Historically, serogroups of V. cholerae have been defined on the basis of the lipopolysaccharide (LPS) O antigen present on the surface of the bacterium, and more than 200 serogroups have now been identified [3]. Most serogroups are found in environmental strains with an ecological niche in coastal waters and estuaries, often in association with zooplankton, mollusks and crustaceans, as for other species from family Vibrionaceae [1]. ...
... Historiquement, différents sérogroupes de V. cholerae ont été décrits en se basant sur l'antigène O du lipopolysaccharide (LPS) présent à la surface de la bactérie et plus de 200 sérogroupes sont actuellement identifiés [3]. La plupart des sérogroupes se retrouvent chez des souches environnementales dont la niche écologique, comme pour les différentes espèces de la famille des Vibrionaceae, est représentée par les eaux côtières et les estuaires, souvent en association avec le zooplancton, les mollusques et les crustacés [1]. ...
In 2022, the burden of cholera-an acute watery diarrheal disease caused by Vibrio cholerae serogroup O1 (or more rarely O139) bacteria, which produce cholera toxin-remains high in many African and Asian countries. In the last few years, microbial genomics has made it possible to define the bacterial populations responsible for cholera more precisely. It has been shown that the current, seventh pandemic is due to a single lineage with a reservoir in the countries of the Bay of Bengal (India and Bangladesh). There have been several transmissions of the causal agent of cholera from this region to Africa, Asia and Latin America, suggesting a human-to-human transmission of the disease. Microbial genetics can help to fight this scourge by providing insight into cholera epidemiology and through its use in disease monitoring, thereby contributing to the achievement of the World Health Organization's goal of reducing cholera deaths by 90% by 2030.
... 17 The difference between the Ogawa and Inaba serotypes lies only in the terminal perosamine sugar of the O1 OPS which is methylated in Ogawa strains and not methylated in Inaba strains ( Figure 1). 18,19 Although Inaba and Ogawa serotypes can be serologically distinguished, they exhibit strong immunologic cross-reactivity such that most of the immune response induced by vaccination with the Inaba O1 LPS recognizes Ogawa O1 LPS and vice versa. Hikojima is the third O1 serotype, which is found to be an unstable phenotype. ...
... The outcome is broadly in agreement with the endorsement of by WHO ECBS. 19 The next steps that are needed to produce the candidate ISs are: 1) Identify a producer for the three serotypes of V. cholerae O LPS a. Analyze the products for structural identity by nuclear magnetic resonance (NMR) and other appropriate methods. ...
Cholera causes an estimated 3 million cases of watery diarrhea and 100,000 deaths globally per year. Although the long-term solution for cholera control lies in universal access to safe drinking water and adequate sanitation, Oral Cholera Vaccines (OCVs) are the most cost-effective measure to contain and prevent the disease and are recommended by the World Health Organization (WHO) as part of an integrated strategy to control cholera. Currently, three OCVs are WHO prequalified and two of these are part of the cholera vaccine stockpile. Other OCVs have been developed for national use or are under development. Lipopolysaccharide (LPS) Inhibition enzyme-linked immunosorbent assays (ELISAs) are used by both manufacturers of OCVs and national regulatory agencies (NRAs) for: in process analysis, as a potency assay for drug substance and drug product batch release, and as an indicator of stability during the shelf life of OCVs. To ensure quality and consistency of this assay performed by different OCV manufacturers and NRAs, harmonization of assay reagents by the introduction of WHO International Standards (IS) for essential reagents is desirable. In May 2018, the International Vaccine Institute (IVI) with assistance from National Institute of Biological Standards and Control (NIBSC) and financial support from the Bill and Melinda Gates Foundation (BMGF) organized a meeting with representatives from vaccine manufacturers, NRAs, leading research institutions and independent experts to discuss and share experiences on potency assays for batch release and select reagents of the LPS Inhibition ELISA as candidate WHO ISs.
... LPS molecules are built from organic components of central carbon metabolism (the O1-antigen is synthesized from malate and D-fructose 6-phosphate) (31). LPS biosynthesis may be impacted by the metabolic state of the cell simply due to different resource availability in different conditions. ...
Bacteriophages (viruses of bacteria) play a pivotal role in shaping both the evolution and dynamics of bacterial populations. Bacteria employ arsenals of genetically encoded phage defense systems, but can alternatively achieve protection by changing the availability of cellular resources that phages rely on for propagation. These physiological changes are often adaptive responses to unique environmental signals. The facultative pathogen Vibrio cholerae adapts to both aquatic and intestinal environments with niche-specific physiological changes that ensure its evolutionary success in such disparate settings. In both niches, V. cholerae is susceptible to predation by lytic phages like ICP1. However, both phages and susceptible bacterial hosts coexist in nature, indicating that environmental cues may modulate V. cholerae cell state to protect against phage infection. This work explores one such modification in response to the intestine-specific signals of bile and anaerobicity. We found that V. cholerae grown in these conditions reduces O1-antigen decoration on its outer membrane lipopolysaccharide. Because the O1-antigen is an essential moiety for ICP1 phage infection, we investigated the effect of partial O1-antigen depletion as a mechanism of phage defense and observed that O1-depletion limits phage adsorption. We identified mechanistic contributions to O1-depletion, including the essentiality of a weak acid tolerance system for O1 production at low pH and alterations in transcriptional profiles indicating limitations in resources for O1-biosynthesis. This analysis illustrates a complex interplay between signals relevant to the intestinal environment and bacterial physiology that provides V. cholerae with protection from phage predation.
IMPORTANCE
Vibrio cholerae is the bacterial pathogen responsible for cholera, a diarrheal disease that impacts people in areas without access to potable water. In regions that lack such infrastructure, cholera represents a large proportion of disease outbreaks. Bacteriophages (phages, viruses that infect bacteria) have recently been examined as potential therapeutic and prophylactic agents to treat and prevent bacterial disease outbreaks like cholera due to their specificity and stability. This work examines the interaction between V. cholerae and vibriophages in consideration for a cholera prophylaxis regimen (M. Yen, L. S. Cairns, and A. Camilli, Nat Commun 8:14187, 2017, https://doi.org/10.1038/ncomms14187 ) in the context of stimuli found in the intestinal environment. We discover that common signals in the intestinal environment induce cell surface modifications in V. cholerae that also restrict some phages from binding and initiating infection. These findings could impact considerations for the design of phage-based treatments, as phage infection appears to be limited by bacterial adaptations to the intestinal environment.
... Since the endemic of Vibrio spp, phenotype variation is frequently used to determine or measure pathogenicity, intraspecies diversity by utilizing metabolizable substrates [45], colony morphotype [46], the presence of membrane proteins and lipopolysaccharide [47], extracellular enzymes such as cytolysins [48,49], siderophores [50], virulence in mice [51], and resistance to animal host defense systems [52,53], genetic divergence remain a prompt strategy for virulence determination. The preliminary phenotypic only provides appreciated evidence about the incidence and occurrence of phenotypic identities among V. strains. ...
Background
Vibrio species are among the autochthonous bacterial populations found in surface waters and associated with various life-threatening extraintestinal diseases, especially in human populations with underlying illnesses and wound infections. Presently, very diminutive information exists regarding these species’ mutational diversity of virulence and resistance genes. This study evaluated variations in endonucleases and mutational diversity of the virulence and resistance genes of Vibrio isolates, harboring virulence-correlated gene ( vcgCPI ), dihydropteroate synthase type 1 and type II genes ( Sul 1 and 11 ), (aadA) aminoglycoside (3′′) (9) adenylyltransferase gene, (aac(3)-IIa, (aacC2)a , aminoglycoside N(3)-acetyltransferase III, and ( strA ) aminoglycoside 3′-phosphotransferase resistance genes.
Methods
Using combinations of molecular biology techniques, bioinformatics tools, and sequence analysis.
Results
Our result revealed various nucleotide variations in virulence determinants of V. vulnificus ( vcgCPI ) at nucleotide positions (codon) 73–75 (A → G) and 300–302 (N → S). The aminoglycosides resistance gene ( aadA ) of Vibrio species depicts a nucleotide difference at position 482 (A → G), while the aminoglycosides resistance gene ( sul 1 and 11 ) showed two variable regions of nucleotide polymorphism (102 and 140). The amino acid differences exist with the nucleotide polymorphism at position 140 (A → E). The banding patterns produced by the restriction enzymes HinP1I , MwoI , and StyD4I showed significant variations. Also, the restriction enzyme digestion of protein dihydropteroate synthase type 1 and type II genes ( Sul 1 and 11 ) differed significantly, while enzymes DpnI and Hinf1 indicate no significant differences. The restriction enzyme NlaIV showed no band compared to reference isolates from the GenBank. However, the resistant determinants show significant point nucleotide mutation, which does not produce any amino acid change with diverse polymorphic regions, as revealed in the restriction digest profile.
Conclusion
The described virulence and resistance determinants possess specific polymorphic locus relevant to pathogenomics studies, pharmacogenomic, and control of such water-associated strains.
... LPS molecules are built from organic components of central carbon metabolism (the O1-antigen is synthesized from malate and D-fructose 6-phosphate) 31 . LPS biosynthesis may be impacted by the metabolic state of the cell simply due to different resource availability in different conditions. ...
Bacteriophages (viruses of bacteria) play a pivotal role in shaping both the evolution and dynamics of bacterial populations. Bacteria employ arsenals of genetically encoded phage defense systems, but can alternatively achieve protection by changing the availability of cellular resources that phages rely on for propagation. These physiological changes are often adaptive responses to unique environmental signals. The facultative pathogen Vibrio cholerae adapts to both aquatic and intestinal environments with niche-specific physiological changes that ensure its evolutionary success in such disparate settings. In both niches, V. cholerae is susceptible to predation by lytic phages like ICP1. However, both phages and susceptible bacterial hosts coexist in nature, indicating that environmental cues may modulate V. cholerae cell state to protect against phage infection. This work explores one such modification in response to the intestine-specific signals of bile and anaerobicity. We found that V. cholerae grown in these conditions reduces O1-antigen decoration on its outer membrane lipopolysaccharide. Because the O1-antigen is an essential moiety for ICP1 phage infection, we investigated the effect of partial O1-antigen depletion as a mechanism of phage defense and observed that O1-depletion limits phage adsorption. We identified mechanistic contributions to O1-depletion, including the essentiality of a weak acid tolerance system for O1 production at low pH, and alterations in transcriptional profiles indicating limitations in resources for O1-biosynthesis. This analysis illustrates a complex interplay between signals relevant to the intestinal environment and bacterial physiology that provide V. cholerae with protection from phage predation.
... The V. cholerae species is highly diverse, evidenced by the fact that over 200 serogroups of V. cholerae have been described [7,8]. In spite of this diversity, genomic evidence has shown that global pandemic cholera since 1961 has been caused by a single lineage of toxigenic serogroup O1 V. cholerae, dubbed 7PET, for seventh pandemic El Tor lineage [9,10]. ...
Between 1965 and 1968, outbreaks of cholera in Sudan and former Czechoslovakia provoked considerable public health concern. These still represent important historical events that need to be linked to the growing genomic evidence describing the aetiological agent of cholera, Vibrio cholerae. Whilst O1 serogroup V. cholerae are canonically associated with epidemic and pandemic cholera, these events were caused by a clone of toxigenic V. cholerae O37 that may be more globally distributed than just to Europe and North Africa. Understanding the biology of these non-O1 strains of V. cholerae is key to understanding how diseases like cholera continue to be globally important. In this article, we consolidate epidemiological, molecular and genomic descriptions of the bacteria responsible for these outbreaks. We attempt to resolve discrepancies in order to summarize the history and provenance of as many commonly used serogroup O37 strains as possible. Finally, we highlight the potential for whole-genome sequencing of V. cholerae O37 isolates from strain collections to shed light on the open questions that we identify.
... There are more than 200 serogroups of V. cholerae identified to date, but only serogroups, O1 and O139 are known to be responsible for pandemic cholera (Feeley, 1965, Chatterjee andChaudhuri, 2003). The O139 serogroup has alterations in both phenotypic and genetic characteristics when compared to the V. cholerae O1 serogroup that was responsible for previous epidemics (Swerdlow, 1993). ...
Vibrio species are natural inhabitants of aquatic environments and have complex interactions with the environment that drive the evolution of traits contributing to their survival. These traits may also contribute to their ability to invade or colonize animal and human hosts. In this review, we attempt to summarize the relationships of Vibrio spp. with other organisms in the aquatic environment and discuss how these interactions could potentially impact colonization of animal and human hosts.Keywords
Vibrio
PredationPathogen persistenceChitinPlankton
... Several extensive studies of model species have revealed the extent of O-antigen diversity. For instance, over 181 O-antigen forms have been identified in E. coli (33) and 46 in S. enterica (31,70), while there are over 208 forms in the Vibrio cholerae species (71). While these numbers are probably close to final for these speciesat least for isolates originating from the currently known niches, novel serotypes are frequently reported for other species due to increased whole genome sequencing efforts (72). ...
The O-antigen, a long polysaccharide that constitutes the distal part of the outer membrane-anchored lipopolysaccharide, is one of the critical components in the protective outer membrane of Gram-negative bacteria. Most species produce one of the structurally diverse O-antigens, with nearly all the polysaccharide components having complex structures made by the Wzx/Wzy pathway. This pathway produces repeat-units of mostly 3-8 sugars on the cytosolic face of the cytoplasmic membrane that is translocated by Wzx flippase to the periplasmic face and polymerized by Wzy polymerase to give long-chain polysaccharides. The Wzy polymerase is a highly diverse integral membrane protein typically containing 10-14 transmembrane segments. Biochemical evidence confirmed that Wzy polymerase is the sole driver of polymerization, and recent progress also began to demystify its interacting partner, Wzz, shedding some light to speculate how the proteins may operate together during polysaccharide biogenesis. However, our knowledge of how the highly variable Wzy proteins work as part of the O-antigen processing machinery remains poor. Here, we discuss the progress to the current understanding of repeat-unit polymerization and propose an updated model to explain the formation of additional short chain O-antigen polymers found in the lipopolysaccharide of diverse Gram-negative species and their importance in the biosynthetic process.
... 238 In addition, N-acylated forms of perosamine (9), the C-2 epimer of viosamine, are found in a number of microbial glycans. 239,240 This section will focus not only on previous syntheses of viosamine but also anthrose because straightforward modification of the routes used for the preparation of the latter could be applied to the viosamine derivative present in APMV glycans. ...
Viruses are a heterogeneous ensemble of entities, all sharing the need for a suitable host to replicate. They are extremely diverse, varying in morphology, size, nature, and complexity of their genomic content. Typically, viruses use host-encoded glycosyltransferases and glycosidases to add and remove sugar residues from their glycoproteins. Thus, the structure of the glycans on the viral proteins have, to date, typically been considered to mimick those of the host. However, the more recently discovered large and giant viruses differ from this paradigm. At least some of these viruses code for an (almost) autonomous glycosylation pathway. These viral genes include those that encode the production of activated sugars, glycosyltransferases, and other enzymes able to manipulate sugars at various levels. This review focuses on large and giant viruses that produce carbohydrate-processing enzymes. A brief description of those harboring these features at the genomic level will be discussed, followed by the achievements reached with regard to the elucidation of the glycan structures, the activity of the proteins able to manipulate sugars, and the organic synthesis of some of these virus-encoded glycans. During this progression, we will also comment on many of the challenging questions on this subject that remain to be addressed.
... Cholera is a disease caused by gram-negative Vibrio cholerae. So far, over 200 serogroups of V. cholerae strains have been identified (Chatterjee and Chaudhuri 2003). Among the strains of V. cholerae, two biochemical varieties have been distinguished: V. cholerae O1 classical type -the etiological factor of six pandemics -and V. cholerae type El Tor -the factor of the seventh most recent cholera pandemic (Harris et al. 2012;Hu et al. 2016). ...
In the second half of the 19th century, various quarters of Poznań differed in terms of infrastructure, including access to clean water. This paper aims to analyse whether these spatial and social inequalities related to the quarter of residence in the city, and thereby whether access to sanitary infrastructure and clean water intake, and in general, differences in living conditions, influenced the probability of death from cholera. Data from four cholera epidemics – 1852, 1855, 1866, and 1873 – were used for this purpose.
In total, 16,285 individual data entries from death registers of Catholic and Protestant parishes were used regarding such information as the date of death, sex, age at death, cause of death, profession, religion, and exact address of residence. There was a significant relationship between socioeconomic factors (quarter of residence, denomination, professions) and biological factors (sex and age at death), and the distributions of deaths due to cholera and other causes. Generalized Linear Models (GLMs) revealed that living in the Old Market Square did not decrease the chance of death from cholera, and on the other hand, living on the right bank of the Warta River did not
increase the chance of death from cholera. In other words, better quarter of residence did not guarantee lower morbidity and did not protect from cholera and vice versa. This work also proves a significant interaction between the quarter of residence and such variables as denomination and occupation on probability of death from specific cause. Virtually until the end of the 19th century, the sanitary conditions in Poznań were so poor that they were conducive to epidemics of infectious diseases
... The causative agent of cholera, Vibrio cholerae is antigenically diverse organism and based on antigenic diversity of their outer membrane lipopolysaccharides, 206 serogroups (O1-O206) have been identified (3,4). V. cholerae serogroup O1 is further classified into two biotypes, classical and El Tor, and two major serotypes, Ogawa and Inaba (5). ...
Background and Objectives: Information on the genetic epidemiology of cholera in Assam, a northeastern state of India is lacking despite cholera being a major public health problem. The study aimed to determine the virulence genes and genes encoding antibiotic resistance in Vibrio cholerae isolates and to determine the prevalent genotypes based on the presence or absence of the virulence genes and ctxB genotype.
Materials and Methods: Twenty-five V. cholerae strains were subjected to conventional biotyping and serotyping followed by multiplex PCR to detect ctxA, ctxB, zot, ace, O1rfb, tcpA, ompU, ompW, rtxC, hly and toxR and antibiotic resistance genes. Cholera toxin B (ctxB) gene was amplified followed by sequencing.
Results: All the V. cholerae O1 isolates were El Tor Ogawa and showed the presence of the core toxin region representing the genome of the filamentous bacteriophage CTXø. The complete cassette of virulence genes was seen in 48% of the isolates which was the predominant genotype. All the isolates possessed amino acid sequences identical to the El Tor ctxB subunit of genotype 3. sulII gene was detected in 68% of the isolates, dfrA1 in 88%, strB in 48% and SXT gene was detected in 36% of the isolates.
Conclusion: Toxigenic V. cholerae O1 El Tor Ogawa strains of ctxB genotype 3 carrying a large pool of virulence genes are prevailing in Assam. Presence of a transmissible genetic element SXT in 36% of the strains is of major concern as it indicates the emergence of multiple drug resistance among the V. cholerae isolates.
... is a diverse species, with more than 200 serogroups being identified based on their surface polysaccharide O antigen [6]. However, only O1 serogroup dominates the pandemic generating (PG) lineage [7,8], which has been responsible for all seven cholera pandemics, and other serogroups mostly represent V. cholerae environmental strains which are generally non-toxigenic [5,9]. ...
Cholera has been endemic to the Ganges Delta for centuries. Although the causative agent, Vibrio cholerae, is autochthonous to coastal and brackish water, cholera occurs continually in Dhaka, the inland capital city of Bangladesh which is surrounded by fresh water. Despite the persistence of this problem, little is known about the environmental abundance and distribution of lineages of V. cholerae, the most important being the pandemic generating (PG) lineage consisting mostly of serogroup O1 strains. To understand spatial and temporal dynamics of PG lineage and other lineages belonging to the V. cholerae species in surface water in and around Dhaka City, we used qPCR and high-throughput amplicon sequencing. Seven different freshwater sites across Dhaka were investigated for six consecutive months, and physiochemical parameters were measured in situ. Total abundance of V. cholerae was found to be relatively stable throughout the 6-month sampling period, with 2 × 10⁵ to 4 × 10⁵ genome copies/L at six sites and around 5 × 10⁵ genome copies/L at the site located in the most densely populated part of Dhaka City. PG O1 V. cholerae was present in high abundance during the entire sampling period and composed between 24 and 92% of the total V. cholerae population, only showing occasional but sudden reductions in abundance. In instances where PG O1 lost its dominance, other lineages underwent a rapid expansion while the size of the total V. cholerae population remained almost unchanged. Intraspecies richness of V. cholerae was positively correlated with salinity, conductivity, and total dissolved solids (TDS), while it was negatively correlated with dissolved oxygen (DO) concentration in water. Interestingly, negative correlation was observed specifically between PG O1 and salinity, even though the changes in this variable were minor (0–0.8 ppt). Observations in this study suggest that at the subspecies level, population composition of naturally occurring V. cholerae can be influenced by fluctuations in environmental factors, which can lead to altered competition dynamics among the lineages.
... In bacteria, the synthesis of glycoconjugates takes place in the series of glycosylation reactions, in which carbohydrates are polymerized or attached to the proteins or lipids, by the action of glycosyltransferase enzymes (GTs). Interestingly, bacterial glycans frequently contain unique monosaccharides such as pseudaminic acid (Pse; Schirm et al., 2003), bacillosamine (Bac; Morrison and Imperiali, 2014), 2,4-diacetamido-2,4,6trideoxygalactose (DATDG; Hartley et al., 2011), N-acetylfucosamine (FucNAc; Horzempa et al., 2008), legionaminic acid (Leg; Morrison and Imperiali, 2014), 3-deoxyd-manno-octulosonic acid (Kdo; Lodowska et al., 2013), rhamnose (Rha; Mistou et al., 2016), and others (Chatterjee and Chaudhuri, 2003;Meeks et al., 2004;Tytgat and Lebeer, 2014; Figure 1A). These carbohydrates are presented in the glycan structures of several clinically relevant pathogens (for instance, Helicobacter pylori, Neisseria meningitidis, Pseudomonas aeruginosa, Campylobacter jejuni, Escherichia coli, among others) and are often important for their virulence (Schirm et al., 2003;Horzempa et al., 2008;Hartley et al., 2011;Hopf et al., 2011;Clark et al., 2016). ...
Glycosylation is a ubiquitous process that is universally conserved in nature. The various products of glycosylation, such as polysaccharides, glycoproteins, and glycolipids, perform a myriad of intra- and extracellular functions. The multitude of roles performed by these molecules is reflected in the significant diversity of glycan structures and linkages found in eukaryotes and prokaryotes. Importantly, glycosylation is highly relevant for the virulence of many bacterial pathogens. Various surface-associated glycoconjugates have been identified in bacteria that promote infectious behavior and survival in the host through motility, adhesion, molecular mimicry, and immune system manipulation. Interestingly, bacterial glycosylation systems that produce these virulence factors frequently feature rare monosaccharides and unusual glycosylation mechanisms. Owing to their marked difference from human glycosylation, bacterial glycosylation systems constitute promising antibacterial targets. With the rise of antibiotic resistance and depletion of the antibiotic pipeline, novel drug targets are urgently needed. Bacteria-specific glycosylation systems are especially promising for antivirulence therapies that do not eliminate a bacterial population, but rather alleviate its pathogenesis. In this review, we describe a selection of unique glycosylation systems in bacterial pathogens and their role in bacterial homeostasis and infection, with a focus on virulence factors. In addition, recent advances to inhibit the enzymes involved in these glycosylation systems and target the bacterial glycan structures directly will be highlighted. Together, this review provides an overview of the current status and promise for the future of using bacterial glycosylation to develop novel antibacterial strategies.
... V. cholerae is ingested via contaminated drinking water or, less commonly, via undercooked seafood [11,12]. Once inside the human host, pathogenic varieties of V. cholerae (typically O1 and O139 serovars [13]) rely on virulence factors to establish infection; the toxin co-regulated pilus (TCP) facilitates attachment to the intestinal wall [14,15] and cholera toxin (CTX) secretion ultimately drives efflux of water and salts from the intestinal epithelium [16]. CTX additionally promotes nutrient competition via depletion of free (i.e., not heme-bound) iron in the intestine [17]. ...
Vibrio cholerae is the causative agent of cholera, a notorious diarrheal disease that is typically transmitted via contaminated drinking water. The current pandemic agent, the El Tor biotype, has undergone several genetic changes that include horizontal acquisition of two genomic islands (VSP-I and VSP-II). VSP presence strongly correlates with pandemicity; however, the contribution of these islands to V . cholerae ’s life cycle, particularly the 26-kb VSP-II, remains poorly understood. VSP-II-encoded genes are not expressed under standard laboratory conditions, suggesting that their induction requires an unknown signal from the host or environment. One signal that bacteria encounter under both host and environmental conditions is metal limitation. While studying V . cholerae ’s zinc-starvation response in vitro , we noticed that a mutant constitutively expressing zinc starvation genes (Δ zur ) congregates at the bottom of a culture tube when grown in a nutrient-poor medium. Using transposon mutagenesis, we found that flagellar motility, chemotaxis, and VSP-II encoded genes were required for congregation. The VSP-II genes encode an AraC-like transcriptional activator (VerA) and a methyl-accepting chemotaxis protein (AerB). Using RNA-seq and lacZ transcriptional reporters, we show that VerA is a novel Zur target and an activator of the nearby AerB chemoreceptor. AerB interfaces with the chemotaxis system to drive oxygen-dependent congregation and energy taxis. Importantly, this work suggests a functional link between VSP-II, zinc-starved environments, and energy taxis, yielding insights into the role of VSP-II in a metal-limited host or aquatic reservoir.
... The protective antibacterial antibodies are mainly, if not exclusively, directed against the O1 LPS [6]. The O1 LPS has a major group-specific epitope(s) "A" shared between the Inaba and Ogawa serotypes and an additional serotype-specific "B" (Ogawa) or "C" (Inaba) epitope; only a methyl group on the B epitope distinguishes Ogawa from the epitope C of Inaba [18]. Both cross-reactive and serotype-specific anti-LPS antibodies contribute to protection [6]. ...
Individual resistance to cholera infection and disease depends on both innate host factors and adaptive immunity acquired by a previous infection or vaccination. Locally produced, intestinal-mucosal secretory IgA (SIgA) antibodies against bacterial surface lipopolysaccharide (LPS) O antigens and/or secreted cholera toxins are responsible for the protective adaptive immunity, in conjunction with an effective mucosal immunologic memory that can elicit a rapid anamnestic SIgA antibody response upon re-exposure to the antigen/pathogen even many years later. Oral cholera vaccines (OCVs), based on inactivated Vibrio cholerae whole-cell components, either together with the cholera toxin B subunit (Dukoral™) or administered alone (Shanchol™/Euvichol-Plus™) were shown to be consistently safe and effective in large field trials in all settings. These OCVs are recommended by the World Health Organisation (WHO) for the control of both endemic cholera and epidemic cholera outbreaks. OCVs are now a cornerstone in WHO’s global strategy found in “Ending Cholera: A Global Roadmap to 2030.” However, the forecasted global demands for OCV, estimated by the Global Alliance for Vaccines and Immunization (GAVI) to 1.5 billion doses for the period 2020–2029, markedly exceed the existing manufacturing capacity. This calls for an increased production capacity of existing OCVs, as well as the rapid introduction of additional and improved vaccines under development.
... Seven cholera pandemics have struck human civilization so far; the rst pandemic of cholera started in 1817 and was followed by six others in the next two hundred years, leaving a devastating human death toll [5]. In the environment, V. cholerae is a diverse species, with more than 200 serogroups being identi ed based on their surface polysaccharide O antigen [6]. However, only O1 serogroup dominates the pandemic generating (PG) lineage [7,8], which has been responsible for all seven cholera pandemics, and other serogroups mostly represent V. cholerae environmental strains which are generally non-pathogenic [5,9]. ...
Cholera has been endemic to the Ganges delta for centuries. Although the causative agent, Vibrio cholerae, is autochthonous to coastal and brackish water, cholera occurs continually in Dhaka, the inland capital city of Bangladesh which is surrounded by fresh water. Despite the persistence of this problem, little is known about the environmental abundance and distribution of lineages of V. cholerae , the most important being the pandemic generating lineage (PG) consisting mostly of serogroup O1 strains. To understand spatial and temporal dynamics of PG and other lineages belonging to the V. cholerae species in surface water in and around Dhaka city, we used qPCR and high throughput amplicon sequencing. Seven different freshwater sites across Dhaka were investigated for six consecutive months and physiochemical parameters were measured in situ . Total abundance of V. cholerae was found to be relatively stable throughout the six months sampling period, with 2×10 ⁵ to 4×10 ⁵ genome copies/L at six sites and around 5 ×10 ⁵ genome copies/L at the site located in the most densely populated part of Dhaka city. PG O1 V. cholerae was present in high abundance during the entire sampling period and composed between 24-92% of the total V. cholerae population, only showing occasional but sudden reductions in abundance. In instances where PG O1 lost its dominance, other lineages underwent a rapid expansion while the size of the total V. cholerae population remained almost unchanged. Intraspecies richness of V. cholerae was positively correlated to salinity, conductivity and total dissolved solids (TDS), while it was negatively correlated to dissolved oxygen (DO) concentration in water. Interestingly, negative correlation was observed specifically between PG O1 and salinity, even though the changes in this variable were minor (0-0.8 ppt). Observations in this study suggest that at the subspecies level, population composition of naturally occurring V. cholerae can be influenced by fluctuations in environmental factors, which can lead to altered competition dynamics among the lineages.
... V. cholerae is represented by more than 200 serogroups that are endemic to sea and brackish waters and often found associated with copepods (5,6). However, only the O1 and O139 serogroups have been associated with cholera, the diarrheal disease in humans (7). ...
Intestinal mucus is the first line of defense against intestinal pathogens. It acts as a physical barrier between epithelial tissues and the lumen that enteropathogens must overcome to establish a successful infection. We investigated the motile behavior of two V. cholerae strains (El Tor C6706 and Classical O395) in mucus using single cell tracking in unprocessed porcine intestinal mucus. We determined that V. cholerae is able to penetrate mucus using flagellar motility and that alkaline pH increases swimming speed, and consequently, improves mucus penetration. Microrheological measurements indicate that changes in pH between 6 and 8 (the physiological range for the human small intestine) had little effect on the viscoelastic properties of mucus. Finally, we determined that acidic pH promotes surface attachment by activating the mannose-sensitive haemagglutinin (MshA) pilus in V. cholerae El Tor C6706 without a measurable change in the total cellular concentration of the secondary messenger cyclic dimeric guanosine monophosphate (c-di-GMP). Overall, our results support that pH is an important factor affecting the motile behavior of V. cholerae and its ability to penetrate mucus. Therefore, changes in pH along the human small intestine may play a role in determining the preferred site for V. cholerae during infection.
IMPORTANCE
The diarrheal disease cholera is still a burden for populations in developing countries with poor sanitation. To develop effective vaccines and prevention strategies against Vibrio cholerae, we must understand the initial steps of infection leading to the colonization of the small intestine. To infect the host and deliver the cholera toxin, V. cholerae has to penetrate the mucus layer protecting the intestinal tissues. However, the interaction of V. cholerae with intestinal mucus has not been extensively investigated. In this report, we demonstrated using single cell tracking that V. cholerae is able to penetrate intestinal mucus using flagellar motility. In addition, we observed that alkaline pH improves the ability of V. cholerae to penetrate mucus. This finding has important implications for understanding the dynamics of infection because pH varies significantly along the small intestine, between individuals, and between species. Blocking mucus penetration by interfering with flagellar motility in V. cholerae, reinforcing the mucosa, controlling intestinal pH, or manipulating the intestinal microbiome, will offer new strategies to fight cholera.
... There are more than 200 serotypes of V. cholera. 3 V. cholerae is a known aetiological agent of cholera, which causes severe outbreaks of diarrhoea. 4 V. cholerae is mainly transmitted through the oro-faecal route or after ingestion of contaminated water or food. ...
Objective:
To study the different epidemiological and polymerase chain reaction-based identification of vibrio cholera.
Methods:
The cross-sectional study was conducted at the Center for Advanced Studies in Vaccinology and Biotechnology, University of Balochistan, Quetta, Pakistan, from January 5 to December 6, 2019, and comprised faecal / rectal swab samples from patients with a history of untreated severe diarrhoea of <12-hour duration. The samples were collected from suspected cholera patients at different hospitals of the province. The isolates were examined and identified on the basis of colony characters on thiosulfate-citrate-bile salts-sucrose agar. Susppected colonies were subjected to gram staining, biochemical tests and polymerase chain reaction-based identification. Data was analysed using SPSS 19.
Results:
Of the 444 samples, 33(7.43%) were positive for vibrio cholera and 411(92.56%) were negative. The incidence was higher in individuals aged 1-20 years 12(2.7%); males 18(4.05%); Balochs 18(4.05%); lower socioeconomic class 18(4.05%); and illiterates 26(5.85%). The incidence was more in summer 19(4.27%) and spring 8(1.80%) seasons. Polymerase chain reaction was highly effective diagnostic approach, with findings showing clear bands of 588bp of ompW gene.
Conclusions:
Surveillance for diarrhoeal disorders is necessary to control future outbreaks of cholera in the region.
... The epidemic and pandemic cholera in various regions was mainly caused by V. cholerae serogroups O1 and O139 (Chatterjee & Chaudhuri, 2003;Sack, Sack, Nair, & Siddiqui, 2004;Siddiqui et al., 2006) producing the major virulence factor cholera toxin (CTX) and toxin-coregulated pilus (TCP) (Faruque et al., 2003). Out of the V. ...
A nanogold‐based immunochromatographic strip test (VCG strip test) for the detection of all 70 isolates of Vibrio cholerae including O1, O139, and non‐O1/O139 (NVCs) was developed using two monoclonal antibodies (MAbs), namely VC‐63 and VC‐201, which bound specifically to 10 and 15 kDa proteins. Direct detection of V. cholerae in experimentally spiked fresh seafood samples such as shrimp, blood clam, mussel, and oyster could be achieved with sensitivities of 10⁷ CFU/ml, which was similar to that of the dot blotting test using each MAb. The detection sensitivity could be improved to 10³ or 10 or 1 CFU/ml of original bacterial content after preincubation of the sample in alkaline peptone water for 6, 12, and 24 hr consecutively. However, the detection sensitivities were also dependent on the content of other bacteria in the sample that might compete or inhibit the growth of V. cholerae during the preenrichment step. Due to its broad specificity, simplicity and rapid result generation, the VCG strip test can be used for the preliminary detection of V. cholerae at point of care for both environmental and clinical strains to assist appropriate decision or epidemiological surveillance of virulent V. cholerae strains (including NVC strains), which are ubiquitous in the environment and seafood.
... The Inaba and Ogawa serotypes of V. cholerae O1 are commonly differentiated by diagnostic laboratories 38 and by epidemiologists 39 as well as in the formulation of cholera vaccines, such as Dukoral 40 , because both serotypes elicit different immunological responses 41 . Methylation of the terminal perosamine sugar on the O1 lipopolysaccharide chain by the WbeT enzyme confers an Ogawa phenotype; lack of methylation by disruption of wbeT confers an Inaba serotype [42][43][44] . There is evidence that reversion from Inaba to Ogawa serotype can occur in vivo, albeit rarely 43,45,46 . ...
In order to control and eradicate epidemic cholera, we need to understand how epidemics begin, how they spread, and how they decline and eventually end. This requires extensive sampling of epidemic disease over time, alongside the background of endemic disease that may exist concurrently with the epidemic. The unique circumstances surrounding the Argentinian cholera epidemic of 1992–1998 presented an opportunity to do this. Here, we use 490 Argentinian V. cholerae genome sequences to characterise the variation within, and between, epidemic and endemic V. cholerae. We show that, during the 1992–1998 cholera epidemic, the invariant epidemic clone co-existed alongside highly diverse members of the Vibrio cholerae species in Argentina, and we contrast the clonality of epidemic V. cholerae with the background diversity of local endemic bacteria. Our findings refine and add nuance to our genomic definitions of epidemic and endemic cholera, and are of direct relevance to controlling current and future cholera epidemics.
... The O-antigen or O-specific polysaccharide (OSP) of the V. cholerae O1 serogroup consists of (1->2)-linked moieties of 4-amino-4,6-dideoxy-alpha-D-mannopyranose (D-perosamine) in which the amino groups are acylated with 3-deoxy-L-glycero-tetronic acid (Kenne et al. 1982). Within the O1 serogroup, there are two serotypes, Ogawa and Inaba, that outer membrane (OM) via a conserved core/lipid A moiety (Chatterjee and Chaudhuri 2003). ...
Following an episode of cholera, a rapidly dehydrating, watery diarrhea caused by the Gram-negative bacterium, Vibrio cholerae O1, humans mount a robust anti-lipopolysaccharide (LPS) antibody response that is associated with immunity to subsequent re-infection. In neonatal mouse and rabbit models of cholera, passively administered anti-LPS polyclonal and monoclonal (MAb) antibodies reduce V. cholerae colonization of intestinal epithelia by inhibiting bacterial motility and promoting vibrio agglutination. Here we demonstrate that human anti-LPS IgG MAbs also arrest V. cholerae motility and induce bacterial paralysis. A subset of those MAbs also triggered V. cholerae to secrete an extracellular matrix (ECM). To identify changes in Downloaded from https://academic.oup.com/femspd/article-abstract/doi/10.1093/femspd/ftaa029/5863188 by Serials Section, Dixson Library user on 28 June 2020
... Within the O1 serogroup, there are two predominant serotypes, Ogawa and Inaba, that differ in the presence (Ogawa) or absence (Inaba) of methylation of the terminal non-reducing D-perosaminyl moiety. The OSP side chain is anchored to the outer leaflet of V. cholerae's outer membrane (OM) via a conserved core/lipid A moiety (Chatterjee and Chaudhuri 2003). ...
Following an episode of cholera, a rapidly dehydrating, watery diarrhea caused by the Gram-negative bacterium, Vibrio cholerae O1, humans mount a robust anti-lipopolysaccharide (LPS) antibody response that is associated with immunity to subsequent re-infection. In neonatal mouse and rabbit models of cholera, passively administered anti-LPS polyclonal and monoclonal (MAb) antibodies reduce V. cholerae colonization of intestinal epithelia by inhibiting bacterial motility and promoting vibrio agglutination. Here we demonstrate that human anti-LPS IgG MAbs also arrest V. cholerae motility and induce bacterial paralysis. A subset of those MAbs also triggered V. cholerae to secrete an extracellular matrix (ECM). To identify changes in gene expression that accompany antibody exposure and that may account for motility arrest and ECM production, we subjected V. cholerae O1 El Tor to RNA-seq analysis after treatment with ZAC-3 IgG, a high affinity MAb directed against core/lipid A region of LPS. We identified > 160 genes whose expression was altered following ZAC-3 IgG treatment, although canonical outer membrane stress regulons were not among them. Two upregulated genes of note were ompS (VCA1028), a porin associated with virulence and indirectly regulated by ToxT, and norR (VCA0182), a σ54-dependent transcription factor involved in late stages of infection.
... It is estimated that each year there are 1.3 to 4 million cholera cases, resulting in 21,000 to 143,000 deaths worldwide [2]. V. cholerae has been classified based on the surface somatic O antigens and more than 200 serogroups are identified to date [3]. Cholera epidemics are caused by isolates of serogroups O1 and O139, with O1 being further differentiated into two biotypes, classical (Cla) and El Tor (ET) [4]. ...
Non-toxigenic Vibrio cholerae isolates have been found associated with diarrheal disease globally, however, the global picture of non-toxigenic infections is largely unknown. Among non-toxigenic V. cholerae, ctxAB negative, tcpA positive (CNTP) isolates have the highest risk of disease. From 2001 to 2012, 71 infectious diarrhea cases were reported in Hangzhou, China, caused by CNTP serogroup O1 isolates. We sequenced 119 V. cholerae genomes isolated from patients, carriers and the environment in Hangzhou between 2001 and 2012, and compared them with 850 publicly available global isolates. We found that CNTP isolates from Hangzhou belonged to two distinctive lineages, named L3b and L9. Both lineages caused disease over a long time period with usually mild or moderate clinical symptoms. Within Hangzhou, the spread route of the L3b lineage was apparently from rural to urban areas, with aquatic food products being the most likely medium. Both lineages had been previously reported as causing local endemic disease in Latin America, but here we show that global spread of them has occurred, with the most likely origin of L3b lineage being in Central Asia. The L3b lineage has spread to China on at least three occasions. Other spread events, including from China to Thailand and to Latin America were also observed. We fill the missing links in the global spread of the two non-toxigenic serogroup O1 V. cholerae lineages that can cause human infection. The results are important for the design of future disease control strategies: surveillance of V. cholerae should not be limited to ctxAB positive strains.
... Most strains of V. cholerae are not pathogenic or only cause local outbreaks of gastroenteritis. Epidemic strains have so far been found in only 2 out of >200 possible somatic O-antigens serogroups (Chatterjee and Chaudhuri, 2003). Epidemic strains are characterized by the capacity to produce cholera toxin, which is responsible for the deadly diarrhoea associated with the disease, and Type IV toxincoregulated pili (TCP), which are essential for small intestine colonization (Herrington et al., 1988). ...
Sister-chromatid cohesion describes the orderly association of newly-replicated DNA molecules behind replication forks. It plays an essential role in the maintenance and the faithful transmission of genetic information. It is created by DNA topological links and proteinaceous bridges, whose formation and deposition could be potentially affected by many different DNA-binding proteins. However, a mean to analyse local variations in the duration of cohesion on a whole genome was lacking. Here, we present a High-throughput methodology to monitor Sister Chromatid Contacts (Hi-SC2), and show that it permits to analyse locus-specific variations in sister-chromatid cohesion over the whole length of chromosomes.
... According to statistical data from the World Health Organization, there are approximately 1.4-4.3 million cases of acute diarrheal diseases worldwide per year, including 28,000-142,000 deaths (Ali et al., 2012). At present, more than 200 serotypes of V. cholerae have been identified based on the different O antigenic lipopolysaccharides (Chatterjee & Chaudhuri, 2003). Only the O1 and O139 serogroups are associated with epidemic and pandemic cholera (Mutreja et al., 2011), while non-O1/non-O139 serogroups can cause sporadic local cholera outbreaks, mild and transient gastrointestinal diseases and extraintestinal infections (Aydanian et al., 2015). ...
Background
DegS is a periplasmic serine protease that is considered to be the initiator of the σ E stress response pathway, and this protein plays an important role in the regulation of the stress response in E. coli . However, knowledge of the biological function and global regulatory network of DegS in Vibrio cholerae remains limited. In this study, we aimed to characterize the molecular functions and further investigate the regulatory network of degS in V. cholerae .
Methods
A deletion mutant of degS was constructed in the V. cholerae HN375 strain. Bacterial colony morphology was observed by a plate-based growth experiment, and bacterial growth ability was observed by a growth curve experiment. High-throughput RNA sequencing (RNA-Seq) technology was used to analyze the differential transcriptomic profiles between the wild-type and degS mutant strains. Gene ontology (GO), pathway analysis and Gene-Act-network analysis were performed to explore the main functions of the differentially expressed genes. Quantitative real-time PCR (qRT-PCR) was performed to validate the reliability and accuracy of the RNA-Seq analysis. The complementation experiments were used to test the roles of degS and ropS in the small colony degS mutant phenotype.
Results
When degS was deleted, the degS mutant exhibited smaller colonies on various media and slower growth than the wild-type strain. A total of 423 differentially expressed genes were identified, including 187 genes that were upregulated in the degS mutant compared to the wild-type strain and 236 genes that were relatively downregulated. GO categories and pathway analysis showed that many differentially expressed genes were associated with various cellular metabolic pathways and the cell cycle. Furthermore, Gene-Act network analysis showed that many differentially expressed genes were involved in cellular metabolic pathways and bacterial chemotaxis. The cAMP-CRP-RpoS signaling pathway and the LuxPQ signal transduction system were also affected by the degS mutant. The expression patterns of nine randomly selected differentially expressed genes were consistent between the qRT-PCR and RNA-seq results. The complementation experiments showed that the small colony degS mutant phenotype could be partially restored by complementation with the pBAD24- degS or pBAD24- rpoS plasmid.
Discussion
These results suggest that the degS gene is important for normal growth of V. cholerae . Some of the differentially expressed genes were involved in various cellular metabolic processes and the cell cycle, which may be associated with bacterial growth. Several new degS -related regulatory networks were identified. In addition, our results suggested that the cAMP-CRP-RpoS signaling pathway may be involved in the small colony degS mutant phenotype. Overall, we believe that these transcriptomic data will serve as useful genetic resources for research on the functions of degS in V. cholerae .
Over the past 200 years since the moment of E. Jenner’s discovery, vaccination continues to be the leading strategy for protection against infectious diseases, but commercially available live attenuated and inactivated vaccines have a number of serious drawbacks. Bacterial strains should be completely attenuated in live vaccines, while maintaining a high degree of immunogenicity. However, the majority of attenuation methods currently used makes potential vaccine strains more susceptible to the action of various host defenses, reducing the ability to persist in the body of the vaccinated individual in quantities and for periods sufficient for formation of long-term and intense immunity. Inactivation of microorganisms underlying the production of killed vaccines, applying various reagents and /or physical factors, can disrupt the native conformation of antigenic epitopes located on bacterial cell surface, which leads to a decrease in immunogenicity. This review examines a promising biotechnological platform for the development of vaccines based on the methodology of regulated delayed gene expression and repression of genes, which was developed to resolve the above-mentioned contradictions.
Vibrio cholerae is a cholera-causing pathogen known to instigate severe contagious diarrhea that affects millions globally. Survival of vibrios depend on a combination of multicellular responses and adapt to changes that prevail in the environment. This process is achieved through a strong communication at the cellular level, the process has been recognized as quorum sensing (QS). The severity of infection is highly dependent on the QS of vibrios in the gut milieu. The quorum may exist in a low/high cell density (LCD/HCD) state to exert a positive or negative response to control the regulatory pathogenic networks. The impact of this regulation reflects on the transition of pathogenic V. cholerae from the environment to infect humans and cause outbreaks or epidemics of cholera. In this context, the review portrays various regulatory processes and associated virulent pathways, which maneuver and control LCD and HCD states for their survival in the host. Although several treatment options are existing, promotion of therapeutics by exploiting the virulence network may potentiate ineffective antibiotics to manage cholera. In addition, this approach is also useful in resource-limited settings, where the accessibility to antibiotics or conventional therapeutic options is limited.
The aim of the study is to analyze the variability of multiple antibiotic resistance caused by variations in the mobile SXT element and chromosomal gene mutations in various strains of the cholera agent isolated in Russia and endemic cholera foci. Based on bioinformatic analysis of the whole genome nucleotide sequences of 110 clinical strains of V. cholerae El Tor isolated in different regions, the structure of their integrative conjugative SXT elements was determined. Mutations in the chromosomal antibiotic-resistance genes gyrA, parC, and carR were identified. Phylogenetic analysis of strains containing different types of SXT was carried out. It was shown that the variability of multiple antibiotic resistance in the studied strains from Russia and Ukraine was associated with the presence in the chromosome of two types of SXT elements with different compositions of resistance genes and spontaneous chromosomal mutations in the gyrA, parC, and carR genes, which determine resistance to nalidixic acid and polymyxin B. At the same time, five types of SXT were found in the studied strains from Asian and African countries, which determined a higher level of variability of their antibiotic resistance. Based on SNP analysis, an assessment of the phylogenetic relationships of isolates with different types of SXT and different chromosomal mutations is given. The data obtained indicate that the variability of cholera-agent resistance to antibiotics is provided by different types of SXT and mutations in chromosomal genes. The revealed high level of SXT genomic diversity in the V. cholerae population in cholera-endemic regions indicates the threat of importation of new cholera-agent strains with previously unknown antibiotic resistance into Russia.
Lipid A plays an important role in the pathogenicity and antimicrobial resistance of Vibrio parahaemolyticus, but little is known about the structure and biosynthesis of lipid A in V. parahaemolyticus. In this study, lipid A species were either directly extracted or obtained by the acid hydrolysis of lipopolysaccharide from V. parahaemolyticus ATCC33846 cells and analyzed by thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry. Several lipid A species in V. parahaemolyticus cells were characterized, and two of these species were not connected to polysaccharides. One free lipid A species has the similar structure as the hexa-acylated lipid A in Escherichia coli, and the other is a hepta-acylated lipid A with an additional secondary C16:0 acyl chain. Three lipid A species were isolated by the acid hydrolysis of lipopolysaccharide: the 1st one has the similar structure as the hexa-acylated lipid A in E. coli, the 2nd one is a hepta-acylated lipid A with an additional secondary C16:0 acyl chain and a secondary 2-OH C12:0 acyl chain, and the 3rd one is equal to the 2nd species with a phosphoethanolamine modification. These results are important for understanding the biosynthesis of lipid A in V. parahaemolyticus.
Cholera, an acute secretory diarrhea, is caused by strains from a phylogenetically confined group within the Vibrio cholerae species, the pandemic cholera group (PCG). To date, the molecular and evolutionary factors that enable the isolated emergence of toxigenic V. cholerae from environmental populations remain mostly enigmatic. Comprehensive analyses of over 1,100 V. cholerae genomes, including novel environmental isolates from this study, reveal that the species consists of four major clades and several minor ones. PCG belongs to a large clade located within a lineage shared with environmental strains, the pandemic cholera lineage. This hierarchical classification provided us with a framework to unravel the eco-evolutionary dynamics of the genetic determinants associated with the emergence of toxigenic V. cholerae . Our analyses indicate that this phenomenon is largely dependent on the acquisition of unique modular gene clusters and allelic variations that confer a competitive advantage during intestinal colonization. We determined that certain PCG-associated alleles are essential for successful colonization whereas others provide a non-linear competitive advantage, acting as a critical bottleneck that elucidates the isolated emergence of PCG. For instance, toxigenic strains encoding non-PCG alleles of a) tcpF or b) a sextuple allelic exchange mutant for genes tcpA , toxT , VC0176 , VC1791 , rfbT and ompU , lose their ability to colonize the intestine. Interestingly, these alleles do not play a role in the colonization of model environmental reservoirs. Our study uncovers the evolutionary roots of toxigenic V. cholerae and offers a tractable approach for investigating the emergence of pathogenic clones within an environmental population.
SIGNIFICANCE
The underlying factors that lead to specific strains within a species to emerge as human pathogens remain mostly enigmatic. Toxigenic clones of the cholera agent, Vibrio cholerae , are encompassed within one phylogenomic clade, the pandemic cholera group (PCG). Here, we investigate the molecular and evolutionary factors that explain the confined nature of this group. Our analyses determined that the emergence of PCG is largely dependent on the acquisition of unique modular gene clusters and allelic variations that confer a competitive advantage during intestinal colonization. These allelic variations act as a critical bottleneck that elucidates the isolated emergence of PCG and provides a tractable blueprint for the study of the emergence of pathogenic clones within an environmental population.
Lipopolysaccharide (LPS) is for most but not all Gram-negative bacteria an essential component of the outer leaflet of the outer membrane. LPS contributes to the integrity of the outer membrane, which acts as an effective permeability barrier to antimicrobial agents and protects against complement-mediated lysis. In commensal and pathogenic bacteria LPS interacts with pattern recognition receptors (e.g LBP, CD14, TLRs) of the innate immune system and thereby plays an important role in determining the immune response of the host. LPS molecules consist of a membrane-anchoring lipid A moiety and the surface-exposed core oligosaccharide and O-antigen polysaccharide. While the basic lipid A structure is conserved among different bacterial species, there is still a huge variation in its details, such as the number, position and chain length of the fatty acids and the decoration of the glucosamine disaccharide with phosphate, phosphoethanolamine or amino sugars. New evidence has emerged over the last few decades on how this lipid A heterogeneity confers distinct benefits to some bacteria because it allows them to modulate host responses in response to changing host environmental factors. Here we give an overview of what is known about the functional consequences of this lipid A structural heterogeneity. In addition, we also summarize new approaches for lipid A extraction, purification and analysis which have enabled analysis of its heterogeneity.
Somatic antigen agglutinable type Vibrio cholerae of 1/139 (SAAT-Vc-1/139) members or O1/O139 Vibrio cholerae have been described by various investigators as choleragenic due to their increase pathogenic potential and production of choleragen. Reported cholera outbreak cases around the World have been associated with these choleragenic Vibrio cholerae with high case fatality involving various educational, human, governmental, animal and financial resources. These Vibrio members have shown genealogical and phylogenetic relationship with the somatic antigen non-agglutinable strains of 1/139 Vibrio cholerae (SANAS- Vc -1/139) or O1/O139 non-agglutinating Vibrio cholerae (O1/O139-NAG-Vc). The O1/O139-NAGVc members have been reported to be implicated in most cholera/cholera-like cases, sporadic cases, diarrhea, production of cholera toxin and transmitted via consumption and/or contact with contaminated water/seafood. Some reported cases of cholera outbreaks, sporadic cases and observed change in nature has also been tracable to these non-agglutinable Vibrio members (O1/O139-NAGVc) yet there is a sustained paucity of reports on the non-agglutinable V. cholerae members implication in cholera outbreak. The emergence of fulminating extraintestinal and systemic Vibriosis is another aspect of SANAS- Vc -1/139 involvement which has received low attention in terms of research driven interest. This review addresses the need to appraise and continue in research based studies on the somatic antigen non-serogroup agglutinable type-1/139 Vibrio cholerae members which are currently prevalent in water bodies, fruits/vegetables, foods and terrestrial environment. Our opinion is a summative of interest in integrated surveillance studies, management/control of cholera outbreaks as well as diarrhea and other disease related cases both in the rural, suburban and urban metropolis.
Somatic antigen agglutinable type-1/139 Vibrio cholerae (SAAT-1/139-Vc) members or O1/O139 V. cholerae have been described by various investigators as pathogenic due to their increasing virulence potential and production of choleragen. Reported cholera outbreak cases around the world have been associated with these choleragenic V. cholerae with high case fatality affecting various human and animals. These virulent Vibrio members have shown genealogical and phylogenetic relationship with the avirulent somatic antigen non-agglutinable strains of 1/139 V. cholerae (SANAS-1/139- Vc) or O1/O139 non-agglutinating V. cholerae (O1/O139-NAG-Vc). Reports on implication of O1/O139-NAGVc members in most sporadic cholera/cholera-like cases of diarrhea, production of cholera toxin and transmission via consumption and/or contact with contaminated water/seafood are currently on the rise. Some reported sporadic cases of cholera outbreaks and observed change in nature has also been tracable to these non-agglutinable Vibrio members (O1/O139-NAGVc) yet there is a sustained paucity of research interest on the non-agglutinable V. cholerae members. The emergence of fulminating extraintestinal and systemic vibriosis is another aspect of SANAS-1/139- Vc implication which has received low attention in terms of research driven interest. This review addresses the need to appraise and continually expand research based studies on the somatic antigen non-serogroup agglutinable type-1/139 V.cholerae members which are currently prevalent in studies of water bodies, fruits/vegetables, foods and terrestrial environment. Our opinion is amassed from interest in integrated surveillance studies, management/control of cholera outbreaks as well as diarrhea and other disease-related cases both in the rural, suburban and urban metropolis.
The rapid advancement of next‐generation sequencing technologies has promoted the use of transcriptomics in non‐model organisms, including species in the aquaculture sector. Compared to that of other crustacean species, portunid crab aquaculture is impeded by the insufficient knowledge of their biological and physiological processes. This systematic review summarised the advances in transcriptomics in cultured portunid crabs using a systematic literature review methodology. The filtered transcriptome dataset comprised 66 articles from four genera: Scylla, Portunus, Callinectes and Charybdis. At the species level, Scylla paramamosain and Portunus trituberculatus were the two most studied species, highlighting their importance in the aquaculture sector. The affordable cost of RNA sequencing (RNA‐Seq) led to an increase in transcriptome‐related research in portunid crabs and made available a huge repertoire of differentially expressed genes (DEGs) and single nucleotide polymorphisms (SNPs). We further discussed the transcriptomic advances based on six main functional categories, that is, ‘growth and moulting’, ‘gonadal development and reproduction’, ‘nutrition metabolism’, ‘disease and immunity’, ‘toxicology and stress’ and ‘general transcriptomic profiling’. In general, transcriptomic studies of portunid crabs, specifically the DEGs and pathways allow an in‐depth understanding of the biological and physiological processes involved at different growth stages or under various conditions based on the difference in gene transcriptional activity. SNPs obtained from the transcriptome data are useful in many genetic improvement‐related downstream applications, including the construction of genetic linkage maps and as population genetic markers. Future directions, such as hybrid approaches of long‐read and common RNA‐Seq, and the incorporation of other omics were also discussed.
Various Gram-negative bacteria possess a specialized membrane-bound protein secretion system known as the Type III secretion system (T3SS), which transports the bacterial effector proteins into the host cytosol thereby helping in bacterial pathogenesis. The T3SS has a special needle-like translocon that can sense the contact with the host cell membrane and translocate effectors. The export apparatus of T3SS recognizes these effector proteins bound to chaperones and translocates them into the host cell. Once in the host cell cytoplasm, these effector proteins result in modulation of the host system and promote bacterial localization and infection. Using molecular biology, bioinformatics, genetic techniques, electron microscopic studies, and mathematical modelling, the structure and function of the T3SS and the corresponding effector proteins in various bacteria have been studied. The strategies used by different human pathogenic bacteria to modulate the host system and thereby enhancing their virulence mechanism using T3SS have also been well studied. Here we review the history, evolution, and general structure of the T3SS highlighting the details on its comparison with the flagellar export machinery. Also, this article provides mechanistic details about the common role of T3SS in subversion and manipulation of host cellular processes. Additionally, this review describes specific T3SS apparatus and the role of their specific effectors in bacterial pathogenesis by considering several human and animal pathogenic bacteria.
Vibrio cholerae is the causative agent of cholera, a notorious diarrheal disease that is typically transmitted via contaminated drinking water. The current pandemic agent, the El Tor biotype, has undergone several genetic changes that include horizontal acquisition of two genomic islands (VSP-I and VSP-II). VSP-I and -2 presence strongly correlates with pandemicity; however, the contribution of these islands to V. cholerae ’s life cycle, particularly the 26-kb VSP-II, remains poorly understood. VSP-II-encoded genes are not expressed under standard laboratory conditions, suggesting that their induction requires an unknown signal from the host or environment. One signal that bacteria encounter under both host and environmental conditions is metal limitation. While studying V. cholerae ’s zinc-starvation response in vitro , we noticed that a mutant constitutively expressing zinc-starvation genes (Δ zur ) aggregates in nutrient-poor media. Using transposon mutagenesis, we found that flagellar motility, chemotaxis, and VSP-II encoded genes are required for aggregation. The VSP-II genes encode an AraC-like transcriptional activator (VerA) and a methyl-accepting chemotaxis protein (AerB). Using RNA-seq and lacZ transcriptional reporters, we show that VerA is a novel Zur target and activator of the nearby AerB chemoreceptor. AerB interfaces with the chemotaxis system to drive oxygen-dependent autoaggregation and energy taxis. Importantly, this work suggests a functional link between VSP-II, zinc-starved environments, and aerotaxis, yielding insights into the role of VSP-II in a metal-limited host or aquatic reservoir.
Author Summary
The Vibrio Seventh Pandemic island was horizontally acquired by El Tor pandemic strain, but its role in pathogenicity or environmental persistence is unknown. A major barrier to VSP-II study was the lack of stimuli favoring its expression. We show that zinc starvation induces expression of this island and describe a transcriptional network that activates a VSP-II encoded aerotaxis receptor. Importantly, aerotaxis may enable V. cholerae to locate more favorable microenvironments, possibly to colonize anoxic portions of the gut or environmental sediments.
Lipopolysaccharides (LPS) present in the outer leaflet of Gram-negative bacterial outer membranes protect the bacteria from external threats and influence antibiotic permeability as well as immune system recognition. The structure of lipid A, the anchor of an LPS molecule to the outer membrane, can make direct influences on membrane properties. Particularly, in Vibrio cholerae, a Gram-negative bacterium responsible for cholera, a severe diarrheal disease, modifications of lipid A structures grant antibiotic resistance and are a primary factor that led to the current cholera pandemic. However, the difference in structural properties incurred by such modifications has not been fully explored. In this work, five symmetric bilayer systems comprised of distinct lipid A structures of Vibrio cholerae LPS with O1 O-antigen were modeled and simulated to explore influences of different lipid A types on membrane properties. All-atom molecular dynamics simulations reveal that membrane properties such as hydrophobic thickness, acyl chain order parameter, and area per lipid are largely impacted by lipid A modifications due to differences in composition and acyl chain distortions. The modified lipid A is also less negatively charged, which possibly reveals a resistance mechanism to cationic antimicrobial peptide evasion. These findings present a possible explanation for Vibrio cholerae's immune system evasion properties and establish the differences between the lipid A types, which should be of use for any future study of the Gram-negative bacteria.
In order to cause disease, pathogenic strains of Vibrio cholerae rely on intricate regulatory networks to orchestrate the transition between their native aquatic environment and the human host. For example, bacteria in a nutrient-starved environment undergo a metabolic shift called the stringent response, which is mediated by the alarmone ppGpp and an RNA-polymerase binding transcriptional factor, DksA. In O1 serogroup strains of V. cholerae, which use the toxin co-regulated pilus (TCP) and cholera toxin (CT) as primary virulence factors, DksA was reported to have additional functions as a mediator of virulence gene expression. However, little is known about the regulatory networks coordinating virulence phenotypes in pathogenic strains that use TCP/CT-independent virulence mechanisms. We therefore investigated whether functions of DksA outside of the stringent response are conserved in type three secretion system (T3SS)-positive V. cholerae . In using the T3SS-positive clinically isolated O39 serogroup strain AM-19226, we observed an increase in dksA expression in the presence of bile at 37 °C. However, DksA was not required for wild-type levels of T3SS structural gene expression, or for colonization in vivo . Rather, data indicate that DksA positively regulates the expression of master regulators in the motility hierarchy. Interestingly, the Δ dksA strain forms a less robust biofilm than the WT parent strain at both 30 and 37 °C. We also found that DksA regulates the expression of hapR , encoding a major regulator of biofilm formation and protease expression. Athough DksA does not appear to modulate T3SS virulence factor expression, its activity is integrated into existing regulatory networks governing virulence-related phenotypes. Strain variations therefore may take advantage of conserved ancestral proteins to expand regulons responding to in vivo signals and thus coordinate multiple phenotypes important for infection.
Of over 200 different identified Vibrio cholerae serogroups only the O1 serogroup is consistently associated with endemic and epidemic cholera disease. The O1 serogroup has two serologically distinguishable variants, the Ogawa and Inaba serotypes, which differ only by a methyl group present on the terminal sugar of the Ogawa O-antigen but absent from Inaba strains. This methylation is catalyzed by a methyltransferase encoded by the wbeT gene, which in Inaba strains is disrupted by mutation. It is currently thought that there is little difference between the two serotypes. However, here we show, using isogenic pairs of O1 El Tor V. cholerae , that Inaba strains show significantly different patterns of gene expression and are significantly less able than the corresponding Ogawa strains to cause cholera in an infant mouse infection model. Our results suggest that changes in gene expression resulting from the loss of the wbeT gene lead to reduced virulence and possibly also reduced survival fitness outside the human host.
Author Summary
The bacterium Vibrio cholerae causes the pandemic diarrheal disease cholera. Despite many identified serotypes of V. cholerae only one, O1, causes pandemic cholera. The O1 serotype of pandemic V. cholerae has two distinguishable variants (called Ogawa and Inaba) long considered to be clinically and epidemiologically equivalent. Cholera outbreaks consist only of one the two variants at any time. In general, Ogawa strains cause the majority of outbreaks with relatively short-lived Inaba outbreaks occurring sporadically. We have suggested earlier that Inaba outbreaks occur during periods of environmental selective pressure against the Ogawa serotype. We demonstrate here that the two variants are not clinically equivalent. The Ogawa serotype is better able to respond to infection in an animal model by up regulating the expression of virulence genes essential for disease development. We suggest that this phenomenon is the result of wider ranging differences in gene expression resulting from the mutation that converts Ogawa into Inaba strains, and may help to explain the dominance of the Ogawa serotype in nature.
Vibrio cholerae, causing the life-threatening diarrheal disease cholerae, can be divided into different serogroups based on the structure of lipopolysaccharide (LPS) that consists of lipid-A, core-polysaccharide and O-antigen polysaccharide (O-PS). The O1 serogroup, the predominant cause of cholera, includes two major serotypes, Inaba and Ogawa. These serotypes are differentiated by the presence of a single 2-O-methyl group in the upstream terminal perosamine of the Ogawa O-PS, which is absent in the Inaba O-PS. To ensure the consistent quality and efficacy of the current cholera vaccines, accurate measurement and characterization of each of these two serotypes is highly important. In this study, we efficiently screened a phage-displayed human synthetic Fab library by bio-panning against Ogawa LPS and finally selected three unique mAbs (D9, E11 and F7) that specifically reacts with Ogawa LPS. The mAbs bound to Vibrio cholerae vaccine in a dose-dependent fashion. Sequence and structure analyses of antibody paratopes suggest that IgG D9 might have the same fine specificity as that of the murine mAbs, which were shown to bind to the upstream terminal perosamine of Ogawa O-PS, whereas IgGs F7 and E11 showed some different characteristics in the paratopes. To our knowledge, this study is the first to show the generation of Ogawa-specific mAbs using phage display technology. The mAbs will be useful for identification and quantification of Ogawa LPS in multivalent V. Cholerae vaccines.
Sister-chromatid cohesion describes the orderly association of newly replicated DNA molecules behind replication forks. It plays an essential role in the maintenance and faithful transmission of genetic information. Cohesion is created by DNA topological links and proteinaceous bridges, whose formation and deposition could be potentially affected by many processes. Current knowledge on cohesion has been mainly gained by fluorescence microscopy observation. However, the resolution limit of microscopy and the restricted number of genomic positions that can be simultaneously visualized considerably hampered progress. Here, we present a high-throughput methodology to monitor sister-chromatid contacts (Hi-SC2). Using the multi-chromosomal Vibrio cholerae bacterium as a model, we show that Hi-SC2 permits to monitor local variations in sister-chromatid cohesion at a high resolution over a whole genome.
Intestinal mucus is the first line of defense against intestinal pathogens. It acts as a physical barrier between the epithelial tissues and luminal microbes. Enteropathogens, such as Vibrio cholerae , must compromise or circumvent the mucus barrier to establish a successful infection. We investigated how motile V. cholerae is able to penetrate mucus using single cell tracking in unprocessed porcine intestinal mucus. We found that changes in pH within the range of what has been measured in the human small intestine indirectly affect V. cholerae flagellar motor torque, and consequently, mucus penetration. Microrheological measurements indicate that the viscoelasticity of mucus does not change substantially within the physiological pH range and that commercially available mucins do not form gels when rehydrated. Finally, we found that besides the reduction in motor torque, El Tor and Classical biotypes have different responses to acidic pH. For El Tor, acidic pH promotes surface attachment that is mediated by activation of the mannose-sensitive haemagglutinin (MshA) pilus without a measurable change in the total cellular concentration of the secondary messenger cyclic dimeric guanosine monophosphate (c-di-GMP). Overall, our results support that the high torque of V. cholerae flagellar motor is critical for mucus penetration and that the pH gradient in the small intestine is likely an important factor in determining the preferred site of infection.
Author summary
The diarrheal disease cholera is still a burden for populations in developing countries with poor sanitation. To develop effective vaccines and prevention strategies against Vibrio cholerae , we must understand the initial steps of infection leading to the colonization of the small intestine. To infect the host and deliver the cholera toxin, V. cholerae has to penetrate the mucus layer protecting the intestinal tissues. However, V. cholerae’s interactions with intestinal mucus has not been extensively investigated. In this report, we demonstrate using single cell tracking that V. cholerae is able to penetrate native intestinal mucus using flagellar motility. In addition, we found that a strong motor torque is required for mucus penetration and, that torque is weakened in acidic environments even though the motor is powered by a sodium potential. This finding has important implications for understanding the dynamics of infection because pH varies significantly along the small intestine, between individuals, and between species. Blocking mucus penetration by interfering with V. cholerae’s flagellar motility, reinforcing the mucosa, controlling intestinal pH, or manipulating the intestinal microbiome, will offer new strategies to fight cholera.
Synthesis of the upstream terminal hexasaccharide part of the lipopolysaccharides (LPS) of Vibrio cholerae O1, serotype Inaba has been improved. The key improvements include but are not limited to optimized conditions for the stereoselectivity of glycosylation reactions involved and fewer number of synthetic steps, compared to previous approaches. Particularly noteworthy is conducting the glycosylation of the very reactive glycosyl acceptor 8-azido-3,6-dioxaoctanol with the fully assembled hexasaccharide trichloroacetimidate under thermodynamic control. It produced the desired α glycoside with an α : β ratio of 7 : 1, compared with the ratio of 1.1 : 1, observed when the coupling was conducted conventionally. Several substances, which were previously obtained in purity acceptable only for synthetic intermediates, were now obtained in the analytically pure state and were fully characterized. The structure of the key trisaccharide glycosyl acceptor was confirmed by single-crystal X-ray structure determination.
Es werden zwei einfache Verfahren zur Extraktion von Bakterien mit Phenol/Wasser angegeben. Nach Behandlung von gramnegativen Bakterien mit Phenol/Wasser-Emulsionen in der Kälte während weniger Minuten erhält man in der wäßrigen Phase die somatischen Glykoproteide der Bakterien (hauptsächlich 0-Antigene) in praktisch quantitativer Ausbeute. Nach Extraktion der Bakterien mit erwärmten, homogenen Phenol/Wasser-Mischungen und Trennen der Phasen in der Kälte finden sich in der wäßrigen Phase die proteinfreien Polysaccharide neben Nucleinsäuren. Einige chemische und immunologische Eigenschaften der nach den beiden Verfahren dargestellten Glykoproteide und Polysaccharide werden beschrieben.
Monoclonal, murine IgG1s S-20-4, A-20-6, and IgA 2D6, directed against Vibrio cholerae O:1 Ogawa-lipopolysaccharide exhibited the same fine specificities and similar affinities for the synthetic methyl α-glycosides of the (oligo)saccharide fragments mimicking the Ogawa O-polysaccharide (O-PS). They did not react with the corresponding synthetic fragments of Inaba O-PS. IgG1s S-20-4 and A-20-6 have absolute affinity constants for synthetic Ogawa mono- to hexasaccharides of from ∼10⁵ to ∼10⁶m ⁻¹. For IgG1s S-20-4, A-20-6, and IgA 2D6, the nonreducing terminal residue of Ogawa O-PS is the dominant determinant, accounting for ∼90% of the maximal binding energy shown by these antibodies. Binding studies of derivatives of the Ogawa monosaccharide and IgGs S-20-4 and A-20-6 revealed that the C-2O-methyl group fits into a somewhat flexible antibody cavity and that hydrogen bonds involving the oxygen and, respectively, the OH at the 2- and 3-position of the sugar moiety as well as the 2′-position in the amide side chain are required.
Monoclonal IgA ZAC-3 and IgG3 I-24-2 are specific forV. cholerae O:1 serotypes Ogawa/Inaba-LPS.¹ The former did not show binding with members of either series of the synthetic ligands related to the O-antigens of the Ogawa or Inaba serotypes, in agreement with its reported specificity for the lipid/core region (1). Inhibition studies revealed that the binding of purified IgG3 I-24-2 to Ogawa-LPS might be mediated by a region in the junction of the OPS to the lipid-core region of the LPS.
cDNA cloning and analysis of the anti-Ogawa antibodies S-20-4, A-20-6, and 2D6 revealed a very high degree of homology among the heavy chains. Among the light chains, no such homology between S-20-4 and A-20-6 on the one hand, and 2D6 on the other hand, exists. For the anti-Inaba/Ogawa antibodies I-24-2 and ZAC-3, their heavy chains are completely different, with some homology among the light chains.
The structure of lipopolysaccharide from a heptose-less mutant of Escherichia coli K-12 has been investigated. Lipopolysaccharide isolated from 32P-labeled cells was treated with mild alkali to yield two separable components: [OH-LPS]-I (approximately 70%) and [OH-LPS]-II (approximately 30%). Mild acidic treatment of [OH-LPS]-I gave mainly a product which was identified as (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine 1-phosphate (Compound I). Further acidic hydrolysis of both [OH-LPS]-I and [OH-LPS]-II yielded as the main product (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine (Compound II). The structures of the above products were deduced by a combination of compositional analyses, sensitivity to phosphomonoesterase, rates of hydrolysis of the phosphate groups and alkali-catalyzed beta elimination of the phosphate residues following appropriate oxidation of hydroxyl groups. These studies together with work reported in the accompanying papers have led to the identification of two species of lipopolysaccharide in the E. coli strain both of which contain a single glucosamine dissacharide unit but differ in having monosubstituted phosphate or pyrophosphate groups at the glycosidic position. Each species of lipopolysaccharide also appeared to be heterogeneous with respect to the number of esterified fatty acyl groups.
Non-O1 Vibrio cholerae produced two distinct colony types, designated as opaque and translucent. NRT36S, a clinical isolate shown to be virulent in volunteers, produced predominantly opaque colonies, but translucent colonies appeared on subculture. Opaque variants were recovered exclusively following exposure to normal human serum or animal passage. A nonreverting translucent mutant of NRT36S, JVB52, was isolated following mutagenesis with the transposon Tn5 IS50L::phoA (TnphoA). Only translucent colonies were produced by a nonpathogenic environmental isolate, A5. Electron microscopic examination of the opaque form of NRT36S revealed thick, electron-dense, fibrous capsules surrounding polycationic ferritin-stained cells. The ferritin-stained material around translucent NRT36S or A5 was patchy or absent. JVB52 had a thin but contiguous capsular layer. The amount of ferritin-stained capsular material correlated with the amount of surface polysaccharide determined by phenol-sulfuric acid assay: opaque NRT36S had approximately three times as much polysaccharide as translucent NRT36S or A5 and four times as much as JVB52. The encapsulated, opaque variant of NRT36S was protected from serum bactericidal activity, while translucent non-O1 V. cholerae was readily killed. The encapsulated form also had increased virulence in mice. Our data provide the first indication that non-O1 V. cholerae strains can have a polysaccharide capsule. This capsule may be important in protecting the organism from host defenses and may contribute to the ability of some non-O1 V. cholerae strains to cause septicemia in susceptible hosts.
Monoclonal antibodies with group and type specificity for lipopolysaccharide antigens were used in combination with protein A-colloidal gold labeling and transmission electron microscopy to demonstrate the presence of lipopolysaccharide antigens on both the sheathed flagellum and the cell surface of Inaba and Ogawa strains of Vibrio cholerae O:1. Labeling was associated with the sheath of the flagellum rather than the core, and flagellar cores were not labeled. Flagellum and cell shared a common set of lipopolysaccharide antigens characteristic of the strain serotype.
Extensive studies on the chemistry of 0-antigenic lipopolysaccharides (LPS) of Enterobacteriaceae have been made (52), and chemotaxonomy based on the sugar composition of LPS was subsequently established for Salmonella and Escherichia coli (22, 24). In contrast, very few studies have been reported with Vibrionaceae from the chemotaxonomical point of view on the basis of the sugar composition of LPS while the chemical properties of LPS from V. cholerae (14, 15, 16, 17, 18, 29, 30, 35, 37, 39, 40) have been relatively well documented. The sugar composition of 0-antigenic LPS from a representative strain of each of 12 0-serotypes of V. parahaemolyticus was elucidated recently by two research groups, Hisatsune and his colleagues (12) and Miwatani’s group (26). Both groups divided V. parahaemolyticus in almost the same fashion, the former into nine and the latter into eight chemotypes. With regard to 0-antigenic LPS from
members of Vibrionaceae other than V. cholerae as well as V. parahaemolyticus, two comparative studies of the sugar composition of LPS were recently carried out, one on group F vibrios and Aeromonas (19) and the other on V. alginolyticus and V. parahaemolyticus (13). Through the latter study, it was found that 2-ket-3-deoxyoctonate (KDO), known generally as a regular component sugar present in gramnegative bacterial LPS, is lacking in the LPS of both V. alginolyticus and V. parahaemolyticus with the exception of V. parahaemolyticus 06 LPS. Furthermore, some KDO-like thiobarbituric acid test-positive substances were shown to be present in LPS from V. parahaemolyticus 07 and 012, and also in three strains of V. alginolyticus.
Members of the genus Vibrio are natural inhabitants of the estuarine and sea environments. Until 1960, only the cholera vibrio was recognized as a human pathogen. Cholera vibrio was first found by Pacini in 1854 in the intestinal contents of patients who had died from cholera and it was given the name Vibrio cholera [sic]. In 1906, Gotschlich isolated organisms closely resembling but not identical to cholera vibrios in their hemolytic activity from pilgrims at El Tor in Sinai. Those hemolytic choleralike vibrios were called V. eltor for many years, but are now included as variants of V. cholerae because they do not differ sufficiently from the latter.
Vibrio cholerae is divided into more than 130 O serogroups; however, only organisms of the O1 serogroup have so far been associated with cholera in humans. V. cholerae O1 strains of both biotypes have been further subdivided into three serotypes, designated Inaba, Ogawa, and Hikojima, grouped according to the structure of the O antigens on the lipopolysaccharide (LPS). The LPS of gram-negative bacteria is the most abundant molecule on the cell surface, where it provides a protective barrier to hydrophobic agents and detergents. The most common sugars found in the O polysaccharide are perosamine and quinovosamine. A number of studies designed to correlate the various O-antigen polysaccharides with particular antigenic specificities have been carried out. The genes involved in O-antigen biosynthesis in V. cholerae O1 strains 569B (Inaba, classical) and O17 (Ogawa, EI Tor) have been cloned and expressed in Escherichia coli K-12. The E. coli RfaD is an ADP-L-glycero-D-mannoheptose epimerase and is one of the critical proteins involved in the synthesis of the core oligosaccharide of the LPS in E. coli. Genetic complementation studies have suggested that the determinant responsible for Ogawa specificity lies at the distal end of the rfb region, an area in which no readily detectable differences could be discerned.
Fifty-seven characteristics of the proposed neotype strains of Vibrio cholerae Pacini 1854 and Vibrio eltor Pribram 1933 are compared. These organisms are so similar that they should be included in the same species under the name which has priority, Vibrio cholerae. Pribram (1933) appears to have been the first to publish validly the name V. eltor.
A comparative study was carried out on the sugar composition of lipopolysaccharides (LPS) isolated from representative strains of members of the family Vibrionaceae including all of the constituting genera, i.e., Vibrio, Aeromonas, Photobacterium, Plesiomonas , and Lucibacterium . More than 100 strains were examined. It was found that, with the exception of Vibrio parahaemolyticus 06, 2‐keto‐3‐deoxyoctonate (KDO), known generally as a component sugar in the core region of usual gram‐negative bacterial LPS, is virtually absent from LPS of the Vibrionaceae strains so far examined. Furthermore, mannose was also lacking in LPS of Vibrionaceae strains with the exception of only one strain, A. anaerogenes (ATCC 15467). Instead, some KDO‐like substances were found in LPS from Vibrio ( “Beneckea” ) nereida (ATCC 25917) and Plesiomonas shigelloides including the type strain (ATCC 14029), the same as those found in LPS from V. parahaemolyticus O7 and O12, and three strains of V. alginolyticus . These substances were strongly positive in the periodate‐thiobarbituric acid test, yielding a color with maximum absorption at 549 nm. The spectra were identical to that of KDO, whereas substances differed from KDO at least in behavior in high‐voltage paper electrophoresis and thin‐layer chromatography.
A particularly interesting feature from the chemotaxonomical point of view was found in the sugar composition of LPS isolated from V. cholerae . Fructose was present exclusively in LPS of V. cholerae (both O1 and non‐O1 groups and classical and eltor biotypes) with the exception of one strain of Photobacterium phosphoreum (NCMB 844). In addition, a pair of rarely occurring amino sugars, perosamine and quinovosamine, was found in LPS from O1 group V. cholerae regardless of either the biotype (classical or eltor) or the serotype (Inaba or Ogawa), whereas this pair was not present in non‐O1 group V. cholerae (the so‐called NAG vibrios). This feature was confirmed with LPS from more than 30 additional strains of O1 group V. cholerae isolated from patients.
The virtual absence of KDO in LPS of the family Vibrionaceae was demonstrated for the first time in this study. These results are compatible with the interpretation that the absence of KDO in LPS can be used as one of the taxonomical characteristics of Vibrionaceae in addition to (G+C) content, DNA (or RNA) homology, and numerical analysis data.
The absolute configuration of 3-hydroxy fatty acids has been studied, which are present in the lipopolysaccharides of the following bacteria: Rhodopseudomonas gelatinosa, Rh. viridis, Rhodospirillum tenue, Chromobacterium violaceum, Pseudomonas aeruginosa, Bordetella pertussis, Vibrio metchnikovii, Vibrio cholerae, Salmonella spp., Escherichia coli, Shigella flexneri, Proteus mirabilis, Yersinia enterocolitica and Fusobacterium nucleatum. The 3-hvdroxy acids were liberated by strong alkaline hydrolysis, converted to 3-methoxy acid l-phenylethylamides and analyzed by gas-liquid chromatography. With the aid of authentic d-3-hydroxy fatty acids it was shown for all lipopolysaccharides that the 3-hydroxy acids, regardless of chain lengths, branching, 3-O-substitution or type of linkage, possess the d-configuration. 2-Hydroxydodecanoic acid, which is present in some lipopolysaccharides, was analyzed in an analogous way and shown to possess the l-configuration.
From the lipopolysaccharides of Vibrio cholerae strains Inaba and Ogawa, an unusual amino sugar was isolated.
By sequential oxidation with sodium metaperiodate and hypoiodide and by oxidation with periodate followed by detection of the acetaldehyde formed, the amino sugar was characterized as a 2-amino-dideoxyaldose.
The hydrochloride of the amino sugar was compared with those of 2-amino-2,6-dideoxy-d-glucose, 2-amino-2,6-dideoxy-l-galactose, 2-amino-2,6-dideoxy-d-mannose, 2-amino-2,6-dideoxy-d-gulose, and 2-amino-2,6-dideoxy-d-allose in an amino acid analyzer. The amino sugar alcohols were studied by paper electrophoresis in molybdate buffer.
From the results obtained the amino sugar was identified as 2-amino-2,6-dideoxyglucose (quinovosamine). Its optical rotation indicated it to be of the d-configuration.
The methyl α-glycosides of 4-amino-4,6-dideoxy-D-mannopyranose (perosamine), N-acylated with either formic, acetic, or (S)-2,4-dihydroxybutanoic acid, have been synthesized. The 1H and 13C n.m.r. spectra of these substances and the parent, non-N-acylated glycoside, demonstrated how the chemical shifts are influenced by the N-acylation. The N-formyl derivative occurred in two conformations, s-cis and s-trans, and the free-energy barrier between these, 86.9 kJ mol–1, was defined by dynamic n.m.r. spectroscopy.
The chemical structure of the lipid A component of the lipopolysaccharide from Vibrio cholerae 95R was studied. After sequential degradation a reduced d-glucosamine disaccharide was isolated from lipid A and, after permethylatiob, shown by combined gas-liquid chromatography,/mass spectrometry to be β,6-linked. The disaccharide is substituted with a phosphate group, ester-bound to the non-reducing glucosamine (GlcN) residue and a pyrophosphorylethanolamine group (PP-Etn) linked to C-1 of the reducing glucosamine residue. This backbone structure is shown in the following formula: P-GlcN(β1-6)GlcN-1-PP-Etn.
The amino groups of the glucosamine disaccharide are substituted by d-3-hydroxytetradecanoic acid; tetradecanoic, hexadecanoic and a d-3-O-(d-3-hydroxydodecanoyl)-dodecanoic acid residue are linked to hydroxyl groups. A similar fatty acid composition was detected in lipopolysaccharides from Inaba, Ogawa and NAG strains of V. cholerae.
Mineral acid hydrolysis of the lipopolysaccharide from Vibrio cholerae 569B (Inaba) gives an oligosaccharide fraction which was shown, by use of 13C NMR and chemical methods, to be a regular α-(1 → 2) linked chain of d-perosamine (4-amino-4,6-dideoxy-d-mannose) units. This chain represents the O-antigen of the lipopolysaccharide, in which the amino functions are acylated with 3-hydroxypropionyl groups. The chromatographic properties of some hydroxamic acids are described and used to characterize these acyl groups.
The structure of the O-antigen from Vibrio cholerae O:2 has been investigated, mainly by methylation analysis, specific degradations, and n.m.r. spectroscopy, and concluded to involve the trisaccharide repeating-unit in which QuiNAc is 2-acetamido-2,6-dideoxyglucose and Sug is tentatively assigned as 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-l-glycero-β-l-manno-nonulosonic acid. The acetamidino group is basic and, consequently, the polysaccharide is neutral. When this group is transformed into an N-acetyl group, by treatment with aqueous triethylamine, the polysaccharide becomes acidic.
The haemolytic properties of El Tor vibrios are usually utilized to differentiate them from the non-haemolytic cholera vibrios. In some freshly isolated Vibrio el tor strains, however, the haemolytic property may be ill-developed and this test may then fail to reveal the true identity of the strains. In order to overcome this difficulty a number of tests have been developed. Investigations reported in this paper indicate that, of these, the bacteriophage-susceptibility test gives the most persistent results. This test is based on the finding that cholera vibrios are uniformly sensitive to a group IV cholera bacteriophage while strains of El Tor vibrios are universally insensitive to it. The author recommends the use of this simple and rapid test as the method of choice for the bacteriological characterization of cholera-like outbreaks.
Considerable amounts of odd-numbered fatty acids, such as non-hydroxy C15 and C17 and 3-hydroxy C11 and C13 acids, were found in lipopolysaccharides from Vibrio cholerae 35A3 (Inaba).
On hydrolysis, the purified lipopolysaccharide (LPS) isolated from Vibrio cholera, Inaba 569 B, yielded glucose, mannose, a heptose behaving like D-glycero-L-manno-heptose and one behaving like D-glycero-L-gluco-heptose, 2-amino-2-deoxyglucose, and glucuronic acid in the molar ratios of approximately 9:4:5:1:2:5. Studies on the LPS, the polysaccharide (PS), and carboxyl-reduced LPS showed that the PS has a branched structure, with (1 leads to 2)-linked mannopyranosyl and a heptopyranosyl, and (1 leads to 4)-linked glucopyranosyluronic and 2-amino-2-deoxyglucopyranosyl residues in the interior part of the molecule, and glucopyranosyl and heptopyranosyl residues as nonreducing end-groups.
Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.
Untreated and partially deacylated lipopolysaccharides from various P− and P+ strains of Salmonella were studied with 31P nuclear magnetic resonance spectroscopy and by conventional analytical methods.
The spectral signals were assigned to various phosphate groups in the lipid A moiety and in the oligosaccharide part. A signal at + 2.3 ppm could be assigned to a phosphodiester linkage formed between 4-amino-4-deoxy-l-arabinose linked via the glycosidic hydroxyl group to the 4′-phosphate group of the glucosamine disaccharide in the lipid A moiety. A strong pyrophosphate signal at + 11 ppm in P− strains was identified as a pyrophosphoryl ethanolamine group at the glycosidic end of this glucosamine disaccharide unit. No evidence was found for phosphodiester or pyrophosphodiester bonds crosslinking lipopolysaccharide ‘subunits’. A revised version of the lipid A structure of Salmonella is presented.
By a combination of 31P nuclear magnetic resonance spectroscopy data and conventional analytical methods the extent to which the lipopolysaccharides are substituted by various phosphate groups on the lipid A and the oligosaccharide moiety could be estimated. It was thus shown that substantial heterogeneity, leading to several molecular species of lipopolysaccharides is caused by addition or omission of certain groups. Since changes in substitution were found to be dependent on the growth conditions, it is thought possible that the overall negative surface charge of Salmonella can be modified by addition or omission of neutralising amino groups from ethanolamine and/or 4-amino-4-deoxy-l-arabinose, and can thus be adapted to the environment.
The purified, specific polysaccharide from Vibrio cholera type NAG, NV 384, O-antigen, 2A, 2B human, contains glucose (5.14%), galactose (4.21%), mannose (64.8%), xylose (3.16%), arabinose (1.98%), fucose (1.50%), mannuronic acid (14.3%), phosphate (0.32%), 2-amino-2-deoxy-D-glucose (2.9%), and 2-amino-2-deoxy-D-galactose (1.0%). Various reactions have shown that the material comprises a phosphoric diester-linked polysaccharide containing mainly ( 1 leads to 2)-linked mannopyranose residues that are highly branched with other sugar residues.
Brief treatment, with 10 M hydrochloric acid, of the lipopolysaccharides of Vibrio cholerae and related organisms led to the release of 4-amino-4, 6-dideoxy-D-mannose (D-perosamine) and 4-amino-4-deoxyl-L-arabinose. These sugar are highly unstable and undergo complex changes in neutral and basic media. Of the strains examined, the lipopolysaccharides from representatives of the Inaba group contain only D-perosamine, while those of the Ogawa biotype have 4-amino-4-deoxyl-L-arabinose as an additional component. The other Vibrios produce lipopolysaccharides which lack these sugars.
A degradation procedure of lipopolysaccharides was worked out which allows the isolation of the reduced backbone of lipid A in a total yield of between 20 and 30%. This procedure was applied to lipopolysaccharides of S forms (Salmonella minnesota, Shigella flexneri 5b, Escherichia coli 086, E. coli 0111, Xanthomonas sinensis, Rhodopseudomonas gelatinosa) and R mutants (Salmonella minnesota, Shigella flexneri, 5b, E. coli BB9 and E. coli EH 100). Chemical analysis, reaction with beta-N-acetyl-glucosaminidase and application of methylation analysis revealed that the lipid A backbone of all strains contains beta 1', 6-linked glucosamine disaccharides carrying two phosphate groups, one in glycosidic and one in ester linkage, a structure, identified previously in the Salmonella minnesota Re mutant.
Cholera and El Tor lipopolysaccharide (LPS) were identical in respect of chemical composition except that the hexosamine content was significantly lower and galactosamine was absent in El Tor LPS. Mukerjee's group IV cholera phage phi149 was inactivated by cholera LPS but was resistant to El Tor LPS.
Lipopolysaccharides were isolated from the cell walls of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba). Chemical analysis revealed the presence of glucose, fructose, mannose, heptose, rhamnose, ethanolamine, fatty acids and glucosamine. The lipopolysaccharides do not contain 2-keto-3-deoxyoctonate, the typical linking sugar of polysaccharide and lipid moieties of enterobacterial lipopolysaccharides. Galactose, a typical core polysaccharide component of many gram-negative bacteria was also absent from lipopolysaccharides of these organisms. By hydrolysis in 1% acetic acid, the lipopolysaccharides have been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Components of degraded polysaccharide and lipid A moiety were identified and determined. The lipid A fractions contained fatty acids, phosphorus and glucosamine. All the neutral sugars detected in lipopolysaccharides were shown to be the constituents of its polysaccharide moiety. The fatty acid analysis of lipopolysaccharide and lipid A showed the presence of both hydroxy and non hydroxy acids. They were different from those of lipids extracted from cell walls before the extraction of lipopolysaccharides. 3-Hydroxylauric and 3-hydroxymyristic acids predominated in lipopolysaccharide and lipid A of Vibrio cholerae and El-tor (Inaba).
After conventional hydrolysis of lipopolysaccharides (LPSs), Kdo was not detectable by the periodate-thiobarbituric acid test in those of any member of Vibrionaceae except the gems Plesiomonas, but phosphorylated Kdo was demonstrated after strong-acid hydrolysis. Dephosphorylation, periodate oxidation, and methylation analysis of LPS preparations from 7 strains selected from all genera of Vibrionaceae, except Plesiomonas, showed that the inner-core region (unlike that in enteric Gram-negative bacteria) contains only one molecule of Kdo 4-phosphate 5-substituted with heptose, a constituent of the distal part of the core region, as in enteric bacteria. The undetectability of Kdo in LPS after conventional hydrolysis and the occurrence of phosphorylated Kdo in strong-acid hydrolysates and of Kdo 4-phosphate in the inner-core region are taxonomic characteristics of the family Vibrionaceae.
The structure of the Vibrio cholerae O:3 O-antigen polysaccharide has been investigated, mainly by n.m.r. spectroscopy, mass spectrometry, sugar and methylation analysis, and specific degradations, and is proposed to involve the following tetrasaccharide repeating-unit. [formula: see text]. In this structure, D-D-Hep is D-glycero-D-manno-heptose, Asc is 3,6-dideoxy-L-arabino-hexose (ascarylose), and Sug is 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine) in which N-2 is acetylated and N-4 is acylated with a 3,5-dihydroxyhexanoic acid. That the 2,4-diamino-2,4,6-trideoxy-D-glucose residue is linked through O-3 and not through one of the hydroxyl groups in the 3,5-dihydroxyhexanoyl group is indicated but not definitely proved. The configuration of the latter group has not been determined. The f.a.b.-mass spectrum of the methylated O-antigen indicates that the structure given above also represents the biological repeating-unit.
The rRNA gene restriction patterns of 89 Vibrio cholerae O1 isolates from different geographic origins were studied. The probe was Escherichia coli 16 + 23S rRNA labelled with "ECL Gene detection system". A total of 17 rRNA gene restriction patterns were observed after BglI cleavage. Four patterns (B1 to B4) were only given by biotype cholerae (14 strains studied). Thirteen patterns (B5 to B17) were only given by biotype El Tor (75 strains studied). There was no correlation between serotypes and rRNA gene restriction patterns. This study provides arguments that (1) strains of biotypes cholerae and El Tor are different clones, (2) a cholera pandemic is not a single world-wide epidemic (due to a single clone) but rather a simultaneous occurrence of several epidemics (several clones involved), and (3) epidemic waves of biotype El Tor could be due to the emergence of new clones.
The O-antigen from Vibrio cholerae O:21 has been investigated, using n.m.r. spectroscopy, methylation analysis, and Smith degradation as the main methods. It is concluded that the O-antigen is composed of tetrasaccharide repeating-units having the following structure (in which Hep = D-glycero-D-manno-heptose). (Formula: see text).
The lipopolysaccharide (LPS) of a rough mutant (95R) of Vibrio cholerae Ogawa has been investigated chemically and serologically. D-Fructose was released from LPS under conditions (10mM trifluoroacetic acid, 60 degrees, 1 h) that liberated no other sugar constituent of the LPS (2-amino-2-deoxy-D-glucose, D-glucose, L-glycero-D-manno-heptose). Upon periodate oxidation, D-fructose and D-glucose were oxidised quantitatively, whereas approximately 50% of heptose was periodate-resistant. The data indicate that D-fructose does not link the polysaccharide and lipid A portion as proposed earlier, and suggest that D-fructose is present as a branch. By passive hemolysis inhibition, it was shown that the release of D-fructose paralleled the exposure of an antigenic determinant cryptic in LPS.
The relationship between the release of fructose from O1 Vibrio cholerae lipopolysaccharides (LPS) by dilute acetic acid hydrolysis and the decrease in their antigenicity was examined. Decrease in the antigenicity of LPS was not parallel with the release of fructose, and occurred very much later than the latter. Periodate oxidation of LPS resulted in the total elimination of the fructose and glucose, and two-thirds of the heptose constituents, but no difference in the antigenicity of LPS was observed before and after oxidation. These findings indicate that the fructose present in O1 V. cholerae LPS is not substantially involved in their specific antigenicity. In the O1 V. cholerae LPS, the fructose is in the branch structure, most probably in the core region.
Naturally elaborated membrane bleb material is frequently observed in cultures of Neisseria gonorrhoeae. This material was purified and analyzed for protein, lipopolysaccharide, and nucleic acid content. The electrophoretic protein profiles of two bleb-rich fractions, called BI and BII, were distinct, with only BII containing lipopolysaccharide and outer membrane proteins I and III. Both fractions contained RNA, circular DNA, and linear DNA. Exogenous pancreatic DNase I appeared to hydrolyze all bleb-associated DNA in fraction BI and the linear DNA in fraction BII. The circular DNA molecules associated with fraction BII resisted digestion. Electron microscopy of the bleb fractions verified their DNA content. Fixing blebs with glutaraldehyde before mounting them for microscopy prevented release of internal DNA. Such fixation produced little change in the micrographs of BI; however, only traces of DNA were observed in fixed BII preparations. Incubation of wild-type gonococci in mixtures of DNase and blebs purified from antibiotic-resistant strains resulted in efficient exchange of penicillinase-specifying R plasmids. Recipients incorporated plasmids independently of endogenous and exogenous chromosomal streptomycin resistance markers. These in vitro results suggest that bleb formation by N. gonorrhoeae may serve to transfer plasmids intercellularly in vivo, perhaps constituting a previously unexplored genetic exchange mechanism in these bacteria.
The sugar composition of the polysaccharide portion of lipopolysaccharides (LPS) was determined for 42 serovars of non-O1 Vibrio cholerae, i.e., from serogroups O2 to O41, O44, and O68. On the basis of their compositional sugar pattern, they were classified into 24 chemotypes. 2-Keto-3-deoxyoctonate (KDO) was totally undetectable by the conventional color test (Weissbach's reaction) under the conventional hydrolysis conditions. Instead, a kind of KDO-like substance, which was positive in the reaction but not identical to KDO, was found in serogroup O19. Fructose, a characteristic sugar constituent of O1 V. cholerae LPS, was found in 33 serogroups but was absent from nine serogroups, approximately 20% of the members of this group so far examined.
A comparative study was made of the quantitative sugar composition of lipopolysaccharides (LPS) isolated from Vibrio cholerae (O1 and non O1 groups), 'V. albensis', 'V. proteus' and V. metschnikovii. The amino sugars 4-amino-4,6-dideoxy-D-mannose (perosamine) and 2-amino-2,6-dideoxy-D-glucose (quinovosamine) were present exclusively in LPS isolated from S-form O1 group of V. cholerae regardless of serotype (i.e. Ogawa or Inaba) and biotype (i.e. classical or eltor). Classical O1 group V. cholerae was distinguishable from eltor O1 group V. cholerae on the basis of the fructose content of the LPS: greater than 3% and less than or equal to 1%, respectively. Distinct differences in the sugar composition of LPS were observed between V. cholerae and 'V. albensis', 'V. proteus' and V. metschnikovii.
The chemical and serological characteristics of lipopolysaccharides (LPS) isolated from Vibrio bio-serogroup 1875 were compared with those of O1 Vibrio cholerae LPS. Vibrio bio-serogroup 1875 LPS contained all the component sugars which were found in O1 V. cholerae LPS, i.e. glucose, L-glycero-D-manno-heptose, fructose, glucosamine, perosamine and quinovosamine, though the amount of perosamine, a characteristic component of O1 V. cholerae LPS, was very low compared with that of O1 V. cholerae LPS. Their LPS additionally contained mannose and two unidentified neutral sugars which are not regular constituents of O1 V. cholerae LPS. Definite serological cross-reactivity in the passive haemolysis test between LPS from Vibrio bio-serogroup 1875 and LPS from O1 V. cholerae was demonstrated.
After treatment with aqueous, 50% hydrofluoric acid, a well-known dephosphorylating agent, the presence of 3-deoxy-D-manno-2-octulosonic acid (KDO), an essential and characteristic constituent of endotoxins, can be readily demonstrated in reportedly KDO-deficient bacterial lipopolysaccharides.