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Schematic map showing chromosome deletion of different CA gene mutations. The mutation Δ(gmd-fcl)26 deletes 2 genes, gmd and fcl. Mutations Δ(wza-wcaL)6 and Δ(wza-wcaL)7 delete 19 genes of the CA gene cluster; the former keeps the SD sequence of wcaM, while the latter does not. Mutation Δ(wza-wcaM)8 deletes all 20 genes in the CA gene cluster.
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Colanic acid (CA) is a common exopolysaccharide produced by many genera in the Enterobacteriaceae. It is critical for biofilm formation on HEp-2 cells and on chicken intestinal tissue by Salmonella. In this study, we generated different CA synthesis gene mutants and evaluated the immune responses induced by these mutants.
One of these mutations, Δ(...
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Colanic acid (CA) is a common exopolysaccharide produced by many genera in the Enterobacteriaceae. It is critical for biofilm formation on HEp-2 cells and on chicken intestinal tissue by Salmonella. In this study, we generated different CA synthesis gene mutants and evaluated the immune responses induced by these mutants. One of these mutations, (w...
Citations
... In combination with the decreased metabolic activity and enhanced stress tolerance of biofilm cells, biofilm communities show an increased tolerance towards antimicrobials, including disinfectants [6,9,10], compared to planktonic bacteria. In Salmonella biofilms, the most common matrix components are amyloid curli fimbriae and cellulose, but other exopolysaccharides, lipopolysaccharides, and surface proteins have been described [11][12][13][14][15]. The biofilm forming ability of individual strains has been observed as an important feature in the persistence of microbial contaminants. ...
Within the European Union, Salmonella is frequently reported in food and feed products. A major route of transmission is upon contact with contaminated surfaces. In nature, bacteria such as Salmonella are often encountered in biofilms, where they are protected against antibiotics and disinfectants. Therefore, the removal and inactivation of biofilms is essential to ensure hygienic conditions. Currently, recommendations for disinfectant usage are based on results of efficacy testing against planktonic bacteria. There are no biofilm-specific standards for the efficacy testing of disinfectants against Salmonella. Here, we assessed three models for disinfectant efficacy testing on Salmonella Typhimurium biofilms. Achievable bacterial counts per biofilm, repeatability, and intra-laboratory reproducibility were analyzed. Biofilms of two Salmonella strains were grown on different surfaces and treated with glutaraldehyde or peracetic acid. Disinfectant efficacy was compared with results for planktonic Salmonella. All methods resulted in highly repeatable cell numbers per biofilm, with one assay showing variations of less than 1 log10 CFU in all experiments for both strains tested. Disinfectant concentrations required to inactivate biofilms were higher compared to planktonic cells. Differences were found between the biofilm methods regarding maximal achievable cell numbers, repeatability, and intra-laboratory reproducibility of results, which may be used to identify the most appropriate method in relation to application context. Developing a standardized protocol for testing disinfectant efficacy on biofilms will help identify conditions that are effective against biofilms.
... Colanic acid is likewise categorized as structural EPS. The colanic acid comprises repeated subunits of D-glucose, L-fucose, D-galactose, and D-glucuronic acid sugars with O-acetyl and pyruvate side chains; it is made in a similar way as lipopolysaccharide Oantigen [67]. The tolerance of S. enterica to desiccation on a leaf surface is linked to colonic acid biosynthesis [68]. ...
Salmonella can form biofilms that contribute to its resistance in food processing environments. Biofilms are a dense population of cells that adhere to the surface, creating a matrix composed of extracellular polymeric substances (EPS) consisting mainly of polysaccharides, proteins, and eDNA. Remarkably, the secreted substances, including cellulose, curli, and colanic acid, act as protective barriers for Salmonella and contribute to its resistance and persistence when exposed to disinfectants. Conventional treatments are mostly ineffective in controlling this problem; therefore, exploring anti-biofilm molecules that minimize and eradicate Salmonella biofilms is required. The evidence indicated that terpenes effectively reduce biofilms and affect their three-dimensional structure due to the decrease in the content of EPS. Specifically, in the case of Salmonella, cellulose is an essential component in their biofilms, and its control could be through the inhibition of glycosyltransferase, the enzyme that synthesizes this polymer. The inhibition of polymeric substances secreted by Salmonella during biofilm development could be considered a target to reduce its resistance to disinfectants, and terpenes can be regarded as inhibitors of this process. However, more studies are needed to evaluate the effectiveness of these compounds against Salmonella enzymes that produce extracellular polymeric substances.
... The Dpmi DwaaL DpagL::TT rhaRS P rhaBAD waaL mutations collectively provide regulated delayed attenuation and cause cessation in the synthesis of LPS O-antigen in the absence of the sugars mannose and rhamnose in vivo (52,74,75). The deletion D(wza-wcaM) enhances complete lysis, blocks the synthesis of cell surface polymers to prevent biofilm formation and enhances induced immunities (76). The DrelA::araC P araBAD lacI TT mutation confers the regulated delayed antigen synthesis phenotype that enhances PIESV colonization and induction of immune responses by the arabinose-dependent regulated LacI synthesis so that P trc -regulated genes on plasmid vectors are gradually expressed as LacI is diluted at each cell division during in vivo growth (77)(78)(79). ...
Helicobacter pylori is a major cause of gastric mucosal inflammation, peptic ulcers, and gastric cancer. Emerging antimicrobial-resistant H. pylori has hampered the effective eradication of frequent chronic infections. Moreover, a safe vaccine is highly demanded due to the absence of effective vaccines against H. pylori. In this study, we employed a new innovative Protective Immunity Enhanced Salmonella Vaccine (PIESV) vector strain to deliver and express multiple H. pylori antigen genes. Immunization of mice with our vaccine delivering the HpaA, Hp-NAP, UreA and UreB antigens, provided sterile protection against H. pylori SS1 infection in 7 out of 10 tested mice. In comparison to the control groups that had received PBS or a PIESV carrying an empty vector, immunized mice exhibited specific and significant cellular recall responses and antigen-specific serum IgG1, IgG2c, total IgG and gastric IgA antibody titers. In conclusion, an improved S. Typhimurium-based live vaccine delivering four antigens shows promise as a safe and effective vaccine against H. pylori infection.
... However, in the same study they show that strains of Klebsiella aerogenes of capsular serotype K21 are resistant to phagocytic uptake. A study on Salmonella enterica serovar Typhimurium where the entire colanic acid operon was deleted also shows no effect on virulence [25]. ...
Multidrug-resistant Klebsiella pneumoniae has emerged as one of the deadliest opportunistic nosocomial pathogens that forms biofilm for the establishment of chronic K. pneumoniae infections. Herein, we made an attempt to identify the genes involved in biofilm formation in the strain K. pneumoniae ATCC13883. To achieve this, we constructed mini-Tn5 transposon insertion mutants and screened them for biofilm production. We observed that the biofilm formation was enhanced in the mutant where the wcaJ gene was disrupted. WcaJ is the initiating enzyme of colanic acid synthesis and loads the first sugar (glucose-1-P) on the lipid carrier undecaprenyl phosphate. The absence of this glycosyltransferase results in the absence of colanic acid, which renders a non-mucoid phenotype to the mutant. Further, to determine the effect of mucoidy on antibiotic susceptibility, we tested the sensitivity of the strains towards different groups of antibiotics. Unlike the mucoid strains, the resistance of the non-mucoid cells was greater for polymyxins, but less for quinolones. Capsular polysaccharides are known to have a protective effect against phagocytosis, therefore we assessed the role of colanic acid in virulence by conducting infection studies on murine macrophages. Surprisingly, the ΔwcaJ strain was less efficient in macrophage activation and was not readily phagocytosed. Thus, the presence of colanic acid appeared to increase the immunogenicity of K. pneumoniae. Overall, the results indicate that the presence of colanic acid increases the vulnerability of K. pneumoniae towards both polymyxins and macrophages, implying that the mucoid strains are less threatening as compared to their high biofilm forming non-mucoid counterparts.
... Recently it has been shown that deletion of wcaJ, another gene whose product is involved in colonic acid (CA) biosynthesis in Edwardsiella tarda, a Gram-negative bacterium, leads to reduced virulence [39]. Colanic acid has also been shown to act as avirulence factor for many Enterobacteriaceae including, E. amylovora, Salmonella [16,40,41] and to also have a high influence in the production of slime layer, capsule, and biofilm [42,43]. Studies in Erwinia amylovora have demonstrated that mutants of two-component rcs regulatory system, rcsB, rcsC, and rcsD are affected in CA biosynthesis and these mutants are non-pathogenic on immature pear fruit [44]. ...
... In Escherichia coli, CA plays a role in protecting the bacterium from unfavorable pH [45]. Consistent with this protective role, unfavorable conditions including low temperature and oxidative stress upregulate CA biosynthesis in Escherichia coli [41]. In this study, we did not study the survival of the mutants and parent under various adverse conditions including pH. ...
Pectobacterium carotovorum is a gram-negative bacterium that, together with other soft rot Enterobacteriaceae causes soft rot disease in vegetables, fruits, and ornamental plants through the action of exoproteins including plant cell wall-degrading enzymes (PCWDEs). Although pathogenicity in these bacteria is complex, virulence levels are proportional to the levels of plant cell wall-degrading exoenzymes (PCWDEs) secreted. Two low enzyme-producing transposon Tn5 mutants were isolated, and compared to their parent KD100, the mutants were less virulent on celery petioles and carrot disks. The inactivated gene responsible for the reduced virulence phenotype in both mutants was identified as wcaG. The gene, wcaG (previously denoted fcl) encodes NAD-dependent epimerase/dehydratase, a homologue of GDP-fucose synthetase of Escherichia coli. In Escherichia coli, GDP-fucose synthetase is involved in the biosynthesis of the exopolysaccharide, colanic acid (CA). The wcaG mutants of P. carotovorum formed an enhanced level of biofilm in comparison to their parent. In the hydrophobicity test the mutants showed more hydrophobicity than the parent in hexane and hexadecane as solvents. Complementation of the mutants with extrachromosomal copies of the wild type gene restored these functions to parental levels. These data indicate that NAD-dependent epimerase/dehydratase plays a vital rule in cell surface properties, exoenzyme production, and virulence in P. carotovorum.
... Colanic Acid (CA) is also classified as a structural EPS (Fig. 2). The colanic acid structure contains repeating subunits of D-glucose, L-fucose, D-galactose, and D-glucuronic acid sugars with O-acetyl and pyruvate side chains and are assembled by an identical processes as lipopolysaccharide O-antigen (Whitfield, 2006;Wang et al., 2013). CA biosynthesis is associated with tolerance to desiccation of S. enterica on the leaf surface (Cowles et al., 2015). ...
When Salmonella enterica is not in a planktonic state, it persists in organised communities encased in extracellular poly-meric substances (EPS), defined as biofilms. Environmental conditions ultimately dictate the key properties of the bio-films such as porosity, density, water content, charge, sorp-tion and ion exchange properties, hydrophobicity and mechanical stability. S. enterica has been extensively studied due to its ability to infect the gastrointestinal environment. However, only during the last decades studies on its persistence and replication in soil, plant and abiotic surfaces have been proposed. S. enterica is an environmental bacterium able to effectively persist outside the human host. It does so by using EPS as tools to cope with environmental fluctuations. We therefore address this mini-review to classify those EPS that are produced by Salmonella with focus on the environment (plant, soil, and abiotic surfaces) by using a classification of EPS proposed by Flemming and collaborators in 2007. The EPS are therefore classified as structural, sorp-tive, surface-active, active, and informative.
... Our subsequent genome sequencing determined that a significant portion of the operon (wca) responsible for colanic acid synthesis is missing in the transient-infection strains. Previous work has shown a connection between motility and colanic acid synthesis (15). In that work, the Rcs signaling system was found to positively regulate colanic acid synthesis and negatively regulate motility. ...
... Our observations of the differences between MPEC strains that cause persistent versus transient infection indicate that persistent-infection strains are more motile, have increased gene expression of the iron acquisition system, and are more resistant to growth inhibition caused by complement. A relationship between iron acquisition and motility has been shown in these strains (8), and a relationship between colanic acid synthesis and motility has been described previously (15). Generation of specific deletion mutants that will test the role of motility, iron acquisition, and colanic acid synthesis in vivo will be necessary to further determine the mechanisms necessary for persistence of E. coli in mammary gland infections. ...
Escherichia coli
is a leading cause of bacterial mastitis in dairy cattle. It is most often transient in nature, causing an infection that lasts 2 to 3 days. However,E. colihas been shown to cause a persistent infection in a minority of cases. Mechanisms that allow for a persistentE. coliinfection are not fully understood. The goal of this work was to determine differences betweenE. colistrains originally isolated from dairy cattle with transient and persistent mastitis. Using RNA sequencing, we show gene expression differences in nearly 200 genes when bacteria from the two clinical phenotypes are compared. We sequenced the genomes of theE. colistrains and report genes unique to the two phenotypes. Differences in thewcaoperon, which encodes colanic acid, were identified by DNA as well as RNA sequencing and differentiated the two phenotypes. Previous work demonstrated thatE. colistrains that cause persistent infections were more motile than those that cause transient infections. Deletion of genes in thewcaoperon from a persistent-infection strain resulted in a reduction of motility as measured in swimming and swarming assays. Furthermore, colanic acid has been shown to protect bacteria from complement-mediated killing. We show that transient-infectionE. colistrains were more sensitive to complement-mediated killing. The deletion of genes from thewcaoperon caused a persistent-infectionE. colistrain to become sensitive to complement-mediated killing. This work identifies important differences betweenE. colistrains that cause persistent and transient mammary infections in dairy cattle.
... All vaccines induced a significantly higher IgG2a response than IgG1. The low level of IgG1/IgG2a ratio indicated that the cellular immunity was biased to Th1-type immune response ( Figure S6 in Supplementary Material), consistent with our and other previous observations that Salmonella induced a predominant Th1-type response to either heterologous antigens or Salmonella own antigens (48,49). Negative control groups (BSG) did not mount a detectable immune response. ...
Salmonella infections remain a big problem worldwide, causing enteric fever by Salmonella Typhi (or Paratyphi) or self-limiting gastroenteritis by non-typhoidal Salmonella (NTS) in healthy individuals. NTS may become invasive and cause septicemia in elderly or immuno-compromised individuals, leading to high mortality and morbidity. No vaccines are currently available for preventing NTS infection in human. As these invasive NTS are restricted to several O-antigen serogroups including B1, D1, C1, and C2, O-antigen polysaccharide is believed to be a good target for vaccine development. In this study, a strategy of O-serotype conversion was investigated to develop live attenuated S. Typhimurium vaccines against the major serovars of NTS infections. The immunodominant O4 serotype of S. Typhimurium was converted into O9, O7, and O8 serotypes through unmarked chromosomal deletion–insertion mutations. O-serotype conversion was confirmed by LPS silver staining and western blotting. All O-serotype conversion mutations were successfully introduced into the live attenuated S. Typhimurium vaccine S738 (Δcrp Δcya) to evaluate their immunogenicity in mice model. The vaccine candidates induced high amounts of heterologous O-polysaccharide-specific functional IgG responses. Vaccinated mice survived a challenge of 100 times the 50% lethality dose (LD50) of wild-type S. Typhimurium. Protective efficacy against heterologous virulent Salmonella challenges was highly O-serotype related. Furthermore, broad-spectrum protection against S. Typhimurium, S. Enteritidis, and S. Choleraesuis was observed by co-vaccination of O9 and O7 O-serotype-converted vaccine candidates. This study highlights the strategy of expressing heterologous O-polysaccharides via genetic engineering in developing live attenuated S. Typhimurium vaccines against NTS infections.
... CA is a surface exopolysaccharide produced by many enteric bacteria that serves as a scaffold for biofilm formation and protects cells from adverse physicochemical and environmental conditions [26][27][28]. Deletion of the entire colanic acid gene cluster does not attenuate S. Typhimurium virulence, but leads to increased synthesis of heterologous antigens [27]. In addition, regulated LPS truncation can enhance induction of cross-reactive immune response against conserved OMPs of enteric bacteria [15,20,29,30], which was further demonstrated by our recent research in which both O-antigen polysaccharide and enterobacterial common antigen (ECA) polysaccharide were synthesized only in the presence of exogenously supplied arabinose in the media via regulation of rfbB gene, a key gene for synthesis of O-antigen and ECA [31]. ...
... CA is a surface exopolysaccharide produced by many enteric bacteria that serves as a scaffold for biofilm formation and protects cells from adverse physicochemical and environmental conditions [26][27][28]. Deletion of the entire colanic acid gene cluster does not attenuate S. Typhimurium virulence, but leads to increased synthesis of heterologous antigens [27]. In addition, regulated LPS truncation can enhance induction of cross-reactive immune response against conserved OMPs of enteric bacteria [15,20,29,30], which was further demonstrated by our recent research in which both O-antigen polysaccharide and enterobacterial common antigen (ECA) polysaccharide were synthesized only in the presence of exogenously supplied arabinose in the media via regulation of rfbB gene, a key gene for synthesis of O-antigen and ECA [31]. ...
Colanic Acid (CA) and lipopolysaccharide (LPS) are two major mannose-containing extracellular polysaccharides of Salmonella. Their presence on the bacterial surface can mask conserved protective outer membrane proteins (OMPs) from the host immune system. The mannose moiety in these molecules is derived from GDP-mannose, which is synthesized in several steps. The first two steps require the action of phosphomannose isomerase, encoded by pmi (manA), followed by phosphomannomutase, encoded by manB. There are two copies of manB present in the Salmonella chromosome, one located in the cps gene cluster (cpsG) responsible for CA synthesis, and the other in the rfb gene cluster (rfbK) involved in LPS O-antigen synthesis. In this study, it was demonstrated that the products of cpsG and rfbK are isozymes. To evaluate the impact of these genes on O-antigen synthesis, virulence and immunogenicity, single mutations (Δpmi, ΔrfbK or ΔcpsG) and a double mutation (ΔrfbK ΔcpsG) were introduced into both wild-type Salmonella enterica and an attenuated Δcya Δcrp vaccine strain. The Δpmi, ΔrfbK and ΔcpsG ΔrfbK mutants were defective in LPS synthesis and attenuated for virulence. In orally inoculated mice, strain S122 (Δcrp Δcya ΔcpsG ΔrfbK) and its parent S738 (Δcrp Δcya) were both avirulent and colonized internal tissues. Strain S122 elicited higher levels of anti-S. Typhimurium OMP serum IgG than its parent strain. Mice immunized with S122 were completely protected against challenge with wild-type virulent S. Typhimurium and partially protected against challenge with either wild-type virulent S. Choleraesuis or S. Enteritidis. These data indicate that deletions in rfbK and cpsG are useful mutations for inclusion in future attenuated Salmonella vaccine strains to induce cross-protective immunity.
... Pectobacterium carotovora also uses a novel regulatory mechanism involving and wzxC (30,31). Mutation of these genes directly affect the virulence activities (32). In E. coli, the gene wcaG from wca operon encodes GDP fucose synthetase, which has a dual function including 3,5-epimerase-4-reductase involved in GDP fucose biosynthesis. ...
... Although we have not investigated the mutants and parent about their ability to survive on various pH condition. In addition unfavorable conditions including low temperature and oxidative stress upregulates CA biosynthesis in E. coli (32). ...
Pectobacterium carotovorum (formerly, Erwinia carotovora subsp. carotovora) is a gram negative bacterium that, together with other soft rot Enterobacteriaceae causes soft rot disease in vegetables, fruits and ornamental plants. Soft rot disease occurs when the pathogen secrets cell wall-degrading exoenzymes resulting in water-soaked tissues and maceration. Virulence levels of soft rot bacteria are proportional to the levels of exoenzymes secretion. Virulence is regulated by many factors including temperature, moisture, host and bacterial chemical signals, and complex network of regulatory genes in the pathogen. Strain KD100 of P. carotovorum was mutagenized using transposon containing a promoterless lacZ (mini-Tn5 lacZ1) and two mutants with reduced levels of production of exoenzymes pectate lyase, protease, polygalacturonase, and cellulase were isolated. We compared to their parent KD100, the mutants were less virulent on celery petioles and carrot disks. Complementation of the mutants also restore the function same as parent. The inactivated gene responsible for the reduced virulent phenotype in both mutants was identified as wcaG. The gene wcaG encodes NAD-dependent epimerase/dehydratase which is homologous to GDP-fucose synthetase of E.coli. The GDP -fucose synthetase is involved in Colanic acid (CA) and lipopolysaccharide (LPS) biosynthesis. The wcaG mutants of P. carotovorum formed enhanced level of biofilm in comparison to their parent. In hydrophobicity test the mutants showed more hydrophobicity than the parent in hexane and hexadecane as solvents. The mutants were also more resistance to hydrogen peroxide in different time duration. These data implicate that NAD-dependent epimerase/dehydratase plays vital rule in cell surface properties, exoenzyme production and virulence in P. carotovorum.
