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A simplified model of the pyruvate metabolism pathway in Salmonella Typhimurium. Genes producing metabolic enzymes (in italics) were deleted in the creation of mutant strains, including pyruvate formate lyase I (pflB) and bifunctional acetaldehyde-CoA/alcohol dehydrogenase (adhE).
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Background
Salmonella enterica serovar Typhimurium is a major foodborne pathogen worldwide. S. Typhimurium encodes type III secretion systems via Salmonella pathogenicity islands (SPI), producing the major effector proteins of virulence. Previously, we identified two genes of Salmonella pyruvate metabolism that were up-regulated during chicken cell...
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Citations
... Pyruvate is an essential metabolite linking the glycolytic pathway with the tricarboxylic acid cycle in living organisms. In Salmonella spp, as well as other bacteria, its metabolism plays an important role in infectivity (Abernathy et al. 2013), pathogenesis, virulence (Jiang et al. 2021) and acid resistance (Wu et al. 2014). The efflux of all these vital molecules is evident in the distortion of membrane permeability which has been reported as a major mechanism of antibacterial activities of many natural products. ...
As a popular plant used traditionally in the treatment of typhoid fever, leaf extracts of Psidium guajava reportedly inhibit typhoidal salmonella in vitro and in vivo. This study explored the antibacterial mechanism involved in the bactericidal activity of n-hexane and ethyl acetate soluble fractions of P. guajava leaves against S. Typhi. S. Typhi was cultured in Mueller Hinton Broth in the presence and absence of the extracts. The time course of bactericidal action was measured by kill-curve analysis. The antibacterial mechanism was elucidated by analyzing the effect of divalent cations (Ca2+ and Mg2+) on the minimum inhibitory concentrations of the fractions against S. Typhi. Cellular leakage was monitored by measuring extracellular levels of protein, 260 nm-absorbing materials, pyruvic acid, potassium ions and inorganic phosphate in treated bacteria cells. DNA damage was measured using single-cell gel electrophoresis. The extracts significantly (P < 0.05) increased the extracellular concentration of protein, 260 nm-absorbing materials, pyruvic acid, potassium ions and inorganic phosphate in bacterial cultures. Scanning electron micrographs showed that the bacterial cells were destroyed. There was also considerable DNA damage. It can be concluded that n-hexane and ethyl acetate soluble fractions of P. guajava exert their bactericidal actions against S. Typhi by distorting the membrane permeability leading to leakages of vital biomolecules as well as morphological and DNA damages.
... Salmonella Typhimurium (Salmonella enterica serovar Typhimurium) was cultured according to Abernathy et al. (2013). Briefly, S. Typhimurium grown at 37°C overnight in Luria-Bertani (LB) broth was sub-cultured for 3 h in pre-warmed (37°C) LB broth before the challenge to ensure log-phase cultures. ...
Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M’s impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein–isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.
... We show the top 5 highly regulated gene groups under virulence inducing conditions as an example (Supplementary Fig. 9b). The gene with highest PTDEX score under virulence condition was adhE, encoding bi-functional acetaldehyde-CoA dehydrogenase and alcohol dehydrogenase, previously shown to be important for E. coli and S. enterica virulence 62,63 . We could also see that adhE is differentially regulated in Gram-positive and -negative bacteria by retriving differential expression levels ( Supplementary Fig. 9c). ...
Bacterial processes necessary for adaption to stressful host environments are potential targets for new antimicrobials. Here, we report large-scale transcriptomic analyses of 32 human bacterial pathogens grown under 11 stress conditions mimicking human host environments. The potential relevance of the in vitro stress conditions and responses is supported by comparisons with available in vivo transcriptomes of clinically important pathogens. Calculation of a probability score enables comparative cross-microbial analyses of the stress responses, revealing common and unique regulatory responses to different stresses, as well as overlapping processes participating in different stress responses. We identify conserved and species-specific ‘universal stress responders’, that is, genes showing altered expression in multiple stress conditions. Non-coding RNAs are involved in a substantial proportion of the responses. The data are collected in a freely available, interactive online resource (PATHOgenex). Bacterial stress responses are potential targets for new antimicrobials. Here, Avican et al. present global transcriptomes for 32 bacterial pathogens grown under 11 stress conditions, and identify common and unique regulatory responses, as well as processes participating in different stress responses.
... In a more indirect fashion, AdhE also regulates gene expression of various virulence factors. For example, in Salmonella typhimurium, AdhE regulates the expression of SPI-1 (Salmonella pathogenicity island À1) virulence genes [9,10], while in Escherichia coli O157:H7, AdhE regulates the expression of flagellar genes thus activating swimming motility. In Streptococcus pneumoniae, AdhE enhances expression of a hemolytic virulence factor, pneumolysin [11]. ...
Aldehyde-alcohol dehydrogenase (AdhE) is a metabolic enzyme and virulence factor in bacteria. E. coli AdhE (eAdhE) multimerizes into spirosomes that are essential for enzymatic activity. However, it is unknown whether AdhE structure is conserved in divergent bacteria. Here, we present the cryo-EM structure of AdhE (vAdhE) from Vibrio cholerae to 4.31 Å resolution. Overall, vAdhE spirosomes are similar to eAdhE with conserved subunit arrangement. However, divergences in key oligomerization residues cause vAdhE to form labile spirosomes with lower enzymatic activity. Mutating the vAdhE oligomerization interface to mimic eAdhE increases spirosome stability and enzymatic activity to levels comparable to eAdhE. These results support the generality of AdhE spirosome structures, and provide a structural basis to target vAdhE to attenuate bacterial virulence.
... S. typhimurium adhE and pflB, which codes for the pyruvate formate lyase I enzyme are up-regulated during bacterial infection. These genes could alter the expression of genes involved in the S. typhimurium T3SS coded by the Salmonella pathogenicity island (SPI) 1. (Abernathy, Corkill, Hinojosa, Li, & Zhou, 2013). ...
Acetaldehyde-alcohol dehydrogenase (AdhE) enzymes is a key metabolic enzyme in bacterial physiology and pathogenicity. They are responsible for the conversion of acetyl-CoA to ethanol via an acetaldehyde intermediate during ethanol fermentation in absence of oxygen. This two-step reaction is associated to NAD+ regeneration, essential for the glycolysis. This protein is composed of two domains: the N-terminal acetaldehyde dehydrogenase (ALDH) domain linked to the C-terminal alcohol dehydrogenase (ADH) domain. This bifunctional AdhE enzyme is well-conserved in all bacterial kingdoms and also in more phylogenetically distant microorganisms such as green microalgae. AdhE is well-studied for its involvement in biofuel production, but as well for its role in bacterial pathogenicity. This enzyme is commonly found as an oligomeric form called spirosomes. Despite the great interest in the AdhE enzyme, the structure and function of these filaments remain unknown since their discovery in the 1970’s.Depending on ligands incubated with, Escherichia coli spirosome are found as two different conformational states.During this thesis, the structure of the extended and the compact conformation have been solved at high-resolution using cryo-electron microscopy.Based on these structures, AdhE mutants have been designed in order to disrupt the filamentation and study its role. We have shown that spirosomes contain active AdhE monomers and the AdhE filamentation is essential for its activity in vitro and in vivo. Again based on the obtained structure of the extended conformation, we identified that a channel within the filament which could be responsible for the channeling of the toxic acetaldehyde intermediate. Like other examples of metabolic enzyme filamentation or micro-compartments encapsulating dehydrogenases, spirosomes are a form of bacterial compartmentalization. In order to look deeper in this compartmentalization, immunolabelling experiments have been performed in order to localize AdhE filaments inside the bacteria. Observations revealed a peripheral localization for E. coli AdhE wild-type and a diffuse localization for the non-filamentous AdhE. These differences are not understood but might suggest a difference of function between oligomeric and monomeric form.The structure of the Streptococcus pneumoniae spirosome has also been solved using cryo-EM. Compared to E.coli spirosomes, AdhE filaments from S. pneumoniae have always been purified as extended conformation. S. pneumoniae spirosomes seem to be naturally found in extended conformation and so active form. This could be due to the differences present in the two homologous AdhE sequences.The determination of the E. coli and S. pneumoniae structures at high-resolution and the analysis of the function reveal that these filaments are required for bacterial physiology in absence of oxygen. The study of the regulation of spirosome activities might lead to the discovery of molecules stimulating or inhibiting conformation change in E. coli AdhE filaments. These molecules would present a great interest in biotechnology or to prevent pathogenic anaerobes.
... AdhE was shown to be a target for antibiotics as the depletion of AdhE in pathogenic bacteria results in nonfunctional flagella and reduces its virulence 6 . Previous studies also report that AdhE acts as a virulence factor in Streptococcus pneumoniae 7 and that AdhE is upregulated during infection in Salmonella typhimurium 8 . The observation that AdhE is important for virulence in several pathogens broadens its appeal as a drug target 9 . ...
Aldehyde-alcohol dehydrogenase (AdhE) is an enzyme responsible for converting acetyl-CoA to ethanol via acetaldehyde using NADH. AdhE is composed of two catalytic domains of aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH), and forms a spirosome architecture critical for AdhE activity. Here, we present the atomic resolution (3.43 Å) cryo-EM structure of AdhE spirosomes in an extended conformation. The cryo-EM structure shows that AdhE spirosomes undergo a structural transition from compact to extended forms, which may result from cofactor binding. This transition leads to access to a substrate channel between ALDH and ADH active sites. Furthermore, prevention of this structural transition by crosslinking hampers the activity of AdhE, suggesting that the structural transition is important for AdhE activity. This work provides a mechanistic understanding of the regulation mechanisms of AdhE activity via structural transition, and a platform to modulate AdhE activity for developing antibiotics and for facilitating biofuel production.
... Salmonella Typhimurium metabolism is very flexible, and T3SS1 expression is also influenced by metabolism. For example, cell invasion increases when pyruvate metabolism is abolished (Abernathy et al., 2013). Here, we determined that ArcA inhibits genes related to pyruvate metabolism (Figure 4). ...
Salmonella Typhimurium, a bacterial pathogen with high metabolic plasticity, can adapt to different environmental conditions; these traits enhance its virulence by enabling bacterial survival. Neutrophils play important roles in the innate immune response, including the production of microbicidal reactive oxygen species (ROS). In addition, the myeloperoxidase in neutrophils catalyzes the formation of hypochlorous acid (HOCl), a highly toxic molecule that reacts with essential biomolecules, causing oxidative damage including lipid peroxidation and protein carbonylation. The bacterial response regulator ArcA regulates adaptive responses to oxygen levels and influences the survival of Salmonella inside phagocytic cells. Here, we demonstrate by whole transcriptomic analyses that ArcA regulates genes related to various metabolic pathways, enabling bacterial survival during HOCl-stress in vitro. Also, inside neutrophils, ArcA controls the transcription of several metabolic pathways by downregulating the expression of genes related to fatty acid degradation, lysine degradation, and arginine, proline, pyruvate, and propanoate metabolism. ArcA also upregulates genes encoding components of the oxidative pathway. These results underscore the importance of ArcA in ATP generation inside the neutrophil phagosome and its participation in bacterial metabolic adaptations during HOCl stress.
... Such association is not surprising given that genes within these functional categories are highly conserved and play critical roles in bacterial physiology (Nei and Kumar, 2000;Rocha and Danchin, 2004;Drummond et al., 2005). Indeed, the functional relevance and evolutionary selection pressure are so central for these PHX genes that their proteins are less prone to mutations that lead to amino acid substitutions (Drummond et al., 2005), allowing their efficient recognition by the most abundant tRNAs and translation from multiple tRNA isoacceptors to ensure appropriate protein synthesis rate (Abernathy et al., 2013). The third most abundant functional category of PHX genes is related to cellular processes and signaling functions (28/161 = 17%), where the majority of genes contribute to cellenvelope or membrane biogenesis (11/28). ...
Chickens are the reservoir host of Salmonella Enteritidis. Salmonella Enteritidis colonizes the gastro-intestinal tract of chickens and replicates within macrophages without causing clinically discernable illness. Persistence of S. Enteritidis in the hostile environments of intestinal tract and macrophages allows it to disseminate extra-intestinally to liver, spleen, and reproductive tract. Extra-intestinal dissemination into reproductive tract leads to contamination of internal contents of eggs, which is a major risk factor for human infection. Understanding the genes that contribute to S. Enteritidis persistence in the chicken host is central to elucidate the genetic basis of the unique pathobiology of this public health pathogen. The aim of this study was to identify a succinct set of genes associated with infection-relevant in vitro environments to provide a rational foundation for subsequent biologically-relevant research. We used in silico prediction of gene expression and RNA-seq technology to identify a core set of 73 S. Enteritidis genes that are consistently highly expressed in multiple S. Enteritidis strains cultured at avian physiologic temperature under conditions that represent intestinal and intracellular environments. These common highly expressed (CHX) genes encode proteins involved in bacterial metabolism, protein synthesis, cell-envelope biogenesis, stress response, and a few proteins with uncharacterized functions. Further studies are needed to dissect the contribution of these CHX genes to the pathobiology of S. Enteritidis in the avian host. Several of the CHX genes could serve as promising targets for studies towards the development of immunoprophylactic and novel therapeutic strategies to prevent colonization of chickens and their environment with S. Enteritidis.
... Within Stsl1 QTL, four genes are strong candidates based on known phenotypes: Cul4a (cullin 4A) deficient animal die in utero and Cul4a is essential for hematopoietic cell survival [52]; Lamp1 (lysosomal-associated membrane protein 1) is highly expressed in macrophages and is involved in autophagy as well as protecting NK cell from degranulation-associated damage [53,54]; Mcf2l (mcf.2 transforming sequence-like) targeted mutant mice (IMPC) have a decreased number of CD8-positive T cells; Pcid2 (PCI domain containing 2) is essential for spleen development and regulation of B cell differentiation [55]. Within Stsl2 QTL one gene is a high-potential candidate: Slc35f1 targeted mutant have a decreased lactate dehydrogenase activity (IMPC, Phenotype MP:0005571), which may alter the pyruvate metabolism pathway in Salmonella [56]. ...
Background:
Salmonella is a Gram-negative bacterium causing a wide range of clinical syndromes ranging from typhoid fever to diarrheic disease. Non-typhoidal Salmonella (NTS) serovars infect humans and animals, causing important health burden in the world. Susceptibility to salmonellosis varies between individuals under the control of host genes, as demonstrated by the identification of over 20 genetic loci in various mouse crosses. We have investigated the host response to S. Typhimurium infection in 35 Collaborative Cross (CC) strains, a genetic population which involves wild-derived strains that had not been previously assessed.
Results:
One hundred and forty-eight mice from 35 CC strains were challenged intravenously with 1000 colony-forming units (CFUs) of S. Typhimurium. Bacterial load was measured in spleen and liver at day 4 post-infection. CC strains differed significantly (P < 0.0001) in spleen and liver bacterial loads, while sex and age had no effect. Two significant quantitative trait loci (QTLs) on chromosomes 8 and 10 and one suggestive QTL on chromosome 1 were found for spleen bacterial load, while two suggestive QTLs on chromosomes 6 and 17 were found for liver bacterial load. These QTLs are caused by distinct allelic patterns, principally involving alleles originating from the wild-derived founders. Using sequence variations between the eight CC founder strains combined with database mining for expression in target organs and known immune phenotypes, we were able to refine the QTLs intervals and establish a list of the most promising candidate genes. Furthermore, we identified one strain, CC042/GeniUnc (CC042), as highly susceptible to S. Typhimurium infection.
Conclusions:
By exploring a broader genetic variation, the Collaborative Cross population has revealed novel loci of resistance to Salmonella Typhimurium. It also led to the identification of CC042 as an extremely susceptible strain.
... 17 An adhE mutant alters the SPI-1 gene expression, supporting crosstalk between metabolism and virulence. 18 In humans, the aldehyde dehydrogenase (ALDH) superfamily includes 19 NAD(P+)-dependent enzymes that irreversibly catalyze the oxidation of endogenous and exogenous aldehydes. Out of these 19 isozymes, there are only drugs developed for 3 of them: ALDH2, involved in alcohol metabolism, and ALDH1A1 and ALDH3A1, involved in cancer. ...
Acetaldehyde dehydrogenase (AdhE) is a bifunctional acetaldehyde–coenzyme A (CoA) dehydrogenase and alcohol dehydrogenase involved in anaerobic metabolism in gram-negative bacteria. This enzyme was recently found to be a key regulator of the type three secretion (T3S) system in Escherichia coli. AdhE inhibitors can be used as tools to study bacterial virulence and a starting point for discovery of novel antibacterial agents. We developed a robust enzymatic assay, based on the acetaldehyde-CoA dehydrogenase activity of AdhE using both absorption and fluorescence detection models (Z′ > 0.7). This assay was used to screen ~11,000 small molecules in 384-well format that resulted in three hits that were confirmed by resynthesis and validation. All three compounds are noncompetitive with respect to acetaldehyde and display a clear dose–response effect with hill slopes of 1–2. These new inhibitors will be used as chemical tools to study the interplay between metabolism and virulence and the role of AdhE in T3S regulation in gram-negative bacteria, and as starting points for the development of novel antibacterial agents.
