Dissecting the invasion of Galleria mellonella by Yersinia enterocolitica reveals metabolic adaptations and a role of a phage lysis cassette in insect killing.

The human pathogen Yersinia enterocolitica strain W22703 is characterized by its toxicity towards invertebrates that requires the insecticidal toxin complex (Tc) proteins encoded by the pathogenicity island Tc-PAIYe. Molecular and pathophysiological details of insect larvae infection and killing by this pathogen, however, have not been dissected. Here, we applied oral infection of Galleria mellonella (Greater wax moth) larvae to study the colonisation, proliferation, tissue invasion, and killing activity of W22703. We demonstrated that this strain is strongly toxic towards the larvae, in which they proliferate by more than three orders of magnitude within six days post infection. Deletion mutants of the genes tcaA and tccC were atoxic for the insect. W22703 ΔtccC, in contrast to W22703 ΔtcaA, initially proliferated before being eliminated from the host, thus confirming TcaA as membrane-binding Tc subunit and TccC as cell toxin. Time course experiments revealed a Tc-dependent infection process starting with midgut colonisation that is followed by invasion of the hemolymph where the pathogen elicits morphological changes of hemocytes and strongly proliferates. The in vivo transcriptome of strain W22703 shows that the pathogen undergoes a drastic reprogramming of central cell functions and gains access to numerous carbohydrate and amino acid resources within the insect. Strikingly, a mutant lacking a phage-related holin/endolysin (HE) cassette, which is located within Tc-PAIYe, resembled the phenotypes of W22703 ΔtcaA, suggesting that this dual lysis cassette may be an example of a phage-related function that has been adapted for the release of a bacterial toxin.

Genome-wide association reveals host-specific genomic traits in Escherichia coli.

BACKGROUND: Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. RESULTS: We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. CONCLUSIONS: This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

Regulation and Functionality of a Holin/Endolysin Pair Involved in Killing of Galleria mellonella and Caenorhabditis elegans by Yersinia enterocolitica.

The insecticidal toxin complex (Tc) proteins are produced by several insect-associated bacteria, including Yersinia enterocolitica strain W22703, which oscillates between two distinct pathogenicity phases in invertebrates and humans. The mechanism by which this high-molecular-weight toxin is released into the extracellular surrounding, however, has not been deciphered. In this study, we investigated the regulation and functionality of a phage-related holin/endolysin (HE) cassette located within the insecticidal pathogenicity island Tc-PAIYe of W22703. Using the Galleria mellonella infection model and luciferase reporter fusions, we revealed that quorum sensing contributes to the insecticidal activity of W22703 upon influencing the transcription of tcaR2, which encodes an activator of the tc and HE genes. In contrast, a lack of the Yersinia modulator, YmoA, stimulated HE gene transcription, and mutant W22703 ΔymoA exhibited a stronger toxicity toward insect larvae than did W22703. A luciferase reporter fusion demonstrated transcriptional activation of the HE cassette in vivo, and a significantly larger extracellular amount of subunit TcaA was found in W22703 ΔymoA relative to its ΔHE mutant. Using competitive growth assays, we demonstrated that at least in vitro, the TcaA release upon HE activity is not mediated by cell lysis of a significant part of the population. Oral infection of Caenorhabditis elegans with a HE deletion mutant attenuated the nematocidal activity of the wild type, similar to the case with a mutant lacking a Tc subunit. We conclude that the dual holin/endolysin cassette of yersiniae is a novel example of a phage-related function adapted for the release of a bacterial toxin. IMPORTANCE Members of the genus Yersinia cause gastroenteritis in humans but also exhibit toxicity toward invertebrates. A virulence factor required for this environmental life cycle stage is the multisubunit toxin complex (Tc), which is distinct from the insecticidal toxin of Bacillus thuringiensis and has the potential to be used in pest control. The mechanism by which this high-molecular-weight Tc is secreted from bacterial cells has not been uncovered. Here, we show that a highly conserved phage-related holin/endolysin pair, which is encoded by the genes holY and elyY located between the Tc subunit genes, is essential for the insecticidal activity of Y. enterocolitica and that its activation increases the amount of Tc subunits in the supernatant. Thus, the dual holY-elyY cassette of Y. enterocolitica constitutes a new example for a type 10 secretion system to release bacterial toxins.

Lon Protease- and Temperature-Dependent Activity of a Lysis Cassette Located in the Insecticidal Island of Yersinia enterocolitica.

The Yersinia genus comprises pathogens that can adapt to an environmental life cycle stage as well as to mammals. Yersinia enterocolitica strain W22703 exhibits both insecticidal and nematocidal activity conferred by the tripartite toxin complex (Tc) that is encoded on the 19-kb pathogenicity island Tc-PAI Ye All tc genes follow a strict temperature regulation in that they are silenced at 37°C but activated at lower temperatures. Four highly conserved phage-related genes, located within the Tc-PAI Ye , were recently demonstrated to encode a biologically functional holin-endolysin gene cassette that lyses its own host W22703 at 37°C. Conditions transcriptionally activating the cassette are not yet known. In contrast to Escherichia coli, the overproduction of holin and endolysin did not result in cell lysis of strain W22703 at 15°C. When the holin-endolysin genes were overexpressed at 15°C in four Y. enterocolitica biovars and in four other Yersinia spp., a heterogenous pattern of phenotypes was observed, ranging from lysis resistance of a biovar 1A strain to the complete growth arrest of a Y. kristensenii strain. To decipher the molecular mechanism underlying this temperature-dependent lysis, we constructed a Lon protease-negative mutant of W22703 in which the overexpression of the lysis cassette leads to cell death at 15°C. Overexpressed endolysin exhibited a high proteolytic susceptibility in strain W22703 but remained stable in the W22703 Δlon strain or in Y. pseudotuberculosis Although artificial overexpression was applied here, the data indicate that Lon protease plays a role in the control of the temperature-dependent lysis in Y. enterocolitica W22703.IMPORTANCE The investigation of the mechanisms that help pathogens survive in the environment is a prerequisite to understanding their evolution and their virulence capacities. In members of the genus Yersinia, many factors involved in virulence, metabolism, motility, or biofilm formation follow a strict temperature-dependent regulation. While the molecular mechanisms underlying the activation of determinants at body temperature have been analyzed in detail, the molecular basis of low-temperature-dependent phenotypes is largely unknown. Here, we demonstrate that a novel phage-related lysis cassette, which is part of the insecticidal and nematocidal pathogenicity island of Y. enterocolitica, does not lyse its own host following overexpression at 15°C and that the Lon protease is involved in this phenotype.

Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli.

BACKGROUND: Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C16H26) produced by selected strains of S. plymuthica. The BGC encoding sodorifen consists of four genes, two of which (sodA, sodB) are homologs of genes encoding enzymes of the non-mevalonate pathway and are thought to enhance the amounts of available farnesyl pyrophosphate (FPP), the precursor of sodorifen. Proceeding from FPP, only two enzymes are necessary to produce sodorifen: an S-adenosyl methionine dependent methyltransferase (SodC) with additional cyclisation activity and a terpene-cyclase (SodD). Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains. This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene. RESULTS: Sequencing and mining of the S. plymuthica WS3236 genome revealed the presence of 38 BGCs according to antiSMASH analysis, including a putative sodorifen BGC. Further genome mining for sodorifen and sodorifen-like BGCs throughout bacteria was performed using SodC and SodD as queries and identified a total of 28 sod-like gene clusters. Using direct pathway cloning (DiPaC) we intercepted the 4.6 kb candidate sodorifen BGC from S. plymuthica WS3236 (sodA-D) and transformed it into Escherichia coli BL21. Heterologous expression under the control of the tetracycline inducible PtetO promoter firmly linked this BGC to sodorifen production. By utilizing this newly established expression system, we increased the production yields by approximately 26-fold when compared to the native producer. In addition, sodorifen was easily isolated in high purity by simple head-space sampling. CONCLUSIONS: Genome mining of all available genomes within the NCBI and JGI IMG databases led to the identification of a wealth of sod-like pathways which may be responsible for producing a range of structurally unknown sodorifen analogs. Introduction of the S. plymuthica WS3236 sodorifen BGC into the fast-growing heterologous expression host E. coli with a very low VOC background led to a significant increase in both sodorifen product yield and purity compared to the native producer. By providing a reliable, high-level production system, this study sets the stage for future investigations of the biological role and function of sodorifen and for functionally unlocking the bioinformatically identified putative sod-like pathways.

Activity of a Holin-Endolysin System in the Insecticidal Pathogenicity Island of Yersinia enterocolitica.

Yersinia enterocolitica is a pathogen that causes gastroenteritis in humans. Because of its low-temperature-dependent insecticidal activity, it can oscillate between invertebrates and mammals as host organisms. The insecticidal activity of strain W22703 is associated with a pathogenicity island of 19 kb (Tc-PAI Ye ), which carries regulators and genes encoding the toxin complex (Tc). The island also harbors four phage-related and highly conserved genes of unknown functions, which are polycistronically transcribed. Two open reading frames showed significant homologies to holins and endolysins and exhibited lytic activity in Escherichia coli cells upon overexpression. When a set of Yersinia strains was tested in an equivalent manner, highly diverse susceptibilities to lysis were observed, and some strains were resistant to lysis. If cell lysis occurred (as demonstrated by membrane staining), it was more pronounced when two accessory elements of the cassette coding for an i-spanin and an o-spanin were included in the overexpression construct. The pore-forming function of the putative holin, HolY, was demonstrated by complementation of the lysis defect of a phage λ S holin mutant. In experiments performed with membrane preparations, ElyY exhibited high specificity for W22703 peptidoglycan, with a cleavage activity resembling that of lysozyme. Although the functionality of the lysis cassette from Tc-PAI Ye was demonstrated in this study, its biological role remains to be elucidated.IMPORTANCE The knowledge of how pathogens survive in the environment is pivotal for our understanding of bacterial virulence. The insecticidal and nematocidal activity of Yersinia spp., by which the bacteria gain access to nutrients and thus improve their environmental fitness, is conferred by the toxin complex (Tc) encoded on a highly conserved pathogenicity island termed Tc-PAI Ye While the regulators and the toxin subunits of the island had been characterized in some detail, the role of phage-related genes within the island remained to be elucidated. Here, we demonstrate that this cassette encodes a holin, an endolysin, and two spanins that, at least upon overexpression, lyse Yersinia strains.

Genetic Characterization of the Galactitol Utilization Pathway of Salmonella enterica Serovar Typhimurium.

Galactitol degradation by salmonellae remains underinvestigated, although this metabolic capability contributes to growth in animals (R. R. Chaudhuri et al., PLoS Genet 9:e1003456, 2013, https://doi.org/10.1371/journal.pgen.1003456). The genes responsible for this metabolic capability are part of a 9.6-kb gene cluster that spans from gatY to gatR (STM3253 to STM3262) and encodes a phosphotransferase system, four enzymes, and a transporter of the major facilitator superfamily. Genome comparison revealed the presence of this genetic determinant in nearly all Salmonella strains. The generation time of Salmonella enterica serovar Typhimurium strain ST4/74 was higher in minimal medium with galactitol than with glucose. Knockout of STM3254 and gatC resulted in a growth-deficient phenotype of S Typhimurium, with galactitol as the sole carbon source. Partial deletion of gatR strongly reduced the lag phase of growth with galactitol, whereas strains overproducing GatR exhibited a near-zero growth phenotype. Luciferase reporter assays demonstrated strong induction of the gatY and gatZ promoters, which control all genes of this cluster except gatR, in the presence of galactitol but not glucose. Purified GatR bound to these two main gat gene cluster promoters as well as to its own promoter, demonstrating that this autoregulated repressor controls galactitol degradation. Surface plasmon resonance spectroscopy revealed distinct binding properties of GatR toward the three promoters, resulting in a model of differential gat gene expression. The cyclic AMP receptor protein (CRP) bound these promoters with similarly high affinities, and a mutant lacking crp showed severe growth attenuation, demonstrating that galactitol utilization is subject to catabolite repression. Here, we provide the first genetic characterization of galactitol degradation in Salmonella, revealing novel insights into the regulation of this dissimilatory pathway. IMPORTANCE: The knowledge of how pathogens adapt their metabolism to the compartments encountered in hosts is pivotal to our understanding of bacterial infections. Recent research revealed that enteropathogens have adapted specific metabolic pathways that contribute to their virulence properties, for example, by helping to overcome limitations in nutrient availability in the gut due to colonization resistance. The capability of Salmonella enterica serovar Typhimurium to degrade galactitol has already been demonstrated to play a role in vivo, but it has not been investigated so far on the genetic level. To our knowledge, this is the first molecular description of the galactitol degradation pathway of a pathogen.

Isotopologue profiling of the listerial N-metabolism.

The nitrogen (N-) sources and the relative contribution of a nitrogenous nutrient to the N-pool of the gram-positive pathogen Listeria monocytogenes are largely unknown. Therefore, (15) N-isotopologue profiling was established to study the N-metabolism of L. monocytogenes. The pathogen was grown in a defined minimal medium supplemented with potential (15) N-labeled nutrients. The bacteria were harvested and hydrolysed under acidic conditions, and the resulting amino acids were analysed by GC-MS, revealing (15) N-enrichments and isotopomeric compositions of amino acids. The differential (15) N-profiles showed the substantial and simultaneous usage of ammonium, glutamine, methionine, and, to a lower extent, the branched-chain amino acids valine, leucine, and isoleucine for anabolic purposes, with a significant preference for ammonium. In contrast, arginine, histidine and cysteine were directly incorporated into proteins. L. monocytogenes is able to replace glutamine with ethanolamine or glucosamine as amino donors for feeding the core N-metabolism. Perturbations of N-fluxes caused by gene deletions demonstrate the involvement of ethanolamine ammonia lyase, and suggest a role of the regulator GlnK of L. monocytogenes distinct from that of Escherichia coli. The metabolism of nitrogenous nutrients reflects the high flexibility of this pathogenic bacterium in exploiting N-sources that could also be relevant for its proliferation during infection.

Parallel independent evolution of pathogenicity within the genus Yersinia.

The genus Yersinia has been used as a model system to study pathogen evolution. Using whole-genome sequencing of all Yersinia species, we delineate the gene complement of the whole genus and define patterns of virulence evolution. Multiple distinct ecological specializations appear to have split pathogenic strains from environmental, nonpathogenic lineages. This split demonstrates that contrary to hypotheses that all pathogenic Yersinia species share a recent common pathogenic ancestor, they have evolved independently but followed parallel evolutionary paths in acquiring the same virulence determinants as well as becoming progressively more limited metabolically. Shared virulence determinants are limited to the virulence plasmid pYV and the attachment invasion locus ail. These acquisitions, together with genomic variations in metabolic pathways, have resulted in the parallel emergence of related pathogens displaying an increasingly specialized lifestyle with a spectrum of virulence potential, an emerging theme in the evolution of other important human pathogens.

Natural-Product-Inspired Aminoepoxybenzoquinones Kill Members of the Gram-Negative Pathogen Salmonella by Attenuating Cellular Stress Response.

Gram-negative bacteria represent a challenging task for antibacterial drug discovery owing to their impermeable cell membrane and restricted uptake of small molecules. We herein describe the synthesis of natural-product-derived epoxycyclohexenones and explore their antibiotic activity against several pathogenic bacteria. A compound with activity against Salmonella Typhimurium was identified, and the target enzymes were unraveled by quantitative chemical proteomics. Importantly, two protein hits were linked to bacterial stress response, and corresponding assays revealed an elevated susceptibility to reactive oxygen species upon compound treatment. The consolidated inhibition of these targets provides a rationale for antibacterial activity and highlights epoxycyclohexenones as natural product scaffolds with suitable properties for killing Gram-negative Salmonella.

YmoA negatively controls the expression of insecticidal genes in Yersinia enterocolitica.

Yersinia enterocolitica is toxic towards invertebrates due to the presence of the toxin complex (tc) genes that are activated by the thermolabile regulator TcaR2. In the search for further regulatory factors involved in insecticidal gene expression, the modulator of yersinial virulence, YmoA, was identified to silence all tc genes of the Y. enterocolitica strain W22703 (biovar 2, serovar O:9). Using promoter fusions with the luciferase reporter, we found that the deletion of ymoA results in elevated transcription of tcaR1, tcaR2, tcaA, tcaB, tcaC, tccC1 and tccC2 at both 15 °C and 37 °C. Complementation by episomal ymoA significantly reduced tc gene expression, thus validating the inhibitory activity of YmoA on the production of insecticidal proteins. YmoA contributes to the binding properties of H-NS to the tc promoters by forming a complex with this nucleoid-associated protein, and this complex not only binds to the upstream regions of all tc genes, but also to intragenic sites of tcaA and tcaB that play an important role in controlling the expression of both genes. At low temperature, the intracellular amount of thermostable YmoA is not reduced, but the repressor is less functional. These data point to H-NS/YmoA as an antagonist of the inducer TcaR2.

The orphan regulator ReiD of Salmonella enterica is essential for myo-inositol utilization.

In Salmonella enterica serovar Typhimurium (S. Typhimurium), the genomic island GEI4417/4436 is responsible for the utilization of myo-inositol (MI) as carbon and energy source. Here, we report the characterization of a novel, island-encoded positive autoregulator termed ReiD (STM4423) that is specific to certain S. enterica strains and Escherichia coli strain ED1a able to use MI. ReiD was essential for growth with this polyol and also contributed to S. Typhimurium proliferation in swine caecum content. Providing higher copy numbers of ReiD reduced the long lag phase of 2 days during growth of S. Typhimurium in MI medium by 50%. In a heterologous host, expression of ReiD activated the transcription from the promoter of iolE/iolG, whose products catalyse the initial two steps in MI degradation. Episomal expression of iolE/iolG1 rescued the otherwise zero growth phenotype of a reiD deletion mutant in MI medium. Gel mobility shift assays with purified ReiD demonstrated directed interaction of ReiD with its own promoter and that of iolE. The repressor IolR bound the reiD promoter, implying that reiD is part of the IolR regulon. Taken together, the regulator ReiD is a trigger to accelerate the switch from more easily accessible nutrients to MI utilization by S. Typhimurium.

The insecticidal toxin genes of Yersinia enterocolitica are activated by the thermolabile LTTR-like regulator TcaR2 at low temperatures.

Temperature-dependent activation of bacterial virulence factors at 37°C is well investigated. The molecular mechanism underlying the expression of toxicity determinants at environmental temperatures, however, has not been characterized. The insecticidal activity of Yersinia enterocolitica strain W22703 requires the toxin complex subunit A (TcaA) encoded on the pathogenicity island Tc-PAIYe . Genes tcaA and tcaB encoding this subunit are maximally produced at low temperatures (10-20°C), but repressed at body temperature. Two further insecticidal genes, tcaC (subunit B) and tccC1 (subunit C), are silent at both temperatures. A novel LysR-type transcriptional regulator (LTTR), TcaR2, revealed to be autoregulated and essential for tcaA and tcaB expression in W22703. Expression of tcaR2 is negatively controlled by a second LTTR-like regulator, TcaR1. Gel mobility shift assays confirmed the interaction of TcaR2 with the tcaR2, tcaA and tcaB promoters. The activity of the tcaA promoter in heterologous hosts in the presence of TcaR2 excludes the requirement of additional, Yersinia-specific (co)factors for toxin gene expression. Overproduced TcaR2 protein is shown to be unstable at 37°C, whereas the mRNA of tcaA and tcaR2 is equally stable at low and high temperature. Thus, TcaR2 is a key player in the induction of insecticidal genes in Y. enterocolitica at low temperatures.

From food to cell: nutrient exploitation strategies of enteropathogens.

Upon entering the human gastrointestinal tract, foodborne bacterial enteropathogens encounter, among numerous other stress conditions, nutrient competition with the host organism and the commensal microbiota. The main carbon, nitrogen and energy sources exploited by pathogens during proliferation in, and colonization of, the gut have, however, not been identified completely. In recent years, a huge body of literature has provided evidence that most enteropathogens are equipped with a large set of specific metabolic pathways to overcome nutritional limitations in vivo, thus increasing bacterial fitness during infection. These adaptations include the degradation of myo-inositol, ethanolamine cleaved from phospholipids, fucose derived from mucosal glycoconjugates, 1,2-propanediol as the fermentation product of fucose or rhamnose and several other metabolites not accessible for commensal bacteria or present in competition-free microenvironments. Interestingly, the data reviewed here point to common metabolic strategies of enteric pathogens allowing the exploitation of nutrient sources that not only are present in the gut lumen, the mucosa or epithelial cells, but also are abundant in food. An increased knowledge of the metabolic strategies developed by enteropathogens is therefore a key factor to better control foodborne diseases.

Hierarchic regulation of a metabolic pathway: H-NS, CRP, and SsrB control myo-inositol utilization by Salmonella enterica.

IMPORTANCE: The capacity to utilize myo-inositol (MI) as sole carbon and energy source is widespread among bacteria, among them the intestinal pathogen S. Typhimurium. This study elucidates the complex and hierarchical regulation that underlies the utilization of MI by S. Typhimurium under substrate limitation. A total of seven regulatory factors have been identified so far, allowing the pathogen an environment-dependent, efficient, and fine-tuned regulation of a metabolic property that provides growth advantages in different environments.

Acid shock of Listeria monocytogenes at low environmental temperatures induces prfA, epithelial cell invasion, and lethality towards Caenorhabditis elegans.

BACKGROUND: The saprophytic pathogen Listeria monocytogenes has to cope with a variety of acidic habitats during its life cycle. The impact of low-temperature coupled with pH decrease for global gene expression and subsequent virulence properties, however, has not been elucidated. RESULTS: qRT-PCR revealed for the first time a transient, acid triggered prfA induction of approximately 4-fold, 5.7-fold, 7-fold and 9.3-fold 60 to 90 min after acid shock of L. monocytogenes at 37°C, 25°C, 18°C, and 10°C, respectively. Comparable data were obtained for seven different L. monocytogenes strains, demonstrating that prfA induction under these conditions is a general response of L. monocytogenes. Transcriptome analysis revealed that the in vivo-relevant genes bsh, clpP, glpD, hfq, inlA, inlB, inlE, lisR, and lplA1 as well as many other genes with a putative role during infection are transiently induced upon acid shock conducted at 25°C and 37°C. Twenty-five genes repressed upon acid shock are known to be down regulated during intracellular growth or by virulence regulators. These data were confirmed by qRT-PCR of twelve differentially regulated genes and by the identification of acid shock-induced genes influenced by σB. To test if up regulation of virulence genes at temperatures below 37°C correlates with pathogenicity, the capacity of L. monocytogenes to invade epithelial cells after acid shock at 25°C was measured. A 12-fold increased number of intracellular bacteria was observed (acid shock, t = 60 min) that was reduced after adaptation to the level of the unshocked control. This increased invasiveness was shown to be in line with the induction of inlAB. Using a nematode infection assay, we demonstrated that Caenorhabditis elegans fed with acid-shocked L. monocytogenes exhibits a shorter time to death of 50% (TD50) of the worms (6.4 days) compared to infection with unshocked bacteria (TD50 = 10.2 days). CONCLUSIONS: PrfA and other listerial virulence genes are induced by an inorganic acid in a temperature-dependent manner. The data presented here suggest that low pH serves as a trigger for listerial pathogenicity at environmental temperatures.

The natural product chlorotonil A preserves colonization resistance and prevents relapsing Clostridioides difficile infection.

Clostridioides difficile infections (CDIs) remain a healthcare problem due to high rates of relapsing/recurrent CDIs (rCDIs). Breakdown of colonization resistance promoted by broad-spectrum antibiotics and the persistence of spores contribute to rCDI. Here, we demonstrate antimicrobial activity of the natural product class of chlorotonils against C. difficile. In contrast to vancomycin, chlorotonil A (ChA) efficiently inhibits disease and prevents rCDI in mice. Notably, ChA affects the murine and porcine microbiota to a lesser extent than vancomycin, largely preserving microbiota composition and minimally impacting the intestinal metabolome. Correspondingly, ChA treatment does not break colonization resistance against C. difficile and is linked to faster recovery of the microbiota after CDI. Additionally, ChA accumulates in the spore and inhibits outgrowth of C. difficile spores, thus potentially contributing to lower rates of rCDI. We conclude that chlorotonils have unique antimicrobial properties targeting critical steps in the infection cycle of C. difficile.

Potentiation of epithelial innate host responses by intercellular communication.

The epithelium efficiently attracts immune cells upon infection despite the low number of pathogenic microbes and moderate levels of secreted chemokines per cell. Here we examined whether horizontal intercellular communication between cells may contribute to a coordinated response of the epithelium. Listeria monocytogenes infection, transfection, and microinjection of individual cells within a polarized intestinal epithelial cell layer were performed and activation was determined at the single cell level by fluorescence microscopy and flow cytometry. Surprisingly, chemokine production after L. monocytogenes infection was primarily observed in non-infected epithelial cells despite invasion-dependent cell activation. Whereas horizontal communication was independent of gap junction formation, cytokine secretion, ion fluxes, or nitric oxide synthesis, NADPH oxidase (Nox) 4-dependent oxygen radical formation was required and sufficient to induce indirect epithelial cell activation. This is the first report to describe epithelial cell-cell communication in response to innate immune activation. Epithelial communication facilitates a coordinated infectious host defence at the very early stage of microbial infection.

Metabolism in the Niche: a Large-Scale Genome-Based Survey Reveals Inositol Utilization To Be Widespread among Soil, Commensal, and Pathogenic Bacteria.

Phytate is the main phosphorus storage molecule of plants and is therefore present in large amounts in the environment and in the diet of humans and animals. Its dephosphorylated form, the polyol myo-inositol (MI), can be used by bacteria as a sole carbon and energy source. The biochemistry and regulation of MI degradation were deciphered in Bacillus subtilis and Salmonella enterica, but a systematic survey of this catabolic pathway has been missing until now. For a comprehensive overview of the distribution of MI utilization, we analyzed 193,757 bacterial genomes, representing a total of 24,812 species, for the presence, organization, and taxonomic prevalence of inositol catabolic gene clusters (IolCatGCs). The genetic capacity for MI degradation was detected in 7,384 (29.8%) of all species for which genome sequences were available. IolCatGC-positive species were particularly found among Actinobacteria and Proteobacteria and to a much lesser extent in Bacteroidetes. IolCatGCs are very diverse in terms of gene number and functions, whereas the order of core genes is highly conserved on the phylum level. We predict that 111 animal pathogens, more than 200 commensals, and 430 plant pathogens or rhizosphere bacteria utilize MI, underscoring that IolCatGCs provide a growth benefit within distinct ecological niches. IMPORTANCE This study reveals that the capacity to utilize inositol is unexpectedly widespread among soil, commensal, and pathogenic bacteria. We assume that this yet-neglected metabolism plays a pivotal role in the microbial turnover of phytate and inositols. The bioinformatic tool established here enables predicting to which extent and genetic variance a bacterial determinant is present in all genomes sequenced so far.