Scalable Reporter Assays to Analyze the Regulation of stx2 Expression in Shiga Toxin-Producing Enteropathogens.

Stx2 is the major virulence factor of EHEC and is associated with an increased risk for HUS in infected patients. The conditions influencing its expression in the intestinal tract are largely unknown. For optimal management and treatment of infected patients, the identification of environmental conditions modulating Stx2 levels in the human gut is of central importance. In this study, we established a set of chromosomal stx2 reporter assays. One system is based on superfolder GFP (sfGFP) using a T7 polymerase/T7 promoter-based amplification loop. This reporter can be used to analyze stx2 expression at the single-cell level using FACSs and fluorescence microscopy. The other system is based on the cytosolic release of the Gaussia princeps luciferase (gluc). This latter reporter proves to be a highly sensitive and scalable reporter assay that can be used to quantify reporter protein in the culture supernatant. We envision that this new set of reporter tools will be highly useful to comprehensively analyze the influence of environmental and host factors, including drugs, small metabolites and the microbiota, on Stx2 release and thereby serve the identification of risk factors and new therapies in Stx-mediated pathologies.

Helicobacter hepaticus is required for immune targeting of bacterial heat shock protein 60 and fatal colitis in mice.

Gut microbiota and the immune system are in constant exchange shaping both host immunity and microbial communities. Here, improper immune regulation can cause inflammatory bowel disease (IBD) and colitis. Antibody therapies blocking signaling through the CD40-CD40L axis showed promising results as these molecules are deregulated in certain IBD patients. To better understand the mechanism, we used transgenic DC-LMP1/CD40 animals with a constitutive CD40-signal in CD11c+ cells, causing a lack of intestinal CD103+ dendritic cells (DCs) and failure to induce regulatory T (iTreg) cells. These mice rapidly develop spontaneous fatal colitis, accompanied by dysbiosis and increased inflammatory IL-17+IFN-γ+ Th17/Th1 and IFN-γ + Th1 cells. In the present study, we analyzed the impact of the microbiota on disease development and detected elevated IgA- and IgG-levels in sera from DC-LMP1/CD40 animals. Their serum antibodies specifically bound intestinal bacteria, and by proteome analysis, we identified a 60 kDa chaperonin GroEL (Hsp60) from Helicobacter hepaticus (Hh) as the main specific antigen targeted in the absence of iTregs. When re-derived to a different Hh-free specific-pathogen-free (SPF) microbiota, mice showed few signs of disease, normal microbiota, and no fatality. Upon recolonization of mice with Hh, the disease developed rapidly. Thus, the present work identifies GroEL/Hsp60 as a major Hh-antigen and its role in disease onset, progression, and outcome in this colitis model. Our results highlight the importance of CD103+ DC- and iTreg-mediated immune tolerance to specific pathobionts to maintain healthy intestinal balance.

Microbiome Analysis from Paired Mucosal and Fecal Samples of a Colorectal Cancer Biobank.

The role of gut microbiota in colorectal cancer is subject to extensive research. Before usage of biorepositories for microbiome studies, it is crucial to evaluate technical feasibility of microbiome profiling from various biospecimens. The aim of this study was to assess the feasibility of DNA-extraction and microbiome profiling of samples from different sample sites, tissue sites and storage duration of a colorectal cancer biobank. Mucosa samples, mucosal scrapings and feces as well as different tissue sites (tumor, normal mucosa) were analyzed. 16S rRNA gene-based microbiome profiling with taxonomic assignment was performed on the Illumina MiSeq (Illumina, San Diego, USA) platform from stored snap frozen samples. For statistical analysis, α- and ß-diversity measures, PCoA, permutational multivariate analysis of variance and graphical representation were performed. Microbiome analysis could be successfully performed in most of the samples (overall 93.3%) with sufficient numbers of high-quality reads. There were no differences between sample sites, while in some measures significant differences were found between tumor and normal mucosa (α-diversity, Shannon/Simpson Indices p = 0.028/0.027, respectively). Samples stored for up to eight years were used and storage conditions had no significant influence on the results. Tumor and tissue samples of a biobank stored long term can be successfully used for microbiome analysis. As large sample sizes are needed for association studies to evaluate microbial impact on tumorigenesis or progression of colorectal cancer, an already established biorepository may be a useful alternative to prospective clinical studies.

Evolutionary Stabilization of Cooperative Toxin Production through a Bacterium-Plasmid-Phage Interplay.

Colicins are toxins produced and released by Enterobacteriaceae to kill competitors in the gut. While group A colicins employ a division of labor strategy to liberate the toxin into the environment via colicin-specific lysis, group B colicin systems lack cognate lysis genes. In Salmonella enterica serovar Typhimurium (S. Tm), the group B colicin Ib (ColIb) is released by temperate phage-mediated bacteriolysis. Phage-mediated ColIb release promotes S. Tm fitness against competing Escherichia coli It remained unclear how prophage-mediated lysis is realized in a clonal population of ColIb producers and if prophages contribute to evolutionary stability of toxin release in S. Tm. Here, we show that prophage-mediated lysis occurs in an S. Tm subpopulation only, thereby introducing phenotypic heterogeneity to the system. We established a mathematical model to study the dynamic interplay of S. Tm, ColIb, and a temperate phage in the presence of a competing species. Using this model, we studied long-term evolution of phage lysis rates in a fluctuating infection scenario. This revealed that phage lysis evolves as bet-hedging strategy that maximizes phage spread, regardless of whether colicin is present or not. We conclude that the ColIb system, lacking its own lysis gene, is making use of the evolutionary stable phage strategy to be released. Prophage lysis genes are highly prevalent in nontyphoidal Salmonella genomes. This suggests that the release of ColIb by temperate phages is widespread. In conclusion, our findings shed new light on the evolution and ecology of group B colicin systems.IMPORTANCE Bacteria are excellent model organisms to study mechanisms of social evolution. The production of public goods, e.g., toxin release by cell lysis in clonal bacterial populations, is a frequently studied example of cooperative behavior. Here, we analyze evolutionary stabilization of toxin release by the enteric pathogen Salmonella The release of colicin Ib (ColIb), which is used by Salmonella to gain an edge against competing microbiota following infection, is coupled to bacterial lysis mediated by temperate phages. Here, we show that phage-dependent lysis and subsequent release of colicin and phage particles occurs only in part of the ColIb-expressing Salmonella population. This phenotypic heterogeneity in lysis, which represents an essential step in the temperate phage life cycle, has evolved as a bet-hedging strategy under fluctuating environments such as the gastrointestinal tract. Our findings suggest that prophages can thereby evolutionarily stabilize costly toxin release in bacterial populations.

Evolutionary Stability of Salmonella Competition with the Gut Microbiota: How the Environment Fosters Heterogeneity in Exploitative and Interference Competition.

Following ingestion, gastrointestinal pathogens compete against the gastrointestinal microbiota and overcome host immune defenses in order to cause infections. Besides employing direct killing mechanisms, the commensal microbiota occupies metabolic niches to outcompete invading pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) uses several strategies to successfully colonize the gut and establish infection, of which an increasing number is based on phenotypic heterogeneity within the S. Typhimurium population. The utilization of myo-inositol (MI) and the production of colicin confer a selective advantage over the microbiota in terms of exploitative and interference competition, respectively. In this review, we summarize the genetic basis underlying bistability of MI catabolism and colicin production. As demonstrated by single-cell analyses, a stochastic switch in the expression of the genes responsible for colicin production and MI degradation constitutes the heterogeneity of the two phenotypes. Both genetic systems are tightly regulated to avoid their expression under non-appropriate conditions and possible detrimental effects on bacterial fitness. Moreover, evolutionary mechanisms underlying formation and stability of these phenotypes in S. Typhimurium are discussed. We propose that both MI catabolism and colicin production create a bet-hedging strategy, which provides an adaptive benefit for S. Typhimurium in the fluctuating environment of the mammalian gut.

Spatially Correlated Gene Expression in Bacterial Groups: The Role of Lineage History, Spatial Gradients, and Cell-Cell Interactions.

Gene expression levels in clonal bacterial groups have been found to be spatially correlated. These correlations can partly be explained by the shared lineage history of nearby cells, although they could also arise from local cell-cell interactions. Here, we present a quantitative framework that allows us to disentangle the contributions of lineage history, long-range spatial gradients, and local cell-cell interactions to spatial correlations in gene expression. We study pathways involved in toxin production, SOS stress response, and metabolism in Escherichia coli microcolonies and find for all pathways that shared lineage history is the main cause of spatial correlations in gene expression levels. However, long-range spatial gradients and local cell-cell interactions also contributed to spatial correlations in SOS response, amino acid biosynthesis, and overall metabolic activity. Together, our data show that the phenotype of a cell is influenced by its lineage history and population context, raising the question of whether bacteria can arrange their activities in space to perform functions they cannot achieve alone.

Temperate phages promote colicin-dependent fitness of Salmonella enterica serovar Typhimurium.

Bacteria employ bacteriocins for interference competition in microbial ecosystems. Colicin Ib (ColIb), a pore-forming bacteriocin, confers a significant fitness benefit to Salmonella enterica serovar Typhimurium (S. Tm) in competition against commensal Escherichia coli in the gut. ColIb is released from S. Tm into the environment, where it kills susceptible competitors. However, colicin-specific release proteins, as they are known for other colicins, have not been identified in case of ColIb. Thus, its release mechanism has remained unclear. In the current study, we have established a new link between ColIb release and lysis activity of temperate, lambdoid phages. By the use of phage-cured S. Tm mutant strains, we show that the presence of temperate phages and their lysis genes is necessary and sufficient for release of active ColIb into the culture supernatant. Furthermore, phage-mediated lysis significantly enhanced S. Tm fitness in competition against a ColIb-susceptible competitor. Finally, transduction with the lambdoid phage 933W rescued the defect of E. coli strain MG1655 with respect to ColIb release. In conclusion, ColIb is released from bacteria in the course of phage lysis. Our data reveal a new mechanism for colicin release and point out a novel function of temperate phages in enhancing colicin-dependent bacterial fitness.

Reporters for Single-Cell Analysis of Colicin Ib Expression in Salmonella enterica Serovar Typhimurium.

Colicins are toxins that mediate interference competition in microbial ecosystems. They serve as a "common good" for the entire producer population but are synthesized by only few members which pay the costs of colicin production. We have previously shown that production of colicin Ib (cib), a group B colicin, confers a competitive advantage to Salmonella enterica serovar Typhimurium (S. Tm) over commensal E. coli strains. Here, we studied regulation of S. Tm cib expression at the single cell level. Comparative analysis of a single- and a multicopy gfp-reporter for the colicin Ib promoter (Pcib) revealed that the latter yielded optimal signal intensity for a diverse range of applications. We further validated this reporter and showed that gfp expression correlated well with colicin Ib (ColIb) protein levels in individual cells. Pcib is negatively controlled by two repressors, LexA and Fur. Only a small fraction of S. Tm expressed cib under non-inducing conditions. We studied Pcib activity in response to mitomycin C mediated DNA damage and iron limitation. Both conditions, if applied individually, lead to an increase in the fraction of GFP+ S. Tm, albeit an overall low fluorescence intensity. When both conditions were applied simultaneously, the majority of S. Tm turned GFP+ and displayed high fluorescence intensity. Thus, both repressors individually confine cib expression to a subset of the population. Taken together, we provide the first thorough characterization of a conventional gfp-reporter to study regulation of a group B colicin at the single cell level. This reporter will be useful to further investigate the costs and benefits of ColIb production in human pathogenic S. Tm and analyze cib expression under environmental conditions encountered in the mammalian gut.