Médéric Diard

Stabilization of cooperative virulence by the expression of an avirulent phenotype

Pathogens often infect hosts through collective actions: they secrete growth-promoting compounds or virulence factors, or evoke host reactions that fuel the colonization of the host. Such behaviours are vulnerable to the rise of mutants that benefit from the collective action without contributing to it; how these behaviours can be evolutionarily stable is not well understood. We address this question using the intestinal pathogen Salmonella enterica serovar Typhimurium (hereafter termed S. typhimurium), which manipulates its host to induce inflammation, and thereby outcompetes the commensal microbiota. Notably, the virulence factors needed for host manipulation are expressed in a bistable fashion, leading to a slow-growing subpopulation that expresses virulence genes, and a fast-growing subpopulation that is phenotypically avirulent. Here we show that the expression of the genetically identical but phenotypically avirulent subpopulation is essential for the evolutionary stability of virulence in this pathogen. Using a combination of mathematical modelling, experimental evolution and competition experiments we found that within-host evolution leads to the emergence of mutants that are genetically avirulent and fast-growing. These mutants are defectors that exploit inflammation without contributing to it. In infection experiments initiated with wild-type S. typhimurium, defectors increase only slowly in frequency. In a genetically modified S. typhimurium strain in which the phenotypically avirulent subpopulation is reduced in size, defectors rise more rapidly, inflammation ceases prematurely, and S. typhimurium is quickly cleared from the gut. Our results establish that host manipulation by S. typhimurium is a cooperative trait that is vulnerable to the rise of avirulent defectors; the expression of a phenotypically avirulent subpopulation that grows as fast as defectors slows down this process, and thereby promotes the evolutionary stability of virulence. This points to a key role of bistable virulence gene expression in stabilizing cooperative virulence and may lead the way to new approaches for controlling pathogens.

Epithelium-Intrinsic NAIP/NLRC4 Inflammasome Drives Infected Enterocyte Expulsion to Restrict Salmonella Replication in the Intestinal Mucosa

The gut mucosal epithelium separates the host from the microbiota, but enteropathogens such as Salmonella Typhimurium (S.Tm) can invade and breach this barrier. Defenses against such acute insults remain incompletely understood. Using a murine model of Salmonella enterocolitis, we analyzed mechanisms limiting pathogen loads in the epithelium during early infection. Although the epithelium-invading S.Tm replicate initially, this intraepithelial replicative niche is restricted by expulsion of infected enterocytes into the lumen. This mechanism is compromised if inflammasome components (NAIP1-6, NLRC4, caspase-1/-11) are deleted, or ablated specifically in the epithelium, resulting in ∼100-fold higher intraepithelial loads and accelerated lymph node colonization. Interestingly, the cytokines downstream of inflammasome activation, interleukin (IL)-1α/β and IL-18, appear dispensable for epithelial restriction of early infection. These data establish the role of an epithelium-intrinsic inflammasome, which drives expulsion of infected cells to restrict the pathogens intraepithelial proliferation. This may represent a general defense mechanism against mucosal infections.

The streptomycin mouse model for Salmonella diarrhea: functional analysis of the microbiota, the pathogen's virulence factors, and the host's mucosal immune response.

Summary:  The mammalian intestine is colonized by a dense microbial community, the microbiota. Homeostatic and symbiotic interactions facilitate the peaceful co‐existence between the microbiota and the host, and inhibit colonization by most incoming pathogens (‘colonization resistance’). However, if pathogenic intruders overcome colonization resistance, a fierce, innate inflammatory defense can be mounted within hours, the adaptive arm of the immune system is initiated, and the pathogen is fought back. The molecular nature of the homeostatic interactions, the pathogen’s ability to overcome colonization resistance, and the triggering of native and adaptive mucosal immune responses are still poorly understood. To study these mechanisms, the streptomycin mouse model for Salmonella diarrhea is of great value. Here, we review how S. Typhimurium triggers mucosal immune responses by active (virulence factor elicited) and passive (MyD88‐dependent) mechanisms and introduce the S. Typhimurium mutants available for focusing on either response. Interestingly, mucosal defense turns out to be a double‐edged sword, limiting pathogen burdens in the gut tissue but enhancing pathogen growth in the gut lumen. This model allows not only studying the molecular pathogenesis of Salmonella diarrhea but also is ideally suited for analyzing innate defenses, microbe handling by mucosal phagocytes, adaptive secretory immunoglobulin A responses, probing microbiota function, and homeostatic microbiota–host interactions. Finally, we discuss the general need for defined assay conditions when using animal models for enteric infections and the central importance of littermate controls.

Bistable expression of virulence genes in Salmonella leads to the formation of an antibiotic-tolerant subpopulation

The bistable expression of virulence genes in Salmonella allows a clonal population to hedge its bets: one subpopulation suffers a growth cost, but is tolerant to antibiotics.

The Impact of 18 Ancestral and Horizontally-Acquired Regulatory Proteins upon the Transcriptome and sRNA Landscape of Salmonella enterica serovar Typhimurium

We know a great deal about the genes used by the model pathogen Salmonella enterica serovar Typhimurium to cause disease, but less about global gene regulation. New tools for studying transcripts at the single nucleotide level now offer an unparalleled opportunity to understand the bacterial transcriptome, and expression of the small RNAs (sRNA) and coding genes responsible for the establishment of infection. Here, we define the transcriptomes of 18 mutants lacking virulence-related global regulatory systems that modulate the expression of the SPI1 and SPI2 Type 3 secretion systems of S. Typhimurium strain 4/74. Using infection-relevant growth conditions, we identified a total of 1257 coding genes that are controlled by one or more regulatory system, including a sub-class of genes that reflect a new level of cross-talk between SPI1 and SPI2. We directly compared the roles played by the major transcriptional regulators in the expression of sRNAs, and discovered that the RpoS (σ38) sigma factor modulates the expression of 23% of sRNAs, many more than other regulatory systems. The impact of the RNA chaperone Hfq upon the steady state levels of 280 sRNA transcripts is described, and we found 13 sRNAs that are co-regulated with SPI1 and SPI2 virulence genes. We report the first example of an sRNA, STnc1480, that is subject to silencing by H-NS and subsequent counter-silencing by PhoP and SlyA. The data for these 18 regulatory systems is now available to the bacterial research community in a user-friendly online resource, SalComRegulon.

Peracetic Acid Treatment Generates Potent Inactivated Oral Vaccines from a Broad Range of Culturable Bacterial Species

Our mucosal surfaces are the main sites of non-vector-borne pathogen entry, as well as the main interface with our commensal microbiota. We are still only beginning to understand how mucosal adaptive immunity interacts with commensal and pathogenic microbes to influence factors such as infectivity, phenotypic diversity, and within-host evolution. This is in part due to difficulties in generating specific mucosal adaptive immune responses without disrupting the mucosal microbial ecosystem itself. Here, we present a very simple tool to generate inactivated mucosal vaccines from a broad range of culturable bacteria. Oral gavage of 1010 peracetic acid-inactivated bacteria induces high-titer-specific intestinal IgA in the absence of any measurable inflammation or species invasion. As a proof of principle, we demonstrate that this technique is sufficient to provide fully protective immunity in the murine model of invasive non-typhoidal Salmonellosis, even in the face of severe innate immune deficiency.

Evolution of bacterial virulence

Bacterial virulence is highly dynamic and context-dependent. For this reason, it is challenging to predict how molecular changes affect the growth of a pathogen in a host and its spread in host population. Two schools of thought have taken quite different directions to decipher the underlying principles of bacterial virulence. While molecular infection biology is focusing on the basic mechanisms of the pathogen-host interaction, evolution biology takes virulence as one of several parameters affecting pathogen spread in a host population. We review both approaches and discuss how they can complement each other in order to obtain a comprehensive understanding of bacterial virulence, its emergence, maintenance and evolution.

Enchained growth and cluster dislocation: a possible mechanism for microbiota homeostasis

Immunoglobulin A is a class of antibodies produced by the adaptive immune system and secreted into the gut lumen to fight pathogenic bacteria. We recently demonstrated that the main physical effect of these antibodies is to enchain daughter bacteria, i.e. to cross-link bacteria into clusters as they divide, preventing them from interacting with epithelial cells, thus protecting the host. These links between bacteria may break over time. We study several models using analytical and numerical calculations. We obtain the resulting distribution of chain sizes, that we compare with experimental data. We study the rate of increase in the number of free bacteria as a function of the replication rate of bacteria. Our models show robustly that at higher replication rates, bacteria replicate before the link between daughter bacteria breaks, leading to growing cluster sizes. On the contrary at low growth rates two daughter bacteria have a high probability to break apart. Thus the gut could produce IgA against all the bacteria it has encountered, but the most affected bacteria would be the fast replicating ones, that are more likely to destabilize the microbiota. Linking the effect of the immune effectors (here the clustering) with a property directly relevant to the potential bacterial pathogeneicity (here the replication rate) could avoid to make complex decisions about which bacteria to produce effectors against. Author Summary Inside the organism, the immune system can fight generically against any bacteria. However, the lumen of the gut is home to a very important microbiota, so the host has to find alternative ways to fight dangerous bacteria while sparing beneficial ones. While many studies have focused on the complex molecular and cellular pathways that trigger an immune response, little is known about how the produced antibodies act once secreted into the intestinal lumen. We recently demonstrated that the main physical effect of these antibodies is to cross-link bacteria into clusters as they divide, preventing them from interacting with epithelial cells, thus protecting the host. These links between bacteria may break over time. Using analytical and numerical calculations, and comparing with experimental data, we studied the dynamics of these clusters. At higher replication rates, bacteria replicate before the link between daughter bacteria breaks, leading to growing cluster sizes, and conversely. Thus the gut could produce IgA against all the bacteria it has encountered, but the most affected bacteria would be the fast replicating ones, that are more likely destabilize the microbiota. Studying the mechanisms of the immune response may uncover more such processes that enable to target properties hard to escape through evolution.

Detection of Mutations Affecting Heterogeneously Expressed Phenotypes by Colony Immunoblot and Dedicated Semi-Automated Image Analysis Pipeline

To understand how bacteria evolve and adapt to their environment, it can be relevant to monitor phenotypic changes that occur in a population. Single cell level analyses and sorting of mutant cells according to a particular phenotypic readout can constitute efficient strategies. However, when the phenotype of interest is expressed heterogeneously in ancestral isogenic populations of cells, single cell level sorting approaches are not optimal. Phenotypic heterogeneity can for instance make no-expression mutant cells indistinguishable from a subpopulation of wild-type cells transiently not expressing the phenotype. The analysis of clonal populations (e.g., isolated colonies), in which the average phenotype is measured, can circumvent this issue. Indeed, no-expression mutants form negative populations while wild-type clones form populations in which average expression of the phenotype yields a positive signal. We present here an optimized colony immunoblot protocol and a semi-automated image analysis pipeline (ImageJ macro) allowing for rapid detection of clones harboring mutations that affect the heterogeneous (i.e., bimodal) expression of the Type Three Secretion System-1 (TTSS-1) in Salmonella enterica serovar Typhimurium. We show that this protocol can efficiently differentiate clones expressing TTSS-1 at various levels in mixed populations. We were able to detect the emergence of hilC mutants in which the proportion of cells expressing TTSS-1 was reduced compared to the ancestor. We could also follow changes in the frequency of different mutants during long-term infections. This demonstrates that our protocol constitutes a tractable approach to assess semi-quantitatively the evolutionary dynamics of heterogeneous phenotypes, such as the expression of virulence genes, in bacterial populations.

Epitope-Tagged Autotransporters as Single-Cell Reporters for Gene Expression by a Salmonella Typhimurium wbaP Mutant

Phenotypic diversity is an important trait of bacterial populations and can enhance fitness of the existing genotype in a given environment. To characterize different subpopulations, several studies have analyzed differential gene expression using fluorescent reporters. These studies visualized either single or multiple genes within single cells using different fluorescent proteins. However, variable maturation and folding kinetics of different fluorophores complicate the study of dynamics of gene expression. Here, we present a proof-of-principle study for an alternative gene expression system in a wbaP mutant of Salmonella Typhimurium (S. Tm) lacking the O-sidechain of the lipopolysaccharide. We employed the hemagglutinin (HA)-tagged inverse autotransporter invasin (invAHA) as a transcriptional reporter for the expression of the type three secretion system 1 (T1) in S. Tm. Using a two-reporter approach with GFP and the InvAHA in single cells, we verify that this reporter system can be used for T1 gene expression analysis, at least in strains lacking the O-antigen (wbaP), which are permissive for detection of the surface-exposed HA-epitope. When we placed the two reporters gfp and invAHA under the control of either one or two different promoters of the T1 regulon, we were able to show correlative expression of both reporters. We conclude that the invAHA reporter system is a suitable tool to analyze T1gene expression in S. Tm and propose its applicability as molecular tool for gene expression studies within single cells.