A two-step activation mechanism enables mast cells to differentiate their response between extracellular and invasive enterobacterial infection.

Mast cells localize to mucosal tissues and contribute to innate immune defense against infection. How mast cells sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other related enterobacteria, here we show that mast cells can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and mast cells colocalize in the mouse gut during acute Salmonella infection. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure lipopolysaccharide, causes a modest induction of cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human mast cells triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to drive the full-blown mast cell response. Supernatants from S.Tm-infected mast cells boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that mast cells can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

Chemoselective bicyclobutane-based mass spectrometric detection of biological thiols uncovers human and bacterial metabolites.

Sulfur is an essential element of life. Thiol-containing metabolites in all organisms are involved in the regulation of diverse biological processes. Especially, the microbiome produces bioactive metabolites or biological intermediates of this compound class. The analysis of thiol-containing metabolites is challenging due to the lack of specific tools, making these compounds difficult to investigate selectively. We have now developed a new methodology comprising bicyclobutane for chemoselective and irreversible capturing of this metabolite class. We utilized this new chemical biology tool immobilized onto magnetic beads for the investigation of human plasma, fecal samples, and bacterial cultures. Our mass spectrometric investigation detected a broad range of human, dietary and bacterial thiol-containing metabolites and we even captured the reactive sulfur species cysteine persulfide in both fecal and bacterial samples. The described comprehensive methodology represents a new mass spectrometric strategy for the discovery of bioactive thiol-containing metabolites in humans and the microbiome.

Barcoded Consortium Infections: A Scalable, Internally Controlled Method to Study Host Cell Binding and Invasion by Pathogenic Bacteria.

Bacterial host cell invasion has routinely been investigated by gentamicin protection assays, which are laborsome and suffer from pronounced experimental noise. This chapter describes an internally controlled, medium- to high-throughput method that resolves the capacity of multiple Salmonella virulence factor mutant strains to bind and invade host cells. The method, widely applicable to also other pathogens, is based on the combination of consortia of genetically tagged isogenic bacterial strains and a modified gentamicin protection assay. These protocols provide a flexible tool box to stringently quantify host cell binding and invasive properties of different mutants. Moreover, the method can be applied to both infections of cultured host cells and in vivo animal models, providing a comparable genetic readout, which greatly facilitates comparisons across experimental models.

The VirF21:VirF30 protein ratio is affected by temperature and impacts Shigella flexneri host cell invasion.

Shigella spp, the etiological agents of bacillary dysentery in humans, have evolved an intricate regulatory strategy to ensure fine-tuned expression of virulence genes in response to environmental stimuli. A key component in this regulation is VirF, an AraC-like transcription factor, which at the host temperature (37°C) triggers, directly or indirectly, the expression of > 30 virulence genes important for invasion of the intestinal epithelium. Previous work identified two different forms of VirF with distinct functions: VirF30 activates virulence gene expression, while VirF21 appears to negatively regulate virF itself. Moreover, VirF21 originates from either differential translation of the virF mRNA or from a shorter leaderless mRNA (llmRNA). Here we report that both expression of the virF21 llmRNA and the VirF21:VirF30 protein ratio are higher at 30°C than at 37°C, suggesting a possible involvement of VirF21 in minimizing virulence gene expression outside the host (30°C). Ectopic elevation of VirF21 levels at 37°C indeed suppresses Shigella´s ability to infect epithelial cells. Finally, we find that the VirF21 C-terminal portion, predicted to contain a Helix-Turn-Helix motif (HTH2), is required for the functionality of this negative virulence regulator.

A motile doublet form of Salmonella Typhimurium diversifies target search behavior at the epithelial surface.

The behaviors of infectious bacteria are commonly studied in bulk. This is effective to define the general properties of a given isolate, but insufficient to resolve subpopulations and unique single-microbe behaviors within the bacterial pool. We here employ microscopy to study single-bacterium characteristics among Salmonella enterica serovar Typhimurium (S.Tm), as they prepare for and launch invasion of epithelial host cells. We find that during the bacterial growth cycle, S.Tm populations switch gradually from fast planktonic growth to a host cell-invasive phenotype, characterized by flagellar motility and expression of the Type-three-secretion-system-1. The indistinct nature of this shift leads to the establishment of a transient subpopulation of S.Tm "doublets"-waist-bearing bacteria anticipating cell division-which simultaneously express host cell invasion machinery. In epithelial cell culture infections, these S.Tm doublets outperform their "singlet" brethren and represent a hyperinvasive subpopulation. Atop both glass and enteroid-derived monolayers, doublets swim along markedly straighter trajectories than singlets, thereby diversifying search patterns and improving the surface exploration capacity of the total bacterial population. The straighter swimming, combined with an enhanced cell-adhesion propensity, suffices to account for the hyperinvasive doublet phenotype. This work highlights bacterial cell length heterogeneity as a key determinant of target search patterns atop epithelia.

Bacterial detection by NAIP/NLRC4 elicits prompt contractions of intestinal epithelial cell layers.

The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Salmonella Typhimurium (STm) within minutes elicited focal contractions with inward movements of up to ∼1,000 IECs. Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin-dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process.

Epithelium-autonomous NAIP/NLRC4 prevents TNF-driven inflammatory destruction of the gut epithelial barrier in Salmonella-infected mice.

The gut epithelium is a critical protective barrier. Its NAIP/NLRC4 inflammasome senses infection by Gram-negative bacteria, including Salmonella Typhimurium (S.Tm) and promotes expulsion of infected enterocytes. During the first ~12-24 h, this reduces mucosal S.Tm loads at the price of moderate enteropathy. It remained unknown how this NAIP/NLRC4-dependent tradeoff would develop during subsequent infection stages. In NAIP/NLRC4-deficient mice, S.Tm elicited severe enteropathy within 72 h, characterized by elevated mucosal TNF (>20 pg/mg) production from bone marrow-derived cells, reduced regeneration, excessive enterocyte loss, and a collapse of the epithelial barrier. TNF-depleting antibodies prevented this destructive pathology. In hosts proficient for epithelial NAIP/NLRC4, a heterogeneous enterocyte death response with both apoptotic and pyroptotic features kept S.Tm loads persistently in check, thereby preventing this dire outcome altogether. Our results demonstrate that immediate and selective removal of infected enterocytes, by locally acting epithelium-autonomous NAIP/NLRC4, is required to avoid a TNF-driven inflammatory hyper-reaction that otherwise destroys the epithelial barrier.

Salmonella enterica Serovar Typhimurium Exploits Cycling through Epithelial Cells To Colonize Human and Murine Enteroids.

Enterobacterial pathogens infect the gut by a multistep process, resulting in colonization of both the lumen and the mucosal epithelium. Due to experimental constraints, it remains challenging to address how luminal and epithelium-lodged pathogen populations cross-feed each other in vivo Enteroids are cultured three-dimensional miniature intestinal organs with a single layer of primary intestinal epithelial cells (IECs) surrounding a central lumen. They offer new opportunities to study enterobacterial infection under near-physiological conditions, at a temporal and spatial resolution not attainable in animal models, but remain poorly explored in this context. We employed microinjection, time-lapse microscopy, bacterial genetics, and barcoded consortium infections to describe the complete infection cycle of Salmonella enterica serovar Typhimurium in both human and murine enteroids. Flagellar motility and type III secretion system 1 (TTSS-1) promoted Salmonella Typhimurium targeting of the intraepithelial compartment and breaching of the epithelial barrier. Strikingly, however, TTSS-1 also potently boosted colonization of the enteroid lumen. By tracing the infection over time, we identified a cycle(s) of TTSS-1-driven IEC invasion, intraepithelial replication, and reemergence through infected IEC expulsion as a key mechanism for Salmonella Typhimurium luminal colonization. These findings suggest a positive feed-forward loop, through which IEC invasion by planktonic bacteria fuels further luminal population expansion, thereby ensuring efficient colonization of both the intraepithelial and luminal niches.IMPORTANCE Pathogenic gut bacteria are common causes of intestinal disease. Enteroids-cultured three-dimensional replicas of the mammalian gut-offer an emerging model system to study disease mechanisms under conditions that recapitulate key features of the intestinal tract. In this study, we describe the full life cycle of the prototype gut pathogen Salmonella enterica serovar Typhimurium within human and mouse enteroids. We map the consecutive steps and define the bacterial virulence factors that drive colonization of luminal and epithelial compartments, as well as breaching of the epithelial barrier. Strikingly, our work reveals how bacterial colonization of the epithelium potently fuels expansion also in the luminal compartment, through a mechanism involving the death and expulsion of bacterium-infected epithelial cells. These findings have repercussions for our understanding of the Salmonella infection cycle. Moreover, our work provides a comprehensive foundation for the use of microinjected enteroids to model gut bacterial diseases.

Salmonella Typhimurium discreet-invasion of the murine gut absorptive epithelium.

Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.

Barcoded Consortium Infections Resolve Cell Type-Dependent Salmonella enterica Serovar Typhimurium Entry Mechanisms.

Bacterial host cell invasion mechanisms depend on the bacterium's virulence factors and the properties of the target cell. The enteropathogen Salmonella enterica serovar Typhimurium (STm) invades epithelial cell types in the gut mucosa and a variety of immune cell types at later infection stages. The molecular mechanism(s) of host cell entry has, however, been studied predominantly in epithelial cell lines. STm uses a type three secretion system (TTSS-1) to translocate effectors into the host cell cytosol, thereby sparking actin ruffle-dependent entry. The ruffles also fuel cooperative invasion by bystander bacteria. In addition, several TTSS-1-independent entry mechanisms exist, involving alternative STm virulence factors, or the passive uptake of bacteria by phagocytosis. However, it remains ill-defined how STm invasion mechanisms vary between host cells. Here, we developed an internally controlled and scalable method to map STm invasion mechanisms across host cell types and conditions. The method relies on host cell infections with consortia of chromosomally tagged wild-type and mutant STm strains, where the abundance of each strain can be quantified by qPCR or amplicon sequencing. Using this methodology, we quantified cooccurring TTSS-1-dependent, cooperative, and TTSS-1-independent invasion events in epithelial, monocyte, and macrophage cells. We found STm invasion of epithelial cells and monocytes to proceed by a similar MOI-dependent mix of TTSS-1-dependent and cooperative mechanisms. TTSS-1-independent entry was more frequent in macrophages. Still, TTSS-1-dependent invasion dominated during the first minutes of interaction also with this cell type. Finally, the combined action of the SopB/SopE/SopE2 effectors was sufficient to explain TTSS-1-dependent invasion across both epithelial and phagocytic cells.IMPORTANCESalmonella enterica serovar Typhimurium (STm) is a widespread and broad-host-spectrum enteropathogen with the capacity to invade diverse cell types. Still, the molecular basis for the host cell invasion process has largely been inferred from studies of a few selected cell lines. Our work resolves the mechanisms that Salmonellae employ to invade prototypical host cell types, i.e., human epithelial, monocyte, and macrophage cells, at a previously unattainable level of temporal and quantitative precision. This highlights efficient bacterium-driven entry into innate immune cells and uncovers a type III secretion system effector module that dominates active bacterial invasion of not only epithelial cells but also monocytes and macrophages. The results are derived from a generalizable method, where we combine barcoding of the bacterial chromosome with mixed consortium infections of cultured host cells. The application of this methodology across bacterial species and infection models will provide a scalable means to address host-pathogen interactions in diverse contexts.

The Intriguing Evolutionary Journey of Enteroinvasive E. coli (EIEC) toward Pathogenicity.

Among the intestinal pathogenic Escherichia coli, enteroinvasive E. coli (EIEC) are a group of intracellular pathogens able to enter epithelial cells of colon, multiplicate within them, and move between adjacent cells with a mechanism similar to Shigella, the ethiological agent of bacillary dysentery. Despite EIEC belong to the same pathotype of Shigella, they neither have the full set of traits that define Shigella nor have undergone the extensive gene decay observed in Shigella. Molecular analysis confirms that EIEC are widely distributed among E. coli phylogenetic groups and correspond to bioserotypes found in many E. coli serogroups. Like Shigella, also in EIEC the critical event toward a pathogenic life-style consisted in the acquisition by horizontal gene transfer of a large F-type plasmid (pINV) containing the genes required for invasion, intracellular survival, and spreading through the intestinal mucosa. In Shigella, the ample gain in virulence determinants has been counteracted by a substantial loss of functions that, although important for the survival in the environment, are redundant or deleterious for the life inside the host. The pathoadaptation process that has led Shigella to modify its metabolic profile and increase its pathogenic potential is still in infancy in EIEC, although maintenance of some features typical of E. coli might favor their emerging relevance as intestinal pathogens worldwide, as documented by recent outbreaks in industrialized countries. In this review, we will discuss the evolution of EIEC toward Shigella-like invasive forms going through the epidemiology, including the emergence of new virulent strains, their genome organization, and the complex interactions they establish with the host.

Role of the SRRz/Rz1 lambdoid lysis cassette in the pathoadaptive evolution of Shigella.

Shigella, the etiological agent of bacillary dysentery (shigellosis), is a highly adapted human pathogen. It evolved from an innocuous ancestor resembling the Escherichia coli strain by gain and loss of genes and functions. While the gain process concerns the acquisition of the genetic determinants of virulence, the loss is related to the adaptation of the genome to the new pathogenic status and occurs by pathoadaptive mutation of antivirulence genes. In this study, we highlight that the SRRz/Rz1 lambdoid lysis cassette, even though stably adopted in E. coli K12 by virtue of its beneficial effect on cell physiology, has undergone a significant decay in Shigella. Moreover, we show the antivirulence nature of the SRRz/Rz1 lysis cassette in Shigella. In fact, by restoring the SRRz/Rz1 expression in this pathogen, we observe an increased release of peptidoglycan fragments, causing an unbalance in the fine control exerted by Shigella on host innate immunity and a mitigation of its virulence. This strongly affects the virulence of Shigella and allows to consider the loss of SRRz/Rz1 lysis cassette as another pathoadaptive event in the life of Shigella.

One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator.

VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF30 (30 kDa), and the shorter VirF21 (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF30 and VirF21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF21 is also translated from a leaderless mRNA (llmRNA) whose 5' end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF21 The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF30 is responsible for activation of the virulence system, VirF21 negatively autoregulates virF expression itself. Since VirF21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA. IMPORTANCE: Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF30 and VirF21, that are functionally distinct. The major form, VirF30, activates the genes necessary for virulence, whereas the minor VirF21, which shares the C-terminal two-thirds of VirF30, negatively autoregulates virF expression itself. VirF21 is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the Shigella invasive process and may help development of new therapies for shigellosis.

The Multifaceted Activity of the VirF Regulatory Protein in the Shigella Lifestyle.

Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV). The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non-invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis.

Multifactor Regulation of the MdtJI Polyamine Transporter in Shigella.

The polyamine profile of Shigella, the etiological agent of bacillary dysentery in humans, differs markedly from that of E. coli, its innocuous commensal ancestor. Pathoadaptive mutations such as the loss of cadaverine and the increase of spermidine favour the full expression of the virulent phenotype of Shigella. Spermidine levels affect the expression of the MdtJI complex, a recently identified efflux pump belonging to the small multi-drug resistance family of transporters. In the present study, we have addressed the regulation of the mdtJI operon in Shigella by asking which factors influence its expression as compared to E. coli. In particular, after identifying the mdtJI promoter by primer extension analysis, in vivo transcription assays and gel-retardation experiments were carried out to get insight on the silencing of mdtJI in E. coli. The results indicate that H-NS, a major nucleoid protein, plays a key role in repressing the mdtJI operon by direct binding to the regulatory region. In the Shigella background mdtJI expression is increased by the high levels of spermidine typically found in this microorganism and by VirF, the plasmid-encoded regulator of the Shigella virulence regulatory cascade. We also show that the expression of mdtJI is stimulated by bile components. Functional analyses reveal that MdtJI is able to promote the excretion of putrescine, the spermidine precursor. This leads us to consider the MdtJI complex as a possible safety valve allowing Shigella to maintain spermidine to a level optimally suited to survival within infected macrophages and, at the same time, prevent toxicity due to spermidine over-accumulation.

Molecular and functional profiling of the polyamine content in enteroinvasive E. coli : looking into the gap between commensal E. coli and harmful Shigella.

Polyamines are small molecules associated with a wide variety of physiological functions. Bacterial pathogens have developed subtle strategies to exploit polyamines or manipulate polyamine-related processes to optimize fitness within the host. During the transition from its innocuous E. coli ancestor, Shigella, the aetiological agent of bacillary dysentery, has undergone drastic genomic rearrangements affecting the polyamine profile. A pathoadaptation process involving the speG gene and the cad operon has led to spermidine accumulation and loss of cadaverine. While a higher spermidine content promotes the survival of Shigella within infected macrophages, the lack of cadaverine boosts the pathogenic potential of the bacterium in host tissues. Enteroinvasive E. coli (EIEC) display the same pathogenicity process as Shigella, but have a higher infectious dose and a higher metabolic activity. Pathoadaption events affecting the cad locus have occurred also in EIEC, silencing cadaverine production. Since EIEC are commonly regarded as evolutionary intermediates between E. coli and Shigella, we investigated on their polyamine profile in order to better understand which changes have occurred along the path to pathogenicity. By functional and molecular analyses carried out in EIEC strains belonging to different serotypes, we show that speG has been silenced in one strain only, favouring resistance to oxidative stress conditions and survival within macrophages. At the same time, we observe that the content of spermidine and putrescine, a relevant intermediate in the synthesis of spermidine, is higher in all strains as compared to E. coli. This may represent an evolutionary response to the lack of cadaverine. Indeed, restoring cadaverine synthesis decreases the expression of the speC gene, whose product affects putrescine production. In the light of these results, we discuss the possible impact of pathoadaptation events on the evolutionary emergence of a polyamine profile favouring to the pathogenic lifestyle of Shigella and EIEC.

Molecular evolution of the nicotinic acid requirement within the Shigella/EIEC pathotype.

Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor in several anabolic and catabolic reactions. NAD derives from quinolinic acid (QUIN) which in Escherichia coli is obtained through a pyridine salvage pathway or a de novo synthesis pathway. In the latter case, two enzymes, L-aspartate oxidase (NadB) and quinolinate synthase (NadA), are required for the synthesis of QUIN. In contrast to its E. coli ancestor, Shigella spp., the causative agent of bacillary dissentery, lacks the de novo pathway and strictly requires nicotinic acid for growth (Nic⁻ phenotype). This phenotype depends on the silencing of the nadB and nadA genes and its pathoadaptive nature is suggested by the observation that QUIN attenuates the Shigella invasive process. Shigella shares the pathogenicity mechanism with enteronvasive E. coli (EIEC), a group of pathogenic E. coli. On the basis of this similarity EIEC and Shigella have been grouped into a single E. coli pathotype. However EIEC strains do not constitute a homogeneous group and do not possess the complete set of characters that define Shigella strains. In this work we have analysed thirteen EIEC strains belonging to different serotypes and originating from different geographic areas. We show that, in contrast to Shigella, only some EIEC strains require nicotinic acid for growth in minimal medium. Moreover, by studying the emergence of the Nic⁻ phenotype in all serotypes of S. flexneri, as well as in S. sonnei and S. dysenteriae, we describe which molecular rearrangements occurred and which mutations are responsible for the inactivation of the nadA and nadB genes. Our data confirm that the genome of Shigella is extremely dynamic and support the hypothesis that EIEC might reflect an earlier stage of the pathoadaptation process undergone by Shigella.

Polyamines: emerging players in bacteria-host interactions.

Polyamines are small polycationic molecules found in almost all cells and associated with a wide variety of physiological processes. In recent years it has become increasingly clear that, in addition to core physiological functions, polyamines play a crucial role in bacterial pathogenesis. Considerable evidence has built up that bacteria have evolved mechanisms to turn these molecules to their own advantage and a novel standpoint to look at host-bacterium interactions emerges from the interplay among polyamines, host cells and infecting bacteria. In this review, we highlight how human bacterial pathogens have developed their own resourceful strategies to exploit polyamines or manipulate polyamine-related processes to optimize their fitness within the host. Besides contributing to a better understanding of the complex relationship between a pathogen and its host, acquisitions in this field have a significant potential towards the development of novel antibacterial therapeutic approaches.

Shedding of genes that interfere with the pathogenic lifestyle: the Shigella model.

Pathoadaptive mutations are evolutionary events leading to the silencing of specific anti-virulence loci. This reshapes the core genome of a novel pathogen, adapts it to the host and boosts its harmful potential. A paradigmatic case is the emergence of Shigella, the causative agent of bacillary dysentery, from its innocuous Escherichia coli ancestor. Here we summarize current views on how pathoadaptation has allowed Shigella to progressively increase its virulence. In this context, modification of the polyamine pattern emerges as a crucial step towards full expression of the virulence program in Shigella.

A new piece of the Shigella Pathogenicity puzzle: spermidine accumulation by silencing of the speG gene [corrected].

The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation of spermidine is a key determinant in the pathogenicity strategy adopted by this microrganism.