Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Gasdermin D is the only Gasdermin that provides protection against acute Salmonella gut infection in mice.

Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1ß (IL1ß) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we used epithelium-specific ablation, bone marrow chimeras, and mouse lines lacking individual Gasdermins, combinations of Gasdermins or even all Gasdermins (GSDMA1-3C1-4DE) at once and performed littermate-controlled oral S.Tm infections in streptomycin-pretreated mice to investigate the impact of all murine Gasdermins. While GSDMA, C, and E appear dispensable, we show that GSDMD i) restricts S.Tm loads in the gut tissue and systemic organs, ii) controls gut inflammation kinetics, and iii) prevents epithelium disruption by 72 h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments as well as 3D-, 2D-, and chimeric enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding IECs, affects extrusion kinetics, and promotes overall mucosal barrier capacity. As such, this work identifies a unique multipronged role of GSDMD among the Gasdermins for mucosal tissue defense against a common enteric pathogen.

Epithelial inflammasomes, gasdermins, and mucosal inflammation - Lessons from Salmonella and Shigella infected mice.

Besides its crucial function in nutrient absorbance and as barrier against the microbiota, the gut epithelium is essential for sensing pathogenic insults and mounting of an appropriate early immune response. In mice, the activation of the canonical NAIP/NLRC4 inflammasome is critical for the defense against enterobacterial infections. Activation of the NAIP/NLRC4 inflammasome triggers the extrusion of infected intestinal epithelial cells (IEC) into the gut lumen, concomitant with inflammasome-mediated lytic cell death. The membrane permeabilization, a hallmark of pyroptosis, is caused by the pore-forming proteins called gasdermins (GSDMs). Recent work has revealed that NAIP/NLRC4-dependent extrusion of infected IECs can, however, also be executed in the absence of GSDMD. In fact, several reports highlighted that various cell death pathways (e.g., pyroptosis or apoptosis) and unique mechanisms specific to particular infection models and stages of gut infection are in action during epithelial inflammasome defense against intestinal pathogens. Here, we summarize the current knowledge regarding the underlying mechanisms and speculate on the putative functions of the epithelial inflammasome activation and cell death, with a particular emphasis on mouse infection models for two prominent enterobacterial pathogens, Salmonella Typhimurium and Shigella flexneri.

Intraluminal neutrophils limit epithelium damage by reducing pathogen assault on intestinal epithelial cells during Salmonella gut infection.

Recruitment of neutrophils into and across the gut mucosa is a cardinal feature of intestinal inflammation in response to enteric infections. Previous work using the model pathogen Salmonella enterica serovar Typhimurium (S.Tm) established that invasion of intestinal epithelial cells by S.Tm leads to recruitment of neutrophils into the gut lumen, where they can reduce pathogen loads transiently. Notably, a fraction of the pathogen population can survive this defense, re-grow to high density, and continue triggering enteropathy. However, the functions of intraluminal neutrophils in the defense against enteric pathogens and their effects on preventing or aggravating epithelial damage are still not fully understood. Here, we address this question via neutrophil depletion in different mouse models of Salmonella colitis, which differ in their degree of enteropathy. In an antibiotic pretreated mouse model, neutrophil depletion by an anti-Ly6G antibody exacerbated epithelial damage. This could be linked to compromised neutrophil-mediated elimination and reduced physical blocking of the gut-luminal S.Tm population, such that the pathogen density remained high near the epithelial surface throughout the infection. Control infections with a ssaV mutant and gentamicin-mediated elimination of gut-luminal pathogens further supported that neutrophils are protecting the luminal surface of the gut epithelium. Neutrophil depletion in germ-free and gnotobiotic mice hinted that the microbiota can modulate the infection kinetics and ameliorate epithelium-disruptive enteropathy even in the absence of neutrophil-protection. Together, our data indicate that the well-known protective effect of the microbiota is augmented by intraluminal neutrophils. After antibiotic-mediated microbiota disruption, neutrophils are central for maintaining epithelial barrier integrity during acute Salmonella-induced gut inflammation, by limiting the sustained pathogen assault on the epithelium in a critical window of the infection.

Transient cell-in-cell formation underlies tumor relapse and resistance to immunotherapy.

Despite the remarkable successes of cancer immunotherapies, the majority of patients will experience only partial response followed by relapse of resistant tumors. While treatment resistance has frequently been attributed to clonal selection and immunoediting, comparisons of paired primary and relapsed tumors in melanoma and breast cancers indicate that they share the majority of clones. Here, we demonstrate in both mouse models and clinical human samples that tumor cells evade immunotherapy by generating unique transient cell-in-cell structures, which are resistant to killing by T cells and chemotherapies. While the outer cells in this cell-in-cell formation are often killed by reactive T cells, the inner cells remain intact and disseminate into single tumor cells once T cells are no longer present. This formation is mediated predominantly by IFNγ-activated T cells, which subsequently induce phosphorylation of the transcription factors signal transducer and activator of transcription 3 (STAT3) and early growth response-1 (EGR-1) in tumor cells. Indeed, inhibiting these factors prior to immunotherapy significantly improves its therapeutic efficacy. Overall, this work highlights a currently insurmountable limitation of immunotherapy and reveals a previously unknown resistance mechanism which enables tumor cells to survive immune-mediated killing without altering their immunogenicity.

Cancer immunotherapies use the body's own immune system to fight off cancer. But, despite some remarkable success stories, many patients only see a temporary improvement before the immunotherapy stops being effective and the tumours regrow. It is unclear why this occurs, but it may have to do with how the immune system attacks cancer cells. Immunotherapies aim to activate a special group of cells known as killer T-cells, which are responsible for the immune response to tumours. These cells can identify cancer cells and inject toxic granules through their membranes, killing them. However, killer T-cells are not always effective. This is because cancer cells are naturally good at avoiding detection, and during treatment, their genes can mutate, giving them new ways to evade the immune system. Interestingly, when scientists analysed the genes of tumour cells before and after immunotherapy, they found that many of the genes that code for proteins recognized by T-cells do not change significantly. This suggests that tumours' resistance to immune attack may be physical, rather than genetic. To investigate this hypothesis, Gutwillig et al. developed several mouse tumour models that stop responding to immunotherapy after initial treatment. Examining cells from these tumours revealed that when the immune system attacks, they reorganise by getting inside one another. This allows some cancer cells to hide under many layers of cell membrane. At this point killer T-cells can identify and inject the outer cell with toxic granules, but it cannot reach the cells inside. This ability of cancer cells to hide within one another relies on them recognising when the immune system is attacking. This happens because the cancer cells can detect certain signals released by the killer T-cells, allowing them to hide. Gutwillig et al. identified some of these signals, and showed that blocking them stopped cancer cells from hiding inside each other, making immunotherapy more effective. This new explanation for how cancer cells escape the immune system could guide future research and lead to new cancer treatments, or approaches to boost existing treatments. Understanding the process in more detail could allow scientists to prevent it from happening, by revealing which signals to block, and when, for best results.

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.

Smart investment of virus RNA testing resources to enhance Covid-19 mitigation.

A variety of mitigation strategies have been employed against the Covid-19 pandemic. Social distancing is still one of the main methods to reduce spread, but it entails a high toll on personal freedom and economic life. Alternative mitigation strategies that do not come with the same problems but are effective at preventing disease spread are therefore needed. Repetitive mass-testing using PCR assays for viral RNA has been suggested, but as a stand-alone strategy this would be prohibitively resource intensive. Here, we suggest a strategy that aims at targeting the limited resources available for viral RNA testing to subgroups that are more likely than the average population to yield a positive test result. Importantly, these pre-selected subgroups include symptom-free people. By testing everyone in these subgroups, in addition to symptomatic cases, large fractions of pre- and asymptomatic people can be identified, which is only possible by testing-based mitigation. We call this strategy smart testing (ST). In principle, pre-selected subgroups can be found in different ways, but for the purpose of this study we analyze a pre-selection procedure based on cheap and fast virus antigen tests. We quantify the potential reduction of the epidemic reproduction number by such a two-stage ST strategy. In addition to a scenario where such a strategy is available to the whole population, we analyze local applications, e.g. in a country, company, or school, where the tested subgroups are also in exchange with the untested population. Our results suggest that a two-stage ST strategy can be effective to curb pandemic spread, at costs that are clearly outweighed by the economic benefit. It is technically and logistically feasible to employ such a strategy, and our model predicts that it is even effective when applied only within local groups. We therefore recommend adding two-stage ST to the portfolio of available mitigation strategies, which allow easing social distancing measures without compromising public health.

CXCL12-abundant reticular cells are the major source of IL-6 upon LPS stimulation and thereby regulate hematopoiesis.

Hematopoiesis is maintained by hematopoietic stem and progenitor cells that are located in the bone marrow (BM) where they are embedded within a complex supportive microenvironment consisting of a multitude of various non-hematopoietic and hematopoietic cell types. The BM microenvironment not only regulates steady-state hematopoiesis by provision of growth factors, cytokines, and cell-cell contact but is also an emerging key player during the adaptation to infectious and inflammatory insults (emergency hematopoiesis). Through a combination of gene expression analyses in prospectively isolated non-hematopoietic BM cell populations and various mouse models, we found that BM CXCL12-abundant reticular (CAR) cells are a major source of systemic and local BM interleukin-6 (IL-6) levels during emergency hematopoiesis after lipopolysaccharide (LPS) stimulation. Importantly, although IL-6 is dispensable during the initial phase of LPS-induced emergency hematopoiesis, it is required to sustain an adequate hematopoietic output during chronic repetitive inflammation. Our data highlight the essential role of the non-hematopoietic BM microenvironment for the sensing and integration of pathogen-derived signals into sustained demand-adapted hematopoietic responses.

Salmonella effector driven invasion of the gut epithelium: breaking in and setting the house on fire.

Salmonella Typhimurium (S.Tm) is a major cause of diarrheal disease. The invasion into intestinal epithelial cells (IECs) is a central step in the infection cycle. It is associated with gut inflammation and thought to benefit S.Tm proliferation also in the intestinal lumen. Importantly, it is still not entirely clear how inflammation is elicited and to which extent it links to IEC invasion efficiency in vivo. In this review, we summarize recent findings explaining IEC invasion by type-three-secretion-system-1 (TTSS-1) effector proteins and discuss their effects on invasion and gut inflammation. In non-polarized tissue culture cells, the TTSS-1 effectors (mainly SopB/E/E2) elicit large membrane ruffles fueling cooperative invasion, and can directly trigger pro-inflammatory signaling. By contrast, in the murine gut, we observe discreet-invasion (mainly via the TTSS-1 effector SipA) and a prominent pro-inflammatory role of the host?"s epithelial inflammasome(s), which sense pathogen associated molecular patterns (PAMPs). We discuss why it has remained a major challenge to tease apart direct and indirect inflammatory effects of TTSS-1 effectors and explain why further research will be needed to fully determine their inflammation-modulating role(s).

Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF.

Intestinal epithelial cell (IEC) NF-κB signaling regulates the balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance and how bacterial exposure elicits the process. Pure LPS induces epithelial NF-κB activation in vivo. However, we found that in mice, IECs do not respond directly to LPS. Instead, tissue-resident lamina propria intercrypt macrophages sense LPS via TLR4 and rapidly secrete TNF to elicit epithelial NF-κB signaling in their immediate neighborhood. This response pattern is relevant also during oral enteropathogen infection. The macrophage-TNF-IEC axis avoids responses to luminal microbiota LPS but enables crypt- or tissue-scale epithelial NF-κB responses in proportion to the microbial threat. Thereby, intercrypt macrophages fulfill important sentinel functions as first responders to Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows the induction of defense mechanisms at an appropriate scale according to the localization and intensity of microbial triggers.

Dynamic modelling to identify mitigation strategies for the COVID-19 pandemic.

Relevant pandemic-spread scenario simulations can provide guiding principles for containment and mitigation policies. We devised a compartmental model to predict the effectiveness of different mitigation strategies with a main focus on mass testing. The model consists of a set of simple differential equations considering the population size, reported and unreported infections, reported and unreported recoveries, and the number of COVID-19-inflicted deaths. We assumed that COVID-19 survivors are immune (e.g., mutations are not considered) and that the virus is primarily passed on by asymptomatic and pre-symptomatic individuals. Moreover, the current version of the model does not account for age-dependent differences in the death rates, but considers higher mortality rates due to temporary shortage of intensive care units. The model parameters have been chosen in a plausible range based on information found in the literature, but it is easily adaptable, i.e., these values can be replaced by updated information any time. We compared infection rates, the total number of people getting infected and the number of deaths in different scenarios. Social distancing or mass testing can contain or drastically reduce the infections and the predicted number of deaths when compared with a situation without mitigation. We found that mass testing alone and subsequent isolation of detected cases can be an effective mitigation strategy, alone and in combination with social distancing. It is of high practical relevance that a relationship between testing frequency and the effective reproduction number of the virus can be provided. However, unless one assumes that the virus can be globally defeated by reducing the number of infected persons to zero, testing must be upheld, albeit at reduced intensity, to prevent subsequent waves of infection. The model suggests that testing strategies can be equally effective as social distancing, though at much lower economic costs. We discuss how our mathematical model may help to devise an optimal mix of mitigation strategies against the COVID-19 pandemic. Moreover, we quantify the theoretical limit of contact tracing and by how much the effect of testing is enhanced, if applied to sub-populations with increased exposure risk or prevalence.

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.

Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection.

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.

Germ-free and microbiota-associated mice yield small intestinal epithelial organoids with equivalent and robust transcriptome/proteome expression phenotypes.

Intestinal epithelial organoids established from gut tissue have become a widely used research tool. However, it remains unclear how environmental cues, divergent microbiota composition and other sources of variation before, during and after establishment confound organoid properties, and how these properties relate to the original tissue. While environmental influences cannot be easily addressed in human organoids, mice offer a controlled assay-system. Here, we probed the effect of donor microbiota differences, previously identified as a confounding factor in murine in vivo studies, on organoids. We analysed the proteomes and transcriptomes of primary organoid cultures established from two colonised and one germ-free mouse colony of C57BL/6J genetic background, and compared them to their tissue of origin and commonly used cell lines. While an imprint of microbiota-exposure was observed on the proteome of epithelial samples, the long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead, stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts and closely mimicked expression patterns in the gut epithelium. This included the inflammasome components ASC, Naip1-6, Nlrc4 and Caspase-1, which were highly expressed in all organoids compared to the reference cell line m-ICc12 or mouse embryonic fibroblasts. Taken together, these results reveal that the donor microbiota has little effect on the organoid phenotype and suggest that organoids represent a more suitable culture model than immortalised cell lines, in particular for studies of intestinal epithelial inflammasomes.

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 Major RNA-Binding Protein ProQ Impacts Virulence Gene Expression in Salmonella enterica Serovar Typhimurium.

FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3'UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs.IMPORTANCE The protein ProQ has recently been discovered as the centerpiece of a previously overlooked "third domain" of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria.

ATP released by intestinal bacteria limits the generation of protective IgA against enteropathogens.

T cell dependent secretory IgA (SIgA) generated in the Peyer's patches (PPs) of the small intestine shapes a broadly diverse microbiota that is crucial for host physiology. The mutualistic co-evolution of host and microbes led to the relative tolerance of host's immune system towards commensal microorganisms. The ATP-gated ionotropic P2X7 receptor limits T follicular helper (Tfh) cells expansion and germinal center (GC) reaction in the PPs. Here we show that transient depletion of intestinal ATP can dramatically improve high-affinity IgA response against both live and inactivated oral vaccines. Ectopic expression of Shigella flexneri periplasmic ATP-diphosphohydrolase (apyrase) abolishes ATP release by bacteria and improves the specific IgA response against live oral vaccines. Antibody responses primed in the absence of intestinal extracellular ATP (eATP) also provide superior protection from enteropathogenic infection. Thus, modulation of eATP in the small intestine can affect high-affinity IgA response against gut colonizing bacteria.

Consequences of Epithelial Inflammasome Activation by Bacterial Pathogens.

Inflammasome signaling impinges on the activation of inflammatory caspases (i.e., caspase-1 and caspase-4/5/11) and endows host cells with a sentinel system to sense microbial intrusion and thereby initiate appropriate immune responses. Lately, it has become evident that mammalian inflammasome-dependent responses to infection are not confined solely to cells of hematopoietic origin. Epithelial cells that line the body's mucosal surfaces use inflammasome signaling to sense and counteract pathogenic microorganisms that compromise barrier integrity. Many of the molecular mechanisms of epithelial inflammasome signaling remain unexplored. However, it now seems clear that epithelial inflammasome activation has a profound impact both on the infected cell itself and on its ability to communicate with other cell types of the mucosa. Here, we summarize current knowledge regarding the output of epithelial inflammasome activation during bacterial infection. Well-established downstream effects include epithelial cell death, release of soluble mediators, and subsequent recruitment of effector cell types, including NK cells, mast cells, and neutrophils, to sites of mucosal infection. We discuss the implications of recent findings for antibacterial defense in the mucosa and sketch out areas for future exploration.