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Severe COVID-19 has been associated with coinfections with bacterial and fungal pathogens. Notably, patients with COVID-19 who develop Staphylococcus aureus bacteremia exhibit higher rates of mortality than those infected with either pathogen alone. To understand this clinical scenario, we collected and examined S. aureus blood and respiratory isolates from a hospital in New York City during the early phase of the pandemic from both SARS-CoV-2+ and SARS-CoV-2- patients. Whole genome sequencing of these S. aureus isolates revealed broad phylogenetic diversity in both patient groups, suggesting that SARS-CoV-2 coinfection was not associated with a particular S. aureus lineage. Phenotypic characterization of the contemporary collection of S. aureus isolates from SARS-CoV-2+ and SARS-CoV-2- patients revealed no notable differences in several virulence traits examined. However, we noted a trend toward overrepresentation of S. aureus bloodstream strains with low cytotoxicity in the SARS-CoV-2+ group. We observed that patients coinfected with SARS-CoV-2 and S. aureus were more likely to die during the acute phase of infection when the coinfecting S. aureus strain exhibited high or low cytotoxicity. To further investigate the relationship between SARS-CoV-2 and S. aureus infections, we developed a murine coinfection model. These studies revealed that infection with SARS-CoV-2 renders mice susceptible to subsequent superinfection with low cytotoxicity S. aureus . Thus, SARS-CoV-2 infection sensitizes the host to coinfections, including S. aureus isolates with low intrinsic virulence., Importance: The COVID-19 pandemic has had an enormous impact on healthcare across the globe. Patients who were severely infected with SARS-CoV-2, the virus causing COVID-19, sometimes became infected with other pathogens, which is termed coinfection. If the coinfecting pathogen is the bacterium Staphylococcus aureus , there is an increased risk of patient death. We collected S. aureus strains that coinfected patients with SARS-CoV-2 to study the disease outcome caused by the interaction of these two important pathogens. We found that both in patients and in mice, coinfection with an S. aureus strain lacking toxicity resulted in more severe disease during the early phase of infection, compared with infection with either pathogen alone. Thus, SARS-CoV-2 infection can directly increase the severity of S. aureus infection., Competing Interests: V.J.T. has consulted for Janssen Research & Development, LLC, and has received honoraria from Genentech and Medimmune. He is also an inventor on patents and patent applications filed by New York University, which are currently under commercial license to Janssen Biotech Inc. Janssen Biotech Inc. had provided research funding and other payments associated with a licensing agreement. B.S. has consulted for Regeneron and MicroGenDx. K.C. has received research support from Pfizer, Takeda, Pacific Biosciences, Genentech, and AbbVie. R.T. completed a NYU-Regeneron postdoctoral training program in laboratory animal medicine.

Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Dissemination to other organ systems is also observed, and is potentially facilitated by macrophages. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the host and parasite during infection. While a small population of infected macrophages mount a response, most remain transcriptionally unchanged, suggesting that the majority of parasites may avoid host detection. The parasite transcriptome reveals large transcriptional changes throughout the life cycle, providing a blueprint for parasite development. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication and dissemination. Together, our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H 2 O 2 , a crucial host defense against S. aureus . We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δ agr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δ agr strains to lethal H 2 O 2 doses. Increased survival of wild-type agr cells during H 2 O 2 exposure required sodA , which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δ agr cells from killing by H 2 O 2 . Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr -mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient ( Cybb -/- ) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage., Competing Interests: MP, AP, GP, AP, JK, EZ, RU, TK, CZ, AH, JS, GW, JK, JC, AR, JW, CN, DL, DP, AP, XZ, KD, IY No competing interests declared, AD Inventor on patents and patent applications (US8431, 687B2; US2019135900 A1; EP4313303A1) filed by New York University, which are currently under commercial license to Janssen Biotech Inc. Janssen Biotech Inc provides research funding and other payments associated with a licensing agreement. These patents pertain solely to the development of vaccines and therapeutics targeting S. aureus toxins and are unrelated to the content presented in this work, VT Has received honoraria from Pfizer and MedImmune, and is an inventor on patents and patent applications filed by New York University,(US8431, 687B2; US2019135900 A1; EP4313303A1) which are currently under commercial license to Janssen Biotech Inc. Janssen Biotech Inc provides research funding and other payments associated with a licensing agreement, BS Has consulted for Basilea Pharmaceutica, (© 2023, Podkowik et al.)

Importance: The prevalence of multidrug-resistant Staphylococcus aureus is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of S. aureus was investigated as a vaccine candidate because of its essential role in bacterial iron acquisition but failed in clinical trials despite strong immunogenicity. Here, we reveal an unexpected second function for IsdB in pathogen-host interaction: the bacterial fitness factor IsdB triggers a strong inflammatory response in innate immune cells via Toll-like receptor 4 and the inflammasome, thus acting as a novel pathogen-associated molecular pattern of S. aureus . Our discovery contributes to a better understanding of how S. aureus modulates the immune response, which is necessary for vaccine development against the sophisticated pathogen., Competing Interests: The authors declare no conflict of interest.

Our previous studies identified a population of stem cell-like proliferating myeloid cells within inflamed tissues that could serve as a reservoir for tissue macrophages to adopt different activation states depending on the microenvironment. By lineage-tracing cells derived from CX3CR1+ precursors in mice during infection and profiling by single-cell RNA sequencing, in this study, we identify a cluster of BIRC5+ myeloid cells that expanded in the liver during chronic infection with either the parasite Schistosoma mansoni or the bacterial pathogen Staphylococcus aureus. In the absence of tissue-damaging toxins, S. aureus infection does not elicit these BIRC5+ cells. Moreover, deletion of BIRC5 from CX3CR1-expressing cells results in improved survival during S. aureus infection. Hence the combination of single-cell RNA sequencing and genetic fate-mapping CX3CR1+ cells revealed a toxin-dependent pathogenic role for BIRC5 in myeloid cells during S. aureus infection., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Innate immune recognition of bacterial pathogens is a key determinant of the ensuing systemic response, and host or pathogen heterogeneity in this early interaction can impact the course of infection. To gain insight into host response heterogeneity, we investigate macrophage inflammatory dynamics using primary human macrophages infected with Group B Streptococcus. Transcriptomic analysis reveals discrete cellular states within responding macrophages, one of which consists of four sub-states, reflecting inflammatory activation. Infection with six additional bacterial species-Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Yersinia pseudotuberculosis, Shigella flexneri, and Salmonella enterica-recapitulates these states, though at different frequencies. We show that modulating the duration of infection and the presence of a toxin impacts inflammatory trajectory dynamics. We provide evidence for this trajectory in infected macrophages in an in vivo model of Staphylococcus aureus infection. Our cell-state analysis defines a framework for understanding inflammatory activation dynamics in response to bacterial infection., Competing Interests: Declaration of interests V.J.T. has consulted for Janssen Research & Development, LLC and has received honoraria from Genentech and Medimmune. V.J.T. is also an inventor on patents and patent applications filed by New York University, which are currently under commercial license to Janssen Biotech Inc. Janssen Biotech Inc. provides research funding and other payments associated with a licensing agreement., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Staphylococcus aureus is an opportunistic pathogen and chief among bloodstream-infecting bacteria. S. aureus produces an array of human-specific virulence factors that may contribute to immune suppression. Here, we defined the response of primary human phagocytes following infection with S. aureus using RNA-sequencing (RNA-Seq). We found that the overall transcriptional response to S. aureus was weak both in the number of genes and in the magnitude of response. Using an ex vivo bacteremia model with fresh human blood, we uncovered that infection with S. aureus resulted in the down-regulation of genes related to innate immune response and cytokine and chemokine signaling. This muted transcriptional response was conserved across diverse S. aureus clones but absent in blood exposed to heat-killed S. aureus or blood infected with the less virulent staphylococcal species Staphylococcus epidermidis . Notably, this signature was also present in patients with S. aureus bacteremia. We identified the master regulator S. aureus exoprotein expression (SaeRS) and the SaeRS-regulated pore-forming toxins as key mediators of the transcriptional suppression. The S. aureus -mediated suppression of chemokine and cytokine transcription was reflected by circulating protein levels in the plasma. Wild-type S. aureus elicited a soluble milieu that was restrictive in the recruitment of human neutrophils compared with strains lacking saeRS . Thus, S. aureus blunts the inflammatory response resulting in impaired neutrophil recruitment, which could promote the survival of the pathogen during invasive infection.

Staphylococcus aureus has evolved into diverse lineages, known as clonal complexes (CCs), which exhibit differences in the coding sequences of core virulence factors. Whether these alterations affect functionality is poorly understood. Here, we studied the highly polymorphic pore-forming toxin LukAB. We discovered that the LukAB toxin variants produced by S. aureus CC30 and CC45 kill human phagocytes regardless of whether CD11b, the previously established LukAB receptor, is present, and instead target the human hydrogen voltage-gated channel 1 (HVCN1). Biochemical studies identified the domain within human HVCN1 that drives LukAB species specificity, enabling the generation of humanized HVCN1 mice with enhanced susceptibility to CC30 LukAB and to bloodstream infection caused by CC30 S. aureus strains. Together, this work advances our understanding of an important S. aureus toxin and underscores the importance of considering genetic variation in characterizing virulence factors and understanding the tug of war between pathogens and the host.

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues., (© 2020 by The American Society of Hematology.)

Myeloid cells are a vital component of innate immunity and comprise monocytes, macrophages, dendritic cells, and granulocytes. How myeloid cell lineage affects activation states in response to cytokines remains poorly understood. The cytokine environment and cellular infiltrate during an inflammatory response may contain prognostic features that predict disease outcome. In this study, we analyzed the transcriptional responses of human monocytes, macrophages, dendritic cells, and neutrophils in response to stimulation by IFN-γ, IFN-β, IFN-λ, IL-4, IL-13, and IL-10 cytokines to better understand the heterogeneity of activation states in inflammatory conditions. This generated a myeloid cell-cytokine-specific response matrix that can infer representation of myeloid cells and the cytokine environment they encounter during infection, in tumors and in whole blood. Neutrophils were highly responsive to type 1 and type 2 cytokine stimulation but did not respond to IL-10. We identified transcripts specific to IFN-β stimulation, whereas other IFN signature genes were upregulated by both IFN-γ and IFN-β. When we used our matrix to deconvolute blood profiles from tuberculosis patients, the IFN-β-specific neutrophil signature was reduced in tuberculosis patients with active disease, whereas the shared response to IFN-γ and IFN-β in neutrophils was increased. When applied to glioma patients, transcripts of neutrophils exposed to IL-4/IL-13 and monocyte responses to IFN-γ or IFN-β emerged as opposing predictors of patient survival. Hence, by dissecting how different myeloid cells respond to cytokine activation, we can delineate biological roles for myeloid cells in different cytokine environments during disease processes, especially during infection and tumor progression., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Staphylococcus aureus is a human pathogen responsible for high morbidity and mortality worldwide. Recurrent infections with this bacterium are common, suggesting that S. aureus thwarts the development of sterilizing immunity. S. aureus strains that cause disease in humans produce up to five different bicomponent toxins (leukocidins) that target and lyse neutrophils, innate immune cells that represent the first line of defense against S. aureus infections. However, little is known about the role of leukocidins in blunting adaptive immunity. Here, we explored the effects of leukocidins on human dendritic cells (DCs), antigen-presenting cells required for the development of adaptive immunity. Using an ex vivo infection model of primary human monocyte-derived dendritic cells, we found that S. aureus , including strains from different clonal complexes and drug resistance profiles, effectively kills DCs despite efficient phagocytosis. Although all purified leukocidins could kill DCs, infections with live bacteria revealed that S. aureus targets and kills DCs primarily via the activity of leukocidin LukAB. Moreover, using coculture experiments performed with DCs and autologous CD4 + T lymphocytes, we found that LukAB inhibits DC-mediated activation and proliferation of primary human T cells. Taken together, the data determined in the study reveal a novel immunosuppressive strategy of S. aureus whereby the bacterium blunts the development of adaptive immunity via LukAB-mediated injury of DCs. IMPORTANCE Antigen-presenting cells such as dendritic cells (DCs) fulfill an indispensable role in the development of adaptive immunity by producing proinflammatory cytokines and presenting microbial antigens to lymphocytes to trigger a faster, specific, and long-lasting immune response. Here, we studied the effect of Staphylococcus aureus toxins on human DCs. We discovered that the leukocidin LukAB hinders the development of adaptive immunity by targeting human DCs. The ability of S. aureus to blunt the function of DCs could help explain the high frequency of recurrent S. aureus infections. Taken together, the results from this study suggest that therapeutically targeting the S. aureus leukocidins may boost effective innate and adaptive immune responses by protecting innate leukocytes, enabling proper antigen presentation and T cell activation., (Copyright © 2019 Berends et al.)

The type of cell death triggered by a particular environmental stimulus influences the outcome of infection or inflammatory disease processes. The ability to identify the cell death pathway that is activated in response to infection is essential for understanding the pathogenesis and host response to infection. Activation of the cysteine protease caspase-1 in various inflammasome complexes indicates that cells are undergoing pyroptosis, a regulated, proinflammatory cell death. Inflammasome assembly and caspase activation can be measured by various methods ranging from detection of inflammasome-dependent cell death, cytokine secretion, cleavage of caspase-1, or the formation of "puncta" within the cell that contain inflammasome components, such as caspase-1 or the adapter protein ASC. Here we describe a method for detecting caspase-1 activation on a single cell level in the context of infection by the Gram-negative pathogen Yersinia using immunofluorescence microscopy. We previously used this approach to quantify caspase-1 puncta formation in cells containing Yersinia translocon components (Zwack et al., MBio 6:e02095-14, 2015). This is a modification of methods used previously by Broz et al. (Cell Host Microbe 8:471-483, 2010) and Case and Roy (MBio 2:e00117-11, 2011). By taking a microscopy-based approach that allows us to quantify puncta as well as other cell-biological features of infection (i.e., number of bacteria associated with a particular cell; levels of bacterial effector or translocon proteins in caspase-1 puncta-containing cells; or levels or localization of host cellular proteins), we can better quantify the heterogeneity between cells undergoing pyroptosis and cells that are not under the same infection conditions. These approaches have the potential to generate hypotheses that can enable further mechanistic insight into activation of pyroptosis in response to bacterial infection.

Gram-negative bacterial pathogens utilize virulence-associated secretion systems to inject, or translocate, effector proteins into host cells to manipulate cellular processes and promote bacterial replication. However, translocated bacterial products are sensed by n ucleotide binding domain and l eucine-rich r epeat-containing proteins (NLRs), which trigger the formation of a multiprotein complex called the inflammasome, leading to secretion of interleukin-1 (IL-1) family cytokines, pyroptosis, and control of pathogen replication. Pathogenic Yersinia bacteria inject effector proteins termed Yops, as well as pore-forming proteins that comprise the translocon itself, into target cells. The Yersinia translocation regulatory protein YopK promotes bacterial virulence by limiting hyperinjection of the translocon proteins YopD and YopB into cells, thereby limiting cellular detection of Yersinia virulence activity. How hyperinjection of translocon proteins leads to inflammasome activation is currently unknown. We found that translocated YopB and YopD colocalized with the late endosomal/lysosomal protein LAMP1 and that the frequency of YopD and LAMP1 association correlated with the level of caspase-1 activation in individual cells. We also observed colocalization between YopD and Galectin-3, an indicator of endosomal membrane damage. Intriguingly, YopK limited the colocalization of Galectin-3 with YopD, suggesting that YopK limits the induction or sensing of endosomal membrane damage by components of the type III secretion system (T3SS) translocon. Furthermore, guanylate binding proteins (GBPs) encoded on chromosome 3 ( Gbp Chr3 ), which respond to pathogen-induced damage or alteration of host membranes, were necessary for inflammasome activation in response to hyperinjected YopB/-D. Our findings indicate that lysosomal damage by Yersinia translocon proteins promotes inflammasome activation and implicate GBPs as key regulators of this process., (Copyright © 2017 American Society for Microbiology.)

Many invasive bacteria establish pathogen-containing vacuoles (PVs) as intracellular niches for microbial growth. Immunity to these infections is dependent on the ability of host cells to recognize PVs as targets for host defense. The delivery of several host defense proteins to PVs is controlled by IFN-inducible guanylate binding proteins (GBPs), which themselves dock to PVs through poorly characterized mechanisms. Here, we demonstrate that GBPs detect the presence of bacterial protein secretion systems as "patterns of pathogenesis" associated with PVs. We report that the delivery of GBP2 to Legionella -containing vacuoles is dependent on the bacterial Dot/Icm secretion system, whereas the delivery of GBP2 to Yersinia- containing vacuoles (YCVs) requires hypersecretion of Yersinia translocon proteins. We show that the presence of bacterial secretion systems directs cytosolic carbohydrate-binding protein Galectin-3 to PVs and that the delivery of GBP1 and GBP2 to Legionella- containing vacuoles or YCVs is substantially diminished in Galectin-3-deficient cells. Our results illustrate that insertion of bacterial secretion systems into PV membranes stimulates Galectin-3-dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program.

Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense., Competing Interests: The authors have declared that no competing interests exist.

The innate immune system plays an essential role in initiating the early response against microbial infection, as well as instructing and shaping subsequent responses. Microbial pathogens are enormously diverse in terms of the niches they occupy, their metabolic properties and requirements, and the cellular pathways that they target. Nevertheless, innate sensing of pathogens triggers a relatively stereotyped set of responses that involve transcriptional induction of key inflammatory mediators, as well as post-translational assembly and activation of a multiprotein inflammatory complex termed 'the inflammasome.' Along with classical Pattern Recognition Receptors, the inflammasome activation pathway has emerged as a key regulator of tissue homeostasis and immune defense. Components of the inflammasome generally exist within the cell in a soluble, monomeric state, and oligomerize in response to diverse enzymatic activities associated with infection or cellular stress. Inflammasome assembly triggers activation of the pro-enzyme caspase-1, resulting in the cleavage of caspase-1 targets. The most extensively studied targets are the cytokines of the IL-1 family, but the recent discovery of Gasdermin D as a novel target of caspase-1 and the related inflammatory caspase, caspase-11, has begun to mechanistically define the links between caspase-1 activation and cell death. Cell death is a hallmark of macrophage infection by many pathogens, including the gram-negative bacterial pathogens of the genus Yersinia. Intriguingly, the activities of the Yersinia-secreted effector proteins and the type III secretion system (T3SS) itself have been linked to both inflammasome activation and evasion during infection. The balance between these activating and inhibitory activities shapes the outcome of Yersinia infection. Here, we describe the current state of knowledge on interactions between Yersinia and the inflammasome system, with the goal of integrating these findings within the general framework of inflammasome responses to microbial pathogens.

Unlabelled: Type III secretion systems (T3SS) translocate effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. However, recognition of T3SS activity by cytosolic pattern recognition receptors (PRRs) of the nucleotide-binding domain leucine rich repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Macrophages infected with Yersinia pseudotuberculosis strains lacking all known effectors or lacking the translocation regulator YopK induce rapid activation of both the canonical NLRP3 and noncanonical caspase-11 inflammasomes. While this inflammasome activation requires a functional T3SS, the precise signal that triggers inflammasome activation in response to Yersinia T3SS activity remains unclear. Effectorless strains of Yersinia as well as ΔyopK strains translocate elevated levels of T3SS substrates into infected cells. To dissect the contribution of pore formation and translocation to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions of the T3SS. Notably, YopD variants that abrogated translocation but not pore-forming activity failed to induce inflammasome activation. Furthermore, analysis of individual infected cells revealed that inflammasome activation at the single-cell level correlated with translocated levels of YopB and YopD themselves. Intriguingly, LcrH mutants that are fully competent for effector translocation but produce and translocate lower levels of YopB and YopD also fail to trigger inflammasome activation. Our findings therefore suggest that hypertranslocation of YopD and YopB is linked to inflammasome activation in response to the Yersinia T3SS., Importance: The innate immune response is critical to effective clearance of pathogens. Recognition of conserved virulence structures and activities by innate immune receptors such as NLRs constitute one of the first steps in mounting the innate immune response. However, pathogens such as Yersinia actively evade or subvert components of host defense, such as inflammasomes. The T3SS-secreted protein YopK is an essential virulence factor that limits translocation of other Yops, thereby limiting T3SS-induced inflammasome activation. However, what triggers inflammasome activation in cells infected by YopK-deficient Yersinia is not clear. Our findings indicate that hypertranslocation of pore complex proteins promotes inflammasome activation and that YopK prevents inflammasome activation by the T3SS by limiting translocation of YopD and YopB themselves., (Copyright © 2015 Zwack et al.)

Toll-like receptor signaling and subsequent activation of NF-κB- and MAPK-dependent genes during infection play an important role in antimicrobial host defense. The YopJ protein of pathogenic Yersinia species inhibits NF-κB and MAPK signaling, resulting in blockade of NF-κB-dependent cytokine production and target cell death. Nevertheless, Yersinia infection induces inflammatory responses in vivo. Moreover, increasing the extent of YopJ-dependent cytotoxicity induced by Yersinia pestis and Yersinia pseudotuberculosis paradoxically leads to decreased virulence in vivo, suggesting that cell death promotes anti-Yersinia host defense. However, the specific pathways responsible for YopJ-induced cell death and how this cell death mediates immune defense against Yersinia remain poorly defined. YopJ activity induces processing of multiple caspases, including caspase-1, independently of inflammasome components or the adaptor protein ASC. Unexpectedly, caspase-1 activation in response to the activity of YopJ required caspase-8, receptor-interacting serine/threonine kinase 1 (RIPK1), and Fas-associated death domain (FADD), but not RIPK3. Furthermore, whereas RIPK3 deficiency did not affect YopJ-induced cell death or caspase-1 activation, deficiency of both RIPK3 and caspase-8 or FADD completely abrogated Yersinia-induced cell death and caspase-1 activation. Mice lacking RIPK3 and caspase-8 in their hematopoietic compartment showed extreme susceptibility to Yersinia and were deficient in monocyte and neutrophil-derived production of proinflammatory cytokines. Our data demonstrate for the first time to our knowledge that RIPK1, FADD, and caspase-8 are required for YopJ-induced cell death and caspase-1 activation and suggest that caspase-8-mediated cell death overrides blockade of immune signaling by YopJ to promote anti-Yersinia immune defense.

Microbial infection triggers assembly of inflammasome complexes that promote caspase-1-dependent antimicrobial responses. Inflammasome assembly is mediated by members of the nucleotide binding domain leucine-rich repeat (NLR) protein family that respond to cytosolic bacterial products or disruption of cellular processes. Flagellin injected into host cells by invading Salmonella induces inflammasome activation through NLRC4, whereas NLRP3 is required for inflammasome activation in response to multiple stimuli, including microbial infection, tissue damage, and metabolic dysregulation, through mechanisms that remain poorly understood. During systemic infection, Salmonella avoids NLRC4 inflammasome activation by down-regulating flagellin expression. Macrophages exhibit delayed NLRP3 inflammasome activation after Salmonella infection, suggesting that Salmonella may evade or prevent the rapid activation of the NLRP3 inflammasome. We therefore screened a Salmonella Typhimurium transposon library to identify bacterial factors that limit NLRP3 inflammasome activation. Surprisingly, absence of the Salmonella TCA enzyme aconitase induced rapid NLRP3 inflammasome activation. This inflammasome activation correlated with elevated levels of bacterial citrate, and required mitochondrial reactive oxygen species and bacterial citrate synthase. Importantly, Salmonella lacking aconitase displayed NLRP3- and caspase-1/11-dependent attenuation of virulence, and induced elevated serum IL-18 in wild-type mice. Together, our data link Salmonella genes controlling oxidative metabolism to inflammasome activation and suggest that NLRP3 inflammasome evasion promotes systemic Salmonella virulence.

Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1β. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1β. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1β, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1β release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1β, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1β release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense.

Unlabelled: Competence for genetic transformation in Streptococcus pneumoniae develops in response to accumulation of a secreted peptide pheromone and was one of the initial examples of bacterial quorum sensing. Activation of this signaling system induces not only expression of the proteins required for transformation but also the production of cellular chaperones and proteases. We have shown here that activity of this pathway is sensitively responsive to changes in the accuracy of protein synthesis that are triggered by either mutations in ribosomal proteins or exposure to antibiotics. Increasing the error rate during ribosomal decoding promoted competence, while reducing the error rate below the baseline level repressed the development of both spontaneous and antibiotic-induced competence. This pattern of regulation was promoted by the bacterial HtrA serine protease. Analysis of strains with the htrA (S234A) catalytic site mutation showed that the proteolytic activity of HtrA selectively repressed competence when translational fidelity was high but not when accuracy was low. These findings redefine the pneumococcal competence pathway as a response to errors during protein synthesis. This response has the capacity to address the immediate challenge of misfolded proteins through production of chaperones and proteases and may also be able to address, through genetic exchange, upstream coding errors that cause intrinsic protein folding defects. The competence pathway may thereby represent a strategy for dealing with lesions that impair proper protein coding and for maintaining the coding integrity of the genome., Importance: The signaling pathway that governs competence in the human respiratory tract pathogen Streptococcus pneumoniae regulates both genetic transformation and the production of cellular chaperones and proteases. The current study shows that this pathway is sensitively controlled in response to changes in the accuracy of protein synthesis. Increasing the error rate during ribosomal decoding induced competence, while decreasing the error rate repressed competence. This pattern of regulation was promoted by the HtrA protease, which selectively repressed competence when translational fidelity was high but not when accuracy was low. Our findings demonstrate that this organism is able to monitor the accuracy of information used for protein biosynthesis and suggest that errors trigger a response addressing both the immediate challenge of misfolded proteins and, through genetic exchange, upstream coding errors that may underlie protein folding defects. This pathway may represent an evolutionary strategy for maintaining the coding integrity of the genome.