Yersinia pestis escapes entrapment in thrombi by targeting platelet function.

BACKGROUND: Platelets are classically recognized for their role in hemostasis and thrombosis. Recent work has demonstrated that platelets can also execute a variety of immune functions. The dual prothrombotic and immunological roles of platelets suggest that they may pose a barrier to the replication or dissemination of extracellular bacteria. However, some bloodborne pathogens, such as the plague bacterium Yersinia pestis, routinely achieve high vascular titers that are necessary for pathogen transmission. OBJECTIVES: It is not currently known how or if pathogens circumvent platelet barriers to bacterial dissemination and replication. We sought to determine whether extracellular bloodborne bacterial pathogens actively interfere with platelet function, using Y  pestis as a model system. METHODS: The interactions and morphological changes of human platelets with various genetically modified Y pestis strains were examined using aggregation assays, immunofluorescence, and scanning electron microscopy. RESULTS: Yersinia pestis directly destabilized platelet thrombi, preventing bacterial entrapment in fibrin/platelet clots. This activity was dependent on two well-characterized bacterial virulence factors: the Y pestis plasminogen activator Pla, which stimulates host-mediated fibrinolysis, and the bacterial type III secretion system (T3SS), which delivers bacterial proteins into the cytoplasm of targeted host cells to reduce or prevent effective immunological responses. Platelets intoxicated by the Y pestis T3SS were unable to respond to prothrombotic stimuli, and T3SS expression decreased the formation of neutrophil extracellular traps in platelet thrombi. CONCLUSIONS: These findings are the first demonstration of a bacterial pathogen using its T3SS and an endogenous protease to manipulate platelet function and to escape entrapment in platelet thrombi.

Redundant and Cooperative Roles for Yersinia pestis Yop Effectors in the Inhibition of Human Neutrophil Exocytic Responses Revealed by Gain-of-Function Approach.

Yersinia pestis causes a rapid, lethal disease referred to as plague. Y. pestis actively inhibits the innate immune system to generate a noninflammatory environment during early stages of infection to promote colonization. The ability of Y. pestis to create this early noninflammatory environment is in part due to the action of seven Yop effector proteins that are directly injected into host cells via a type 3 secretion system (T3SS). While each Yop effector interacts with specific host proteins to inhibit their function, several Yop effectors either target the same host protein or inhibit converging signaling pathways, leading to functional redundancy. Previous work established that Y. pestis uses the T3SS to inhibit neutrophil respiratory burst, phagocytosis, and release of inflammatory cytokines. Here, we show that Y. pestis also inhibits release of granules in a T3SS-dependent manner. Moreover, using a gain-of-function approach, we discovered previously hidden contributions of YpkA and YopJ to inhibition and that cooperative actions by multiple Yop effectors are required to effectively inhibit degranulation. Independent from degranulation, we also show that multiple Yop effectors can inhibit synthesis of leukotriene B4 (LTB4), a potent lipid mediator released by neutrophils early during infection to promote inflammation. Together, inhibition of these two arms of the neutrophil response likely contributes to the noninflammatory environment needed for Y. pestis colonization and proliferation.

Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death.

Limited proteolysis of gasdermin D (GSDMD) generates an N-terminal pore-forming fragment that controls pyroptosis in macrophages. GSDMD is processed via inflammasome-activated caspase-1 or -11. It is currently unknown whether macrophage GSDMD can be processed by other mechanisms. Here, we describe an additional pathway controlling GSDMD processing. The inhibition of TAK1 or IκB kinase (IKK) by the Yersinia effector protein YopJ elicits RIPK1- and caspase-8-dependent cleavage of GSDMD, which subsequently results in cell death. GSDMD processing also contributes to the NLRP3 inflammasome-dependent release of interleukin-1ß (IL-1ß). Thus, caspase-8 acts as a regulator of GSDMD-driven cell death. Furthermore, this study establishes the importance of TAK1 and IKK activity in the control of GSDMD cleavage and cytotoxicity.

Gain-of-Function Analysis Reveals Important Virulence Roles for the Yersinia pestis Type III Secretion System Effectors YopJ, YopT, and YpkA.

Virulence of Yersinia pestis in mammals requires the type III secretion system, which delivers seven effector proteins into the cytoplasm of host cells to undermine immune responses. All seven of these effectors are conserved across Y. pestis strains, but three, YopJ, YopT, and YpkA, are apparently dispensable for virulence. Some degree of functional redundancy between effector proteins would explain both observations. Here, we use a combinatorial genetic approach to define the minimal subset of effectors required for full virulence in mice following subcutaneous infection. We found that a Y. pestis strain lacking YopJ, YopT, and YpkA is attenuated for virulence in mice and that addition of any one of these effectors to this strain increases lethality significantly. YopJ, YopT, and YpkA likely contribute to virulence via distinct mechanisms. YopJ is uniquely able to cause macrophage cell death in vitro and to suppress accumulation of inflammatory cells to foci of bacterial growth in deep tissue, whereas YopT and YpkA cannot. The synthetic phenotypes that emerge when YopJ, YopT, and YpkA are removed in combination provide evidence that each effector enhances Y. pestis virulence and that YopT and YpkA act through a mechanism distinct from that of YopJ.

Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis.

Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

The Yersinia pestis Effector YopM Inhibits Pyrin Inflammasome Activation.

Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1ß and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1ß/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1ß/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1ß activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1ß generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1ß/IL-18 release suggests that these effects are essential for maximal control of innate immunity during plague.

Manipulation of Interleukin-1ß and Interleukin-18 Production by Yersinia pestis Effectors YopJ and YopM and Redundant Impact on Virulence.

Innate immunity plays a central role in resolving infections by pathogens. Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1ß and IL-18. These cytokines are produced by a two-step mechanism involving NF-κB-mediated pro-cytokine production and inflammasome-driven maturation into bioactive inflammatory mediators. Because of the anti-microbial effects induced by IL-1ß/IL-18, it may be desirable for pathogens to manipulate their production. Y. pestis type III secretion system effectors YopJ and YopM can interfere with different parts of this process. Both effectors have been reported to influence inflammasome caspase-1 activity; YopJ promotes caspase-8-dependent cell death and caspase-1 cleavage, whereas YopM inhibits caspase-1 activity via an incompletely understood mechanism. However, neither effector appears essential for full virulence in vivo Here we report that the sum of influences by YopJ and YopM on IL-1ß/IL-18 release is suppressive. In the absence of YopM, YopJ minimally affects caspase-1 cleavage but suppresses IL-1ß, IL-18, and other cytokines and chemokines. Importantly, we find that Y. pestis containing combined deletions of YopJ and YopM induces elevated levels of IL-1ß/IL-18 in vitro and in vivo and is significantly attenuated in a mouse model of bubonic plague. The reduced virulence of the YopJ-YopM mutant is dependent on the presence of IL-1ß, IL-18, and caspase-1. Thus, we conclude that Y. pestis YopJ and YopM can both exert a tight control of host IL-1ß/IL-18 production to benefit the bacteria, resulting in a redundant impact on virulence.

Reversion From Methicillin Susceptibility to Methicillin Resistance in Staphylococcus aureus During Treatment of Bacteremia.

Approximately 3% of Staphylococcus aureus strains that, according to results of conventional phenotypic methods, are highly susceptible to methicillin-like antibiotics also have polymerase chain reaction (PCR) results positive for mecA. The genetic nature of these mecA-positive methicillin-susceptible S. aureus (MSSA) strains has not been investigated. We report the first clearly defined case of reversion from methicillin susceptibility to methicillin resistance among mecA-positive MSSA within a patient during antibiotic therapy. We describe the mechanism of reversion for this strain and for a second clinical isolate that reverts at a similar frequency. The rates of reversion are of the same order of magnitude as spontaneous resistance to drugs like rifampicin. When mecA is detected by PCR in the clinical laboratory, current guidelines recommend that these strains be reported as resistant. Because combination therapy using both a ß-lactam and a second antibiotic suppressing the small revertant population may be superior to alternatives such as vancomycin, the benefits of distinguishing between mecA-positive MSSA and MRSA in clinical reports should be evaluated.

Genome-wide mutant fitness profiling identifies nutritional requirements for optimal growth of Yersinia pestis in deep tissue.

UNLABELLED: Rapid growth in deep tissue is essential to the high virulence of Yersinia pestis, causative agent of plague. To better understand the mechanisms underlying this unusual ability, we used transposon mutagenesis and high-throughput sequencing (Tn-seq) to systematically probe the Y. pestis genome for elements contributing to fitness during infection. More than a million independent insertion mutants representing nearly 200,000 unique genotypes were generated in fully virulent Y. pestis. Each mutant in the library was assayed for its ability to proliferate in vitro on rich medium and in mice following intravenous injection. Virtually all genes previously established to contribute to virulence following intravenous infection showed significant fitness defects, with the exception of genes for yersiniabactin biosynthesis, which were masked by strong intercellular complementation effects. We also identified more than 30 genes with roles in nutrient acquisition and metabolism as experiencing strong selection during infection. Many of these genes had not previously been implicated in Y. pestis virulence. We further examined the fitness defects of strains carrying mutations in two such genes-encoding a branched-chain amino acid importer (brnQ) and a glucose importer (ptsG)-both in vivo and in a novel defined synthetic growth medium with nutrient concentrations matching those in serum. Our findings suggest that diverse nutrient limitations in deep tissue play a more important role in controlling bacterial infection than has heretofore been appreciated. Because much is known about Y. pestis pathogenesis, this study also serves as a test case that assesses the ability of Tn-seq to detect virulence genes. IMPORTANCE: Our understanding of the functions required by bacteria to grow in deep tissues is limited, in part because most growth studies of pathogenic bacteria are conducted on laboratory media that do not reflect conditions prevailing in infected animal tissues. Improving our knowledge of this aspect of bacterial biology is important as a potential pathway to the development of novel therapeutics. Yersinia pestis, the plague bacterium, is highly virulent due to its rapid dissemination and growth in deep tissues, making it a good model for discovering bacterial adaptations that promote rapid growth during infection. Using Tn-seq, a genome-wide fitness profiling technique, we identified several functions required for fitness of Y. pestis in vivo that were not previously known to be important. Most of these functions are needed to acquire or synthesize nutrients. Interference with these critical nutrient acquisition pathways may be an effective strategy for designing novel antibiotics and vaccines.

Caspase-8 and RIP kinases regulate bacteria-induced innate immune responses and cell death.

A number of pathogens cause host cell death upon infection, and Yersinia pestis, infamous for its role in large pandemics such as the "Black Death" in medieval Europe, induces considerable cytotoxicity. The rapid killing of macrophages induced by Y. pestis, dependent upon type III secretion system effector Yersinia outer protein J (YopJ), is minimally affected by the absence of caspase-1, caspase-11, Fas ligand, and TNF. Caspase-8 is known to mediate apoptotic death in response to infection with several viruses and to regulate programmed necrosis (necroptosis), but its role in bacterially induced cell death is poorly understood. Here we provide genetic evidence for a receptor-interacting protein (RIP) kinase-caspase-8-dependent macrophage apoptotic death pathway after infection with Y. pestis, influenced by Toll-like receptor 4-TIR-domain-containing adapter-inducing interferon-ß (TLR4-TRIF). Interestingly, macrophages lacking either RIP1, or caspase-8 and RIP3, also had reduced infection-induced production of IL-1ß, IL-18, TNF, and IL-6; impaired activation of the transcription factor NF-κB; and greatly compromised caspase-1 processing. Cleavage of the proform of caspase-1 is associated with triggering inflammasome activity, which leads to the maturation of IL-1ß and IL-18, cytokines important to host responses against Y. pestis and many other infectious agents. Our results identify a RIP1-caspase-8/RIP3-dependent caspase-1 activation pathway after Y. pestis challenge. Mice defective in caspase-8 and RIP3 were also highly susceptible to infection and displayed reduced proinflammatory cytokines and myeloid cell death. We propose that caspase-8 and the RIP kinases are key regulators of macrophage cell death, NF-κB and inflammasome activation, and host resistance after Y. pestis infection.

The NLRP12 inflammasome recognizes Yersinia pestis.

Yersinia pestis, the causative agent of plague, is able to suppress production of inflammatory cytokines IL-18 and IL-1ß, which are generated through caspase-1-activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. Here, we sought to elucidate the role of NLRs and IL-18 during plague. Lack of IL-18 signaling led to increased susceptibility to Y. pestis, producing tetra-acylated lipid A, and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. We found that the NLRP12 inflammasome was an important regulator controlling IL-18 and IL-1ß production after Y. pestis infection, and NLRP12-deficient mice were more susceptible to bacterial challenge. NLRP12 also directed interferon-γ production via induction of IL-18, but had minimal effect on signaling to the transcription factor NF-κB. These studies reveal a role for NLRP12 in host resistance against pathogens. Minimizing NLRP12 inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis.

Involvement of CD8+ T cell-mediated immune responses in LcrV DNA vaccine induced protection against lethal Yersinia pestis challenge.

Yersinia pestis (Y. pestis) is the causative pathogen of plague, a highly fatal disease for which an effective vaccine, especially against mucosal transmission, is still not available. Like many bacterial infections, antigen-specific antibody responses have been traditionally considered critical, if not solely responsible, for vaccine-induced protection against Y. pestis. Studies in recent years have suggested the importance of T cell immune responses against Y. pestis infection but information is still limited about the details of Y. pestis antigen-specific T cell immune responses. In current report, studies are conducted to identify the presence of CD8+ T cell epitopes in LcrV protein, the leading antigen of plague vaccine development. Furthermore, depletion of CD8+ T cells in LcrV DNA vaccinated Balb/C mice led to reduced protection against lethal intranasal challenge of Y. pestis. These findings establish that an LcrV DNA vaccine is able to elicit CD8+ T cell immune responses against specific epitopes of this key plague antigen and that a CD8+ T cell immune response is involved in LcrV DNA vaccine-elicited protection. Future studies in plague vaccine development will need to examine if the presence of detectable T cell immune responses, in particular CD8+ T-cell immune responses, will enhance the protection against Y. pestis in higher animal species or humans.

An active site water network in the plasminogen activator pla from Yersinia pestis.

The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 A. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

No better time to FRET: shedding light on host pathogen interactions.

Understanding the spatio-temporal subversion of host cell signaling by bacterial virulence factors is key to combating infectious diseases. Following a recent study by Buntru and co-workers published in BMC Biology, we review how fluorescence (Forster) resonance energy transfer (FRET) has been applied to studying host-pathogen interactions and consider the prospects for its future application.

Antigen engineering can play a critical role in the protective immunity elicited by Yersinia pestis DNA vaccines.

The use of a DNA immunization approach to deliver protective antigens against Yersinia pestis (Y. pestis) has been successful in previously reported studies. In the current study, the gene designs for V and F1, two well-studied virulent factors serving as main targets for vaccine development, were altered to explore additional options in hopes of improving the protective immunity of DNA vaccines expressing these two antigens. Compared to the wild type V gene DNA vaccines, the use of codon optimized V gene sequences was effective in improving the antigen expression, titers of anti-V antibody responses, and survival against a mucosal lethal challenge. For the F1 DNA vaccine, removal of the N-terminal hydrophobic region was able to improve protective immunity. However, adding a mammalian signal peptide sequence to F1 actually led to reduced protection despite it inducing slightly higher anti-F1 antibody responses. The F1 gene can be fused with a gene coding for YscF, a newly confirmed partial protective antigen for Y. pestis, to produce DNA vaccines that express fused F1 and YscF antigens. One design, in particular, that had YscF fused to the downstream sequence of F1, produced better protection than separate F1 or YscF DNA vaccines, suggesting a potential synergistic effect between these two antigens. Findings from the above studies indicated that there are multiple approaches to optimize the protective immunity for plague DNA vaccines. Most importantly, proper antigen engineering to produce optimal antigen gene inserts in DNA vaccines can clearly play a major role in the future designs of a wide range of DNA vaccines.

Targeting type III secretion in Yersinia pestis.

Yersinia pestis, the causative agent of plague, utilizes a plasmid-encoded type III secretion system (T3SS) to aid it with its resistance to host defenses. This system injects a set of effector proteins known as Yops (Yersinia outer proteins) into the cytosol of host cells that come into contact with the bacteria. T3SS is absolutely required for the virulence of Y. pestis, making it a potential target for new therapeutics. Using a novel and simple high-throughput screening method, we examined a diverse collection of chemical libraries for small molecules that inhibit type III secretion in Y. pestis. The primary screening of 70,966 compounds and mixtures yielded 421 presumptive inhibitors. We selected eight of these for further analysis in secondary assays. Four of the eight compounds effectively inhibited Yop secretion at micromolar concentrations. Interestingly, we observed differential inhibition among Yop species with some compounds. The compounds did not inhibit bacterial growth at the concentrations used in the inhibition assays. Three compounds protected HeLa cells from type III secretion-dependent cytotoxicity. Of the eight compounds examined in secondary assays, four show good promise as leads for structure-activity relationship studies. They are a diverse group, with each having a chemical scaffold not only distinct from each other but also distinct from previously described candidate type III secretion inhibitors.

Relative immunogenicity and protection potential of candidate Yersinia Pestis antigens against lethal mucosal plague challenge in Balb/C mice.

Yersinia Pestis outer proteins, plasminogen activator protease and Yop secretion protein F are necessary for the full virulence of Yesinia pestis and have been proposed as potential protective antigens for vaccines against plague. In the current study, we used DNA immunization as a tool to study the relative protective immunity of these proteins with a standardized intranasal challenge system in mice. While the natural full-length gene sequences for most of these Y. pestis proteins did not display a good level of protein expression in vitro when delivered by a DNA vaccine vector, the overall immunogenicity of these wild type gene DNA vaccines was low in eliciting antigen-specific antibody responses and gene sequence modifications improved both of these parameters. However, even modified YopD, YopO and YscF antigens were only able to partially protect immunized mice at various levels against lethal challenge with Y. pestis KIM 1001 strain while no protection was observed with either the YopB or Pla antigens. These results demonstrate that DNA immunization is effective in screening, optimizing and comparing optimal antigen designs and immunogenicity of candidate antigens for the development of a subunit-based plague vaccine.

High throughput screening for small-molecule inhibitors of type III secretion in Yersinia pestis.

Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, utilize a plasmid encoded type III secretion system (T3SS) to promote infection by delivering Yersinia outer proteins (Yops) into the cytosol of mammalian cells. This T3SS is absolutely required for Yersinia virulence, which makes T3SS an attractive target in the development of novel therapeutics for treatment of plague and other Yersinia infections. In this study, a new method for high throughput screening (HTS) of small molecules for the ability to inhibit type III secretion (T3S) in Y. pestis has been developed. In comparison with screening assays employed by others, this method is very simple and rapid, and thus well suited for examining very large compound sets. Using this method, we screened a diverse collection of libraries at the US National Screening Laboratory. The initial examination of 70,966 compounds and mixtures from 13 libraries resulted in 431 primary hits. Strong positive indications of inhibition were observed at a rate of 0.01%, while moderate and weak but potentially meaningful signals were observed at rates of 0.056% and 0.54% respectively. Further characterizations were conducted on selected primary hits in Y. pestis. Of the eight compounds examined in secondary assays, four show good promise as leads for structure activity relationship studies. They are a diverse group, each having chemical scaffolds not only distinct from one another, but also distinct from previously described candidate T3S inhibitors.