Yersiniabactin contributes to overcoming zinc restriction during Yersinia pestis infection of mammalian and insect hosts.

Yersinia pestis causes human plague and colonizes both a mammalian host and a flea vector during its transmission cycle. A key barrier to bacterial infection is the host's ability to actively sequester key biometals (e.g., iron, zinc, and manganese) required for bacterial growth. This is referred to as nutritional immunity. Mechanisms to overcome nutritional immunity are essential virulence factors for bacterial pathogens. Y. pestis produces an iron-scavenging siderophore called yersiniabactin (Ybt) that is required to overcome iron-mediated nutritional immunity and cause lethal infection. Recently, Ybt has been shown to bind to zinc, and in the absence of the zinc transporter ZnuABC, Ybt improves Y. pestis growth in zinc-limited medium. These data suggest that, in addition to iron acquisition, Ybt may also contribute to overcoming zinc-mediated nutritional immunity. To test this hypothesis, we used a mouse model defective in iron-mediated nutritional immunity to demonstrate that Ybt contributes to virulence in an iron-independent manner. Furthermore, using a combination of bacterial mutants and mice defective in zinc-mediated nutritional immunity, we identified calprotectin as the primary barrier for Y. pestis to acquire zinc during infection and that Y. pestis uses Ybt to compete with calprotectin for zinc. Finally, we discovered that Y. pestis encounters zinc limitation within the flea midgut, and Ybt contributes to overcoming this limitation. Together, these results demonstrate that Ybt is a bona fide zinc acquisition mechanism used by Y. pestis to surmount zinc limitation during the infection of both the mammalian and insect hosts.

Modification of the Pulmonary MyD88 Inflammatory Response Underlies the Role of the Yersinia pestis Pigmentation Locus in Primary Pneumonic Plague.

Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing bronchopneumonia involving focal bacterial growth, neutrophilic congestion, and alveolar necrosis. Within a short time after inhalation of Y. pestis, inflammatory cytokines are expressed via the Toll/interleukin-1 (IL-1) adaptor myeloid differentiation primary response 88 (MyD88), which facilitates the primary lung infection. We previously showed that Y. pestis lacking the 102-kb chromosomal pigmentation locus (pgm) is unable to cause inflammatory damage in the lungs, whereas the wild-type (WT) strain induces the toxic MyD88 pulmonary inflammatory response. In this work, we investigated the involvement of the pgm in skewing the inflammatory response during pneumonic plague. We show that the early MyD88-dependent and -independent cytokine responses to pgm- Y. pestis infection of the lungs are similar yet distinct from those that occur during pgm+ infection. Furthermore, we found that MyD88 was necessary to prevent growth of the iron-starved pgm- Y. pestis despite the presence of iron chelators lactoferrin and transferrin. However, while this induced neutrophil recruitment, there was no hyperinflammatory response, and pulmonary disease was mild without MyD88. In contrast, growth in blood and tissues progressed rapidly in the absence of MyD88, due to an almost total loss of serum interferon gamma (IFN-γ). We further show that the expression of MyD88 by myeloid cells is important to control bacteremia but not the primary lung infection. The combined data indicate distinct roles for myeloid and nonmyeloid MyD88 and suggest that expression of the pgm is necessary to skew the inflammatory response in the lungs to cause pneumonic plague.

Yersinia pestis Plasminogen Activator.

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

Ancient familial Mediterranean fever mutations in human pyrin and resistance to Yersinia pestis.

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by homozygous or compound heterozygous gain-of-function mutations in MEFV, which encodes pyrin, an inflammasome protein. Heterozygous carrier frequencies for multiple MEFV mutations are high in several Mediterranean populations, suggesting that they confer selective advantage. Among 2,313 Turkish people, we found extended haplotype homozygosity flanking FMF-associated mutations, indicating evolutionarily recent positive selection of FMF-associated mutations. Two pathogenic pyrin variants independently arose >1,800 years ago. Mutant pyrin interacts less avidly with Yersinia pestis virulence factor YopM than with wild-type human pyrin, thereby attenuating YopM-induced interleukin (IL)-1ß suppression. Relative to healthy controls, leukocytes from patients with FMF harboring homozygous or compound heterozygous mutations and from asymptomatic heterozygous carriers released heightened IL-1ß specifically in response to Y. pestis. Y. pestis-infected MefvM680I/M680I FMF knock-in mice exhibited IL-1-dependent increased survival relative to wild-type knock-in mice. Thus, FMF mutations that were positively selected in Mediterranean populations confer heightened resistance to Y. pestis.

Difference in Strain Pathogenicity of Septicemic Yersinia pestis Infection in a TLR2-/- Mouse Model.

Yersinia pestis is the causative agent of bubonic, pneumonic, and septicemic plague. We demonstrate that Toll-like receptor 2-deficient (TLR2-/-) mice are resistant to septicemic infection by the KIM5 strain of Y. pestis but not to infection by the CO92 Δpgm strain. This resistance is dependent on TLR2, the route of infection, and the isoform of YopJ. Elevated bacterial burdens were found in the spleens of CO92 Δpgm-infected animals by 24 h postinfection and in the livers by 4 days. The YopJ isoform present contributed directly to cytotoxicity and inflammatory cytokine production of bone marrow-derived macrophages from TLR2-/- mice. Immune cell trafficking is altered in CO92 Δpgm infections, with an increased neutrophil infiltration to the spleen 5 days postinfection. Immune cell infiltration to the liver was greater and earlier in KIM5-infected TLR2-/- mice. The functionality of the immune cells was assessed by the ability to develop reactive oxygen and nitrogen species. Our data suggest an inhibition of granulocytes in forming these species in CO92 Δpgm-infected TLR2-/- mice. These findings suggest that resistance to KIM5 in TLR2-/- mice is dependent on early immune cell trafficking and functionality.

The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis during Planktonic Growth and Growth in Biofilms.

Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δcrp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis-infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection.IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

Robust Th1 cellular and humoral responses generated by the Yersinia pestis rF1-V subunit vaccine formulated to contain an agonist of the CD137 pathway do not translate into increased protection against pneumonic plague.

Yersinia pestis is the causative agent of plague and is a re-emerging pathogen that also has the potential as a biological weapon, necessitating the development of a preventive vaccine. Despite intense efforts for the last several decades, there is currently not a vaccine approved by the FDA. The rF1-V vaccine adjuvanted with Alhydrogel is a lead candidate subunit vaccine for plague and generates a strong Th2-mediate humoral response with a modest Th1 cellular response. As immune protection against Y. pestis requires both humoral and Th1 cellular responses, modifying the rF1-V subunit vaccine formulation to include a robust inducer of Th1 responses may improve efficacy. Thus, we reformulated the subunit vaccine to include SA-4-1BBL, an agonist of the CD137 costimulatory pathway and a potent inducer of Th1 response, and assessed its protective efficacy against pneumonic plague. We herein show for the first time a sex bias in the prophylactic efficacy of the Alhydrogel adjuvanted rF1-V vaccine, with female mice showing better protection against pneumonic plague than male. The sex bias for protection was irrespective of the generation of comparable levels of rF1-V-specific antibody titers and Th1 cellular responses in both sexes. The subunit vaccine reformulated with SA-4-1BBL generated robust Th1 cellular and humoral responses. A prime-boost vaccination scheme involving prime with rF1-V + Alhydrogel and boost with the rF1-V + SA-4-1BBL provided protection in male mice against pneumonic plague. In marked contrast, prime and boost with rF1-V reformulated with both adjuvants resulted in the loss of protection against pneumonic plague, despite generating high levels of humoral and Th1 cellular responses. While unexpected, these findings demonstrate the complexity of immune mechanisms required for protection. Elucidating mechanisms responsible for these differences in protection will help to guide the development of better prophylactic subunit vaccines effective against pneumonic plague.

Yersinia pestis Exploits Early Activation of MyD88 for Growth in the Lungs during Pneumonic Plague.

Yersinia pestis causes bubonic, pneumonic, and septicemic plague. Although no longer responsible for pandemic outbreaks, pneumonic plague continues to be a challenge for medical treatment and has been classified as a reemerging disease in some parts of the world. In the early stage of infection, inflammatory responses are believed to be suppressed by Y. pestis virulence factors in order to prevent clearance, while later, the hyperactivation of inflammation contributes to the progression of disease. In this work, we sought to identify the host factors that mediate this process and studied the role of the Toll/interleukin 1 (IL-1) receptor adapter and major inflammatory mediator myeloid differentiation primary response 88 (MyD88) in pneumonic plague. We show that pulmonary challenge of Myd88-/- mice with wild-type (WT) Y. pestis results in significant loss of pro- and anti-inflammatory cytokines and chemokines, especially gamma interferon (IFN-γ) and KC, in the lungs compared to that in WT mice. Bacterial growth in the lungs occurred more rapidly in the WT mice, however, indicating a role for the MyD88 response in facilitating the primary lung infection. Nevertheless, Myd88-/- mice were more sensitive to lethality from secondary septicemic plague. Together these findings indicate a central role for MyD88 during the biphasic inflammatory response to pulmonary Y. pestis infection. In the early phase, low-level MyD88-dependent chemokine expression limits initial growth but facilitates Y. pestis access to a protected replicative niche. The later hyperinflammatory phase is partially MyD88 dependent and ineffective in the lungs but controls systemic infection and reduces the progression of secondary septicemic plague.

Of mice and men: Traces of life in the death registries of the 1630 plague in Milano.

The death registries of the plague epidemic of 1630, stored at the Archivio di Stato of Milano, have been interrogated via the EVA film technology (ethyl vinyl acetate film studded with crushed strong anion and cation exchangers as well as C8 resins). The EVA diskettes have been left in contact with the lower right margins of 11 different pages pertaining to the peak months of the raging disease (June through end of September) for 60-90min and then the captured material, after elution and digestion, analysed by mass spectrometry. The main findings: 17 Yersiniaceae family proteins, 31 different human keratins, 22 unique mouse keratins, about 400 peptides from different bacterial strains, 58 human tissue proteins and 130 additional mouse and rat tissue proteins. In addition, >60 plant proteins (notably potato, corn, rice, carrot and chickpeas), likely representing the meagre meals of the scribes, contaminating the pages, were detected. The significance of these unique findings is amply illustrated in the body of the article. SIGNIFICANCE: Archivists, historians, librarians usually explore the texts of ancient and modern manuscript in order to extract the meaning of the writing and understand the mood, feelings, political, philosophical and/or religious ideas therein expressed by the authors. With the present EVA methodology (the only one, at present, able to access our Cultural Heritage without damaging or contaminating it) we interrogate, instead, the support, be it paper, parchment, wood panel, cloth, canvas and the like, in order to extract invisible data, such as the presence of drugs, medicaments, infectious pathogens, human and environmental contaminants. Metabolites, proteins and peptides thus captured are then analysed via mass spectrometry. The unique data mined by this technology should considerably enlarge the (so far) restricted horizon of the writing exploration and add new insight on the environmental conditions in which such documents were produced as well as, importantly, on the health/pathological conditions of the authors. It is believed that the present technology, as here reported, will become the officially accepted one for exploring the world Cultural Heritage.

Paleoproteomics of the Dental Pulp: The plague paradigm.

Chemical decomposition and fragmentation may limit the detection of ancient host and microbial DNA while some proteins can be detected for extended periods of time. We applied paleoproteomics on 300-year-old dental pulp specimens recovered from 16 individuals in two archeological funeral sites in France, comprising one documented plague site and one documented plague-negative site. The dental pulp paleoproteome of the 16 teeth comprised 439 peptides representative of 30 proteins of human origin and 211 peptides representative of 27 proteins of non-human origin. Human proteins consisted of conjunctive tissue and blood proteins including IgA immunoglobulins. Four peptides were indicative of three presumable Yersinia pestis proteins detected in 3/8 dental pulp specimens from the plague-positive site but not in the eight dental pulp specimens collected in the plague-negative site. Paleoproteomics applied to the dental pulp is a new and innovative approach to screen ancient individuals for the detection of blood-borne pathogens and host inflammatory response.

Centrality in the host-pathogen interactome is associated with pathogen fitness during infection.

To perform their functions proteins must interact with each other, but how these interactions influence bacterial infection remains elusive. Here we demonstrate that connectivity in the host-pathogen interactome is directly related to pathogen fitness during infection. Using Y. pestis as a model organism, we show that the centrality-lethality rule holds for pathogen fitness during infection but only when the host-pathogen interactome is considered. Our results suggest that the importance of pathogen proteins during infection is directly related to their number of interactions with the host. We also show that pathogen proteins causing an extensive rewiring of the host interactome have a higher impact in pathogen fitness during infection. Hence, we conclude that hubs in the host-pathogen interactome should be explored as promising targets for antimicrobial drug design.

The Yersinia Type III secretion effector YopM Is an E3 ubiquitin ligase that induced necrotic cell death by targeting NLRP3.

Yersinia pestis uses type III effector proteins to target eukaryotic signaling systems. The Yersinia outer protein (Yop) M effector from the Y. pestis strain is a critical virulence determinant; however, its role in Y. pestis pathogenesis is just beginning to emerge. Here we first identify YopM as the structural mimic of the bacterial IpaH E3 ligase family in vitro, and establish that the conserved CLD motif in its N-terminal is responsible for the E3 ligase function. Furthermore, we show that NLRP3 is a novel target of the YopM protein. Specially, YopM associates with NLRP3, and its CLD ligase motif mediates the activating K63-linked ubiquitylation of NLRP3; as a result, YopM modulates NLRP3-mediated cell necrosis. Mutation of YopM E3 ligase motif dramatically reduces the ability of Y. pestis to induce HMGB1 release and cell necrosis, which ultimately contributes to bacterial virulence. In conclusion, this study has identified a previously unrecognized role for YopM E3 ligase activity in the regulation of host cell necrosis and plague pathogenesis.

New Role for FDA-Approved Drugs in Combating Antibiotic-Resistant Bacteria.

Antibiotic resistance in medically relevant bacterial pathogens, coupled with a paucity of novel antimicrobial discoveries, represents a pressing global crisis. Traditional drug discovery is an inefficient and costly process; however, systematic screening of Food and Drug Administration (FDA)-approved therapeutics for other indications in humans offers a rapid alternative approach. In this study, we screened a library of 780 FDA-approved drugs to identify molecules that rendered RAW 264.7 murine macrophages resistant to cytotoxicity induced by the highly virulent Yersinia pestis CO92 strain. Of these compounds, we identified 94 not classified as antibiotics as being effective at preventing Y. pestis-induced cytotoxicity. A total of 17 prioritized drugs, based on efficacy in in vitro screens, were chosen for further evaluation in a murine model of pneumonic plague to delineate if in vitro efficacy could be translated in vivo Three drugs, doxapram (DXP), amoxapine (AXPN), and trifluoperazine (TFP), increased animal survivability despite not exhibiting any direct bacteriostatic or bactericidal effect on Y. pestis and having no modulating effect on crucial Y. pestis virulence factors. These findings suggested that DXP, AXPN, and TFP may modulate host cell pathways necessary for disease pathogenesis. Finally, to further assess the broad applicability of drugs identified from in vitro screens, the therapeutic potential of TFP, the most efficacious drug in vivo, was evaluated in murine models of Salmonella enterica serovar Typhimurium and Clostridium difficile infections. In both models, TFP treatment resulted in increased survivability of infected animals. Taken together, these results demonstrate the broad applicability and potential use of nonantibiotic FDA-approved drugs to combat respiratory and gastrointestinal bacterial pathogens.

Reciprocal regulation of Yersinia pestis biofilm formation and virulence by RovM and RovA.

RovA is known to enhance Yersinia pestis virulence by directly upregulating the psa loci. This work presents a complex gene regulatory paradigm involving the reciprocal regulatory action of RovM and RovA on the expression of biofilm and virulence genes as well as on their own genes. RovM and RovA enhance and inhibit Y. pestis biofilm production, respectively, whereas RovM represses virulence in mice. RovM directly stimulates the transcription of hmsT, hmsCDE and rovM, while indirectly enhancing hmsHFRS transcription. It also indirectly represses hmsP transcription. By contrast, RovA directly represses hmsT transcription and indirectly inhibits waaAE-coaD transcription, while RovM inhibits psaABC and psaEF transcription by directly repressing rovA transcription. rovM expression is significantly upregulated at 26°C (the temperature of the flea gut) relative to 37°C (the warm-blooded host temperature). We speculate that upregulation of rovM together with downregulation of rovA in the flea gut would promote Y. pestis biofilm formation while inhibiting virulence gene expression, leading to a more transmissible infection of this pathogen in fleas. Once the bacterium shifts to a lifestyle in the warm-blooded hosts, inhibited RovM production accompanied by recovered RovA synthesis would encourage virulence factor production and inhibit biofilm gene expression.

Circumventing Y. pestis Virulence by Early Recruitment of Neutrophils to the Lungs during Pneumonic Plague.

Pneumonic plague is a fatal disease caused by Yersinia pestis that is associated with a delayed immune response in the lungs. Because neutrophils are the first immune cells recruited to sites of infection, we investigated the mechanisms responsible for their delayed homing to the lung. During the first 24 hr after pulmonary infection with a fully virulent Y. pestis strain, no significant changes were observed in the lungs in the levels of neutrophils infiltrate, expression of adhesion molecules, or the expression of the major neutrophil chemoattractants keratinocyte cell-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2) and granulocyte colony stimulating factor (G-CSF). In contrast, early induction of chemokines, rapid neutrophil infiltration and a reduced bacterial burden were observed in the lungs of mice infected with an avirulent Y. pestis strain. In vitro infection of lung-derived cell-lines with a YopJ mutant revealed the involvement of YopJ in the inhibition of chemoattractants expression. However, the recruitment of neutrophils to the lungs of mice infected with the mutant was still delayed and associated with rapid bacterial propagation and mortality. Interestingly, whereas KC, MIP-2 and G-CSF mRNA levels in the lungs were up-regulated early after infection with the mutant, their protein levels remained constant, suggesting that Y. pestis may employ additional mechanisms to suppress early chemoattractants induction in the lung. It therefore seems that prevention of the early influx of neutrophils to the lungs is of major importance for Y. pestis virulence. Indeed, pulmonary instillation of KC and MIP-2 to G-CSF-treated mice infected with Y. pestis led to rapid homing of neutrophils to the lung followed by a reduction in bacterial counts at 24 hr post-infection and improved survival rates. These observations shed new light on the virulence mechanisms of Y. pestis during pneumonic plague, and have implications for the development of novel therapies against this pathogen.

The role of transition metal transporters for iron, zinc, manganese, and copper in the pathogenesis of Yersinia pestis.

Yersinia pestis, the causative agent of bubonic, septicemic and pneumonic plague, encodes a multitude of Fe transport systems. Some of these are defective due to frameshift or IS element insertions, while others are functional in vitro but have no established role in causing infections. Indeed only 3 Fe transporters (Ybt, Yfe and Feo) have been shown to be important in at least one form of plague. The yersiniabactin (Ybt) system is essential in the early dermal/lymphatic stages of bubonic plague, irrelevant in the septicemic stage, and critical in pneumonic plague. Two Mn transporters have been characterized (Yfe and MntH). These two systems play a role in bubonic plague but the double yfe mntH mutant is fully virulent in a mouse model of pneumonic plague. The same in vivo phenotype occurs with a mutant lacking two (Yfe and Feo) of four ferrous transporters. A role for the Ybt siderophore in Zn acquisition has been revealed. Ybt-dependent Zn acquisition uses a transport system completely independent of the Fe-Ybt uptake system. Together Ybt components and ZnuABC play a critical role in Zn acquisition in vivo. Single mutants in either system retain high virulence in a mouse model of septicemic plague while the double mutant is completely avirulent.

Hexa-acylated LPS-lipid A deploys the appropriate level of fibrin to confer protection through MyD88.

OBJECTIVES: Fibrin has been demonstrated to function protectively against pathogens in our previous studies, but we observed that a very high level of fibrin played a negative role during infection. We performed this research to address the complication. METHODS: After infection, mice were monitored daily and harvested on day 4. The fibrin levels within the tissue samples were quantified by Western-blot. The in situ assay was used to detect plasminogen activators, protein C-ase and prothrombinase activation. PT-PCR was used to test coagulation factors expression. RESULTS: Mice treated with Coumadin showed that the protection correlates with fibrin levels. By interacting with Toll-like receptor 4, the hexa-acylated lipopolysaccharide, although not the tetra-acylated lipopolysaccharide, activates coagulation and regulates plasminogen activator inhibitor 1, thrombin activatable fibrinolysis inhibitor and thrombomodulin expression through myeloid differentiation factor 88, leading to plasminogen activators, protein C-ase and prothrombinase activation and fibrin formation. Because of the regulation, fibrin formation was controlled to deposit appropriate levels and confer protection. CONCLUSIONS: We demonstrated that the appropriate level of fibrin formation was deployed by hexa-acylated LPS-lipid A through myeloid differentiation factor 88 to confer protection.

Caspase-3 mediates the pathogenic effect of Yersinia pestis YopM in liver of C57BL/6 mice and contributes to YopM's function in spleen.

The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G(+), F4/80(+), and iNOS(+) cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.

Effect of nanovaccine chemistry on humoral immune response kinetics and maturation.

Acute respiratory infections represent a significant portion of global morbidity and mortality annually. There is a critical need for efficacious vaccines against respiratory pathogens. To vaccinate against respiratory disease, pulmonary delivery is an attractive route because it mimics the route of natural infection and can confer both mucosal and systemic immunity. We have previously demonstrated that a single dose, intranasal vaccine based on polyanhydride nanoparticles elicited a protective immune response against Yersinia pestis for at least 40 weeks after immunization with F1-V. Herein, we investigate the effect of nanoparticle chemistry and its attributes on the kinetics and maturation of the antigen-specific serum antibody response. We demonstrate that manipulation of polyanhydride nanoparticle chemistry facilitated differential kinetics of development of antibody titers, avidity, and epitope specificity. The results provide new insights into the underlying role(s) of nanoparticle chemistry in providing long-lived humoral immunity and aid in the rational design of nanovaccine formulations to induce long-lasting and mature antibody responses.

A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.