Proteogenomic discovery of sORF-encoded peptides associated with bacterial virulence in Yersinia pestis.

Plague caused by Yersinia pestis is one of the deadliest diseases. However, many molecular mechanisms of bacterial virulence remain unclear. This study engaged in the discovery of small open reading frame (sORF)-encoded peptides (SEPs) in Y. pestis. An integrated proteogenomic pipeline was established, and an atlas containing 76 SEPs was described. Bioinformatic analysis indicated that 20% of these SEPs were secreted or localized to the transmembrane and that 33% contained functional domains. Two SEPs, named SEPs-yp1 and -yp2 and encoded in noncoding regions, were selected by comparative peptidomics analysis under host-specific environments and high-salinity stress. They displayed important roles in the regulation of antiphagocytic capability in a thorough functional assay. Remarkable attenuation of virulence in mice was observed in the SEP-deleted mutants. Further global proteomic analysis indicated that SEPs-yp1 and -yp2 affected the bacterial metabolic pathways, and SEP-yp1 was associated with the bacterial virulence by modulating the expression of key virulence factors of the Yersinia type III secretion system. Our study provides a rich resource for research on Y. pestis and plague, and the findings on SEP-yp1 and SEP-yp2 shed light on the molecular mechanism of bacterial virulence.

Immunotropic Properties of an Experimental Synthetic Selenium-Organic Compound.

We studied immunotropic properties of synthetic selenium-organic preparation 2,6-dipyridinium-9-selenabicyclo[3.3.1]nonyl dibromide (974zh). The experimental preparation reduced the cAMP/cGMP ratio, which indicated an increase in proliferative activity of cells of immunocompetent organs (thymus and spleen) in experimental animals. It was shown that 974zh intensified the immune response to Yersinia pestis EV thereby increasing the resistance to the plague agent.

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.

FPR1 is the plague receptor on host immune cells.

The causative agent of plague, Yersinia pestis, uses a type III secretion system to selectively destroy immune cells in humans, thus enabling Y. pestis to reproduce in the bloodstream and be transmitted to new hosts through fleabites. The host factors that are responsible for the selective destruction of immune cells by plague bacteria are unknown. Here we show that LcrV, the needle cap protein of the Y. pestis type III secretion system, binds to the N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors. Plague infection in mice is characterized by high mortality; however, Fpr1-deficient mice have increased survival and antibody responses that are protective against plague. We identified FPR1R190W as a candidate resistance allele in humans that protects neutrophils from destruction by the Y. pestis type III secretion system. Thus, FPR1 is a plague receptor on immune cells in both humans and mice, and its absence or mutation provides protection against Y. pestis. Furthermore, plague selection of FPR1 alleles appears to have shaped human immune responses towards other infectious diseases and malignant neoplasms.

A Recombinant Attenuated Yersinia pseudotuberculosis Vaccine Delivering a Y. pestis YopENt138-LcrV Fusion Elicits Broad Protection against Plague and Yersiniosis in Mice.

In this study, a novel recombinant attenuated Yersinia pseudotuberculosis PB1+ strain (χ10069) engineered with ΔyopK ΔyopJ Δasd triple mutations was used to deliver a Y. pestis fusion protein, YopE amino acid 1 to 138-LcrV (YopENt138-LcrV), to Swiss Webster mice as a protective antigen against infections by yersiniae. χ10069 bacteria harboring the pYA5199 plasmid constitutively synthesized the YopENt138-LcrV fusion protein and secreted it via the type 3 secretion system (T3SS) at 37°C under calcium-deprived conditions. The attenuated strain χ10069(pYA5199) was manifested by the establishment of controlled infection in different tissues without developing conspicuous signs of disease in histopathological analysis of microtome sections. A single-dose oral immunization of χ10069(pYA5199) induced strong serum antibody titers (log10 mean value, 4.2), secretory IgA in bronchoalveolar lavage (BAL) fluid from immunized mice, and Yersinia-specific CD4+ and CD8+ T cells producing high levels of tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin 2 (IL-2), as well as IL-17, in both lungs and spleens of immunized mice, conferring comprehensive Th1- and Th2-mediated immune responses and protection against bubonic and pneumonic plague challenges, with 80% and 90% survival, respectively. Mice immunized with χ10069(pYA5199) also exhibited complete protection against lethal oral infections by Yersinia enterocolitica WA and Y. pseudotuberculosis PB1+. These findings indicated that χ10069(pYA5199) as an oral vaccine induces protective immunity to prevent bubonic and pneumonic plague, as well as yersiniosis, in mice and would be a promising oral vaccine candidate for protection against plague and yersiniosis for human and veterinary applications.

Predatory bacteria can protect SKH-1 mice from a lethal plague challenge.

With the rise of antimicrobial resistance, novel ways to treat bacterial infections are required and the use of predatory bacteria may be one such approach. Bdellovibrio species have been shown in vitro to predate on a wide range of other Gram-negative bacteria, including CDC category A/B pathogens such as Yersinia pestis. The data reported here show that treatment of SKH-1 mice with Bdellovibrio bacteriovorus HD100 provided significant protection from a lethal challenge of Yersinia pestis CO92. This is the first report of protection conferred by predation in vivo against a systemic pathogen challenge. However, this protective effect was not observed in a preliminary study with Balb/c mice. Therefore the effects of the predatory bacteria are complex and may be dependent on immune status/genetics of the host. Overall, predatory bacteria may have utility as a therapeutic modality but further work is required to understand the predator-host interaction.

Necroptosis of infiltrated macrophages drives Yersinia pestis dispersal within buboes.

When draining lymph nodes become infected by Yersinia pestis (Y. pestis), a massive influx of phagocytic cells occurs, resulting in distended and necrotic structures known as buboes. The bubonic stage of the Y. pestis life cycle precedes septicemia, which is facilitated by trafficking of infected mononuclear phagocytes through these buboes. However, how Y. pestis convert these immunocytes recruited by host to contain the pathogen into vehicles for bacterial dispersal and the role of immune cell death in this context are unknown. We show that the lymphatic spread requires Yersinia outer protein J (YopJ), which triggers death of infected macrophages by downregulating a suppressor of receptor-interacting protein kinase 1-mediated (RIPK1-mediated) cell death programs. The YopJ-triggered cell death was identified as necroptotic, which released intracellular bacteria, allowing them to infect new neighboring cell targets. Dying macrophages also produced chemotactic sphingosine 1-phosphate, enhancing cell-to-cell contact, further promoting infection. This necroptosis-driven expansion of infected macrophages in buboes maximized the number of bacteria-bearing macrophages reaching secondary lymph nodes, leading to sepsis. In support, necrostatins confined bacteria within macrophages and protected mice from lethal infection. These findings define necrotization of buboes as a mechanism for bacterial spread and a potential target for therapeutic intervention.

Uropathogenic enterobacteria use the yersiniabactin metallophore system to acquire nickel.

Invasive Gram-negative bacteria often express multiple virulence-associated metal ion chelators to combat host-mediated metal deficiencies. Escherichia coli, Klebsiella, and Yersinia pestis isolates encoding the Yersinia high pathogenicity island (HPI) secrete yersiniabactin (Ybt), a metallophore originally shown to chelate iron ions during infection. However, our recent demonstration that Ybt also scavenges copper ions during infection led us to question whether it might be capable of retrieving other metals as well. Here, we find that uropathogenic E. coli also use Ybt to bind extracellular nickel ions. Using quantitative MS, we show that the canonical metal-Ybt import pathway internalizes the resulting Ni-Ybt complexes, extracts the nickel, and releases metal-free Ybt back to the extracellular space. We find that E. coli and Klebsiella direct the nickel liberated from this pathway to intracellular nickel enzymes. Thus, Ybt may provide access to nickel that is inaccessible to the conserved NikABCDE permease system. Nickel should be considered alongside iron and copper as a plausible substrate for Ybt-mediated metal import by enterobacteria during human infections.

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.

A Single Amino Acid Change in the Response Regulator PhoP, Acquired during Yersinia pestis Evolution, Affects PhoP Target Gene Transcription and Polymyxin B Susceptibility.

Yersinia pestis, the causative agent of plague, evolved from the closely related pathogen Yersinia pseudotuberculosis During its emergence, Y. pestis is believed to have acquired its unique pathogenic characteristics through numerous gene gains/losses, genomic rearrangements, and single nucleotide polymorphism (SNP) changes. One such SNP creates a single amino acid variation in the DNA binding domain of PhoP, the response regulator in the PhoP/PhoQ two-component system. Y. pseudotuberculosis and the basal human-avirulent strains of Y. pestis harbor glycines at position 215 of PhoP, whereas the modern human-virulent strains (e.g., KIM and CO92) harbor serines at this residue. Since PhoP plays multiple roles in the adaptation of Y. pestis to stressful host conditions, we tested whether this amino acid substitution affects PhoP activity or the ability of Y. pestis to survive in host environments. Compared to the parental KIM6+ strain carrying the modern allele of phoP (phoP-S215), a derivative carrying the basal allele (phoP-G215) exhibited slightly defective growth under a low-Mg2+ condition and decreased transcription of a PhoP target gene, ugd, as well as an ∼8-fold increase in the susceptibility to the antimicrobial peptide polymyxin B. The phoP-G215 strain showed no apparent defect in flea colonization, although a phoP-null mutant showed decreased flea infectivity in competition experiments. Our results suggest that the amino acid variation at position 215 of PhoP causes subtle changes in the PhoP activity and raise the possibility that the change in this residue have contributed to the evolution of increased virulence in Y. pestisIMPORTANCEY. pestis acquired a single nucleotide polymorphism (SNP) in phoP when the highly human-virulent strains diverged from less virulent basal strains, resulting in an amino acid substitution in the DNA binding domain of the PhoP response regulator. We show that Y. pestis carrying the modern phoP allele has an increased ability to induce the PhoP-regulated ugd gene and resist antimicrobial peptides compared to an isogenic strain carrying the basal allele. Given the important roles PhoP plays in host adaptation, the results raise an intriguing possibility that this amino acid substitution contributed to the evolution of increased virulence in Y. pestis Additionally, we present the first evidence that phoP confers a survival fitness advantage to Y. pestis inside the flea midgut.

Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages.

Yersinia pestis has evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis, Y. pestis prevents phagolysosome maturation and establishes a modified compartment termed the Yersinia-containing vacuole (YCV). Y. pestis actively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requires Y. pestis interactions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required for Y. pestis survival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival of Y. pestis Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated that Y. pestis actively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV by Y. pestis to resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered that Y. pestis disrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence that Y. pestis targets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV.IMPORTANCEYersinia pestis can infect and survive within macrophages. However, the mechanisms that the bacterium use to subvert killing by these phagocytes have not been defined. To provide a better understanding of these mechanisms, we used an RNAi approach to identify host factors required for intracellular Y. pestis survival. This approach revealed that the host endocytic recycling pathway is essential for Y. pestis to avoid clearance by the macrophage. We further demonstrate that Y. pestis remodels the phagosome to resemble a recycling endosome, allowing the bacterium to avoid the normal phagolysosomal maturation pathway. Moreover, we show that infection with Y. pestis disrupts normal recycling in the macrophage and that disruption is required for bacterial replication. These findings provide the first evidence that Y. pestis targets the host endocytic recycling pathway in order to evade killing by macrophages.

Identification of New Virulence Factors and Vaccine Candidates for Yersinia pestis.

Earlier, we reported the identification of new virulence factors/mechanisms of Yersinia pestis using an in vivo signature-tagged mutagenesis (STM) screening approach. From this screen, the role of rbsA, which encodes an ATP-binding protein of ribose transport system, and vasK, an essential component of the type VI secretion system (T6SS), were evaluated in mouse models of plague and confirmed to be important during Y. pestis infection. However, many of the identified genes from the screen remained uncharacterized. In this study, in-frame deletion mutants of ypo0815, ypo2884, ypo3614-3168 (cyoABCDE), and ypo1119-1120, identified from the STM screen, were generated. While ypo0815 codes for a general secretion pathway protein E (GspE) of the T2SS, the ypo2884-encoded protein has homology to the ßγ crystallin superfamily, cyoABCDE codes for the cytochrome o oxidase operon, and the ypo1119-1120 genes are within the Tol-Pal system which has multiple functions. Additionally, as our STM screen identified three T6SS-associated genes, and, based on in silico analysis, six T6SS clusters and multiple homologs of the T6SS effector hemolysin-coregulated protein (Hcp) exist in Y. pestis CO92, we also targeted these T6SS clusters and effectors for generating deletion mutants. These deletion mutant strains exhibited varying levels of attenuation (up to 100%), in bubonic or pneumonic murine infection models. The attenuation could be further augmented by generation of combinatorial deletion mutants, namely ΔlppΔypo0815, ΔlppΔypo2884, ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3. We earlier showed that deletion of the lpp gene, which encodes Braun lipoprotein (Lpp) and activates Toll-like receptor-2, reduced virulence of Y. pestis CO92 in murine models of bubonic and pneumonic plague. The surviving mice infected with ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3 mutant strains were 55-100% protected upon subsequent re-challenge with wild-type CO92 in a pneumonic model. Further, evaluation of the attenuated T6SS mutant strains in vitro revealed significant alterations in phagocytosis, intracellular survival in murine macrophages, and their ability to induce cytotoxic effects on macrophages. The results reported here provide further evidence of the utility of the STM screening approach for the identification of novel virulence factors and to possibly target such genes for the development of novel live-attenuated vaccine candidates for plague.

Induction of Type I Interferon through a Noncanonical Toll-Like Receptor 7 Pathway during Yersinia pestis Infection.

Yersinia pestis causes bubonic, pneumonic, and septicemic plague, diseases that are rapidly lethal to most mammals, including humans. Plague develops as a consequence of bacterial neutralization of the host's innate immune response, which permits uncontrolled growth and causes the systemic hyperactivation of the inflammatory response. We previously found that host type I interferon (IFN) signaling is induced during Y. pestis infection and contributes to neutrophil depletion and disease. In this work, we show that type I IFN expression is derived from the recognition of intracellular Y. pestis by host Toll-like receptor 7 (TLR7). Type I IFN expression proceeded independent of myeloid differentiation factor 88 (MyD88), which is the only known signaling adaptor for TLR7, suggesting that a noncanonical mechanism occurs in Y. pestis-infected macrophages. In the murine plague model, TLR7 was a significant contributor to the expression of serum IFN-ß, whereas MyD88 was not. Furthermore, like the type I IFN response, TLR7 contributed to the lethality of septicemic plague and was associated with the suppression of neutrophilic inflammation. In contrast, TLR7 was important for defense against disease in the lungs. Together, these data demonstrate that an atypical TLR7 signaling pathway contributes to type I IFN expression during Y. pestis infection and suggest that the TLR7-driven type I IFN response plays an important role in determining the outcome of plague.

Characterization of Yersinia pestis Interactions with Human Neutrophils In vitro.

Yersinia pestis is a gram-negative, zoonotic, bacterial pathogen, and the causative agent of plague. The bubonic form of plague occurs subsequent to deposition of bacteria in the skin by the bite of an infected flea. Neutrophils are recruited to the site of infection within the first few hours and interactions between neutrophils and Y. pestis have been demonstrated in vivo. In contrast to macrophages, neutrophils have been considered non-permissive to Y. pestis intracellular survival. Several studies have shown killing of the vast majority of Y. pestis ingested by human neutrophils. However, survival of 10-15% of Y. pestis after phagocytosis by neutrophils is consistently observed. Furthermore, these surviving bacteria eventually replicate within and escape from the neutrophils. We set out to further characterize the interactions between Y. pestis and human neutrophils by (1) determining the effects of known Y. pestis virulence factors on bacterial survival after uptake by neutrophils, (2) examining the mechanisms employed by the neutrophil to kill the majority of intracellular Y. pestis, (3) determining the activation phenotype of Y. pestis-infected neutrophils, and (4) characterizing the Y. pestis-containing phagosome in neutrophils. We infected human neutrophils in vitro with Y. pestis and assayed bacterial survival and uptake. Deletion of the caf1 gene responsible for F1 capsule production resulted in significantly increased uptake of Y. pestis. Surprisingly, while the two-component regulator PhoPQ system is important for survival of Y. pestis within neutrophils, pre-induction of this system prior to infection did not increase bacterial survival. We used an IPTG-inducible mCherry construct to distinguish viable from non-viable intracellular bacteria and determined the association of the Y. pestis-containing phagosome with neutrophil NADPH-oxidase and markers of primary, secondary and tertiary granules. Additionally, we show that inhibition of reactive oxygen species (ROS) production or Src family kinases increased survival of intracellular bacteria indicating that both ROS and granule-phagosome fusion contribute to neutrophil killing of Y. pestis. The data presented here further our understanding of the Y. pestis neutrophil interactions and suggest the existence of still unknown virulence factors involved in Y. pestis survival within neutrophils.

Host Iron Nutritional Immunity Induced by a Live Yersinia pestis Vaccine Strain Is Associated with Immediate Protection against Plague.

Prompt and effective elicitation of protective immunity is highly relevant for cases of rapidly deteriorating fatal diseases, such as plague, which is caused by Yersinia pestis. Here, we assessed the potential of a live vaccine to induce rapid protection against this infection. We demonstrated that the Y. pestis EV76 live vaccine protected mice against an immediate lethal challenge, limiting the multiplication of the virulent pathogen and its dissemination into circulation. Ex vivo analysis of Y. pestis growth in serum derived from EV76-immunized mice revealed that an antibacterial activity was produced rapidly. This activity was mediated by the host heme- and iron-binding proteins hemopexin and transferrin, and it occurred in strong correlation with the kinetics of hemopexin induction in vivo. We suggest a new concept in which a live vaccine is capable of rapidly inducing iron nutritional immunity, thus limiting the propagation of pathogens. This concept could be exploited to design novel therapeutic interventions.

Glutathionylation of Yersinia pestis LcrV and Its Effects on Plague Pathogenesis.

Glutathionylation, the formation of reversible mixed disulfides between glutathione and protein cysteine residues, is a posttranslational modification previously observed for intracellular proteins of bacteria. Here we show that Yersinia pestis LcrV, a secreted protein capping the type III secretion machine, is glutathionylated at Cys273 and that this modification promotes association with host ribosomal protein S3 (RPS3), moderates Y. pestis type III effector transport and killing of macrophages, and enhances bubonic plague pathogenesis in mice and rats. Secreted LcrV was purified and analyzed by mass spectrometry to reveal glutathionylation, a modification that is abolished by the codon substitution Cys273Ala in lcrV Moreover, the lcrVC273A mutation enhanced the survival of animals in models of bubonic plague. Investigating the molecular mechanism responsible for these virulence attributes, we identified macrophage RPS3 as a ligand of LcrV, an association that is perturbed by the Cys273Ala substitution. Furthermore, macrophages infected by the lcrVC273A variant displayed accelerated apoptotic death and diminished proinflammatory cytokine release. Deletion of gshB, which encodes glutathione synthetase of Y. pestis, resulted in undetectable levels of intracellular glutathione, and we used a Y. pestis ΔgshB mutant to characterize the biochemical pathway of LcrV glutathionylation, establishing that LcrV is modified after its transport to the type III needle via disulfide bond formation with extracellular oxidized glutathione.IMPORTANCEYersinia pestis, the causative agent of plague, has killed large segments of the human population; however, the molecular bases for the extraordinary virulence attributes of this pathogen are not well understood. We show here that LcrV, the cap protein of bacterial type III secretion needles, is modified by host glutathione and that this modification contributes to the high virulence of Y. pestis in mouse and rat models for bubonic plague. These data suggest that Y. pestis exploits glutathione in host tissues to activate a virulence strategy, thereby accelerating plague pathogenesis.

Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages.

Non-coding small RNAs (sRNAs) are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.

Immunology of Yersinia pestis Infection.

As a pathogen of plague, Yersinia pestis caused three massive pandemics in history that killed hundreds of millions of people. Yersinia pestis is highly invasive, causing severe septicemia which, if untreated, is usually fatal to its host. To survive in the host and maintain a persistent infection, Yersinia pestis uses several stratagems to evade the innate and the adaptive immune responses. For example, infections with this organism are biphasic, involving an initial "noninflammatory" phase where bacterial replication occurs initially with little inflammation and following by extensive phagocyte influx, inflammatory cytokine production, and considerable tissue destruction, which is called "proinflammatory" phase. In contrast, the host also utilizes its immune system to eliminate the invading bacteria. Neutrophil and macrophage are the first defense against Yersinia pestis invading through phagocytosis and killing. Other innate immune cells also play different roles, such as dendritic cells which help to generate more T helper cells. After several days post infection, the adaptive immune response begins to provide organism-specific protection and has a long-lasting immunological memory. Thus, with the cooperation and collaboration of innate and acquired immunity, the bacterium may be eliminated from the host. The research of Yersinia pestis and host immune systems provides an important topic to understand pathogen-host interaction and consequently develop effective countermeasures.

Yersinia YopJ negatively regulates IRF3-mediated antibacterial response through disruption of STING-mediated cytosolic DNA signaling.

The Yersinia outer protein J (YopJ) plays a pivotal role in evading the host immune response and establishes a persistent infection in host cells after bacterial infection. YopJ is a cysteine protease and can act as a deubiquitinating enzyme that deubiquitinates several targets in multiple signaling pathways. Stimulator of interferon genes (STING) is a critical adapter for the induction of interferon regulatory factor 3 (IRF3) phosphorylation and subsequent production of the cytokines in response to nucleic acids in the cytoplasm. Our studies demonstrate that YopJ targets STING to inhibit IRF3 signaling. Specially, YopJ interacts with STING to block its ER-to-Golgi traffic and remove its K63-linked ubiquitination chains. Deubiquited STING perturbs the formation of STING-TBK1 complex and the activation of IRF3. The 172th cysteine of YopJ mediated STING deubiquitination and IRF3 signaling inhibition. Consequently, mice infected with WT and ΔYopJ/YopJ bacteria induced lower levels of IRF3 and IFN-ß, decreased inflammation and reduced staining of STING as compared to ΔYopJ and ΔYopJ/YopJ C172A strains infection. The data herein reveal a previously unrecognized mechanism by which YopJ modulates innate immune signaling.

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.