Influx of podoplanin-expressing inflammatory macrophages into the genital tract following Chlamydia infection.

Genital Chlamydia trachomatis infection remains a major health issue as it causes severe complications including pelvic inflammatory disease, ectopic pregnancy and infertility in females as a result of infection-associated chronic inflammation. Podoplanin, a transmembrane receptor, has been previously reported on inflammatory macrophages. Thus, strategies that specifically target podoplanin might be able to reduce local inflammation. This study investigated the expression level and function of podoplanin in a C. trachomatis infection model. C57BL/6 mice infected with the mouse pathogen Chlamydia muridarum were examined intermittently from days 1 to 60 using flow cytometry analysis. Percentages of conventional macrophages (CD11b+ CD11c- F4/80+ ) versus inflammatory macrophages (CD11b+ CD11c+ F4/80+ ), and the expression of podoplanin in these cells were investigated. Subsequently, a podoplanin-knockout RAW264.7 cell was used to evaluate the function of podoplanin in C. trachomatis infection. Our findings demonstrated an increased CD11b+ cell volume in the spleen at day 9 after the infection, with augmented podoplanin expression, especially among the inflammatory macrophages. A large number of podoplanin-expressing macrophages were detected in the genital tract of C. muridarum-infected mice. Furthermore, analysis of the C. trachomatis-infected patients demonstrated a higher percentage of podoplanin-expressing monocytes than that in the noninfected controls. Using an in vitro infection in a transwell migration assay, we identified that macrophages deficient in podoplanin displayed defective migratory function toward C. trachomatis-infected HeLa 229 cells. Lastly, using immunoprecipitation-mass spectrometry method, we identified two potential podoplanin interacting proteins, namely, Cofilin 1 and Talin 1 actin-binding proteins. The present study reports a role of podoplanin in directing macrophage migration to the chlamydial infection site. Our results suggest a potential for reducing inflammation in individuals with chronic chlamydial infections by targeting podoplanin.

Chlamydia trachomatis plasmid-encoding Pgp3 protein induces secretion of distinct inflammatory signatures from HeLa cervical epithelial cells.

BACKGROUND: Genital Chlamydia trachomatis infection is the most common bacterial sexual transmitted disease that causes severe complications including pelvic inflammatory disease, ectopic pregnancy, and infertility in females. The Pgp3 protein encoded by C. trachomatis plasmid has been speculated to be an important player in chlamydial pathogenesis. However, the precise function of this protein is unknown and thus remains to be thoroughly investigated. METHODS: In this study, we synthesized Pgp3 protein for in vitro stimulation in the Hela cervical carcinoma cells. RESULTS AND CONCLUSION: We showed that Pgp3 induced prominent expression of host inflammatory cytokine genes including interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha-induced protein 3 (TNFAIP3), and chemokine C-X-C motif ligand 1 (CXCL1), implying a possible role of Pgp3 in modulating the inflammatory reaction in the host.

Gastrointestinal Chlamydia-Induced CD8+ T Cells Promote Chlamydial Pathogenicity in the Female Upper Genital Tract.

Chlamydia is known to both ascend to the upper genital tract and spread to the gastrointestinal tract following intravaginal inoculation. Gastrointestinal Chlamydia was recently reported to promote chlamydial pathogenicity in the genital tract since mice intravaginally inoculated with an attenuated Chlamydia strain, which alone failed to develop pathology in the genital tract, were restored to develop hydrosalpinx by intragastric coinoculation with wild-type Chlamydia. Gastrointestinal Chlamydia promoted hydrosalpinx via an indirect mechanism since Chlamydia in the gut did not directly spread to the genital tract lumen. In the current study, we further investigated the role of CD8+ T cells in the promotion of hydrosalpinx by gastrointestinal Chlamydia. First, we confirmed that intragastric coinoculation with wild-type Chlamydia promoted hydrosalpinx in mice that were inoculated with an attenuated Chlamydia strain in the genital tract 1 week earlier. Second, the promotion of hydrosalpinx by intragastrically coinoculated Chlamydia was blocked by depleting CD8+ T cells. Third, adoptive transfer of gastrointestinal Chlamydia-induced CD8+ T cells was sufficient for promoting hydrosalpinx in mice that were intravaginally inoculated with an attenuated Chlamydia strain. These observations have demonstrated that CD8+ T cells induced by gastrointestinal Chlamydia are both necessary and sufficient for promoting hydrosalpinx in the genital tract. The study has laid a foundation for further revealing the mechanisms by which Chlamydia-induced T lymphocyte responses (as a 2nd hit) promote hydrosalpinx in mice with genital Chlamydia-triggered tubal injury (as a 1st hit), a continuing effort in testing the two-hit hypothesis as a chlamydial pathogenic mechanism.

Adoptive Transfer of Group 3-Like Innate Lymphoid Cells Restores Mouse Colon Resistance to Colonization of a Gamma Interferon-Susceptible Chlamydia muridarum Mutant.

The obligate intracellular bacterium Chlamydia muridarum can colonize the mouse colon for a long period, but a gamma interferon (IFN-γ)-susceptible mutant clone fails to do so. Nevertheless, the mutant's colonization is rescued in mice deficient in interleukin-7 receptor (IL-7R) (lacking both lymphocytes and innate lymphoid cells [ILCs]) or IFN-γ but not in mice lacking recombination-activated gene 1 (Rag1-/- mice) (lacking adaptive immunity lymphocytes), indicating a critical role of ILC-derived IFN-γ in regulating chlamydial colonization. In the current study, we have used an adoptive transfer approach for further characterizing the responsible ILCs. First, intestinal ILCs isolated from Rag1-/- mice were able to rescue IL-7R-deficient mice to restrict the colonization of the IFN-γ-susceptible Chlamydia muridarum mutant. Second, the responsible ILCs were localized to the intestinal lamina propria since ILCs from the lamina propria but not the intraepithelial compartment conferred the restriction. Third, lamina propria ILCs enriched for RORγt expression but not those negative for RORγt rescued the IL-7R-deficient mice to restrict mutant colonization, indicating a critical role of group 3-like ILCs (ILC3s) since RORγt is a signature transcriptional factor of ILC3s. Fourth, a portion of the ILC3s expressed IFN-γ, thus defined as ex-ILC3s, and the transfer of the ex-ILC3s conferred colon resistance to mutant Chlamydia muridarum colonization in IFN-γ-deficient mice. Finally, genetically labeled RORγt-positive (RORγt+) ILCs were able to inhibit mutant colonization. Thus, we have demonstrated that ILC3s are sufficient for regulating chlamydial colonization, laying a foundation for further revealing the mechanisms by which an obligate intracellular bacterium activates colonic ILC3s.

The Cryptic Plasmid Improves Chlamydia Fitness in Different Regions of the Gastrointestinal Tract.

The cryptic plasmid is important for chlamydial colonization in the gastrointestinal tract. We used a combination of intragastric, intrajejunal, and intracolon inoculations to reveal the impact of the plasmid on chlamydial colonization in distinct regions of gastrointestinal tract. Following an intragastric inoculation, the plasmid significantly improved chlamydial colonization. At the tissue level, plasmid-positive Chlamydia produced infectious progenies throughout gastrointestinal tract. However, to our surprise, plasmid-deficient Chlamydia failed to produce infectious progenies in small intestine, although infectious progenies were eventually detected in large intestine, indicating a critical role of the plasmid in chlamydial differentiation into infectious particles in small intestine. The noninfectious status may represent persistent infection, since Chlamydia genomes proliferated in the same tissues. Following an intrajejunal inoculation that bypasses the gastric barrier, plasmid-deficient Chlamydia produced infectious progenies in small intestine but was 530-fold less infectious than plasmid-positive Chlamydia, suggesting that (i) the noninfectious status developed after intragastric inoculation might be induced by a combination of gastric and intestinal effectors and (ii) chlamydial colonization in small intestine was highly dependent on plasmid. Finally, following an intracolon inoculation, the dependence of chlamydial colonization on plasmid increased over time. Thus, we have demonstrated that the plasmid may be able to improve chlamydial fitness in different gut regions via different mechanisms, which has laid a foundation to further reveal the specific mechanisms.

Antigen-Specific CD4+ T Cell-Derived Gamma Interferon Is Both Necessary and Sufficient for Clearing Chlamydia from the Small Intestine but Not the Large Intestine.

The genital tract pathogen Chlamydia trachomatis is frequently detected in the gastrointestinal tract, but the host immunity that regulates chlamydial colonization in the gut remains unclear. In a Chlamydia muridarum-C57 mouse model, chlamydial organisms are cleared from the genital tract in ∼4 weeks, but the genital organisms can spread to the gastrointestinal tract. We found that the gastrointestinal chlamydial organisms were cleared from the small intestine by day 28, paralleling their infection course in the genital tract, but persisted in the large intestine for long periods. Mice deficient in α/ß T cells or CD4+ T cells but not CD8+ T cells showed chlamydial persistence in the small intestine, indicating a critical role for CD4+ T cells in clearing Chlamydia from the small intestine. The CD4+ T cell-dependent clearance is likely mediated by gamma interferon (IFN-γ), since mice deficient in IFN-γ but not interleukin 22 (IL-22) signaling pathways rescued chlamydial colonization in the small intestine. Furthermore, exogenous IFN-γ was sufficient for clearing Chlamydia from the small intestine but not the large intestine. Mice deficient in developing Chlamydia-specific Th1 immunity showed chlamydial persistence in the small intestine. Finally, IFN-γ-producing CD4+ but not CD8+ T cells from immunized donor mice were sufficient for eliminating Chlamydia from the small intestine but not the large intestine of recipient mice. Thus, we have demonstrated a critical role for Th1 immunity in clearing Chlamydia from the small intestine but not the large intestine, indicating that chlamydial colonization in different regions of the gastrointestinal tract is regulated by distinct immune mechanisms.

Combination of Cystatins 9 and C Modulates Serum Biomarkers Associated with Inflammation and Provides Prophylactic as Well as Long-Term Protection against Multidrug-Resistant Klebsiella pneumoniae.

We have identified recombinant human cystatins 9 (rCST9) and C (rCSTC) as a combination immunotherapeutic treatment against multidrug-resistant (MDR) New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae We evaluated the lasting protection of rCST9/rCSTC treatment against MDR NDM-1 K. pneumoniae pneumonia. Results showed that rCST9/rCSTC treatment modulated endogenous serum biomarkers, cystatins 9 and C and amyloid A, associated with poor patient outcomes and provided prophylactic and long-term protection in a murine model of pneumonia.

Platelet enhancement of bacterial growth during room temperature storage: mitigation through refrigeration.

INTRODUCTION: Due to high risk of septic transfusion reactions arising from bacterial contamination, US Food and Drug Administration regulations currently limit platelet storage to 5 days at room temperature (RT). However, blood culturing methods can take up to 7 days to detect bacteria, allowing transfusion of potentially contaminated units. Thus, cold storage (CS) may be a viable means of extending shelf life and improving safety. STUDY DESIGN AND METHODS: Platelets and fresh plasma (FP) were collected by apheresis from healthy donors, aliquoted, and challenged with Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Staphylococcus epidermidis. Aliquots were then stored at either RT or CS. RESULTS: Significant (p < 0.05) bacterial growth was detected at RT for most bacteria as early as Day 1 after collection, with peak growth occurring between Days 3 and 4. Growth remained static during CS. Additionally, platelets appeared to enhance bacterial replication with growth significantly lower (p < 0.05) in FP relative to RT-stored platelets. Lactic acid promoted bacterial growth when added to FP at RT. Bacterial challenge also resulted in significantly increased platelet activation (p < 0.05) and significantly reduced platelet function (p < 0.05) in RT storage relative to uninfected controls by Day 5 after collection. Conversely, CS ablated bacteria growth, limited platelet metabolism, and preserved platelet function throughout the study. CONCLUSION: These data suggest that CS presents an attractive alternative to RT to both extend storage life and reduce the risk of transfusion-related sepsis.

Cystatins 9 and C as a Novel Immunotherapy Treatment That Protects against Multidrug-Resistant New Delhi Metallo-Beta-Lactamase-1-Producing Klebsiella pneumoniae.

Multidrug-resistant (MDR) bacterial pneumonia can induce dysregulated pulmonary and systemic inflammation leading to morbidity and mortality. Antibiotics to treat MDR pathogens do not function to modulate the extent and intensity of inflammation and can have serious side effects. Here we evaluate the efficacy of two human cysteine proteinase inhibitors, cystatin 9 (CST9) and cystatin C (CSTC), as a novel immunotherapeutic treatment to combat MDR New Delhi metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae Our results showed that mice infected intranasally (i.n.) with a 90% lethal dose (LD90) challenge of NDM-1 K. pneumoniae and then treated with the combination of human recombinant CST9 (rCST9) and rCSTC (rCSTs; 50 pg of each i.n. at 1 h postinfection [p.i.] and/or 500 pg of each intraperitoneally [i.p.] at 3 days p.i.) had significantly improved survival compared to that of infected mice alone or infected mice treated with individual rCSTs (P < 0.05). Results showed that both of our optimal rCST treatment regimens modulated pulmonary and systemic proinflammatory cytokine secretion in the serum, lungs, liver, and spleen in infected mice (P < 0.05). Treatment also significantly decreased the bacterial burden (P < 0.05) while preserving lung integrity, with reduced inflammatory cell accumulation compared to that in infected mice. Further, rCST treatment regimens reduced lipid peroxidation and cell apoptosis in the lungs of infected mice. Additionally, in vitro studies showed that rCSTs (50 or 500 pg of each) directly decreased the viability of NDM-1 K. pneumoniae In conclusion, the data showed that rCST9/rCSTC worked synergistically to modulate host inflammation against MDR NDM-1 K. pneumoniae pneumonia, which significantly improved survival. Therefore, rCST9/rCSTC is a promising therapeutic candidate for the treatment of bacterial pneumonia.

Chlamydial protease-like activity factor mediated protection against C. trachomatis in guinea pigs.

We have comprehensively demonstrated using the mouse model that intranasal immunization with recombinant chlamydial protease-like activity factor (rCPAF) leads to a significant reduction in bacterial burden, genital tract pathology and preserves fertility following intravaginal genital chlamydial challenge. In the present report, we evaluated the protective efficacy of rCPAF immunization in guinea pigs, a second animal model for genital chlamydial infection. Using a vaccination strategy similar to the mouse model, we intranasally immunized female guinea pigs with rCPAF plus CpG deoxynucleotides (CpG; as an adjuvant), and challenged intravaginally with C. trachomatis serovar D (CT-D). Immunization with rCPAF/CpG significantly reduced vaginal CT-D shedding and induced resolution of infection by day 24, compared with day 33 in CpG alone treated and challenged animals. Immunization induced robust anti-rCPAF serum IgG 2 weeks following the last immunization, and was sustained at a high-level 4 weeks post challenge. Upregulation of antigen-specific IFN-γ gene expression was observed in rCPAF/CpG-vaccinated splenocytes. Importantly, a significant reduction in inflammation in the genital tissue in rCPAF/CpG-immunized guinea pigs compared with CpG-immunized animals was observed. Taken together, this study provides evidence of the protective efficacy of rCPAF as a vaccine candidate in a second animal model of genital chlamydial infection.

Temporal proteomic profiling of Chlamydia trachomatis-infected HeLa-229 human cervical epithelial cells.

Chlamydia trachomatis is the leading causative agent of bacterial sexually transmitted infections worldwide which can lead to female pelvic inflammatory disease and infertility. A greater understanding of host response during chlamydial infection is essential to design intervention technique to reduce the increasing incidence rate of genital chlamydial infection. In this study, we investigated proteome changes in epithelial cells during C. trachomatis infection by using an isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique coupled with a liquid chromatography-tandem mass spectrometry (LC-MS(3) ) analysis. C. trachomatis (serovar D, MOI 1)-infected HeLa-229 human cervical carcinoma epithelial cells (at 2, 4 and 8 h) showed profound modifications of proteome profile which involved 606 host proteins. MGST1, SUGP2 and ATXN10 were among the top in the list of the differentially upregulated protein. Through pathway analysis, we suggested the involvement of eukaryotic initiation factor 2 (eIF2) and mammalian target of rapamycin (mTOR) in host cells upon C. trachomatis infection. Network analysis underscored the participation of DNA repair mechanism during C. trachomatis infection. In summary, intense modifications of proteome profile in C. trachomatis-infected HeLa-229 cells indicate complex host-pathogen interactions at early phase of chlamydial infection.

Live attenuated Francisella novicida vaccine protects against Francisella tularensis pulmonary challenge in rats and non-human primates.

Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt), leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD) protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP). The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform.

Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection among women who visited obstetrics and gynecology clinics in Malaysia.

BACKGROUND: The 7.5 kb cryptic plasmid of Chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. The aim of this study was to investigate the prevalence and potential involvement of the C. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome (PCOS) among genital C. trachomatis-infected patients. RESULTS: A total of 180 female patients of child bearing age (mean 30.9 years old, IQR:27-35) with gynecological complications and subfertility issues, who visited Obstetrics and Gynecology clinics in Kuala Lumpur, Malaysia were recruited for the study. Prevalence of genital chlamydial infection among these patients was alarmingly high at 51.1% (92/180). Of the 92 chlamydia-infected patients, 93.5% (86/92) were infected with plasmid-bearing (+) C. trachomatis while the remaining 6.5% (6/92) were caused by the plasmid-free (-) variant. Our data showed that genital C. trachomatis infection was associated with infertility issues, inflammation in the reproductive tract (mucopurulent cervicitis or endometriosis), irregular menstrual cycles and polycystic ovarian syndrome (PCOS). However, no statistical significance was detected among patients with plasmid (+) versus plasmid (-) C. trachomatis infection. Interestingly, plasmid (+) C. trachomatis was detected in all patients with PCOS, and the plasmid copy numbers were significantly higher among PCOS patients, relative to non-PCOS patients. CONCLUSION: Our findings show a high incidence of C. trachomatis infection among women with infertility or gynecological problems in Malaysia. However, due to the low number of plasmid (-) C. trachomatis cases, a significant role of the plasmid in causing virulence in human requires further investigation of a larger cohort.

Murine MicroRNA-214 regulates intracellular adhesion molecule (ICAM1) gene expression in genital Chlamydia muridarum infection.

The hallmark of chlamydial infection is the development of upper genital pathology in the form of hydrosalpinx and oviduct and/or tubal dilatation. Although molecular events leading to genital tissue presentation and cellular architectural remodelling are unclear, early-stage host immune responses are believed to contribute to these long-term sequelae. Recently, we reported the contribution of selected infection-associated microRNAs (miRs) in the generation of host immunity at early-stage infection (day 6 after intravaginal Chlamydia muridarum challenge in C57BL/6 mice). In this report, we describe the contribution of an infection-associated microRNA, i.e. miR-214, to host immunity. Chlamydia muridarum infection in the C57BL/6 mouse genital tract significantly down-regulated miR-214 while up-regulating intracellular adhesion molecule 1 (ICAM1) gene expression. These in vivo observations were confirmed by establishing direct regulation of ICAM-1 by miR-214 in ex vivo genital cell cultures in the presence of miR-214 mimic and inhibitor. Because, ICAM-1 contributes to recruitment of neutrophils following infection, we also demonstrated that alteration of ICAM1 by miR-214 in interleukin-17A-deficient (IL-17A(-/-) ) mice correlated with reduction of neutrophils infiltrating genital tissue at day 6 after challenge. Additionally, these early-stage events resulted in significantly decreased genital pathology in IL-17A(-/-) mice compared with C57BL/6 mice. This report provides evidence for early-stage regulation of ICAM1 by microRNAs, resulting in reduction of genital pathology associated with chlamydial infection.

Chlamydia pneumoniae promotes dysfunction of pancreatic beta cells.

The human pathogen Chlamydia pneumoniae has been implicated in chronic inflammatory diseases including type 2 diabetes. Therefore, we designed a study to evaluate pancreatic beta cells and mast cells during chlamydial infection. Our study revealed that C. pneumoniae infected mast cells significantly (p<0.005) decreased beta cell ATP and insulin production, in contrast to uninfected mast cells co-cultured with beta cells. Infected mast cells exhibited pyknotic nuclei and active caspase-3 and caspase-1 expression. Additionally, ex vivo analyses of tissues collected from C. pneumoniae infected mice showed increased interleukin-1ß production in splenocytes and pancreatic tissues as was observed with in vitro mast cell-beta cell co-cultures during C. pneumoniae infection. Notably, infected mast cells promoted beta cell destruction. Our findings reveal the negative effect of C. pneumoniae on mast cells, and the consequential impact on pancreatic beta cell function and viability.

Severe Acinetobacter baumannii sepsis is associated with elevation of pentraxin 3.

Multidrug-resistant Acinetobacter baumannii is among the most prevalent bacterial pathogens associated with trauma-related wound and bloodstream infections. Although septic shock and disseminated intravascular coagulation have been reported following fulminant A. baumannii sepsis, little is known about the protective host immune response to this pathogen. In this study, we examined the role of PTX3, a soluble pattern recognition receptor with reported antimicrobial properties and stored within neutrophil granules. PTX3 production by murine J774a.1 macrophages was assessed following challenge with A. baumannii strains ATCC 19606 and clinical isolates (CI) 77, 78, 79, 80, and 86. Interestingly, only CI strains 79, 80, and 86 induced PTX3 synthesis in murine J774a.1 macrophages, with greatest production observed following CI 79 and 86 challenge. Subsequently, C57BL/6 mice were challenged intraperitoneally with CI 77 and 79 to assess the role of PTX3 in vivo. A. baumannii strain CI 79 exhibited significantly (P < 0.0005) increased mortality, with an approximate 50% lethal dose (LD50) of 10(5) CFU, while an equivalent dose of CI 77 exhibited no mortality. Plasma leukocyte chemokines (KC, MCP-1, and RANTES) and myeloperoxidase activity were also significantly elevated following challenge with CI 79, indicating neutrophil recruitment/activation associated with significant elevation in serum PTX3 levels. Furthermore, 10-fold-greater PTX3 levels were observed in mouse serum 12 h postchallenge, comparing CI 79 to CI 77 (1,561 ng/ml versus 145 ng/ml), with concomitant severe pathology (liver and spleen) and coagulopathy. Together, these results suggest that elevation of PTX3 is associated with fulminant disease during A. baumannii sepsis.

Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis.

TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the live vaccine strain were used to investigate the contribution of mast cell/TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHC class II and lysosomal-associated membrane protein 2. Infected TLR2(-/-) mast cells, in contrast to wild-type and TLR4(-/-) cells, lacked detectable IL-4 and displayed increased cell death with a 2-3 log increase of F. tularensis replication, but could be rescued with rIL-4 treatment. Importantly, MHC class II and lysosomal-associated membrane protein 2 localization with labeled F. tularensis in the lungs was greater in wild-type than in TLR2(-/-) mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses.

Development and Evaluation of an Immunoinformatics-Based Multi-Peptide Vaccine against Acinetobacter baumannii Infection.

Multi-drug-resistant (MDR) Acinetobacter baumannii is an opportunistic pathogen associated with hospital-acquired infections. Due to its environmental persistence, virulence, and limited treatment options, this organism causes both increased patient mortality and incurred healthcare costs. Thus, prophylactic vaccination could be ideal for intervention against MDR Acinetobacter infection in susceptible populations. In this study, we employed immunoinformatics to identify peptides containing both putative B- and T-cell epitopes from proteins associated with A. baumannii pathogenesis. A novel Acinetobacter Multi-Epitope Vaccine (AMEV2) was constructed using an A. baumannii thioredoxin A (TrxA) leading protein sequence followed by five identified peptide antigens. Antisera from A. baumannii infected mice demonstrated reactivity to rAMEV2, and subcutaneous immunization of mice with rAMEV2 produced high antibody titer against the construct as well as peptide components. Immunization results in increased frequency of IL-4-secreting splenocytes indicative of a Th2 response. AMEV2-immunized mice were protected against intranasal challenge with a hypervirulent strain of A. baumannii and demonstrated reduced bacterial burden at 48 h. In contrast, all mock vaccinated mice succumbed to infection within 3 days. Results presented here provide insight into the effectiveness of immunoinformatic-based vaccine design and its potential as an effective strategy to combat the rise of MDR pathogens.

Francisella DnaK inhibits tissue-nonspecific alkaline phosphatase.

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

Immunomodulatory and antibacterial effects of cystatin 9 against Francisella tularensis.

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.