Immunization with Dendritic Cells Pulsed ex vivo with Recombinant Chlamydial Protease-Like Activity Factor Induces Protective Immunity Against Genital Chlamydia muridarum Challenge

We have shown that immunization with soluble recombinant chlamydial protease-like activity factor (rCPAF) and a T helper 1 type adjuvant can induce significantly enhanced bacterial clearance and protection against Chlamydia-induced pathological sequelae in the genital tract. In this study, we investigated the use of bone marrow derived dendritic cells (BMDCs) pulsed ex vivo with rCPAF + CpG in an adoptive subcutaneous immunization for the ability to induce protective immunity against genital chlamydial infection. We found that BMDCs pulsed with rCPAF + CpG efficiently up-regulated the expression of activation markers CD86, CD80, CD40, and major histocompatibility complex class II (MHC II), and secreted interleukin-12, but not IL-10 and IL-4. Mice adoptively immunized with rCPAF + CpG-pulsed BMDCs or UV-EB + CpG-pulsed BMDCs produced elevated levels of antigen-specific IFN-γ and enhanced IgG1 and IgG2a antibodies. Moreover, mice immunized with rCPAF + CpG-pulsed BMDCs or UV-EB + CpG-pulsed BMDCs exhibited significantly reduced genital Chlamydia shedding, accelerated resolution of infection, and reduced oviduct pathology when compared to infected mock-immunized animals. These results suggest that adoptive subcutaneous immunization with ex vivo rCPAF-pulsed BMDCs is an effective approach, comparable to that induced by UV-EB–BMDCs, for inducing robust anti-Chlamydia immunity

Mannose-Containing Oligosaccharides of Non-Specific Human Secretory Immunoglobulin A Mediate Inhibition of Vibrio cholerae Biofilm Formation

The role of antigen-specific secretory IgA (SIgA) has been studied extensively, whereas there is a limited body of evidence regarding the contribution of non-specific SIgA to innate immune defenses against invading pathogens. In this study, we evaluated the effects of non-specific SIgA against infection with Vibrio cholerae O139 strain MO10 and biofilm formation. Seven day old infant mice deficient in IgA (IgA-/- mice) displayed significantly greater intestinal MO10 burden at 24 hr post-challenge when compared to IgA+/+ pups. Importantly, cross-fostering of IgA-/- pups with IgA+/+ nursing dams reversed the greater susceptibility to MO10 infection, suggesting a role for non-specific SIgA in protection against the infection. Since biofilm formation is associated with virulence of MO10, we further examined the role of human non-specific SIgA on this virulence phenotype of the pathogen. Human non-specific SIgA, in a dose-dependent fashion, significantly reduced the biofilm formation by MO10 without affecting the viability of the bacterium. Such an inhibitory effect was not induced by human serum IgA, IgG, or IgM, suggesting a role for the oligosaccharide-rich secretory component (SC) of SIgA. This was supported by the demonstration that SIgA treated with endoglycosidase H, to cleave the high-mannose containing terminal chitobiose residues, did not induce a reduction in biofilm formation by MO10. Furthermore, the addition of free mannose per se, across a wide dose range, induced significant reduction in MO10 biofilm formation. Collectively, these results suggest that mannose containing oligosacchardies within human non-specific secretory IgA can alter important virulence phenotypes of Vibrio cholerae such as biofilm formation, without affecting viability of the microorganism. Such effects may contribute significantly to innate immune defenses against invading pathogens in vivo in the gastrointestinal tract

Microbial Co-Infection Alters Macrophage Polarization, Phagosomal Escape, and Microbial Killing

Macrophages are important innate immune cells that respond to microbial insults. In response to multi-bacterial infection, the macrophage activation state may change upon exposure to nascent mediators, which results in different bacterial killing mechanism(s). In this study, we utilized two respiratory bacterial pathogens, Mycobacterium bovis (Bacillus Calmette Guẻrin, BCG) and Francisella tularensis live vaccine strain (LVS) with different phagocyte evasion mechanisms, as model microbes to assess the influence of initial bacterial infection on the macrophage response to secondary infection. Non-activated (M0) macrophages or activated M2-polarized cells (J774 cells transfected with the mouse IL-4 gene) were first infected with BCG for 24–48 h, subsequently challenged with LVS, and the results of inhibition of LVS replication in the macrophages was assessed. BCG infection in M0 macrophages activated TLR2-MyD88 and Mincle-CARD9 signaling pathways, stimulating nitric oxide (NO) production and enhanced killing of LVS. BCG infection had little effect on LVS escape from phagosomes into the cytosol in M0 macrophages. In contrast, M2-polarized macrophages exhibited enhanced endosomal acidification, as well as inhibiting LVS replication. Pre-infection with BCG did not induce NO production and thus did not further reduce LVS replication. This study provides a model for studies of the complexity of macrophage activation in response to multi-bacterial infection

The Fischer 344 Rat Reflects Human Susceptibility to Francisella Pulmonary Challenge and Provides a New Platform for Virulence and Protection Studies

Background: The pathogenesis of Francisella tularensis, the causative agent of tularemia, has been primarily characterized in mice. However, the high degree of sensitivity of mice to bacterial challenge, especially with the human virulent strains of F. tularensis, limits this animal model for screening of defined attenuated vaccine candidates for protection studies. Methods and Findings: We analyzed the susceptibility of the Fischer 344 rat to pulmonary (intratracheal) challenge with three different subspecies (subsp) of F. tularensis that reflect different levels of virulence in humans, and characterized the bacterial replication profile in rat bone marrow-derived macrophages (BMDM). In contrast to the mouse, Fischer 344 rats exhibit a broader range of sensitivity to pulmonary challenge with the human virulent subsp. tularensis and holarctica. Unlike mice, Fischer rats exhibited a high degree of resistance to pulmonary challenge with LVS (an attenuated derivative o

Evasion of IFN-γ Signaling by Francisella novicida Is Dependent upon Francisella Outer Membrane Protein C

Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918.The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components

Vaginal Chlamydial Clearance Following Primary or Secondary Infection in Mice Occurs Independently of TNF-α

The role of TNF-α in chlamydial clearance is uncertain. Antibody-mediated depletion of TNF-α in mice and guinea pigs has been shown not to significantly affect chlamydial clearance, whereas production of TNF-α in addition to IFN-γ from T cells has been shown to correlate with enhanced clearance. The aim of our study is to evaluate the mechanistic role of TNF-α in clearance of primary and secondary chlamydial infection from the genital tract (GT) using C57BL/6 TNF-α deficient (TNF-α-/-) and wild type (WT) mice. Chlamydial shedding from the lower GT was evaluated following primary and secondary intravaginal challenge. Also, antibody and antigen specific cytokine responses were analyzed from the infected GT and spleens, and oviduct pathology determined to analyze the role of TNF-α in upper GT pathological sequelae. MHC II-/- mice, known to display muted adaptive immune responses and failure to resolve genital chlamydial infections, were used as a negative control. Following both primary and secondary genital chlamydial infection, TNF-α-/- mice exhibited elevated granzyme B production, but similar IFN-γ and antibody responses. Importantly, absence of TNF-α did not significantly alter the resolution of infection. However, TNF-α-/- mice displayed significantly reduced upper GT pathology compared to WT mice. This study demonstrates mechanistically that optimal chlamydial clearance following primary and secondary chlamydial genital infection can occur in the complete absence of TNF-α, and considered with the reduction of upper GT pathology in TNF-α-/- mice, suggests that targeted induction of anti-chlamydial TNF-α responses by vaccination may be unnecessary, and moreover could be potentially pathogenic

Thioredoxin-A is a virulence factor and mediator of the type IV pilus system in Acinetobacter baumannii.

The Gram-negative pathogen, Acinetobacter baumannii has emerged as a global nosocomial health threat affecting the majority of hospitals in the U.S. and abroad. The redox protein thioredoxin has been shown to play several roles in modulation of cellular functions affecting various virulence factors in Gram-negative pathogens. This study aims to explore the role of thioredoxin-A protein (TrxA) in A. baumannii virulence. We determined that deletion of the TrxA gene did not significantly affect resistance to environmental stressors such as temperature, salt, and pH. However, TrxA was critical for survival in the presence of elevated levels of hydrogen peroxide. Lack of TrxA was associated with decreased expression of type IV pili related genes and an inability to undergo normal twitching motility. Interestingly, the TrxA-null mutant was able to form biofilms better than the wildtype (WT) and was observed to be significantly less virulent than the WT in a pulmonary infection model. These results are supportive of thioredoxin playing a key role in A. baumannii virulence