Vesiculation from Pseudomonas aeruginosa under SOS

Bacterial infections can be aggravated by antibiotic treatment that induces SOS response and vesiculation. This leads to a hypothesis concerning association of SOS with vesiculation. To test it, we conducted multiple analyses of outer membrane vesicles (OMVs) produced from the Pseudomonas aeruginosa wild type in which SOS is induced by ciprofloxacin and from the LexA noncleavable (lexAN) strain in which SOS is repressed. The levels of OMV proteins, lipids, and cytotoxicity increased for both the treated strains, demonstrating vesiculation stimulation by the antibiotic treatment. However, the further increase was suppressed in the lexAN strains, suggesting the SOS involvement. Obviously, the stimulated vesiculation is attributed by both SOS-related and unrelated factors. OMV subproteomic analysis was performed to examine these factors, which reflected the OMV-mediated cytotoxicity and the physiology of the vesiculating cells under treatment and SOS. Thus, SOS plays a role in the vesiculation stimulation that contributes to cytotoxicity

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

Alpha-1 Antitrypsin is Markedly Decreased Following Pulmonary F. tularensis Challenge

Neutrophils form the first line of defense during infection and are indispensable in this function. The neutrophil elastase is a key effector molecule of the innate immune system with potent antimicrobial activity against Gram-negative bacteria, spirochaetes, and fungi. However, the release of neutrophil elastase during bacterial infection must be checked otherwise its release in the extracellular milieu will result in damage to surrounding tissues. Alpha-1 antitrypsin is a small glycoprotein clade A serpine serine protease inhibitor and has been shown to increase in humans following bacterial and viral infection. Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of tularemia. Type A strains are the most virulent with an infectious dose as low as 10 colony forming units and a mortality rate of 30–60% among untreated cases of pneumonic tularemia. We report here significant reduction of this major inhibitor of the neutrophil elastase in plasma of F. tularensis LVS and F. tularensis (type A) SCHU S4 infected animals following pulmonary challenge. Associated with an imbalance of protease–antiprotease function at the alveolar level in lungs of infected animals, increased elastase activity was observed in lung lavage fluids accompanied by decrease lung function, i.e., loss of lung elastance with concomitant increase of pulmonary hysteresivity. Consistent with a competent acute phase response following F. tularensis LVS and F. tularensis (type A) SCHU S4 pulmonary challenge and proposed up-regulation of plasma haptoglobin during the course of the acute phase reaction, haptoglobin was observed significantly increased. These data suggest that unchecked neutrophil serine protease activity may arise from F. tularensis targeted reduction of plasma α(1)-antitrysin promoting lung tissue damage facilitating increased dissemination of this bacterium in infected animals

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

Hydrodynamic Regulation of Monocyte Inflammatory Response to an Intracellular Pathogen

Systemic bacterial infections elicit inflammatory response that promotes acute or chronic complications such as sepsis, arthritis or atherosclerosis. Of interest, cells in circulation experience hydrodynamic shear forces, which have been shown to be a potent regulator of cellular function in the vasculature and play an important role in maintaining tissue homeostasis. In this study, we have examined the effect of shear forces due to blood flow in modulating the inflammatory response of cells to infection. Using an in vitro model, we analyzed the effects of physiological levels of shear stress on the inflammatory response of monocytes infected with chlamydia, an intracellular pathogen which causes bronchitis and is implicated in the development of atherosclerosis. We found that chlamydial infection alters the morphology of monocytes and trigger the release of pro-inflammatory cytokines TNF-α, IL-8, IL-1β and IL-6. We also found that the exposure of chlamydia-infected monocytes to short durations of arterial shear stress significantly enhances the secretion of cytokines in a time-dependent manner and the expression of surface adhesion molecule ICAM-1. As a functional consequence, infection and shear stress increased monocyte adhesion to endothelial cells under flow and in the activation and aggregation of platelets. Overall, our study demonstrates that shear stress enhances the inflammatory response of monocytes to infection, suggesting that mechanical forces may contribute to disease pathophysiology. These results provide a novel perspective on our understanding of systemic infection and inflammation

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

Production of a Proteolytically Active Protein, Chlamydial Protease/Proteasome-Like Activity Factor, by Five Different Chlamydia Species

We have previously identified a chlamydial protein, chlamydial protease/proteasome-like activity factor (CPAF), for degrading host transcription factors in cells infected with the human chlamydial species Chlamydia trachomatis or Chlamydia pneumoniae. We now report that functional CPAF was also produced during infection with the species Chlamydia muridarum, Chlamydia psittaci, and Chlamydia caviae, which primarily infect nonhuman hosts