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

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

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

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

[[alternative]]Involvement of New Synthesis of Enzyme Molecules in the Induction

[[volume]]24

A Recombinant β-1,3-Glucanosyltransferase Homolog of Coccidioides posadasii Protects Mice against Coccidioidomycosis

Coccidioides posadasii is a fungal respiratory pathogen which is responsible for recurrent epidemics of San Joaquin Valley fever (coccidioidomycosis) in desert regions of the southwestern United States. Numerous studies have revealed that the cell wall of the parasitic phase of the fungus is a reservoir of immunoreactive macromolecules and a potential source of a vaccine against this mycosis. A 495-bp fragment of a C. posadasii gene which encodes a putative wall-associated, glycosylphosphatidylinositol (GPI)-anchored β-1,3-glucanosyltransferase was identified by computational analysis of the partially sequenced genome of this pathogen. The translated, full-length gene (GEL1) showed high sequence homology to a reported β-1,3-glucanosyltransferase of Aspergillus fumigatus (70% identity, 90% similarity) and was selected for further study. The GEL1 mRNA of C. posadasii was detected at the highest level during the endosporulation stage of the parasitic cycle, and the mature protein was immunolocalized to the surface of endospores. BALB/c or C57BL/6 mice were immunized subcutaneously with the bacterium-expressed recombinant protein (rGel1p) to evaluate its protective efficacy against a lethal challenge of C. posadasii by either the intraperitoneal or intranasal route. In both cases, rGel1p-immune mice infected with the pathogen showed a significant reduction in fungal burden and increased survival compared to nonimmune mice. The recombinant β-1,3-glucanosyltransferase is a valuable addition to an arsenal of immunoreactive proteins which could be incorporated into a human vaccine against coccidioidomycosis

A Parasitic Phase-Specific Adhesin of Coccidioides immitis Contributes to the Virulence of This Respiratory Fungal Pathogen

We report the isolation of a Coccidioides immitis gene (SOWgp) which encodes an immunodominant, spherule outer wall glycoprotein that is presented as a component of a parasitic phase-specific, membranous layer at the cell surface. The open reading frame of the gene from C. immitis isolate C735 translates a 422-amino-acid (aa) polypeptide that contains 6 copies of a 41- to 47-residue tandem repeat enriched in proline (20.4 mol%) and aspartate (19.7%). Two additional isolates of C. immitis produce SOWgps of different molecular sizes (328 and 375 aa) and show a corresponding difference in the number of tandem repeats (four and five, respectively). The accurate molecular sizes of these proline-rich antigens, as determined by surface-enhanced laser desorption/ionization mass spectrometry, are comparable to the predicted sizes from the translated protein sequences rather than the estimated sizes based on gel-electrophoretic separation. The results of Northern hybridization confirmed that SOWgp expression is parasitic phase specific, and immunoblot studies showed that elevated levels of production of this antigen occurred during early spherule development. The recombinant polypeptide (rSOWp) was shown to bind to mammalian extracellular matrix (ECM) proteins in an in vitro assay (laminin > fibronectin > collagen type IV), suggesting that the parasitic cell surface antigen may function as an adhesin. Deletion of the SOWgp gene by using a targeted gene replacement strategy resulted in partial loss of the ability of intact spherules to bind to ECM proteins and a significant reduction in virulence of the mutant strain. The wild-type gene was restored in the mutant by homologous recombination, and the revertant strain was shown to be as virulent as the parental isolate in our murine model of coccidioidomycosis. The parasitic cell surface glycoprotein encoded by the SOWgp gene appears to function as an adhesin and contributes to the virulence of C. immitis

Repurposing Auranofin, Ebselen, and PX-12 as Antimicrobial Agents Targeting the Thioredoxin System

As microbial resistance to drugs continues to rise at an alarming rate, finding new ways to combat pathogens is an issue of utmost importance. Development of novel and specific antimicrobial drugs is a time-consuming and expensive process. However, the re-purposing of previously tested and/or approved drugs could be a feasible way to circumvent this long and costly process. In this review, we evaluate the U.S. Food and Drug Administration tested drugs auranofin, ebselen, and PX-12 as antimicrobial agents targeting the thioredoxin system. These drugs have been shown to act on bacterial, fungal, protozoan, and helminth pathogens without significant toxicity to the host. We propose that the thioredoxin system could serve as a useful therapeutic target with broad spectrum antimicrobial activity