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Mice lacking expression of the homologous phosphatases Sts-1 and Sts-2 (Sts−/− mice) are resistant to disseminated candidiasis caused by the fungal pathogen Candida albicans. To better understand the immunological mechanisms underlying the enhanced resistance of Sts−/− mice, we examined the kinetics of fungal clearance at early time points. In contrast to the rapid C. albicans growth seen in normal kidneys during the first 24 h postinfection, we observed a reduction in kidney fungal CFU within Sts−/− mice beginning at 12 to 18 h postinfection. This corresponds to the time period when large numbers of innate leukocytes enter the renal environment to counter the infection. Because phagocytes of the innate immune system are important for host protection against pathogenic fungi, we evaluated responses of bone marrow leukocytes. Relative to wild-type cells, Sts−/− marrow monocytes and bone marrow-derived dendritic cells (BMDCs) displayed a heightened ability to inhibit C. albicans growth ex vivo. This correlated with significantly enhanced production of reactive oxygen species (ROS) by Sts−/− BMDCs downstream of Dectin-1, a C-type lectin receptor that plays a critical role in stimulating host responses to fungi. We observed no visible differences in the responses of other antifungal effector pathways, including cytokine production and inflammasome activation, despite enhanced activation of the Syk tyrosine kinase downstream of Dectin-1 in Sts−/− cells. Our results highlight a novel mechanism regulating the immune response to fungal infections. Further understanding of this regulatory pathway could aid the development of therapeutic approaches to enhance protection against invasive candidiasis., IMPORTANCE Systemic candidiasis caused by fungal Candida species is becoming an increasingly serious medical problem for which current treatment is inadequate. Recently, the Sts phosphatases were established as key regulators of the host antifungal immune response. In particular, genetic inactivation of Sts significantly enhanced survival of mice infected intravenously with Candida albicans. The Sts−/− in vivo resistance phenotype is associated with reduced fungal burden and an absence of inflammatory lesions. To understand the underlying mechanisms, we studied phagocyte responses. Here, we demonstrate that Sts−/− phagocytes have heightened responsiveness to C. albicans challenge relative to wild-type cells. Our data indicate the Sts proteins negatively regulate phagocyte activation via regulating selective elements of the Dectin-1–Syk tyrosine kinase signaling axis. These results suggest that phagocytes lacking Sts respond to fungal challenge more effectively and that this enhanced responsiveness partially underlies the profound resistance of Sts−/− mice to systemic fungal challenge.

Candida albicans is a common human fungal pathogen capable of causing serious systemic infections that can progress to become lethal. Current therapeutic approaches have limited effectiveness, especially once a systemic infection is established, in part due to the lack of an effective immune response. Boosting the immune response to C. albicans has been the goal of immunotherapy, but it has to be done selectively to prevent deleterious hyperinflammation (sepsis). Although an efficient inflammatory response is necessary to fight infection, the typical response to C. albicans results in collateral damage to tissues thereby exacerbating the pathological effects of infection. For this reason, identifying specific ways of modulating the immune system holds promise for development of new improved therapeutic approaches. This review will focus on recent studies that provide insight using mutant strains of mice that are more resistant to bloodstream infection by C. albicans. These mice are deficient in signal transduction proteins including the Jnk1 MAP kinase, the Cbl-b E3 ubiquitin ligase, or the Sts phosphatases. Interestingly, the mutant mice display a different response to C. albicans that results in faster clearance of infection without hyper-inflammation and collateral damage. A common underlying theme between the resistant mouse strains is loss of negative regulatory proteins that are known to restrain activation of cell surface receptor-initiated signaling cascades. Understanding the cellular and molecular mechanisms that promote resistance to C. albicans in mice will help to identify new approaches for improving antifungal therapy.

N-acetylglucosamine (GlcNAc) has long been known to play important roles in cell surface structure. Recent studies are now revealing new functions for GlcNAc in cell signaling. Exposure to GlcNAc regulates virulence functions in the human fungal pathogen Candida albicans and in pathogenic bacteria. These signaling pathways sense exogenous GlcNAc and are distinct from the O-GlcNAc signaling pathways in mammalian cells in which increased levels of intracellular GlcNAc synthesis leads to post-translational modification of proteins by attachment of O-GlcNAc. The novel roles of GlcNAc in cell signaling will be the subject of this mini-review.

Contagious Bovine Pleuropneumonia caused by Mycoplasma mycoides subsp. mycoides small colony type is a respiratory disease of considerable economic importance in sub-Saharan Africa; control of the disease in Africa is hampered by diagnostic tests which are suited for herd-level but not for individual animal diagnostics. In the work presented we identified 22 potential immunogenic antigens of the Kenyan outbreak strain B237 by using phage display technology. We determined the relative strength of immunogenicity, the discriminatory capacity between bovine positive and negative sera, and the cross-reactivity with rabbit hyperimmune sera directed against 15 different mycoplasmal species. The three best-performing antigens, a conserved hypothetical protein (MSC_0636), a glycosyl transferase (MSC_0108), and an acyl carrier protein phosphodiesterase (MSC_0029) were considered candidate diagnostic proteins. They were expressed as GST-fusion proteins in Escherichia coli, purified, and used in an ELISA as solid phase antigens. The diagnostic potential of the recombinant antigens was tested using the sera of ten experimentally infected animals and six control animals. This prototype test resulted in 100% diagnostic sensitivity and specificity. In comparison, the complement fixation test and the competitive ELISA performed with a diagnostic sensitivity of 70% and 60%, respectively.

A balanced immune response to infection is essential to prevent the pathology and tissue damage that can occur from an unregulated or hyperactive host defense. Interferons (IFNs) are critical mediators of the innate defense to infection, and in this study we evaluated the contribution of a specific gene coding for IFIT2 induced by type I IFNs in a murine model of disseminated Candida albicans. Invasive candidiasis is a frequent challenge during immunosuppression or surgical medical interventions, and C. albicans is a common culprit that leads to high rates of mortality. When IFIT2 knockout mice were infected systemically with C. albicans, they were found to have improved survival and reduced fungal burden compared to wild-type mice. One of the mechanisms by which IFIT2 increases the pathological effects of invasive C. albicans appears to be suppression of NADPH oxidase activation. Loss of IFIT2 increases production of reactive oxygen species by leukocytes, and we demonstrate that IFIT2 is a binding partner of a critical regulatory subunit of NADPH oxidase, p67phox. Since the administration of IFN has been used therapeutically to combat viral infections, cancer, and multiple sclerosis, we evaluated administration of IFN-β to mice prior to C. albicans infection. IFN-β treatment promoted pathology and death from C. albicans infection. We provide evidence that IFIT2 increases the pathological effects of invasive C. albicans and that administration of IFN-β has deleterious effects during infection., IMPORTANCE The attributable mortality associated with systemic C. albicans infections in health care settings is significant, with estimates greater than 40%. This life-threatening disease is common in patients with weakened immune systems, either due to disease or as a result of therapies. Type I interferons (IFN) are cytokines of the innate defense response that are used as immune modulators in the treatment of specific cancers, viral infections, and multiple sclerosis. In this study, we show using a murine model that the loss of a specific IFN-stimulated gene coding for IFIT2 improves survival following systemic C. albicans infection. This result infers a harmful effect of IFN during C. albicans infection and is supported by our finding that administration of IFN-β prior to invasive infection promotes fatal pathology. The findings contribute to our understanding of the innate immune response to C. albicans, and they suggest that IFN therapies present a risk factor for disseminated candidiasis.

The fungal pathogen Candida albicans causes lethal systemic infections in humans. To better define how pathogens resist oxidative attack by the immune system, we examined a family of four Flavodoxin-Like Proteins (FLPs) in C. albicans. In agreement with previous studies showing that FLPs in bacteria and plants act as NAD(P)H quinone oxidoreductases, a C. albicans quadruple mutant lacking all four FLPs (pst1Δ, pst2Δ, pst3Δ, ycp4Δ) was more sensitive to benzoquinone. Interestingly, the quadruple mutant was also more sensitive to a variety of oxidants. Quinone reductase activity confers important antioxidant effects because resistance to oxidation was restored in the quadruple mutant by expressing either Escherichia coli wrbA or mammalian NQO1, two distinct types of quinone reductases. FLPs were detected at the plasma membrane in C. albicans, and the quadruple mutant was more sensitive to linolenic acid, a polyunsaturated fatty acid that can auto-oxidize and promote lipid peroxidation. These observations suggested that FLPs reduce ubiquinone (coenzyme Q), enabling it to serve as an antioxidant in the membrane. In support of this, a C. albicans coq3Δ mutant that fails to synthesize ubiquinone was also highly sensitive to oxidative stress. FLPs are critical for survival in the host, as the quadruple mutant was avirulent in a mouse model of systemic candidiasis under conditions where infection with wild type C. albicans was lethal. The quadruple mutant cells initially grew well in kidneys, the major site of C. albicans growth in mice, but then declined after the influx of neutrophils and by day 4 post-infection 33% of the mice cleared the infection. Thus, FLPs and ubiquinone are important new antioxidant mechanisms that are critical for fungal virulence. The potential of FLPs as novel targets for antifungal therapy is further underscored by their absence in mammalian cells., Author Summary Oxidative damage is a fundamental problem for cells and a particular challenge for microbial pathogens, which require special mechanisms to resist the oxidative attack by the host immune system. We identified four proteins in the human fungal pathogen Candida albicans that belong to a large family of enzymes in bacteria and plants that reduce quinone molecules to detoxify them. Interestingly, mutational studies in C. albicans showed that these enzymes also confer resistance to a wide range of oxidants, suggesting they may have broader impact by reducing the major quinone present in cells (ubiquinone or coenzyme Q). In support of this, we found that mutating the COQ3 gene to block ubiquinone synthesis rendered cells highly sensitive to oxidative stress, revealing that it plays a very important antioxidant function in addition to its well known role in energy metabolism. These quinone reductases play a critical role in vivo, as they were required for virulence in mouse infections studies. Since mammalian cells lack this type of quinone reductase, this difference could be exploited to develop much needed novel therapeutic approaches for fungal and bacterial pathogens.

Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [ N ′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo- N ′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5 , COS111 , MKK1 , and STE2 , which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

A t otal of 100-day-old broiler chicken were randomly divided into five groups and kept under elevated temperature (95-98.6 F) to observe the effect of potassium chloride and sodium bicarbonate on the o weight gain, feed conversion ratio (FCR), serum potassium and serum bicarbonate level. Thermostress lead to significant in decrease (P

This study was undertaken to evaluate the effects of probiotics (Protexin) on the immune response of broiler chicks. The parameters of investigation were hemagglutination inhibition HI titer of antibodie s against avian influenza virus and post field AIV challenge. The findings were compared with the cyclophosphamide treated AIV-vaccinated; untreated and AIV-vaccinated and unvaccinated and control birds this investigation revealed that protexin treated chicks have higher AIV-HI antibody and no AIV post challenge mortality compared to the cyclophosphamide treated and untreated chicks the overall finding of this study clearly demonstrate that the use of this multistrain probiotics has good effect on immune response of broiler chicks.

The human fungal pathogen Candida albicans causes invasive candidiasis, characterized by fatal organ failure due to disseminated fungal growth and inflammatory damage. The s uppressor of T CR s ignaling 1 (Sts-1) and Sts-2 are two homologous phosphatases that negatively regulate signaling pathways in a number of hematopoietic cell lineages, including T lymphocytes, mast cells, and platelets. Functional inactivation of both Sts enzymes leads to profound resistance to systemic infection by C. albicans , such that greater than 80% of mice lacking Sts-1 and -2 survive a dose of C. albicans (2.5 × 10 5 CFU/mouse) that is uniformly lethal to wild-type mice within 10 days. Restriction of fungal growth within the kidney occurs by 24 h postinfection in the mutant mice. This occurs without induction of a hyperinflammatory response, as evidenced by the decreased presence of leukocytes and inflammatory cytokines that normally accompany the antifungal immune response. Instead, the absence of the Sts phosphatases leads to the rapid induction of a unique immunological environment within the kidney, as indicated by the early induction of a proinflammatory cytokine (CXL10). Mice lacking either Sts enzyme individually display an intermediate lethality phenotype. These observations identify an opportunity to optimize host immune responses toward a deadly fungal pathogen.

There is growing evidence that the sugar N-acetylglucosamine (GlcNAc) plays diverse roles in cell signaling pathways that impact the virulence properties of microbes and host cells. GlcNAc is already well known as a ubiquitous structural component at the cell surface that forms part of bacterial cell wall peptidoglycan, cell wall chitin in fungi and parasites, and extracellular matrix glycosaminoglycans of animal cells. Chitin and peptidoglycan have been previously linked to cell signaling as they can stimulate responses in plant and animal host cells [1–3]. Recent studies now indicate that GlcNAc released from these polymers can also activate cell signaling via several different mechanisms [4–6]. The role of these new GlcNAc signaling pathways in the regulation of virulence factors will be the focus of this review.