N-Acetylglucosamine (GlcNAc) stimulates important signaling pathways in a wide range of organisms. In the human fungal pathogen Candida albicans, GlcNAc stimulates hyphal cell morphogenesis, virulence genes, and the genes needed to catabolize GlcNAc. Previous studies on the GlcNAc transporter (NGT1) indicated that GlcNAc has to be internalized to induce signaling. Therefore, the role of GlcNAc catabolism was examined by deleting the genes required to phosphorylate, deacetylate, and deaminate GlcNAc to convert it to fructose-6-PO 4 (HXK1, NAG1, and DAC1). As expected, the mutants failed to utilize GlcNAc. Surprisingly, GlcNAc inhibited the growth of the nag1⌬ and dac1⌬ mutants in the presence of other sugars, suggesting that excess GlcNAc-6-PO 4 is deleterious. Interestingly, both hxk1⌬ and an hxk1⌬ nag1⌬ dac1⌬ triple mutant could be efficiently stimulated by GlcNAc to form hyphae. These mutants could also be stimulated to express GlcNAcregulated genes. Because GlcNAc must be phosphorylated by Hxk1 to be catabolized, and also for it to enter the anabolic pathways that form chitin, N-linked glycosylation, and glycosylphosphatidylinositol anchors, the mutant phenotypes indicate that GlcNAc metabolism is not needed to induce signaling in C. albicans. Thus, these studies in C. albicans reveal a novel role for GlcNAc in cell signaling that may also regulate critical pathways in other organisms.

Since its discovery 40 years ago, rotavirus (RV) is considered to be a major cause of infant and childhood morbidity and mortality particularly in developing countries. Nearly every child in the world under 5 years of age is at the risk of RV infection. It is estimated that 90% of RV-associated mortalities occur in developing countries of Africa and Asia. Two live oral vaccines, RotaTeq (RV5, Merck) and Rotarix (RV1, GlaxoSmithKline) have been successfully deployed to scale down the disease burden in Europe and America, but they are less effective in Africa and Asia. In April 2009, the World Health Organization recommended the inclusion of RV vaccination in national immunization programs of all countries with great emphasis in developing countries. To date, 86 countries have included RV vaccines into their national immunization programs including 41 Global Alliance for Vaccines and Immunization eligible countries. The predominant RV genotypes circulating all over the world are G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8], while G12[P6] and G12[P8] are emerging genotypes. On account of the segmented genome, RV shows an enormous genetic diversity that leads to the evolution of new genotypes that can influence the efficacy of current vaccines. The current need is for a global RV surveillance program to monitor the prevalence and antigenic variability of new genotypes to formulate future vaccine development planning. In this review, we will summarize the previous and recent insights into RV structure, classification, and epidemiology and current status of RV vaccination around the globe and will also cover the status of RV research and vaccine policy in Pakistan.

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.

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.

The monosaccharide N-acetylglucosamine (GlcNAc) is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc stimulates developmental pathways in the fungal pathogen Candida albicans, which is a commensal organism that colonizes the mammalian gut and causes disease in the setting of host immunodeficiency. Here we investigate GlcNAc signaling in thermally dimorphic human fungal pathogens, a group of fungi that are highly evolutionarily diverged from C. albicans and cause disease even in healthy individuals. These soil organisms grow as polarized, multicellular hyphal filaments that transition into a unicellular, pathogenic yeast form when inhaled by a human host. Temperature is the primary environmental cue that promotes reversible cellular differentiation into either yeast or filaments; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. We found GlcNAc to be a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. In addition to increasing the rate of filamentous growth, micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift that was independent of GlcNAc catabolism, indicating that fungal cells sense GlcNAc to promote filamentation. Whole-genome expression profiling to identify candidate genes involved in establishing the filamentous growth program uncovered two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes.