Paromomycin Affects Translation and Vesicle-Mediated Trafficking as Revealed by Proteomics of Paromomycin –Susceptible –Resistant Leishmania donovani

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL) and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr) strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC) followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr) strain identified a total of 226 proteins at ≥95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani

Deploying FLAREs to Visualize Functional Outcomes of Host-Pathogen Encounters.

One of the most exciting features of fluorescent probes is their rapid evolution from simple tracers to functional indicators in diverse research fields [1]. In mammalian cells, fluorescence has been coupled to synaptic transmission [2,3], neuronal differentiation [4], apoptotic cell death [5], fate mapping in different immune cells [6,7], and functional changes in cell physiol-ogy, for example, the induction of cytokines [8]. In microbial cells, fluorescence has long been utilized as a tracer for pathogenic microbes, revealing microbial localization, residence, and spread in host tissues [9]. However, measuring functional outcomes during individual encoun-ters with host immune cells remains challenging. In this Pearl, we describe fluorescence-based approaches, which we term “functional micro-bial reporters, ” designed to relay changes in microbial physiology that occur in host cell and tis-sue environments. This class of microbial reporters typically harnesses fluorescence emission at two wavelengths. One of these signals functions as an invariant tracer of microbial cells and is generally unaffected by hostile conditions that are encountered during interactions with host cells, such as reactive oxygen species or acidified compartments. The other signal varies accord-ing to a change in microbial physiology or residence in the host. This second signal may act as

Paromomycin: uptake and resistance in Leishmania donovani

Paromomycin is currently in phase IV clinical trials against leishmaniasis. In the present work we elucidate the effect and mechanism of uptake of paromomycin in Leishmania donovani. The in vitro sensitivities of both promastigotes and amastigotes were determined to this aminoglycoside. Association of paromomycin with L. donovani involved a rapid initial phase that was non-saturable up to 1 mM of the drug. This initial phase was largely independent of temperature and not affected by metabolic inhibitors. Poly-lysine, a membrane impermeant polycation, caused profound inhibition of this association of the drug with the parasite indicating that it represented a binding of the cationic paromomycin to the negatively charged leishmanial glycocalyx. After 72 h of exposure to the drug the mitochondrial membrane potential was significantly decreased, indicating that this organelle might be the ultimate target of the drug. Both cytoplasmic and mitochondrial protein synthesis were inhibited following paromomycin exposure. A line selected for resistance to the drug showed reduced paromomycin accumulation associated with a significant reduction in the initial binding to the cell surface. The drug induced reduction in membrane potential and inhibition of protein synthesis were less pronounced in the resistant strain in comparison to the wild-type

Comparative systems analysis of the secretome of the opportunistic pathogen Aspergillus fumigatus and other Aspergillus species

Abstract Aspergillus fumigatus and multiple other Aspergillus species cause a wide range of lung infections, collectively termed aspergillosis. Aspergilli are ubiquitous in environment with healthy immune systems routinely eliminating inhaled conidia, however, Aspergilli can become an opportunistic pathogen in immune-compromised patients. The aspergillosis mortality rate and emergence of drug-resistance reveals an urgent need to identify novel targets. Secreted and cell membrane proteins play a critical role in fungal-host interactions and pathogenesis. Using a computational pipeline integrating data from high-throughput experiments and bioinformatic predictions, we have identified secreted and cell membrane proteins in ten Aspergillus species known to cause aspergillosis. Small secreted and effector-like proteins similar to agents of fungal-plant pathogenesis were also identified within each secretome. A comparison with humans revealed that at least 70% of Aspergillus secretomes have no sequence similarity with the human proteome. An analysis of antigenic qualities of Aspergillus proteins revealed that the secretome is significantly more antigenic than cell membrane proteins or the complete proteome. Finally, overlaying an expression dataset, four A. fumigatus proteins upregulated during infection and with available structures, were found to be structurally similar to known drug target proteins in other organisms, and were able to dock in silico with the respective drug

Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection

Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein which possesses anti-microbial activity primarily due to its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9 (−/−) mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro, and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9(−/−) mice by injecting recombinant calprotectin. Further, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin’s anti-hyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro, or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatus ΔzafA mutant, which demonstrates deficient zinc transport, exhibits impaired growth in infected corneas and following incubation with neutrophils or calprotectin in vitro as compared to wild-type. Collectively, these studies demonstrate a novel stage - specific susceptibility of A. fumigatus to zinc and manganese chelation by neutrophil-derived calprotectin