Acetate Metabolism in the Fungal Pathogen Cryptococcus Neoformans

Cryptococcus neoformans is an environmental basidiomycetous fungus with a worldwide distribution and a wide range of habitats. Inhalation of the desiccated yeasts or spores of C. neoformans often leads to opportunistic pulmonary infections in immunocompromised individuals, and in severe cases causes lethal meningitis following hematogenous dissemination. During infection, depending on the tissue and disease state, the invading fungi experience a range of nutrient microenvironments within the host body. As a result, rapid metabolic adaptations geared towards efficient utilization of carbon sources alternative to glucose become one of the prime determinants of survival and growth for the pathogen. Incidentally, cryptococcal infection has a long-standing association with acetate metabolism as underlined by previous cryptococcal transcriptomic studies in infection models that revealed an increased activity of alternative carbon metabolism during infection, and highlighted by the observation that cryptococcomas (infectious granulomas) are enriched with the two-carbon metabolite acetate, a physiologically important alternative carbon source. In this work I investigated the direct transcriptomic impacts of acetate utilization compared to glucose utilization via RNA-sequencing, and noted that cryptococcal transcriptome bears signatures of nutrient starvation and metabolic adaptations in acetate-grown conditions, as well as a remarkable upregulation of virulence-associated genes. Moreover, to investigate the importance of acetate production in C. neoformans we biochemically and kinetically characterized acetate kinase (Ack), an enzyme involved in acetate metabolism, and found that kinetically Ack has a preference in the acetate forming direction compared acetyl phosphate forming direction. This observation is consistent with the previously discovered biochemical role of another cryptococcal enzyme xylulose-5-phosphate/ fructose-6-phosphate phosphoketolase (Xfp2), which produces acetyl phosphate from phosphoketose sugars. Taken together, here I propose that Ack and Xfp2 forms a pathway of acetate production in C. neoformans, and hence are likely to be involved in acetate homeostasis. Possibility of the Xfp-Ack pathway of acetate production in the fungal species C. neoformans prompted us to search for Ack sequences in other eukaryotic genomes. Here I employed a hidden Markov model based strategy to predict Ack sequences in publicly available and curated eukaryotic genomes, and found that an overwhelming majority of the predicted Ack sequences can be observed in dikaryotic fungi. Maximum likelihood based phylogeny of the predicted eukaryotic Ack sequences suggest an evolutionary trajectory involving divergence from a common ancestor and loss in most eukaryotic lineages. Finally, to interpret the observation that acetate utilization appears to be connected with the upregulation of virulence-associated genes in C. neoformans, I have proposed a new theoretical framework to explain the emergence of virulence in fungal pathogens. The hypothesis posits that molecular response to various environmental stresses and virulence phenotypes are modular in nature and hence are often not connected in most fungal species. Occasionally, species may co-opt virulence modules, and hierarchically nest it with stress-response modules, thereby acquiring the ability of opportunistic pathogenicity in mammalian hosts. Overall, this work furthers our current understanding of the impacts of acetate metabolism in the human fungal pathogen C. neoformans

The Roles of Coenzyme A Binding Pocket Residues in Short and Medium Chain Acyl-CoA Synthetases

Short- and medium-chain acyl-CoA synthetases catalyze similar two-step reactions in which acyl substrate and ATP bind to form an enzyme-bound acyl-adenylate, then CoA binds for formation of the acyl-CoA product. We investigated the roles of active site residues in CoA binding in acetyl-CoA synthetase (Acs) and a medium-chain acyl-CoA synthetase (Macs) that uses 2-methylbutyryl-CoA. Three highly conserved residues, Arg193, Arg528, and Arg586 of Methanothermobacter thermautotrophicus Acs (AcsMt), are predicted to form important interactions with the 5′- and 3′-phosphate groups of CoA. Kinetic characterization of AcsMt variants altered at each of these positions indicates these Arg residues play a critical role in CoA binding and catalysis. The predicted CoA binding site of Methanosarcina acetivorans Macs (MacsMa) is structurally more closely related to that of 4-chlorobenzoate:coenzyme A ligase (CBAL) than Acs. Alteration of MacsMa residues Tyr460, Arg490, Tyr525, and Tyr527, which correspond to CoA binding pocket residues in CBAL, strongly affected CoA binding and catalysis without substantially affecting acyl-adenylate formation. Both enzymes discriminate between 3′-dephospho-CoA and CoA, indicating interaction between the enzyme and the 3′-phosphate group is important. Alteration of MacsMa residues Lys461 and Lys519, located at positions equivalent to AcsMt Arg528 and Arg586, respectively, had only a moderate effect on CoA binding and catalysis. Overall, our results indicate the active site architecture in AcsMt and MacsMa differs even though these enzymes catalyze mechanistically similar reactions. The significance of this study is that we have delineated the active site architecture with respect to CoA binding and catalysis in this important enzyme superfamily

Draft genome sequence of Grammothele lineata SDL-CO-2015-1, a jute endophyte with a potential for paclitaxel biosynthesis

Grammothele lineata strain SDL-CO-2015-1, a basidiomycete fungus, was identified as an endophyte from a jute species, Corchorus olitorius var. 2015, and found to produce paclitaxel, a diterpenic polyoxygenated pseudoalkaloid with antitumor activity. Here, we report the draft genome sequence (42.8 Mb with 9,395 genes) of this strain

Genome of Tenualosa ilisha from the river Padma, Bangladesh

Abstract Objective Hilsa shad (Tenualosa ilisha), is a popular fish of Bangladesh belonging to the Clupeidae family. An anadromous species, like the salmon and many other migratory fish, it is a unique species that lives in the sea and travels to freshwater rivers for spawning. During its entire life, Tenualosa ilisha migrates both from sea to freshwater and vice versa. Data description The genome of Tenualosa ilisha collected from the river Padma of Rajshahi, Bangladesh has been sequenced and its de novo hybrid assembly and structural annotations are being reported here. Illumina and PacBio sequencing platforms were used for high depth sequencing and the draft genome assembly was found to be 816 MB with N50 size of 188 kb. MAKER gene annotation tool predicted 31,254 gene models. Benchmarking Universal Single-Copy Orthologs refer 95% completeness of the assembled genome