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Diversification of Fungal Chitinases and Their Functional Differentiation in Histoplasma capsulatum.

ABSTRACT: Chitinases enzymatically hydrolyze chitin, a highly abundant and utilized polymer of N-acetyl-glucosamine. Fungi are a rich source of chitinases; however, the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C) as chitinases previously assigned to the "C" clade are not resolved as distinct from the "A" clade. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the "B" clade. Furthermore, chitin-binding domains (including the LysM domain) do not define specific clades, but instead are found more broadly across clades of chitinases. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade, one B clade, and one formerly classified C clade chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. These results suggest chitinase activity is variable even within subclades and that predictions of functionality require more sophisticated models.

SUBMITTER: Goughenour KD

PROVIDER: S-EPMC8042737 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Phenotypic switching between 2 opposing cellular states is a fundamental aspect of biology, and fungi provide facile systems to analyze the interactions between regulons that control this type of switch. A long-standing mystery in fungal pathogens of humans is how thermally dimorphic fungi switch their developmental form in response to temperature. These fungi, including the subject of this study, Histoplasma capsulatum, are temperature-responsive organisms that utilize unknown regulatory pathways to couple their cell shape and associated attributes to the temperature of their environment. H. capsulatum grows as a multicellular hypha in the soil that switches to a pathogenic yeast form in response to the temperature of a mammalian host. These states can be triggered in the laboratory simply by growing the fungus either at room temperature (RT; which promotes hyphal growth) or at 37 °C (which promotes yeast-phase growth). Prior worked revealed that 15% to 20% of transcripts are differentially expressed in response to temperature, but it is unclear which transcripts are linked to specific phenotypic changes, such as cell morphology or virulence. To elucidate temperature-responsive regulons, we previously identified 4 transcription factors (required for yeast-phase growth [Ryp]1-4) that are required for yeast-phase growth at 37 °C; in each ryp mutant, the fungus grows constitutively as hyphae regardless of temperature, and the cells fail to express genes that are normally induced in response to growth at 37 °C. Here, we perform the first genetic screen to identify genes required for hyphal growth of H. capsulatum at RT and find that disruption of the signaling mucin MSB2 results in a yeast-locked phenotype. RNA sequencing (RNAseq) experiments reveal that MSB2 is not required for the majority of gene expression changes that occur when cells are shifted to RT. However, a small subset of temperature-responsive genes is dependent on MSB2 for its expression, thereby implicating these genes in the process of filamentation. Disruption or knockdown of an Msb2-dependent mitogen-activated protein (MAP) kinase (HOG2) and an APSES transcription factor (STU1) prevents hyphal growth at RT, validating that the Msb2 regulon contains genes that control filamentation. Notably, the Msb2 regulon shows conserved hyphal-specific expression in other dimorphic fungi, suggesting that this work defines a small set of genes that are likely to be conserved regulators and effectors of filamentation in multiple fungi. In contrast, a few yeast-specific transcripts, including virulence factors that are normally expressed only at 37 °C, are inappropriately expressed at RT in the msb2 mutant, suggesting that expression of these genes is coupled to growth in the yeast form rather than to temperature. Finally, we find that the yeast-promoting transcription factor Ryp3 associates with the MSB2 promoter and inhibits MSB2 transcript expression at 37 °C, whereas Msb2 inhibits accumulation of Ryp transcripts and proteins at RT. These findings indicate that the Ryp and Msb2 circuits antagonize each other in a temperature-dependent manner, thereby allowing temperature to govern cell shape and gene expression in this ubiquitous fungal pathogen of humans.

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Diversification of Fungal Chitinases and Their Functional Differentiation in Histoplasma capsulatum.

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