Biology subjects

Kalem, M. C.

Publications and source records attributed to Kalem, M. C..

7 recordsLinked to original sources

bioRxiv2026

Temperature-driven isoform switching reprograms developmental gene expression in a human fungal pathogen

Post-transcriptional regulation is key to development, and yet little is known about how RNA isoform choice contributes to developmental choices in fungi. Here we assemble the first isoform-level transcriptome of Histoplasma, a ubiquitous human fungal pathogen that grows as an infectious environmental form (hyphae) or a pathogenic host form (yeast) in response to temperature. We find extensive morphology-associated longer leader and trailer isoforms, rapid temperature-driven transcription start site remodeling, and inclusion of regulatory elements in longer leaders. We observe isoform-specific patterns of ribosome and polysome association, indicating that isoform switching regulates the proteome. Our results suggest a model in which isoform diversity and rapid isoform switching are central to post-transcriptional regulation during thermal dimorphism, enabling precise and timely translation of factors needed to establish and maintain hyphal and yeast forms. These studies illuminate how eukaryotic systems utilize post- transcriptional regulation to determine developmental states in response to external stimuli.

microbiology↗

bioRxiv2026

Optimization of conidial production in the thermally dimorphic fungal pathogen Histoplasma

Sporulation is an integral process in the lifecycle of many fungal pathogens, including Histoplasma, a primary human pathogen that causes respiratory infections. Histoplasma conidia, or asexual spores, are the primary infectious particle but very little is known about them, in part due to the need for Biosafety Level 3 containment and inconsistency in generating viable conidia under laboratory conditions. Here, we identify media that consistently promote Histoplasma conidiation, yielding both micro- and macroconidia, and conditions that promote high levels of germination. We show that conidiation media and duration affect the proportion of macroconidia produced, and we demonstrate that Histoplasma strains vary in their response to these conidiation parameters. Finally, imaging studies of chitin, exposed chitin, and cell wall mannoproteins show that while micro- and macroconidia have similar cell wall compositions, strain type and conidiation media variation result in qualitative differences in staining. These optimized methods for Histoplasma conidial preparations will enable more detailed investigations into this understudied aspect of the biology of an important human fungal pathogen.

microbiology↗

bioRxiv2025

Host aging induces a senescent-like phenotype in neutrophils and altered transcriptional responses to Streptococcus pneumoniae

Aging drives increased susceptibility to respiratory infections by Streptococcus pneumoniae (pneumococci). Polymorphonuclear leukocytes (PMNs) are among the first responders in the lung following pneumococcal infection and are required for bacterial clearance. However, PMN antimicrobial function declines with age. To identify mechanisms underlying this decline, we performed RNA sequencing on PMNs in the lungs of young and old mice following pulmonary infection with S. pneumoniae. We observed significant transcriptomic differences across host age. Transcriptional analysis followed by functional validation revealed that in infected mice, PMNs from aged hosts failed to upregulate several effector activities including glycolysis and subsequent mitochondrial reactive oxygen species (ROS) production, which are necessary for bacterial killing by PMNs. Analysis of potential transcription factors controlling these changes indicated differential regulation by E2f2 in aged mice, which was linked to lower PMN differentiation resulting in more immature PMNs in the lungs of aged mice compared to young controls. Conversely, PMNs in aged mice displayed a higher senescence-associated secretory phenotype (SASP) score and upregulated pathways involved in cellular senescence. Follow-up functional characterization found that in uninfected hosts, PMNs in aged mice expressed higher levels of SASP factors IL-10, TNF, and ROS, had lower incidence of apoptosis, and had a higher proportion of cells positive for senescence-associated {beta}-galactosidase, features of a senescent-like phenotype. In conclusion, host aging is associated with altered PMN phenotypes, including a shift toward senescent-like energy-deficient cells, which may contribute to impaired host defense and represent potential targets for improved interventions against infection in older adults.

immunology↗

bioRxiv2022

Contributions of Ccr4 and Gcn2 to the translational response of C. neoformans to host-relevant stressors and Integrated Stress Response induction

In response to the host environment, Cryptococcus neoformans must rapidly reprogram its translatome from one which promotes growth to one which is responsive to host temperature and oxidative stress. This reprogramming is primarily driven through the Gcn2-mediated repression of translation initiation and Ccr4-mediated removal of abundant pro-growth mRNAs from the translating pool. Here we investigate the contributions of these two pathways to the translational response to stress, and show that the response to oxidative stress is primarily driven by Gcn2 whereas temperature and oxidative stress both require Ccr4. Temperature stress, but not oxidative stress, result in an increase in RNase I resistant disome. Further, eIF2 phosphorylation varies in response to the type and magnitude of stress, yet all tested conditions induce translation of integrated stress response (ISR) transcription factor Gcn4, but not necessarily the Gcn4-dependent transcription. Finally, we define the ISR regulon in response to oxidative stress in C. neoformans. Together this study identifies the differential response to host-relevant stressors in an environmental fungus which can adapt to the environment inside the human host.

molecular biology↗

bioRxiv2022

Arginine methylation of Puf4 drives diverse protein functions

The evolutionarily conserved Pumilio domain-containing RNA binding proteins (RBPs) are involved in many steps of post-transcriptional gene regulation, including RNA stability, polyadenylation, deadenylation, and translation. RBPs are post-translationally modified by methylation of arginine/glycine-rich domains, though the consequences of these modifications are not well known. We determined the arginine methylation and phosphorylation landscape of the Pumilio domain-containing RBP Puf4 from the basidiomycete fungus Cryptococcus neoformans. We found that methyl-deficient Puf4 mutants do not complement critical PUF4 deletion phenotypes, such as resistances to endoplasmic reticulum stress and antifungals, and cell wall remodeling. Methyl-deficient mutants also exhibit unique RNA and protein interactions. Lastly, we identified intra-protein cross talk between post-translationally modified methylated and phosphorylated residues. Overall, we show that post-translational modifications, particularly arginine methylation, of Puf4 regulate the functions of this RBP.

molecular biology↗

bioRxiv2022

The interactome of Cryptococcus neoformans Rmt5 reveals multiple regulatory points in fungal cell biology and pathogenesis

Protein arginine methylation is a key post-translational modification in eukaryotes that modulates core cellular processes, including translation, morphology, transcription, and RNA fate. However, this has not been explored in Cryptococcus neoformans, a human-pathogenic basidiomycetous encapsulated fungus. We characterized the five protein arginine methyltransferases in C. neoformans and highlight Rmt5 as critical regulator of cryptococcal morphology and virulence. An rmt5{Delta} mutant was defective in thermotolerance, had a remodeled cell wall, and exhibited enhanced growth in an elevated carbon dioxide atmosphere and in chemically induced hypoxia. We revealed that Rmt5 interacts with post-transcriptional gene regulators, such as RNA-binding proteins and translation factors. Further investigation of the rmt5{Delta} mutant showed that Rmt5 is critical for the homeostasis of eIF2 and its phosphorylation state following 3-amino-1,2,4-triazole-induced ribosome stalling. RNA sequencing of one rmt5{Delta} clone revealed stable chromosome 9 aneuploidy that was ameliorated by complementation but did not impact the rmt5{Delta} phenotype. As a result of these diverse interactions and functions, loss of RMT5 enhanced phagocytosis by murine macrophages and attenuated disease progression in mice. Taken together, our findings link arginine methylation to critical cryptococcal cellular processes that impact pathogenesis, including post-transcriptional gene regulation by RNA-binding proteins. SignificanceThe fungal pathogen Cryptococcus neoformans is a huge threat for people living with immune deficits, especially HIV/AIDS. Its virulence potential is dependent on virulence factors, stress adaptation, and thermotolerance. Post-transcriptional gene regulation is important for these pathogenic processes, but the mechanisms that govern post-transcriptional regulator function are unexplored. Protein arginine methylation is a major modification of post-transcriptional regulators that has not been investigated in pathogenic fungi. Here we investigated the role of arginine methylation by arginine methyltransferases on the biology and virulence of C. neoformans. Phenotypic characterization of deletion mutants revealed pleiotropic functions for RMTs in this pathogen. Further investigation of the Rmt5 interactome using proximity-dependent biotinylation revealed interactions with RNA binding proteins and translation factors, thereby impacting virulence-associated processes.

microbiology↗

bioRxiv2020

Puf4 Mediates Post-transcriptional Regulation of Caspofungin Resistance in Cryptococcus neoformans

Echinocandins have been on the market for 20 years, yet they are the newest class of antifungal drugs. The human fungal pathogen Cryptococcus neoformans is intrinsically resistant to the echinocandin antifungal drug caspofungin, which targets the {beta}-1,3-glucan synthase encoded by the FKS1. Analysis of a C. neoformans puf4{Delta} mutant, lacking the pumilio/FBF RNA binding protein family member Puf4, revealed exacerbated caspofungin resistance. In contrast, overexpression of PUF4 resulted in caspofungin sensitivity. The FKS1 mRNA contains three Puf4-binding elements (PBEs) in its 5 untranslated region. Puf4 binds with specificity to this region of the FKS1. The FKS1 mRNA was destabilized in the puf4{Delta} mutant, and the abundance of the FKS1 mRNA was reduced compared to wild type, suggesting that Puf4 is a positive regulator FKS1 mRNA stability. In addition to FKS1, the abundance of additional cell wall biosynthesis genes, including chitin synthases (CHS3, CHS4, CHS6) and deacetylases (CDA1, CDA2, CDA3) as well as a {beta}-1,6-glucan synthase gene (SKN1) was regulated by Puf4 during a caspofungin time course. The use of fluorescent dyes to quantify cell wall components revealed that the puf4{Delta} mutant had increased chitin content, suggesting a cell wall composition that is less reliant on {beta}-1,3-glucan. Overall, our findings suggest a mechanism by which caspofungin resistance, and more broadly, cell wall biogenesis, is regulated post-transcriptionally by Puf4. ImportanceCryptococcus neoformans is an environmental fungus that causes pulmonary and central nervous system infections. It is also responsible for 15% of AIDS-related deaths. A major contributor to the high morbidity and mortality statistics is the lack of safe and effective antifungal therapies, especially in resource-poor settings. Yet, antifungal drug development has stalled in the pharmaceutical industry. Therefore, it is of importance to understand the mechanism by which C. neoformans is resistant to caspofungin in order to design adjunctive therapies to potentiate its activity toward this important pathogen.

microbiology↗