Project description:Cryptococcus neoformans and Cryptococcus gattii are both known urease producers and have the potential to cause hyperammonemia. We hypothesized that the risk of hyperammonemia is increased by renal failure, burden of cryptococcal infection, and fungal strain characteristics. We performed a retrospective review of plasma ammonia levels in patients with cryptococcal infections. Risk factors for hyperammonemia were statistically compared between patients with and without hyperammonemia (>53 µmol/L). Cryptococcal cells from three patients included in the study were recovered from our biorepository. Strain characteristics including urease activity, ammonia production, growth curves, microscopy, melanin production, and M13 molecular typing were analyzed and compared with a wild-type (WT) C. neoformans strain. We included 29 patients, of whom 37.9% had hyperammonemia, 59% had disseminated cryptococcal infection (DCI), and 41% had isolated central nervous system infection. Thirty-eight percent of patients had renal failure and 28% had liver disease. Renal failure was associated with 4.4 times (95% confidence interval [CI] 1.5, 13.0) higher risk of hyperammonemia. This risk was higher in DCIs (RR 6.2, 95% CI 1.0, 40.2) versus isolated cryptococcal meningitis (RR 2.5, 95% CI, 0.40, 16.0). Liver disease and cryptococcal titers were not associated with hyperammonemia. C. neoformans from one patient with extreme hyperammonemia demonstrated a 4- to 5-fold increase in extracellular urease activity, slow growth, enlarged cell size phenotypes, and diminished virulence factors. Hyperammonemia was strongly associated with renal failure in individuals with DCI, surpassing associations with liver failure or cryptococcal titers. However, profound hyperammonemia in one patient was attributable to high levels of urease secretion unique to that cryptococcal strain. Prospective studies are crucial to exploring the significance of this association.IMPORTANCECryptococcus produces and secretes the urease enzyme to facilitate its colonization of the host. Urease breaks down urea into ammonia, overwhelming the liver's detoxification process and leading to hyperammonemia in some hosts. This underrecognized complication exacerbates organ dysfunction alongside the infection. Our study investigated this intricate relationship, uncovering a strong association between the development of hyperammonemia and renal failure in patients with cryptococcal infections, particularly those with disseminated infections. We also explore mechanisms underlying increased urease activity, specifically in strains associated with extreme hyperammonemia. Our discoveries provide a foundation for advancing research into cryptococcal metabolism and identifying therapeutic targets to enhance patient outcomes.

Project description:Living organisms are constantly exposed to DNA damage stress caused by endogenous and exogenous events. Eukaryotic cells have evolutionarily conserved DNA damage checkpoint surveillance systems. We previously reported that a unique transcription factor, Bdr1, whose expression is strongly induced by the protein kinase Rad53 governs DNA damage responses by controlling the expression of DNA repair genes in the basidiomycetous fungus Cryptococcus neoformans However, the regulatory mechanism of the Rad53-dependent DNA damage signal cascade and its function in pathogenicity remain unclear. Here, we demonstrate that Rad53 is required for DNA damage response and is phosphorylated by two phosphatidylinositol 3-kinase (PI3K)-like kinases, Tel1 and Mec1, in response to DNA damage stress. Transcriptome analysis revealed that Rad53 regulates the expression of several DNA repair genes in response to gamma radiation. We found that expression of CHK1, another effector kinase involved in the DNA damage response, is regulated by Rad53 and that CHK1 deletion rendered cells highly susceptible to DNA damage stress. Nevertheless, BDR1 expression is regulated by Rad53, but not Chk1, indicating that DNA damage signal cascades mediated by Rad53 and Chk1 exhibit redundant and distinct functions. We found that perturbation of both RAD53 and CHK1 attenuated the virulence of C. neoformans, perhaps by promoting phagosome maturation within macrophage, reducing melanin production, and increasing susceptibility to oxidative stresses. Furthermore, deletion of both RAD53 and CHK1 increased susceptibility to certain antifungal drugs such as amphotericin B. This report provides insight into the regulatory mechanism of fungal DNA damage repair systems and their functional relationship with fungal virulence and antifungal drug susceptibility.IMPORTANCE Genome instability is detrimental for living things because it induces genetic disorder diseases and transfers incorrect genome information to descendants. Therefore, living organisms have evolutionarily conserved signaling networks to sense and repair DNA damage. However, how the DNA damage response pathway is regulated for maintaining the genome integrity of fungal pathogens and how this contributes to their pathogenicity remain elusive. In this study, we investigated the DNA damage response pathway in the basidiomycete pathogen Cryptococcus neoformans, which causes life-threatening meningoencephalitis in immunocompromised individuals, with an average of 223,100 infections leading to 181,100 deaths reported annually. Here, we found that perturbation of Rad53- and Chk1-dependent DNA damage response pathways attenuated the virulence of C. neoformans and increased its susceptibility to certain antifungal drugs, such as amphotericin B and flucytosine. Therefore, our work paves the way to understanding the important role of human fungal DNA damage networks in pathogenesis and antifungal drug susceptibility.

Project description:Quantifying the frequency of shifts to new host plants within diverse clades of specialist herbivorous insects is critically important to understand whether and how host shifts contribute to the origin of species. Oak gall wasps (Hymenoptera: Cynipidae: Cynipini) comprise a tribe of ∼1000 species of phytophagous insects that induce gall formation on various organs of trees in the family Fagacae-primarily the oaks (genus Quercus; ∼435 sp.). The association of oak gall wasps with oaks is ancient (∼50 my), and most oak species are galled by one or more gall wasp species. Despite the diversity of both gall wasp species and their plant associations, previous phylogenetic work has not identified the strong signal of host plant shifting among oak gall wasps that has been found in other phytophagous insect systems. However, most emphasis has been on the Western Palearctic and not the Nearctic where both oaks and oak gall wasps are considerably more species rich. We collected 86 species of Nearctic oak gall wasps from most of the major clades of Nearctic oaks and sequenced >1000 Ultraconserved Elements (UCEs) and flanking sequences to infer wasp phylogenies. We assessed the relationships of Nearctic gall wasps to one another and, by leveraging previously published UCE data, to the Palearctic fauna. We then used phylogenies to infer historical patterns of shifts among host tree species and tree organs. Our results indicate that oak gall wasps have moved between the Palearctic and Nearctic at least four times, that some Palearctic wasp clades have their proximate origin in the Nearctic, and that gall wasps have shifted within and between oak tree sections, subsections, and organs considerably more often than previous data have suggested. Given that host shifts have been demonstrated to drive reproductive isolation between host-associated populations in other phytophagous insects, our analyses of Nearctic gall wasps suggest that host shifts are key drivers of speciation in this clade, especially in hotspots of oak diversity. Although formal assessment of this hypothesis requires further study, two putatively oligophagous gall wasp species in our dataset show signals of host-associated genetic differentiation unconfounded by geographic distance, suggestive of barriers to gene flow associated with the use of alternative host plants.

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Correction: Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response.

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