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Cellular responses to external stress allow microorganisms to adapt to a vast array of environmental conditions, including infection sites. The molecular mechanisms behind these responses are studied to gain insight into microbial pathogenesis, which could lead to new antimicrobial therapies. Here, we explore a role for arrestin protein-mediated ubiquitination in stress response and pathogenesis in the pathogenic fungus Cryptococcus neoformans . In a previous study, we identified four arrestin-like proteins in C. neoformans and found that one of these is required for efficient membrane synthesis, likely by directing interaction between fatty acid synthases and the Rsp5 E3 ubiquitin ligase. Here, we further explore Cn Rsp5 function and determine that this single Ub ligase is absolutely required for pathogenesis and survival in the presence of cellular stress. Additionally, we show that a second arrestin-like protein, Ali2, similarly facilitates interaction between Rsp5 and some of its protein targets. Of the four postulated C. neoformans arrestin-like proteins, Ali2 appears to contribute the most to C. neoformans pathogenesis, likely by directing Rsp5 to pathogenesis-related ubiquitination targets. A proteomics-based differential ubiquitination screen revealed that several known cell surface proteins are ubiquitinated by Rsp5 and a subset also requires Ali2 for their ubiquitination. Rsp5-mediated ubiquitination alters the stability and the localization of these proteins. A loss of Rsp5-mediated ubiquitination results in cell wall defects that increase susceptibility to external stresses. These findings support a model in which arrestin-like proteins guide Rsp5 to ubiquitinate specific target proteins, some of which are required for survival during stress., Importance: Microbial proteins involved in human infectious diseases often need to be modified by specific chemical additions to be fully functional. Here, we explore the role of a particular protein modification, ubiquitination, in infections due to the human fungal pathogen Cryptococcus neoformans . We identified a complex of proteins responsible for adding ubiquitin groups to fungal proteins, and this complex is required for virulence. These proteins are fungal specific and might be targets for novel anti-infection therapy., Competing Interests: The authors declare no conflict of interest.

Cryptococcosis, caused by fungi of the genus Cryptococcus , manifests in a broad range of clinical presentations, including severe pneumonia and disease of the central nervous system (CNS) and other tissues (bone and skin). Immune deficiency or development of overexuberant inflammatory responses can result in increased susceptibility or host damage, respectively, during fungal encounters. Leukotrienes help regulate inflammatory responses against fungal infections. Nevertheless, studies showed that Cryptococcus exploits host 5-lipoxygenase (5-LO), an enzyme central to the metabolism of arachidonic acid into leukotrienes, to facilitate transmigration across the brain-blood barrier. To investigate the impact of host 5-LO on the development of protective host immune responses and mortality during cryptococcosis, wild-type (C57BL/6) and 5-lipoxygenase-deficient (5-LO -/- ) mice were given experimental pulmonary and systemic Cryptococcus sp., infections. Our results showed that 5-LO -/- mice exhibited reduced pathology and better disease outcomes (i.e., no mortality or signs associated with cryptococcal meningoencephalitis) following pulmonary infection with C. deneoformans, despite having detectable yeast in the brain tissues. In contrast, C57BL/6 mice exhibited classical signs associated with cryptococcal meningoencephalitis. Additionally, brain tissues of 5-LO -/- mice exhibited lower levels of cytokines (CCL2 and CCL3) clinically associated with Cryptococcus -related immune reconstitution inflammatory syndrome (C-IRIS). In a systemic mouse model of cryptococcosis, 5-LO -/- mice and those treated with a Federal Drug Administration (FDA)-approved 5-LO synthesis inhibitor, zileuton, displayed significantly reduced mortality compared to C57BL/6 infected mice. These results suggest that therapeutics designed to inhibit host 5-LO signaling could reduce disease pathology and mortality associated with cryptococcal meningoencephalitis., Importance: Cryptococcosis is a mycosis with worldwide distribution and has a broad range of clinical manifestations, including diseases of the CNS. Globally, there is an estimated 179,000 cases of cryptococcal meningitis, resulting in approximately 112,000 fatalities per annum and 19% of AIDS-related deaths. Understanding how host immune responses are modulated during cryptococcosis is central to mitigating the morbidity and mortality associated with cryptococcosis. Leukotrienes (LTs) have been shown to modulate inflammatory responses during infection. In this study, we show that mice deficient in 5-lipoxygenase (5-LO), an enzyme central to the metabolism of arachidonic acid into leukotrienes, exhibit reduced pathology, disease, and neurological signs associated with cryptococcal meningitis. Additionally, mice given an experimental cryptococcal infection and subsequently treated with an FDA-approved 5-LO synthesis inhibitor exhibited significantly reduced mortality rates. These results suggest that therapeutics designed to inhibit host 5-LO activity could significantly reduce pathology and mortality rates associated with cryptococcal meningitis., Competing Interests: The authors declare no conflict of interest.