Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance

Abstract

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall PLOS Genetics | https://doi.contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options. Author summaryA. fumigatus is the main ethiological agent of one of the most important life-threatening fungal diseases in immuno-compromised humans, invasive aspergillosis. Treatment commonly involves echinocandin antifungal drugs that inhibit cell wall β-1,3-glucan polysaccharide biosynthesis. Calcium is an important messenger for many signaling pathways regulating the fungal response to stress and antifungal drugs. The calcium responsive calcineurin phosphatase influences the localization and activity of the CrzA transcription factor (TF), regulating the activation of several stress responses and cell wall modifications. For many years, CrzA has been recognized as the sole calcium/calcineurin-dependent TF. Here, we identify nine TFs involved in the calcium/calcineurin metabolism, including a novel A. fumigatus calcium/calcineurin dependent TF named ZipD. Transcriptional profiling of the response of A. fumigatus wild-type, plus the ΔcrzA and ΔzipD mutant, strains shows CrzA and ZipD to have shared and unique functions. ZipD was found to be important for not only calcium metabolism, but also for the cell wall organization, osmotic stress and echinocandin tolerance. During host infection, ZipD plays an important role in modulating fungal cell walls, promoting evasion of the host pro-inflammatory immune responses and virulence. Our work emphasize...

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

“…Here, we demonstrated that there were increased levels of chitin composition in the cell wall of the ΔcbfA and ΔnctA mutants but not in that of the ΔnctC mutant. We previously demonstrated that the ΔzipD and ΔrlmA mutants have altered chitin composition (22,23). It remains to be determined if the chitin concentration is altered in the ΔnctB, ΔfhdA, ΔznfA, ΔatfA, ΔzfpA, and ΔAFUB_054000 mutants.…”

“…Six of them (ΔcbfA, ΔzipD, ΔnctC, ΔznfA, ΔatfA, and ΔrlmA) have been previously identified as being associated with calcium sensitivity and caspofungin sensitivity and displaying reduced CPE (22,23,28,42). ZipD and RlmA are important for osmotic and cell wall stresses, collaborating for cell wall construction and the activation of several pathways important for osmotic stability (22,23). Here, we demonstrated that there were increased levels of chitin composition in the cell wall of the ΔcbfA and ΔnctA mutants but not in that of the ΔnctC mutant.…”

“…To assess if any other TF plays a role in CPE, a library of 484 A. fumigatus TF null mutants (25) was screened for sensitivity to high concentrations of caspofungin (16 g/ml). At this concentration, the CPE is induced in the CEA17 parental strain (22,23). Eleven null mutants, including the ΔzipD mutant (ΔAFUB_020350), exhibited increased sensitivity to caspofungin at a concentration of 16 g/ml (the other 10 mutants included…”

“…In Candida albicans, aneuploidy also enables the CPE by altering the copy number of several genes involved in cell wall remodeling or calcium metabolism (20,21). Recently, our group identified a novel basic leucine zipper ZipD transcription factor (TF) that was found to play a role in the calcium-calcineurin pathway and to be involved in CPE (22,23). Moreover, it has been suggested that reactivation of ␤-1,3-glucan synthase is essential for CPE maintenance (24).…”

Pleiotropy and epistasis within and between signaling pathways defines the genetic architecture of fungal virulence

“…The propensity of fungal pathogens to cause disease is a complex outcome dependent on a variety of underlying physiological features that facilitate survival in stressful host niches, such as the ability to forage and acquire nutrients (Crawford and Wilson, 2015), to tolerate bombardment from reactive oxygen species (Komalapriya et al, 2015), and to mount a successful defense against (or evade) the host immune system (Jimenez-Lopez and Lorenz, 2013;Hernández-Chávez et al, 2017). Significant progress has been made with respect to understanding the cell and molecular biology of fungal pathogenesis; numerous virulence-related traits and key genes and pathways that regulate these traits have been identified for many fungal pathogens (Navarro-García et al, 2001;Hull and Heitman, 2002;Casadevall, 2006;Liu et al, 2008;Botts et al, 2009;Saputo et al, 2012;Bahn and Jung, 2013;O'Meara et al, 2014;Brown et al, 2014;Bultman et al, 2016;Zaragoza, 2019; de Castro et al, 2019). Similarly, the availability of low-cost, high-throughput genome sequencing has greatly advanced the understanding of genetic variation and population structure for many fungal pathogens (Hu et al, 2008;Fedorova et al, 2008;Butler et al, 2009;Ngamskulrungroj et al, 2009;D'Souza et al, 2011;Hirakawa et al, 2015;Mixão et al, 2019).…”