Biology subjects

McCracken, C.

Publications and source records attributed to McCracken, C..

2 recordsLinked to original sources

bioRxiv2020

Determining Aspergillus fumigatus transcription factor expression and function during invasion of the mammalian lung

To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which governs the ability of the organism to proliferate in the lung. The other was ace1, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the {Delta}ace1 mutant is due to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators. Author summaryAlthough A. fumigatus causes the majority of cases of invasive aspergillosis, the function of most of the genes in its genome remains unknown. To identify genes encoding transcription factors that may be important for virulence, we used a NanoString nCounter to measure the mRNA levels of A. fumigatus transcription factor genes in the lungs of mice with invasive aspergillosis. The transcriptional profiling data indicate that the organism is exposed to nutrient limitation and stress during growth in the lungs, and that it responds by up-regulating genes that encode mycotoxins and secondary metabolites. In vitro, this response was most closely mimicked by growth in medium that was deficient in nitrogen, iron and/or zinc. Using the transcriptional profiling data, we identified two transcription factors that govern A. fumigatus virulence. These were RlmA, which is governs proliferation in the lung and Ace1, which controls the production of mycotoxins and secondary metabolites.

microbiology↗

bioRxiv2020

Tornadic shear stress induces a transient, calcineurin-dependent hyper-virulent phenotype in Mucorales molds

Trauma-related necrotizing myocutaneous mucormycosis (NMM) has a high morbidity and mortality in victims of combat-related injuries, geo-meteorological disasters, and severe burns. Inspired by the observation that several recent clusters of NMM have been associated with extreme mechanical forces (e.g. during tornados), we studied the impact of mechanical stress on Mucoralean biology and virulence in a Drosophila melanogaster infection model. In contrast to other experimental procedures to exert mechanical stress, tornadic shear challenge (TSC) by magnetic stirring induced a hyper-virulent phenotype in several clinically relevant Mucorales species but not in Aspergillus or Fusarium. Whereas fungal growth rates, morphogenesis, and susceptibility to noxious environments or phagocytes were not altered by TSC, soluble factors released in the supernatant of shear-challenged R. arrhizus spores rendered static spores hyper-virulent. Consistent with a rapid decay of TSC-induced hyper-virulence, minimal transcriptional changes were revealed by comparative RNA sequencing analysis of static and shear-challenged Rhizopus arrhizus. However, inhibition of the calcineurin/heat shock protein 90 (hsp90) stress response circuitry by cyclosporine A (CsA) and tanespimycin abrogated the increased pathogenicity of R. arrhizus spores following TSC. Similarly, calcineurin loss-of-function mutants of Mucor circinelloides displayed no increased virulence capacity in flies after undergoing TSC. Collectively, these results establish that TSC induces hyper-virulence specifically in Mucorales and point out the calcineurin/hsp90 pathway as a key orchestrator of this phenotype. Our findings invite future studies of topical calcineurin inhibitor treatment of wounds as an adjunct mitigation strategy for NMM following high-energy trauma. SignificanceGiven the limited efficacy of current medical treatments in trauma-related necrotizing mucormycosis, there is a dire need to better understand the Mucoralean pathophysiology in order to develop novel strategies to counteract fungal tissue invasion following severe trauma. Here, we describe that tornadic shear stress challenge transiently induces a hyper-virulent phenotype in various pathogenic Mucorales species but not in other molds known to cause wound infections. Our data support a model whereby shear stress-induced hyper-virulence is primarily driven by soluble factors and orchestrated by the calcineurin/hsp90 pathway. Importantly, pharmacological and genetic inhibition of calcineurin signaling abrogated hyper-virulence in shear stress-challenged Mucorales, encouraging further evaluation of (topical) calcineurin inhibitors to improve therapeutic outcomes of NMM after combat-related blast injuries or violent storms.

microbiology↗