Abstract
Purpose :
The ability of fungi to infect the cornea relies upon their assimilation of carbon and nitrogen from a complex and highly dynamic nutrient environment. In this study, we identify regulators of primary metabolism in mold Aspergillus fumigatus as critical virulence factors in a murine model of fungal keratitis (FK).
Methods :
A. fumigatus genes encoding phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-responsive transcription factor (creA) were deleted via Cas9-mediated recombination and strains were verified by genomic PCR. Strains were assessed in vitro for alterations in growth rate, cell wall composition and sensitivity to antifungals. To assess relative virulence in vivo, corneas of C57BL/6J mice were abraded with an algerbrush and topically inoculated with germinated spores of the various strains. Corneal disease was assessed longitudinally by slit-lamp and optical coherence tomography (OCT), and at endpoint by histological staining (PASH) or homogenization to quantify fungal burden (colony forming units, CFUs).
Results :
We hypothesized that the naïve cornea is limiting in free glucose, thereby rendering fungal gluconeogenesis important for the synthesis of the polysaccharide-rich cell wall. Deletion of the A. fumigatus gene encoding PEPCK, the first essential step in the pathway, resulted in both a glucose-dependent growth defect in vitro as well as reduced infection rate and/or fungal load in vivo. We next reasoned that a broad perturbation in metabolic pathway expression would further impact pathogenic fitness, which we accomplished through deletion of the carbon catabolite repression (CCR) transcription factor, CreA. Cell wall composition and antifungal sensitivities were broadly altered in the creA knock out, and corneas inoculated with the mutant failed to establish signs of disease (opacification), maintained normal corneal structure (OCT, histology) and harbored no viable fungus (CFUs) at 72 h post-inoculation.
Conclusions :
These results suggest that A. fumigatus must tightly regulate its metabolic programming in order to initiate and maintain invasive growth in the cornea. As a result, the targeting of specific enzymes or metabolic regulators during FK may represent a novel strategy to directly inhibit fungal growth, alter the host-pathogen interaction at the level of fungal cell wall, or influence sensitivity to conventional antifungals.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.