One Small Step for a Yeast - Microevolution within Macrophages Renders Candida glabrata Hypervirulent Due to a Single Point Mutation

Abstract

Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures – a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.

Innate Inflammatory Response and Immunopharmacologic Activity of Micafungin, Caspofungin, and Voriconazole against Wild-Type and FKS Mutant Candida glabrata Isolates

“…We also observed macrophage activation levels to be similar between the persistent WT (or persistent FKS mutant) and index WT isolate obtained from patients with persistent candidemia in the absence of antifungals. While the limited number of strains included in this study precludes a firm conclusion, our results are consistent with previous studies and suggest that the innate immune response to C. glabrata may be strain dependent, with some strains having enhanced immune evasion capabilities that could favor their persistence and the development of antifungal resistance in vivo (9,17). Further in vivo studies that include a larger number of clinical isolates with acquired echinocandin resistance will be required.…”

“…Direct or indirect interactions of antifungals with the host immune response have been shown to play a role in shaping their efficacy against fungal pathogens in vitro and in vivo (3,9,16,17). Similarly, acquired gene mutations conferring antifungal resistance and/or adaptive cell wall stress responses may diminish innate immune cell recognition and response, which may promote the survival and proliferation of resistant strains in vivo (11,14).…”

“…Although this indirect immunopharmacologic activity may play an important role in shaping their efficacy in vivo, these mechanisms are not often considered to be a component of their antifungal activity. A better understanding of how echinocandins modulate the innate immune response can improve our efforts to optimize the use of these antifungals, especially against C. glabrata, which has a high incidence of antifungal resistance and propensity for immunoevasion (7)(8)(9)(10)(11)(12)(13). It may also provide insight on how specific antifungal resistance mutations (such as FKS gene mutations conferring resistance to echinocandins) parallel immunoevasive strategies and contribute to the persistence of resistant strains in vivo (11,14).…”

When green and red mycology meet: Impressions from an interdisciplinary forum on virulence mechanisms of phyto- and human-pathogenic fungi

“…Di Pietro (S) 31,32 Fungal spore components determine the interaction with immune cells A. Brakhage (S) 38 Regulatory networks of C. albicans morphogenesis are complex C. D'Enfert (S) 41 Experimental evolution reveals unexpected factors of fungal virulence S. Brunke (Y) 75,76 Candida albicans produces a toxic surprise: Candidalysin J. Naglik (S) 56 Coprinopsis cinerea produces potent fungal defense effectors M. K€ unzler (S) [59][60][61] The fungal cell wall acts as immunological & drug target N. Gow (S) 70 Copper detoxification supports A. fumigatus virulence N. Osherov (Po) 77 Note. a P: plenary lecture, S: session talk, Y: young researcher presentation, Po: poster plant pathogenic fungi and might reveal natural products that are active in either host, be it plant or human.…”

Beyond Candida albicans: Mechanisms of immunity to non-albicans Candida species

“…Some strains of C. tropicalis can also form true hyphae (Table 1), although many do not in vitro [22, 23]. Similarly, in vitro studies have shown that C. parapsilosis , C. krusei and C. glabrata do not form hyphae, though they have been reported to form pseudohyphae [24–27]. C. albicans can also switch between normal white yeast cell morphology and mating-competent opaque cell growth.…”