Experimental Evolution of Candida by Serial Passaging in Host Cells.

Experimental evolution is an experiment class of its own; instead of requiring an a priori hypothesis, the genetic adaptation of microbes to defined environments tells us about the underlying pathways and mechanisms. Such experiments are often deceptively simple in their design, based on a single abiotic stressor and what is in essence a long-term continuous culture. However, they generally provide a starting point to thorough follow-up analyses (which are specific for the organism at hand and not part of this method chapter). In this chapter, we describe a method to use a biotic stressor which is frequently encountered by pathogenic fungi-macrophage-like cells-in a serial passaging regime. Experimental evolution under such conditions can reveal new virulence attributes and mechanisms by selecting for adaptive mutations against the host cell-induced stress.It is important to note that every evolution experiment is different, and these techniques should be taken as a general guideline to be adapted to different organisms and questions. Then, it is a powerful tool with many potential applications in pathobiology research.

Fungal biotin homeostasis is essential for immune evasion after macrophage phagocytosis and virulence.

Biotin is an important cofactor for multiple enzymes in central metabolic processes. While many bacteria and most fungi are able to synthesise biotin de novo, Candida spp. are auxotrophic for this vitamin and thus require efficient uptake systems to facilitate biotin acquisition during infection. Here we show that Candida glabrata and Candida albicans use a largely conserved system for biotin uptake and regulation, consisting of the high-affinity biotin transporter Vht1 and the transcription factor Vhr1. Both species induce expression of biotin-metabolic genes upon in vitro biotin depletion and following phagocytosis by macrophages, indicating low biotin levels in the Candida-containing phagosome. In line with this, we observed reduced intracellular proliferation of both Candida cells pre-starved of biotin and deletion mutants lacking VHR1 or VHT1 genes. VHT1 was essential for the full virulence of C. albicans during systemic mouse infections, and the lack of VHT1 led to reduced fungal burden in C. glabrata-infected brains and C. albicans-infected brains and kidneys. Together, our data suggest a critical role of Vht1-mediated biotin acquisition for C. glabrata and C. albicans during intracellular growth in macrophages and systemic infections.

A three-dimensional immunocompetent intestine-on-chip model as in vitro platform for functional and microbial interaction studies.

Alterations of the microbial composition in the gut and the concomitant dysregulation of the mucosal immune response are associated with the pathogenesis of opportunistic infections, chronic inflammation, and inflammatory bowel disease. To create a platform for the investigation of the underlying mechanisms, we established a three-dimensional microphysiological model of the human intestine. This model resembles organotypic microanatomical structures and includes tissue resident innate immune cells exhibiting features of mucosal macrophages and dendritic cells. The model displays the physiological immune tolerance of the intestinal lumen to microbial-associated molecular patterns and can, therefore, be colonised with living microorganisms. Functional studies on microbial interaction between probiotic Lactobacillus rhamnosus and the opportunistic pathogen Candida albicans show that pre-colonization of the intestinal lumen of the model by L. rhamnosus reduces C. albicans-induced tissue damage, lowers its translocation, and limits fungal burden. We demonstrate that microbial interactions can be efficiently investigated using the in vitro model creating a more physiological and immunocompetent microenvironment. The intestinal model allows a detailed characterisation of the immune response, microbial pathogenicity mechanisms, and quantification of cellular dysfunction attributed to alterations in the microbial composition.