Usingin vivotranscriptomics and RNA enrichment to identify genes involved in virulence ofCandida glabrata

Abstract

Candida species are the most commonly isolated opportunistic fungal pathogens in humans. Candida albicans causes most of the diagnosed infections, closely followed by Candida glabrata . C. albicans is well studied, and many genes have been shown to be important for infection and colonization of the host. It is however less clear how C. glabrata infects the host. With the help of fungal RNA enrichment, we here investigated for the first time the transcriptomic profile of C. glabrata during urinary tract infection (UTI) in mice. In the UTI model, bladders and kidneys are major target organs and therefore fungal transcriptomes were addressed in these organs. Our results showed that, next to adhesins and proteases, nitrogen metabolism and regulation play a vital role during C. glabrata UTI. Genes involved in nitrogen metabolism were upregulated and among them we show that DUR1,2 (urea amidolyase) and GAP1 (amino acid permease) were important for virulence. Furthermore, we confirmed the importance of the glyoxylate cycle in the host and identified MLS1 (malate synthase) as an important gene necessary for C. glabrata virulence. In conclusion, our study shows with the support of in vivo transcriptomics how C. glabrata adapts to host conditions.

A novel Candida glabrata doxycycline-inducible system for in vitro/in vivo use

“…Uptake, metabolism, and specific regulation of nitrogen was shown to be of high importance for C. glabrata virulence (Schrevens et al 2022 ). Nitrogen is one of the most important essential nutrient sources, and probably available in limited amounts extracellularly inside a host (Zhang et al 2018 ).…”

“…The general amino acid permease Gap1 is well-studied in S. cerevisiae and was shown to have a broad substrate specificity and high substrate affinity (Jauniaux and Grenson 1990 ). Gap1 was shown to be important for virulence in C. glabrata during urinary tract infection (Schrevens et al 2022 ). Due to its potentially central role in the uptake and regulation of amino acid metabolism, similarly to S. cerevisiae , Gap1 may also functions during invasive infection.…”

“…The reverse transactivator sequence was ordered as a synthetic gene (Eurofins Genomics, Germany) and cloned into the pVS29 backbone (Schrevens et al 2022 ) using Spe I and Bam HI restriction sites, thus resulting in pSSa26. Upon digestions with Nhe I (a restriction site situated in the CgCEN sequence of pSSa26), it was transformed into DSY562, resulting in strain SSY40.…”

“…Unlike S. cerevisiae , the interest in C. glabrata stems only from the significant morbidity and mortality it causes in the clinic. However, C. glabrata pathogenesis is still not well-understood and only few specific genes or gene families have been linked to virulence (Kaur et al 2007 , Lopez-Fuentes et al 2018 , Schrevens et al 2022 ). This is due to the limited existing in vivo studies on this organism (Ferrari et al 2011a , b , Nakayama et al 2011 , Schrevens et al 2022 ).…”

“…However, C. glabrata pathogenesis is still not well-understood and only few specific genes or gene families have been linked to virulence (Kaur et al 2007 , Lopez-Fuentes et al 2018 , Schrevens et al 2022 ). This is due to the limited existing in vivo studies on this organism (Ferrari et al 2011a , b , Nakayama et al 2011 , Schrevens et al 2022 ). A tetracycline-dependent gene expression system (Tet-off) has been described in C. glabrata that uses a tetracycline transactivator (tTA) coupled with TRE-containing promoters to decrease gene expression (Nakayama et al 1998 ).…”