Genetic adaptive mechanisms mediating response and tolerance to acetic acid stress in the human pathogen Candida glabrata: role of the CgHaa1-dependent signaling pathway

The increased resilience of Candida glabrata to azoles and the continuous emergence of strains resistant to other antifungals demands the development of new therapeutic approaches focused on non-conventional biological targets. Genes contributing to increase C. glabrata competitiveness in the different infection sites are an interesting and unexplored cohort of therapeutic targets. To thrive in the vaginal tract and avoid exclusion C. glabrata cells have evolved dedicated responses rendering them capable of tolerating multiple environmental challenges, including the presence of acetic and lactic acids produced by the commensal microbiota. In this work a cohort of vaginal clinical isolates were phenotyped for their tolerance to acetic acid stress at a low pH as well as for several traits that are known to influence sensitivity to this organic acid, including the structure of the cell envelope and the ability to consume the acid in the presence of glucose. The role played by the ORF CAGL0L09339g, an homologue of the ScHaa1, a critical regulator of acetic acid resistance in S. cerevisiae[1], in C. glabrata response and tolerance to acetic acid stress at pH 4 was also scrutinized using a transcriptomic analysis. The role of CgHaa1 as well as of several of its target genes in mediating virulence of C. glabrata against epithelial vaginal cells was also studied.Funding received by the Institute for Bioengineering and Biosciences from the Portuguese Foundation for Science and Technology (FCT) (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (project no. 007317) is acknowledged. FCT is also acknowledged for funding the Centre of Biological Engineering through contracts FCOMP-01-0124-FEDER- 020243 and PTDC/EBB-EBI/120495/2010info:eu-repo/semantics/publishedVersio

The CgHaa1-dependent pathway mediates Candida glabrata response and tolerance to acetic acid thereby enhancing colonization of vaginal epithelium

To successfully colonize the vaginal tract Candida glabrata has to cope with various stresses including the presence of acetic acid at a low pH that is produced by the bacteria that co-colonize this niche. The genes/pathways involved in C. glabrata tolerance and response to acetic acid are largely unknown, although these are a highly interesting set of novel targets to control vaginal infections caused by this yeast. Saccharomyces cerevisae response and tolerance to acetic acid was found to be largely mediated by the ScHaa1 transcription factor [1,2,3]. In this work the involvement of CgHaa1 in C. glabrata tolerance and response to acetic acid is demonstrated. Elimination of CgHAA1 gene from C. glabrata genome dramatically increased susceptibility of this pathogenic yeast to acetic acid (30 mM at pH 4.0). Around 140 genes were found to be up-regulated, directly or indirectly, by CgHaa1 in response to acetic acid stress, based on results of a transcriptomic analysis. Functional clustering of the genes activated by CgHaa1 under acetic acid stress shows an enrichment of those involved in carbohydrate metabolism, transport, cell wall maintenance, regulation of internal pH and nucleic acid processing. At least five of the CgHaa1-regulated genes were found to increase C. glabrata tolerance to acetic acid including CgGAD1, encoding a glutamate decarboxylase; CgTPO2/3, encoding a drug efflux pump of the Major Facilitator Superfamily; CgYPS1, encoding a cell wall aspartyl protease; and CAGL0H04851 and CAGL0E03740, encoding two uncharacterized ORFs. Altogether our results are consistent with the concept that the CgHaa1- signalling pathway increases C. glabrata tolerance to acetic acid by reducing the internal accumulation of the acid and by up-regulating the activity of the plasma membrane proton pump H+-ATPase CgPma1, two essential features for a robust weak acid response. The role exerted by CgHaa1 in the ability of C. glabrata to colonize reconstituted vaginal human epithelium (RVHE) in the presence of acetic acid (30 mM at pH 4.0) was also investigated in this work. In the absence of acetic acid wild-type and DCgHaa1 mutant cells were able to colonize RVHE at a similar rate, however, in the presence of acetic acid colonization of the vaginal tissue was markedly reduced in the mutant background. The reduced colonizing capacity of DCgHaa1 mutant cells was correlated with a reduced expression of the adhesin-encoding genes EPA6, EPA7 and EPA1 and with a lower adhesiveness to the extracellular matrix proteins fibronectin and vitronectin

Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes

For Assembly 20 of the Candida albicans genome, the sequence of each of the eight chromosomes was determined, revealing new insights into gene family creation and dispersion, subtelomere organization, and chromosome evolution

Genetic adaptive mechanisms mediating response and tolerance to acetic acid stress in the human pathogen Candida glabrata: role of the CgHaa1-dependent signaling pathway

C. glabrata is a commensal found in the human genitourinary tract but under certain conditions this harmless colonization evolves to a mucosal infection and, in more serious cases, to disseminated mycosis. To thrive in the acidic vaginal tract C. glabrata has to cope with the presence of a competing commensal microbiota known to restrain the overgrowth of pathogens through the production of acetic and lactic acids, among other interference effects. The persistent emergence of C. glabrata strains resistant to currently used antifungals demands the implementation of novel therapeutic strategies based on non-conventional targets. Genes contributing to increase C. glabrata competitiveness in the vaginal tract by mediating tolerance to the organic acids found therein are a cohort of interesting and yet unexplored therapeutic targets. Tolerance mechanisms of C. glabrata to acetic acid at low pH are poorly studied but much knowledge was gathered in Saccharomyces cerevisiae (Mira et al 2010a; 2010b; 2011; 2010c). In particular, the central role of the ScHaa1 transcription factor in mediating S. cerevisiae tolerance to acetic acid stress was demonstrated (Mira et al 2010b; 2011; 2010c). In this work it is shown that CgHaa1, an orthologue of ScHaa1, controls an acetic acid-responsive system in C. glabrata. The mechanisms by which the CgHaa1 pathway mediate tolerance to acetic acid in C. glabrata were further dissected, exploring a transcriptomics approach, being of notice the involvement of this regulatory system in the control of internal pH and in reducing the internal accumulation of the acid. In the presence of acetic acid CgHaa1 enhanced adhesion and colonization of reconstituted vaginal human epithelium by C. glabrata, this correlating with a positive effect of CgHaa1 over the expression of adhesinencoding genes. The results obtained show similarities, but also remarkable differences, in the way by which the ScHaa1 and CgHaa1 pathways mediate tolerance to acetic acid in S. cerevisiae and in C. glabrata, indicating a functional expansion of the network in the later species. The role of the CgHaa1-pathway in the extreme acetic acid-tolerance exhibited by vaginal C. glabrata isolates will also be discussed, along with other uncovered mechanistic insights

Mild Heat Stress Affects on the Cell Wall Structure in Candida albicans Biofilm

福岡歯科大学2018年

The CgHaa1-regulon mediates response and tolerance to acetic acid stress in the human pathogen Candida glabrata

To thrive in the acidic vaginal tract C. glabrata has to cope with high concentrations of acetic acid. The mechanisms underlying C. glabrata tolerance to acetic acid at low pH remain largely uncharacterized. In this work it is demonstrated the essential role of the CgHaa1 transcription factor (encoded by ORF CAGL0L09339g) in the response and tolerance of C. glabrata to acetic acid. Transcriptomic analysis showed that CgHaa1 regulates, directly or indirectly, the expression of about 75% of the genes activated under acetic acid stress. CgHaa1-activated targets are involved in multiple physiological functions including membrane transport, metabolism of carbohydrates and amino acids, regulation of the activity of the plasma membrane H+-ATPase and adhesion. Under acetic acid stress CgHaa1 increased the activity and the expression of the CgPma1 proton pump and contributed to increased colonization of vaginal epithelial cells by C. glabrata. CgHAA1, CgTPO3 and CgHSP30, two identified CgHaa1-activated targets, are herein demonstrated to be determinants of C. glabrata tolerance to acetic acid. The protective effect of CgTpo3 and of CgHaa1 was linked to a role of these proteins in reducing the accumulation of acetic acid inside C. glabrata cells. In response to acetic acid stress, marked differences were found in the regulons controlled by CgHaa1 and by its S. cerevisiae ScHaa1 ortholog, demonstrating a clear divergent evolution of the two regulatory networks. The results gathered in this study significantly advance the understanding of the molecular mechanisms underlying the success of C. glabrata as a vaginal colonizer.Cristoph Schueller (University of Natural Resources and Life Sciences, Austria) and Ken Haynes (University of Exeter, UK) are acknowledged for sharing strains. Funding received by the Institute for Bioengineering and Biosciences from the Portuguese Foundation for Science and Technology (FCT) (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (project no. 007317)is acknowledged. FCT is also acknowledged for funding the Centre of Biological Engineering through contracts FCOMP-01-0124-FEDER020243 and PTDC/EBB-EBI/120495/2010. Science Foundation Ireland and the Wellcome Trust are acknowledged for funding G.B

Intestinal Resident Yeast Candida glabrata Requires Cyb2p-Mediated Lactate Assimilation to Adapt in Mouse Intestine

The intestinal resident Candida glabrata opportunistically infects humans. However few genetic factors for adaptation in the intestine are identified in this fungus. Here we describe the C. glabrata CYB2 gene encoding lactate dehydrogenase as an adaptation factor for survival in the intestine. CYB2 was identified as a virulence factor by a silkworm infection study. To determine the function of CYB2, we analysed in vitro phenotypes of the mutant Δcyb2. The Δcyb2 mutant grew well in glucose medium under aerobic and anaerobic conditions, was not supersensitive to nitric oxide which has fungicidal-effect in phagocytes, and had normal levels of general virulence factors protease, lipase and adherence activities. A previous report suggested that Cyb2p is responsible for lactate assimilation. Additionally, it was speculated that lactate assimilation was required for Candida virulence because Candida must synthesize glucose via gluconeogenesis under glucose-limited conditions such as in the host. Indeed, the Δcyb2 mutant could not grow on lactate medium in which lactate is the sole carbon source in the absence of glucose, indicating that Cyb2p plays a role in lactate assimilation. We hypothesized that Cyb2p-mediated lactate assimilation is necessary for proliferation in the intestinal tract, as the intestine is rich in lactate produced by bacteria flora, but not glucose. The Δcyb2 mutant showed 100-fold decreased adaptation and few cells of Saccharomyces cerevisiae can adapt in mouse ceca. Interestingly, C. glabrata could assimilate lactate under hypoxic conditions, dependent on CYB2, but not yeast S. cerevisiae. Because accessible oxygen is limited in the intestine, the ability for lactate assimilation in hypoxic conditions may provide an advantage for a pathogenic yeast. From those results, we conclude that Cyb2p-mediated lactate assimilation is an intestinal adaptation factor of C. glabrata