In order to redefine the mannitol pathway in the necrotrophic plant pathogen Botrytis cinerea, we used a targeted deletion strategy of genes encoding two proteins of mannitol metabolism, BcMTDH (B. cinerea mannitol dehydrogenase) and BcMPD (B. cinerea mannitol-1-phosphate dehydrogenase). Mobilization of mannitol and quantification of Bcmpd and Bcmtdh gene transcripts during development and osmotic stress confirmed a role for mannitol as a temporary and disposable carbon storage compound. In order to study metabolic fluxes, we followed conversion of labelled hexoses in wild-type and ΔBcmpd and ΔBcmtdh mutant strains by in vivo NMR spectroscopy. Our results revealed that glucose and fructose were metabolized via the BcMPD and BcMTDH pathways respectively. The existence of a novel mannitol phosphorylation pathway was also suggested by the NMR investigations. This last finding definitively challenged the existence of the originally postulated mannitol cycle in favour of two simultaneously expressed pathways. Finally, physiological and biochemical studies conducted on double deletion mutants (ΔBcmpdΔBcmtdh) showed that mannitol was still produced despite a complete alteration of both mannitol biosynthesis pathways. This strongly suggests that one or several additional undescribed pathways could participate in mannitol metabolism in B. cinerea.

We have investigated the antimicrobial effects of photocatalysis on the yeast model Saccharomyces cerevisiae . To accurately study the antimicrobial mechanisms of the photocatalytic process, we focused our investigations on two questions: the entry of the nanoparticles in treated cells and the fate of the intracellular environment. Transmission electronic microscopy did not reveal any entry of nanoparticles within the cells, even for long exposure times, despite degradation of the cell wall space and deconstruction of cellular compartments. In contrast to proteins located at the periphery of the cells, intracellular proteins did not disappear uniformly. Disappearance or persistence of proteins from the pool of oxidized intracellular isoforms was not correlated to their functions. Altogether, our data suggested that photocatalysis induces the establishment of an intracellular oxidative environment. This hypothesis was sustained by the detection of an increased level of superoxide ions (O ₂ ° ⁻ ) in treated cells and by greater cell cultivability for cells expressing oxidant stress response genes during photocatalytic exposure. The increase in intracellular ROS, which was not connected to the entry of nanoparticles within the cells or to a direct contact with the plasma membrane, could be the result of an imbalance in redox status amplified by chain reactions. Moreover, we expanded our study to other yeast and filamentous fungi and pointed out that, in contrast to the laboratory model S. cerevisiae , some environmental strains are very resistant to photocatalysis. This could be related to the cell wall composition and structure.

The pathogenic yeast Candida albicans is both a powerful commensal and a pathogen of humans that can infect wide range of organs and body sites. Metabolic flexibility promotes infection and commensal colonization by this opportunistic pathogen. Yeast cell survival depends upon assimilation of fermentable and non-fermentable locally available carbon sources. Physiologically relevant sugars like glucose and fructose are present at low levels in host niches. However, because glucose is the preferred substrate for energy and biosynthesis of structural components, its efficient detection and metabolism are fundamental for the metabolic adaptation of the pathogen. We explored and characterized the C. albicans hexose kinase system composed of one hexokinase (CaHxk2) and two glucokinases (CaGlk1 and CaGlk4). Using a set of mutant strains, we found that hexose phosphorylation is mostly performed by CaHxk2, which sustains growth on hexoses. Our data on hexokinase and glucokinase expression point out an absence of cross regulation mechanisms at the transcription level and different regulatory pathways. In the presence of glucose, CaHxk2 migrates in the nucleus and contributes to the glucose repression signaling pathway. In addition, CaHxk2 participates in oxidative, osmotic and cell wall stress responses, while glucokinases are overexpressed under hypoxia. Hexose phosphorylation is a key step necessary for filamentation that is affected in the hexokinase mutant. Virulence of this mutant is clearly impacted in the Galleria mellonella and macrophage models. Filamentation, glucose phosphorylation and stress response defects of the hexokinase mutant prevent host killing by C. albicans. By contributing to metabolic flexibility, stress response and morphogenesis, hexose kinase enzymes play an essential role in the virulence of C. albicans.

In Kluyveromyces lactis , the expression of the major glucose permease gene RAG1 is controlled by extracellular glucose through a signaling cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 pathway. We have identified a key component of the K. lactis glucose signaling pathway by characterizing a new mutation, rag20-1 , which impairs the regulation of RAG1 and hexokinase RAG5 genes by glucose. Functional complementation of the rag20-1 mutation identified the KlSNF2 gene, which encodes a protein 59% identical to S. cerevisiae Snf2, the major subunit of the SWI/SNF chromatin remodeling complex. Reverse transcription-quantitative PCR and chromatin immunoprecipitation analyses confirmed that the KlSnf2 protein binds to RAG1 and RAG5 promoters and promotes the recruitment of the basic helix-loop-helix Sck1 activator. Besides this transcriptional effect, KlSnf2 is also implicated in the glucose signaling pathway by controlling Sms1 and KlRgt1 posttranscriptional modifications. When KlSnf2 is absent, Sms1 is not degraded in the presence of glucose, leading to constitutive RAG1 gene repression by KlRgt1. Our work points out the crucial role played by KlSnf2 in the regulation of glucose transport and metabolism in K. lactis , notably, by suggesting a link between chromatin remodeling and the glucose signaling pathway.

Survival of the pathogenic yeast Candida albicans depends upon assimilation of fermentable and non-fermentable carbon sources detected in host microenvironments. Among the various carbon sources encountered in a human body, glucose is the primary source of energy. Its effective detection, metabolism and prioritization via glucose repression are primordial for the metabolic adaptation of the pathogen. In C. albicans, glucose phosphorylation is mainly performed by the hexokinase 2 (CaHxk2). In addition, in the presence of glucose, CaHxK2 migrates in the nucleus and contributes to the glucose repression signaling pathway. Based on the known dual function of the Saccharomyces cerevisiae hexokinase 2 (ScHxk2), we intended to explore the impact of both enzymatic and regulatory functions of CaHxk2 on virulence, using a site-directed mutagenesis approach. We show that the conserved aspartate residue at position 210, implicated in the interaction with glucose, is essential for enzymatic and glucose repression functions but also for filamentation and virulence in macrophages. Point mutations and deletion into the N-terminal region known to specifically affect glucose repression in ScHxk2 proved to be ineffective in CaHxk2. These results clearly show that enzymatic and regulatory functions of the hexokinase 2 cannot be unlinked in C. albicans.