Coordinate regulation of virulence and metabolic genes by the transcription factor HbhR in Mycobacterium marinum.

The heparin-binding hemagglutinin (HBHA) is a multifunctional protein involved in adherence of Mycobacterium tuberculosis to non-phagocytic cells and in the formation of intracytosolic lipid inclusions. We demonstrate that the expression of hbhA is regulated by a transcriptional repressor, named HbhR, in Mycobacterium marinum. The hbhR gene, located upstream of hbhA, was identified by screening a transposon insertion library and detailed analysis of a mutant overproducing HBHA. HbhR was found to repress both hbhA and hbhR transcription by binding to the promoter regions of both genes. Complementation restored production of HBHA. RNA-seq analyses comparing the mutant and parental strains uncovered 27 genes, including hbhA, that were repressed and 20 genes activated by HbhR. Among the former, the entire locus of genes coding for a type-VII secretion system, including esxA, esxB and pe-ppe paralogs, as well as the gene coding for PspA, present in intracellular lipid vesicles, was identified, as was katG, a gene involved in the sensitivity to isoniazid. The latter category contains genes that play a role in diverse functions, such as metabolism and resistance to oxidative conditions. Thus, HbhR appears to be a master regulator, linking the transcriptional regulation of virulence, metabolic and antibiotic sensitivity genes in M. marinum.

ß-1,2-Mannosyltransferases 1 and 3 Participate in Yeast and Hyphae O- and N-Linked Mannosylation and Alter Candida albicans Fitness During Infection.

ß-1,2-mannosylation of Candida albicans glycoconjugates has been investigated through the identification of enzymes involved in the addition of ß-1,2-oligomannosides (ß-Mans) to phosphopeptidomannan and phospholipomannan. ß-1,2-oligomannosides are supposed to have virulence properties that they confer to these glycoconjugates. In a previous study, we showed that cell wall mannoproteins (CWMPs) harbor ß-Mans in their O-mannosides; therefore, we analyzed their biosynthesis and impact on virulence. In this study, we demonstrate that O-mannans are heterogeneous and that α-mannosylated O-mannosides, which are biosynthesized by Mnt1 and Mnt2 α-1,2-mannosyltransferases, can be modified with ß-Mans but only at the nonreducing end of α-1,2-mannotriose. ß-1,2-mannosylation of this O-mannotriose depends on growth conditions, and it involves 2 ß-1,2-mannosyltransferases, Bmt1 and Bmt3. These Bmts are essential for ß-1,2-mannosylation of CWMPs and expression of ß-Mans on germ tubes. A bmt1Δ mutant and a mutant expressing no ß-Mans unexpectedly disseminated more in BALB/c mice, whereas they had neither attenuated nor enhanced virulence in C57BL/6 mice. In galectin (Gal)3 knockout mice, the reference strain was more virulent than in C57BL/6 mice, suggesting that the ß-Mans innate receptor Gal3 is involved in C. albicans fitness during infection.

Members 5 and 6 of the Candida albicans BMT family encode enzymes acting specifically on ß-mannosylation of the phospholipomannan cell-wall glycosphingolipid.

A family of nine genes encoding proteins involved in the synthesis of ß-1,2 mannose adhesins of Candida albicans has been identified. Four of these genes, BMT1-4, encode enzymes acting stepwise to add ß-mannoses on to cell-wall phosphopeptidomannan (PPM). None of these acts on phospholipomannan (PLM), a glycosphingolipid member of the mannose-inositol-phosphoceramide family, which contributes with PPM to ß-mannose surface expression. We show that deletion of BMT5 and BMT6 led to a dramatic reduction of PLM glycosylation and accumulation of PLM with a truncated ß-oligomannoside chain, respectively. Disruptions had no effect on sphingolipid biosynthesis and on PPM ß-mannosylation. ß-Mannose surface expression was not affected, confirming that ß-mannosylation is a process based on specificity of acceptor molecules, but liable to global regulation.

Candida albicans cell wall glycosylation may be indirectly required for activation of epithelial cell proinflammatory responses.

Oral epithelial cells discriminate between the yeast and hyphal forms of Candida albicans via the mitogen-activated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether ß-glucan and N- or O-glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of ß-glucan and N- and O-mannan mutants, we found that N-mannosylation (via Δoch1 and Δpmr1 mutants) and O-mannosylation (via Δpmt1 and Δmnt1 Δmnt2 mutants), but not phosphomannan (via a Δmnn4 mutant) or ß-1,2 mannosylation (via Δbmt1 to Δbmt6 mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, the N- and O-mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the α-1,6-mannose backbone of the N-linked outer chain (corresponding to a Δmnn9 mutant) may be required for epithelial adhesion, while the α-1,2-mannose component of phospholipomannan (corresponding to a Δmit1 mutant) may contribute to epithelial cell damage. ß-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary, N- and O-mannosylation defects affect the ability of C. albicans to induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/MKP1/c-Fos hypha-mediated immune response.

Molecular phenotyping of mannosyltransferases-deficient Candida albicans cells by high-resolution magic angle spinning NMR.

The yeast Candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. Recognition of yeasts by host cells is directly mediated by cell wall components of the yeast, including a wide range of abundantly expressed glycoconjugates. Of particular interest in C. albicans are the beta-mannosylated epitopes that show a complex expression pattern on N-glycan moiety of phosphopeptidomannans and are absent in the non-pathogenic species Saccharomyces cerevisiae. Being known as potent antigens for the adaptive immune response and elicitors of specific infection-protective antibodies, the exact delineation of beta-mannosides regulation and expression pathways has lately become a major milestone toward the comprehension of host-pathogen interplay. Using the newly developed HR-MAS NMR methodology, we demonstrate the possibility of assessing the general profiles of cell-surface-exposed glycoconjugates from intact living yeast cells without any prior purification step. This technique permitted to directly observe structural modifications of surface expressed phosphodiester-linked beta-mannosides on a series of deletion strains in beta-mannosyltransferases and phospho-mannosyltransferases compared with their parental strains.

Beta-1,2 oligomannose adhesin epitopes are widely distributed over the different families of Candida albicans cell wall mannoproteins and are associated through both N- and O-glycosylation processes.

Beta-1,2-linked mannosides (beta-Mans) are believed to contribute to Candida albicans virulence. The presence of beta-Mans has been chemically established for two molecules (phosphopeptidomannan [PPM] and phospholipomannan) that are noncovalently linked to the cell wall, where they correspond to specific epitopes. However, a large number of cell wall mannoproteins (CWMPs) also express beta-Man epitopes, although their nature and mode of beta-mannosylation are unknown. We therefore used Western blotting to map beta-Man epitopes for the different families of mannoproteins gradually released from the cell wall according to their mode of anchorage (soluble, released by dithiothreitol, beta-1,3 glucan linked, and beta-1,6 glucan linked). Reduction of beta-Man epitope expression occurred after chemical and enzymatic deglycosylation of the different cell wall fractions, as well as in a secreted form of Hwp1, a representative of the CWMPs linked by glycosylphosphatidylinositol remnants. Enzyme-linked immunosorbent assay inhibition tests were performed to assess the presence of beta-Man epitopes in released oligomannosides. A comparison of the results obtained with CWMPs to the results obtained with PPM and the use of mutants with mutations affecting O and N glycosylation demonstrated that both O glycosylation and N glycosylation participate in the association of beta-Mans with the protein moieties of CWMPs. This process, which can alter the function of cell wall molecules and their recognition by the host, is therefore more important and more complex than originally thought, since it differs from the model established previously with PPM.

Identification of a new family of genes involved in beta-1,2-mannosylation of glycans in Pichia pastoris and Candida albicans.

Structural studies of cell wall components of the pathogenic yeast Candida albicans have demonstrated the presence of beta-1,2-linked oligomannosides in phosphopeptidomannan and phospholipomannan. During C. albicans infection, beta-1,2-oligomannosides play an important role in host/pathogen interactions by acting as adhesins and by interfering with the host immune response. Despite the importance of beta-1,2-oligomannosides, the genes responsible for their synthesis have not been identified. The main reason is that the reference species Saccharomyces cerevisiae does not synthesize beta-linked mannoses. On the other hand, the presence of beta-1,2-oligomannosides has been reported in the cell wall of the more genetically tractable C. albicans relative, P. pastoris. Here we present the identification, cloning, and characterization of a novel family of fungal genes involved in beta-mannose transfer. Employing in silico analysis, we identified a family of four related new genes in P. pastoris and subsequently nine homologs in C. albicans. Biochemical, immunological, and structural analyses following deletion of four genes in P. pastoris and deletion of four genes acting specifically on C. albicans mannan demonstrated the involvement of these new genes in beta-1,2-oligomannoside synthesis. Phenotypic characterization of the strains deleted in beta-mannosyltransferase genes (BMTs) allowed us to describe the stepwise activity of Bmtps and acceptor specificity. For C. albicans, despite structural similarities between mannan and phospholipomannan, phospholipomannan beta-mannosylation was not affected by any of the CaBMT1-4 deletions. Surprisingly, depletion in mannan major beta-1,2-oligomannoside epitopes had little impact on cell wall surface beta-1,2-oligomannoside antigenic expression.

Candida albicans is an immunogen for anti-Saccharomyces cerevisiae antibody markers of Crohn's disease.

BACKGROUND AND AIMS: Antibodies directed against oligomannose sequences alpha-1,3 Man (alpha-1,2 Man alpha-1,2 Man)(n) (n = 1 or 2), termed anti-Saccharomyces cerevisiae antibodies (ASCAs) are markers of Crohn's disease (CD). S. cerevisiae mannan, which expresses these haptens, is used to detect ASCA, but the exact immunogen for ASCA is unknown. Structural and genetic studies have shown that Candida albicans produces mannosyltransferase enzymes that can synthesize S cerevisiae oligomannose sequences depending on growth conditions. This study investigated whether C. albicans could act as an immunogen for ASCA. METHODS: Sequential sera were collected from patients with CD, systemic candidiasis, and rabbits infected with C. albicans. Antibodies were purified by using chemically synthesized (Sigma) ASCA major epitopes. These affinity-purified antibodies and lectins were then used to analyze the expression of ASCA epitopes on molecular extracts and cell walls of C. albicans and S cerevisiae grown in various conditions. RESULTS: In humans and rabbits, generation of ASCA was shown to be associated with the generation of anti-C. albicans antibodies resulting specifically from infection. By using affinity-purified antibodies, C. albicans was shown to express ASCA epitopes on mannoproteins similar to those of S. cerevisiae. By changing the growth conditions, C. albicans mannan was also able to mimic S. cerevisiae mannan in its ability to detect ASCA associated with CD. This overexpression of ASCA epitopes was achieved when C. albicans grew in human tissues. CONCLUSIONS: C. albicans is one of several immunogens for ASCA and may be at the origin of an aberrant immune response in CD.

Candida albicans serotype B strains synthesize a serotype-specific phospholipomannan overexpressing a beta-1,2-linked mannotriose.

Candida albicans strains consist of serotypes A and B depending on the presence of terminal beta-1,2-linked mannose residues in the acid-stable part of serotype A phosphopeptidomannan (PPM). The distribution of C. albicans serotypes varies according to country and human host genetic and infectious backgrounds. However, these epidemiological traits have not yet been related to a phenotypically stable molecule as cell surface expression of the serotype A epitope depends on the growth conditions. We have shown that C. albicans serotype A associates beta-mannose residues with another molecule, phospholipomannan (PLM), which is a member of the mannoseinositolphosphoceramide family. In this study, PLM from serotype B strains was analysed in order to provide structural bases for the differences in molecular mass and antigenicity observed between PLMs from both serotypes. Through these analyses, carbon 10 was shown to be the location of a second hydroxylation of fatty acids previously unknown in fungal sphingolipids. Minor differences observed in the ceramide moiety appeared to be strain-dependent. More constant features of PLM from serotype B strains were the incorporation of greater amounts of phytosphingosine C20, a twofold reduced glycosylation of PLM and overexpression of a beta-1,2 mannotriose, the epitope of protective antibodies. This specific beta-mannosylation was observed even when growth conditions altered serotype A PPM-specific epitopes, confirming the potential of PLM as a phenotypically stable molecule for serotyping. This study also suggests that the regulation of beta-mannosyltransferases, which define specific immunomodulatory adhesins whose activity depends on the mannosyl chain length, are part of the genetic background that differentiates serotypes.

Inactivation of CaMIT1 inhibits Candida albicans phospholipomannan beta-mannosylation, reduces virulence, and alters cell wall protein beta-mannosylation.

Studies on Candida albicans phospholipomannan have suggested a novel biosynthetic pathway for yeast glycosphingolipids. This pathway is thought to diverge from the usual pathway at the mannose-inositol-phospho-ceramide (MIPC) step. To confirm this hypothesis, a C. albicans gene homologue for the Saccharomyces cerevisiae SUR1 gene was identified and named MIT1 as it coded for GDP-mannose:inositol-phospho-ceramide mannose transferase. Two copies of this gene were disrupted. Western blots of cell extracts revealed that strain mit1Delta contained no PLM. Thin layer chromatography and mass spectrometry confirmed that mit1Delta did not synthesize MIPC, demonstrating a role of MIT1 in the mannosylation of C. albicans IPCs. As MIT1 disruption prevented downstream beta-1,2 mannosylation, mit1Delta represents a new C. albicans mutant affected in the expression of these specific virulence attributes, which act as adhesins/immunomodulators. mit1Delta was less virulent during both the acute and chronic phases of systemic infection in mice (75 and 50% reduction in mortality, respectively). In vitro, mit1Delta was not able to escape macrophage lysis through down-regulation of the ERK1/2 phosphorylation pathway previously shown to be triggered by PLM. Phenotypic analysis also revealed pleiotropic effects of MIT1 disruption. The most striking observation was a reduced beta-mannosylation of phosphopeptidomannan. Increased beta-mannosylation of mannoproteins was observed under growth conditions that prevented the association of beta-oligomannosides with phosphopeptidomannan, but not with PLM. This suggests that C. albicans has strong regulatory mechanisms associating beta-oligomannoses with different cell wall carrier molecules. These mechanisms and the impact of the different presentations of beta-oligomannoses on the host response need to be defined.