Alteration of ß-glucan in the emerging fungal pathogen Candida auris leads to immune evasion and increased virulence.

Candida auris is an emerging pathogenic yeast that has been categorized as a global public health threat and a critical priority among fungal pathogens. Despite this, the immune response against C. auris infection is still not well understood. Hosts fight Candida infections through the immune system that recognizes pathogen-associated molecular patterns such as ß-glucan, mannan, and chitin on the fungal cell wall. In this study, levels of ß-glucan and mannan exposures in C. auris grown under different physiologically relevant stimuli were quantified by flow cytometry-based analysis. Lactate, hypoxia, and sublethal concentration of fluconazole trigger a decrease in surface ß-glucan while low pH triggers an increase in ß-glucan. There is no inverse pattern between exposure levels of ß-glucan and mannan in the cell wall architecture among the three clades. To determine the effect of cell wall remodeling on the immune response, a phagocytosis assay was performed, followed by quantification of released cytokines by ELISA. Lactate-induced decrease in ß-glucan leads to reduced uptake of C. auris by PMA-differentiated THP-1 and RAW 264.7 macrophages. Furthermore, reduced production of CCL3/MIP-1⍺ but not TNF-⍺ and IL-10 were observed. An in vivo infection analysis using silkworms reveals that a reduction in ß-glucan triggers an increase in the virulence of C. auris. This study demonstrates that ß-glucan alteration occurs in C. auris and serves as an escape mechanism from immune cells leading to increased virulence.

Protective Effects of Inulin on Stress-Recurrent Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the digestive tract and is closely associated with the homeostasis of the gut microbiota. Inulin, as a natural prebiotic, displays anti-inflammatory activity and maintains equilibrium of the intestinal microbiota. In this study, our research aimed to explore the potential of inulin in enhancing intestinal immunity and reducing inflammation in stress-recurrent IBD. In this study, a co-culture intestinal epithelium model and a stress-recurrent IBD mouse model was used to examine the protective effects of inulin. It was observed that inulin digesta significantly reduced pro-inflammatory cytokine expression (CXCL8/IL8 and TNFA) and increased MUC2 expression in intestinal epithelial cells. In vivo, our findings showed that Inulin intake significantly prevented IBD symptoms. This was substantiated by a decrease in serum inflammatory markers (IL-6, CALP) and a downregulation of inflammatory cytokine (Il6) in colon samples. Additionally, inulin intake led to an increase in short-chain fatty acids (SCFAs) in cecal contents and a reduction in the expression of endoplasmic reticulum (ER) stress markers (CHOP, BiP). Our results highlight that inulin can improve stress-recurrent IBD symptoms by modulating microbiota composition, reducing inflammation, and alleviating ER stress. These findings suggested the therapeutic potential of inulin as a dietary intervention for ameliorating stress-recurrent IBD.

Complete genome sequence of Aquitalea pelogenes USM4 (JCM19919), a polyhydroxyalkanoate producer.

Polyhydroxyalkanoate (PHA) is a type of biopolymer produced by most bacteria and archaea, resembling thermoplastic with biodegradability and biocompatibility features. Here, we report the complete genome of a PHA producer, Aquitalea sp. USM4, isolated from Perak, Malaysia. This bacterium possessed a 4.2 Mb circular chromosome and a 54,370 bp plasmid. A total of 4067 predicted protein-coding sequences, 87 tRNA genes, and 25 rRNA operons were identified using PGAP. Based on ANI and dDDH analysis, the Aquitalea sp. USM4 is highly similar to Aquitalea pelogenes. We also identified genes, including acetyl-CoA (phaA), acetoacetyl-CoA (phaB), PHA synthase (phaC), enoyl-CoA hydratase (phaJ), and phasin (phaP), which play an important role in PHA production in Aquitalea sp. USM4. The heterologous expression of phaC1 from Aquitalea sp. USM4 in Cupriavidus necator PHB-4 was able to incorporate six different types of PHA monomers, which are 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV), 3-hydroxyhexanoate (3HHx) and isocaproic acid (3H4MV) with suitable precursor substrates. This is the first complete genome sequence of the genus Aquitalea among the 22 genome sequences from 4 Aquitalea species listed in the GOLD database, which provides an insight into its genome evolution and molecular machinery responsible for PHA biosynthesis.

A Putative NADPH Oxidase Gene in Unicellular Pathogenic Candida glabrata Is Required for Fungal ROS Production and Oxidative Stress Response.

Most previous studies on fungal NADPH oxidases (Nox) focused on multicellular fungi and highlighted the important roles of Nox-derived reactive oxygen species (ROS) in cellular differentiation and signaling communication. However, there are few reports about Nox in unicellular fungi. A novel NOX ortholog, CAGL0K05863g (named CgNOX1), in Candida glabrata was investigated in this study. Deletion of CgNOX1 led to a decrease in both intracellular and extracellular ROS production. In addition, the Cgnox1∆ mutant exhibited hypersensitivity to hydrogen peroxide and menadione. Also, the wild-type strain showed higher levels of both CgNOX1 mRNA expression and ROS production under oxidative stress. Moreover, the absence of CgNOX1 resulted in impaired ferric reductase activity. Although there was no effect on in vitro biofilm formation, the CgNOX1 mutant did not produce hepatic apoptosis, which might be mediated by fungal Nox-derived ROS during co-incubation. Together, these results indicated that the novel NOX gene plays important roles in unicellular pathogenic C. glabrata and its interaction with host cells.

Curcumin affects function of Hsp90 and drug efflux pump of Candida albicans.

Candida albicans is a pathogenic yeast that causes candidiasis in immunocompromised patients. The overuse of antifungal drugs has led to the development of resistance to such drugs by this fungus, which is a major challenge in antifungal chemotherapy. One approach to this problem involves the utilization of new natural products as an alternative source of antifungals. Curcumin, one such natural product, has been widely studied as a drug candidate and is reported to exhibit antifungal activity against C. albicans. Although studies of the mechanism of curcumin against human cancer cells have shown that it inhibits heat shock protein 90 (Hsp90), little is known about its function against C. albicans. In this paper, using a doxycycline-mediated HSP90 strain and an HSP90-overexpressing strain of C. albicans, we demonstrated that the curcumin triggered a decrease in Hsp90 by affecting it at the post-transcriptional level. This also led to the downregulation of HOG1 and CDR1, resulting in a reduction of the stress response and efflux pump activity of C. albicans. However, the inhibition of HSP90 by curcumin was not due to the inhibition of transcription factors HSF1 or AHR1. We also found that curcumin can not only decrease the transcriptional expression of CDR1, but also inhibit the efflux pump activity of Cdr1. Hence, we conclude that disruption of HSP90 by curcumin could impair cell growth, stress responses and efflux pump activity of C. albicans.

Erg25 Controls Host-Cholesterol Uptake Mediated by Aus1p-Associated Sterol-Rich Membrane Domains in Candida glabrata.

The uptake of cholesterol from the host is closely linked to the proliferation of pathogenic fungi and protozoa during infection. For some pathogenic fungi, cholesterol uptake is an important strategy for decreasing susceptibility to antifungals that inhibit ergosterol biosynthesis. In this study, we show that Candida glabrata ERG25, which encodes an enzyme that demethylates 4,4-dimethylzymosterol, is required for cholesterol uptake from host serum. Based on the screening of C. glabrata conditional knockdown mutants for each gene involved in ergosterol biosynthesis, ERG25 knockdown was found to decrease lethality of infected mice. ERG25 knockdown impairs the plasma membrane localization of the sterol importer Aus1p, suggesting that the accumulated 4,4-dimethylzymosterol destabilizes the lipid domain with which Aus1p functionally associates. ERG25 knockdown further influences the structure of the membrane compartment of Can1p (MCC)/eisosomes (ergosterol-rich lipid domains), but not the localization of the membrane proteins Pma1p and Hxt1p, which localize to sterol-poor domains. In the sterol-rich lipid domain, Aus1p-contining domain was mostly independent of MCC/eisosomes, and the nature of these domains was also different: Ausp1-contining domain was a dynamic network-like domain, whereas the MCC/eisosomes was a static dot-like domain. However, deletion of MCC/eisosomes was observed to influence the localization of Aus1p after Aus1p was transported from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane. These findings suggest that ERG25 plays a key role in stabilizing sterol-rich lipid domains, constituting a promising candidate target for antifungal therapy.

Vph1 is associated with the copper homeostasis of Cryptococcus neoformans serotype D.

We clarified the roles of VPH1 in Cryptococcus neoformans serotype D by examining the detailed phenotypes of VPH1-deficient cells (Δvph1) in terms of their capability to grow in acidic and alkaline pH, at a high temperature, and under high osmotic conditions, in addition to the involvement of VPH1 in copper (Cu) homeostasis and the expression of some C. neoformans virulence factors. Δvph1 could grow well on minimal medium (YNB) but exhibited hypersensitivity to 20 µM Cu due to the failure to induce Cu-detoxifying metallothionein genes (CMT1 and CMT2). In contrast, Δvph1 exhibited defective growth on rich medium (YPD), and the induction of Cu transporter genes (CTR1 and CTR4) did not occur in this medium, implying that this strain was incapable of the uptake of Cu ions for growth. However, the addition of excess Cu promoted CTR gene expression and supported Δvph1 growth. These results suggested that the lack of the VPH1 gene disturbed Cu homeostasis in C. neoformans. Moreover, the loss of Vph1 function influenced the urease activity of C. neoformans.

Triphenilphosphonium Analogs of Chloramphenicol as Dual-Acting Antimicrobial and Antiproliferating Agents.

In the current work, in continuation of our recent research, we synthesized and studied new chimeric compounds, including the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP), which are linked by alkyl groups of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly reduce membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP in bacterial ribosomes differs from that of CHL. By simulating the dynamics of CAM-Cn-TPP complexes with bacterial ribosomes, we proposed a possible explanation for the specificity of the action of these analogs in the translation process. CAM-C10-TPP and CAM-C14-TPP more strongly inhibit the growth of the Gram-positive bacteria, as compared to CHL, and suppress some CHL-resistant bacterial strains. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds targeting both the ribosomes and cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds has an inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells possess qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.

The Lack of SNARE Protein Homolog Syn8 Influences Biofilm Formation of Candida glabrata.

Biofilm formation of Candida species is considered to be a pathogenic factor of host infection. Since biofilm formation of Candida glabrata has not been as well studied as that of Candida albicans, we performed genetic screening of C. glabrata, and three candidate genes associated with biofilm formation were identified. Candida glabrata SYN8 (CAGL0H06325g) was selected as the most induced gene in biofilm cells for further research. Our results indicated that the syn8Δ mutant was defective not only in biofilm metabolic activity but also in biofilm morphological structure and biomass. Deletion of SYN8 seemed to have no effect on extracellular matrix production, but it led to a notable decrease in adhesion ability during biofilm formation, which may be linked to the repression of two adhesin genes, EPA10 and EPA22. Furthermore, hypersensitivity to hygromycin B and various ions in addition to the abnormal vacuolar morphology in the syn8Δ mutant suggested that active vacuolar function is required for biofilm formation of C. glabrata. These findings enhance our understanding of biofilm formation in this fungus and provide information for the development of future clinical treatments.

Candida albicans Bgl2p, Ecm33p, and Als1p proteins are involved in adhesion to saliva-coated hydroxyapatite.

Introduction: Candida albicans is an opportunistic pathogen that causes oral candidiasis. A previous study showed that Bgl2p and Ecm33p may mediate the interaction between the yeast and saliva-coated hydroxyapatite (SHA; a model for the tooth surface). This study investigated the roles of these cell wall proteins in the adherence of C. albicans to SHA beads. Methods: C. albicans BGL2 and ECM33 null mutants were generated from wild-type strain SC5314 by using the SAT1-flipper gene disruption method. A novel method based on labelling the yeast with Nile red, was used to investigate the adherence. Results: Adhesion of bgl2Δ and ecm33Δ null mutants to SHA beads was 76.4% and 64.8% of the wild-type strain, respectively. Interestingly, the adhesion of the bgl2Δ, ecm33Δ double mutant (87.7%) was higher than that of both single mutants. qRT-PCR analysis indicated that the ALS1 gene was over-expressed in the bgl2Δ, ecm33Δ strain. The triple null mutant showed a significantly reduced adherence to the beads, (37.6%), compared to the wild-type  strain. Conclusion: Bgl2p and Ecm33p contributed to the interaction between C. albicans and SHA beads. Deletion of these genes triggered overexpression of the ALS1 gene in the bgl2Δ/ecm33Δ mutant strain, and deletion of all three genes caused a significant decrease in adhesion.

Acyl-CoA synthetases, Aal4 and Aal7, are involved in the utilization of exogenous fatty acids in Yarrowia lipolytica.

The yeast Yarrowia lipolytica assimilates hydrophobic compounds, such as n-alkanes and fatty acids, as sole carbon and energy sources. It has been shown that the acyl-CoA synthetase (ACS) genes, FAT1 and FAA1, are involved in the activation of fatty acids produced during the metabolism of n-alkanes, but the ACS genes that are involved in the metabolism of fatty acids from the culture medium remains to be identified. In this paper, we have identified the ACS genes involved in the utilization of exogenous fatty acids. RNA-seq analysis and qRT-PCR revealed that the transcript levels of the peroxisomal ACS-like protein-encoding genes AAL4 and AAL7 were increased in the presence of oleic acid. The single deletion mutant of AAL4 or AAL7 and double deletion mutant of AAL4 and AAL7 did not show any defects in the growth on the medium containing glucose, glycerol, n-alkanes, or fatty acids. In contrast, the mutant with deletion of seven genes, FAA1, FAT1-FAT4, AAL4, and AAL7, showed severe growth defects on the medium containing dodecanoic acid or oleic acid. These results suggest that Aal4p and Aal7p play important roles in the metabolism of exogenous fatty acids in collaboration with Faa1p and Fat1p-Fat4p.

Draft Genome Sequence of Naganishia liquefaciens Strain N6, Isolated from the Japan Trench.

The draft genome sequence of the deep-sea yeast Naganishia liquefaciens strain N6, isolated from the Japan Trench, is reported here. This strain was previously classified into a Cryptococcus clade. Phylogenetic analysis using the presented sequence suggests that strain N6 is in the clade of the genus Naganishia.

Identification and functional characterization of Penicillium marneffei major facilitator superfamily (MFS) transporters.

PENICILLIUM MARNEFFEI: is a thermally dimorphic fungus that causes penicilliosis, and become the third-most-common opportunistic fungal infection in immunocompromised patients in Southeast Asia. Azoles and amphotericin B have been introduced for the treatment, however, it is important to investigate possible mechanisms of azole resistance for future treatment failure. We identified 177 putative MFS transporters and classified into 17 subfamilies. Among those, members of the Drug:H+ antiporter 1 subfamily are known to confer resistance to antifungals. Out of 39 paralogs, three (encoded by PmMDR1, PmMDR2, and PmMDR3) were heterologously overexpressed in S. cerevisiae AD∆ conferred resistance to various drugs and compounds including azoles, albeit to different degrees. PmMDR1-expressing strain showed resistance to the broadest range of drugs, followed by the PmMDR3, and PmMDR2 conferred weak resistance to a limited range of drugs. We conclude that PmMDR1 and PmMDR3, may be able to serve as multidrug efflux pumps.

Fungal NOX is an essential factor for induction of TG2 in human hepatocytes.

NADPH oxidases (Nox) generate reactive oxygen species (ROS) such as superoxide anion radical (O2-) and hydrogen peroxide (H2O2). The pathogenic fungi Candida albicans and Candida glabrata enhance cellular transglutaminase 2 (TG2) activity levels in co-cultured human hepatic cells in a ROS-mediated manner. Deletion of NOX1 (CgNOX1) in C. glabrata blocks the ability of C. glabrata to induce TG2 activity. Here, we investigated whether Nox proteins from C. albicans and Saccharomyces cerevisiae are related with induction of TG2 activity in hepatic cells. C. albicans CFL11 (CaCFL11) was identified as a key factor in this fungus for hepatic TG2 induction in the co-cultures. The cfl11 mutant of C. albicans did not induce TG2 activity in hepatocytes. In addition, overexpression of YNO1, a homolog of CgNOX1, in S. cerevisiae led to induction of ROS generation and TG2 activity in hepatic cells in co-incubation experiments. These findings indicated that a fungal Nox plays a role in enhancing TG2 activity in human hepatocytes and leads to apoptosis.

Deletion of CDR1 reveals redox regulation of pleiotropic drug resistance in Candida glabrata.

Microbial cells sense the presence of xenobiotics and, in response, upregulate genes involved in pleiotropic drug resistance (PDR). In yeast, PDR activation to a major extent relies on the transcription factor Pdr1. In addition, many xenobiotics induce oxidative stress, which may upregulate PDR independently of Pdr1 activity. Mitochondria are important sources of reactive oxygen species under stressful conditions. To evaluate the relevance of this redox pathway, we studied the activation of PDR in the yeast Candida glabrata, which we treated with a mitochondrially targeted antioxidant plastoquinonyl-decyl-triphenylphosphonium and dodecyltriphenylphosphonium (C12TPP) as a control. We found that both compounds induced activation of PDR genes and decreased the intracellular concentration of the PDR transporter substrate Nile red. Interestingly, the deletion of PDR transporter gene CDR1 inhibited the decrease in Nile red accumulation induced by antioxidant plastoquinonyl-decyl-triphenylphosphonium but not that by C12TPP. Moreover, antioxidant alpha-tocopherol inhibited C12TPP-mediated activation of PDR in Δcdr1 but not in the wild-type strain. Furthermore, pre-incubation of yeast cells with low concentrations of hydrogen peroxide induced a decrease in the intracellular concentration of Nile red in Δcdr1 and Δpdr1 as well as in control cells. Deletion of PDR1 inhibited the C12TPP-induced activation of CDR1 but not that of FLR1, which is a redox-regulated PDR transporter gene. It appears that disruption of the PDR1/CDR1 regulatory circuit makes auxiliary PDR regulation mechanisms crucial. Our data suggest that redox regulation of PDR is dispensable in wild-type cells because of redundancy in the activation pathways, but is manifested upon deletion of CDR1.

Long-Chain Acyl-CoA Synthetase is Associated with the Growth of Malassezia spp.

The lipophilic fungal pathogen Malassezia spp. must acquire long-chain fatty acids (LCFAs) from outside the cell. To clarify the mechanism of LCFA acquisition, we investigated fatty acid uptake by this fungus and identified the long-chain acyl-CoA synthetase (ACS) gene FAA1 in three Malassezia spp.: M. globosa, M. pachydermatis, and M. sympodialis. These FAA1 genes could compensate for the double mutation of FAA1 and FAA4 in Saccharomyces cerevisiae, suggesting that Malassezia Faa1 protein recognizes exogenous LCFAs. MgFaa1p and MpFaa1p utilized a medium-chain fatty acid, lauric acid (C12:0). Interestingly, the ACS inhibitor, triacsin C, affected the activity of the Malassezia Faa1 proteins but not that of S. cerevisiae. Triacsin C also reduced the growth of M. globosa, M. pachydermatis, and M. sympodialis. These results suggest that triacsin C and its derivatives are potential compounds for the development of new anti-Malassezia drugs.

Role of major facilitator superfamily transporter Qdr2p in biofilm formation by Candida glabrata.

Candida glabrata represents the second-most frequent cause of candidiasis infections of the mucosa, bloodstream and genito-urinary tract in immunocompromised individuals. The incidence of C glabrata infection has increased significantly in the last two decades, mainly due to this species' abilities to resist various antifungal drugs and to form biofilms. We focused on the relationship between biofilm formation and the product of QDR2, a C glabrata member of the major facilitator superfamily (MFS) gene family, given that fungal biofilm formation limits drug penetration and is associated with persistent infection. The fungal cells in biofilms were compared between a C glabrata ∆qdr2 mutant and its wild-type strain. Cells were analysed for metabolism activity and drug susceptibility (using tetrazolium assay), adhesion activity, growth assay and intracellular pH (using flow cytometry). Compared to the wild type, the C glabrata ∆qdr2 showed lower adhesion activity and higher fluconazole susceptibility when assessed as a biofilm. The mutant also showed decreased metabolic activity during biofilm formation. Furthermore, the mutant grew more slowly under neutral-basic pH conditions. The qdr2 deletion in C glabrata resulted in an impaired ability to maintain pH homeostasis, which led in turn to a reduction of cell growth and of adherence to an artificial matrix. These results suggested that the Qdr2p function is needed for proper biofilm formation and biofilm maintenance in C glabrata as well as biofilm drug resistance towards fluconazole. Qdr2p may play an important role in C glabrata's ability to form biofilms on implanted medical devices in human bodies.

Identification and functional characterization of Candida albicans mannose-ethanolamine phosphotransferase (Mcd4p).

Glycosylphosphatidylinositol (GPI) is an important compound for the growth of fungi, because GPI-anchored proteins including glycosyltransferases and adhesins are involved in cell-wall integrity, adhesion, and nutrient uptake in this organism. In this study, we examined orf19.5244 in the genome database of the pathogenic fungus Candida albicans, a homologue of the Saccharomyces cerevisiae mannose-ethanolamine phosphotransferase gene, MCD4, which plays a role in GPI synthesis. Expression of this homologue, designated CaMCD4, restored cell growth in a defective conditional mcd4 mutant of S. cerevisiae, Scmcd4t, in which expression of native MCD4 was repressed in the presence of doxycycline (Dox). Analysis of radiolabeled lipids showed that the accumulation of abnormal GPI anchor precursors in Scmcd4t decreased markedly upon expression of CaMCD4. Moreover, we constructed a single mutant (Camcd4/CaMCD4) and a conditional double mutant (Camcd4/Camcd4t) at the MCD4 locus of C. albicans. Repression of CaMCD4 expression by Dox led to a decrease in growth and appearance of abnormal morphology in C. albicans, both in vitro and in a silkworm infection model. These results suggest that CaMcd4p is indispensable for growth of C. albicans both in vitro and in infected hosts and a candidate target for the development of new antifungals.

Secreted Hydrolytic and Haemolytic Activities of Malassezia Clinical Strains.

Malassezia yeasts are opportunistic pathogens associated with a number of skin diseases in animals and humans. The free fatty acids released through these organisms' lipase and phospholipase activities trigger inflammation in the host; thus, these lipase and phospholipase activities are widely recognised as some of the most important factors in Malassezia pathogenesis. In this study, we sought to investigate and examine the relationship between these secreted hydrolytic activities and haemolytic activity in newly isolated Malassezia clinical strains. This characterisation was expected to elucidate pathogenicity of this fungus. We isolated 35 clinical strains of Malassezia spp.; the most frequently isolated species were M. sympodialis and M. furfur. Next, we analysed the hydrolytic activities of all of these clinical isolates; all of these strains (except for one M. dermatis isolate) showed detectable lipase and phospholipase activities against 4-nitrophenyl palmitate and L-α-phosphatidylcholine, dipalmitoyl, respectively. Most of the M. globosa isolates showed higher lipase activities than isolates of other Malassezia species. In terms of phospholipase activity, no significant difference was observed among species of Malassezia, although one isolate of M. globosa showed considerably higher phospholipase activity than the others. All tested strains also exhibited haemolytic activity, both as determined using 5% (v/v) sheep blood agar (halo assay) and by quantitative assay. Although all tested strains showed detectable haemolytic activity, we did not observe an apparent correlation between the secreted lipase and phospholipase activities and haemolytic activity. We infer that the haemolytic activities of Malassezia spp. are mediated by non-enzymatic factor(s) that are present in the secreted samples.