Candida albicans and Candida glabrata triosephosphate isomerase - a moonlighting protein that can be exposed on the candidal cell surface and bind to human extracellular matrix proteins.

BACKGROUND: Triosephosphate isomerase (Tpi1) is a glycolytic enzyme that has recently been reported also to be an atypical proteinaceous component of the Candida yeast cell wall. Similar to other known candidal "moonlighting proteins", surface-exposed Tpi1 is likely to contribute to fungal adhesion during the colonization and infection of a human host. The aim of our present study was to directly prove the presence of Tpi1 on C. albicans and C. glabrata cells under various growth conditions and characterize the interactions of native Tpi1, isolated and purified from the candidal cell wall, with human extracellular matrix proteins. RESULTS: Surface plasmon resonance measurements were used to determine the dissociation constants for the complexes of Tpi1 with host proteins and these values were found to fall within a relatively narrow range of 10- 8-10- 7 M. Using a chemical cross-linking method, two motifs of the Tpi1 molecule (aa 4-17 and aa 224-247) were identified to be directly involved in the interaction with vitronectin. A proposed structural model for Tpi1 confirmed that these interaction sites were at a considerable distance from the catalytic active site. Synthetic peptides with these sequences significantly inhibited Tpi1 binding to several extracellular matrix proteins suggesting that a common region on the surface of Tpi1 molecule is involved in the interactions with the host proteins. CONCLUSIONS: The current study provided structural insights into the interactions of human extracellular matrix proteins with Tpi1 that can occur at the cell surface of Candida yeasts and contribute to the host infection by these fungal pathogens.

Epitope-Based Peptide Vaccine Design against Fructose Bisphosphate Aldolase of Candida glabrata: An Immunoinformatics Approach.

BACKGROUND: Candida glabrata is a human opportunistic pathogen that can cause life-threatening systemic infections. Although there are multiple effective vaccines against fungal infections and some of these vaccines are engaged in different stages of clinical trials, none of them have yet been approved by the FDA. AIM: Using immunoinformatics approach to predict the most conserved and immunogenic B- and T-cell epitopes from the fructose bisphosphate aldolase (Fba1) protein of C. glabrata. Material and Method. 13 C. glabrata fructose bisphosphate aldolase protein sequences (361 amino acids) were retrieved from NCBI and presented in several tools on the IEDB server for prediction of the most promising epitopes. Homology modeling and molecular docking were performed. RESULT: The promising B-cell epitopes were AYFKEH, VDKESLYTK, and HVDKESLYTK, while the promising peptides which have high affinity to MHC I binding were AVHEALAPI, KYFKRMAAM, QTSNGGAAY, RMAAMNQWL, and YFKEHGEPL. Two peptides, LFSSHMLDL and YIRSIAPAY, were noted to have the highest affinity to MHC class II that interact with 9 alleles. The molecular docking revealed that the epitopes QTSNGGAAY and LFSSHMLDL have the lowest binding energy to MHC molecules. CONCLUSION: The epitope-based vaccines predicted by using immunoinformatics tools have remarkable advantages over the conventional vaccines in that they are more specific, less time consuming, safe, less allergic, and more antigenic. Further in vivo and in vitro experiments are needed to prove the effectiveness of the best candidate's epitopes (QTSNGGAAY and LFSSHMLDL). To the best of our knowledge, this is the first study that has predicted B- and T-cell epitopes from the Fba1 protein by using in silico tools in order to design an effective epitope-based vaccine against C. glabrata.

The involvement of the Candida glabrata trehalase enzymes in stress resistance and gut colonization.

Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.

Roles of Elm1 in antifungal susceptibility and virulence in Candida glabrata.

Elm1 is a serine/threonine kinase involved in multiple cellular functions, including cytokinesis, morphogenesis, and drug resistance in Saccharomyces cerevisiae; however, its roles in pathogenic fungi have not been reported. In this study, we created ELM1-deletion, ELM1-reconstituted, ELM1-overexpression, and ELM1-kinase-dead strains in the clinically important fungal pathogen Candida glabrata and investigated the roles of Elm1 in cell morphology, stress response, and virulence. The elm1Δ strain showed elongated morphology and a thicker cell wall, with analyses of cell-wall components revealing that this strain exhibited significantly increased chitin content relative to that in the wild-type and ELM1-overexpression strains. Although the elm1Δ strain exhibited slower growth than the other two strains, as well as increased sensitivity to high temperature and cell-wall-damaging agents, it showed increased virulence in a Galleria mellonella-infection model. Moreover, loss of Elm1 resulted in increased adhesion to agar plates and epithelial cells, which represent important virulence factors in C. glabrata. Furthermore, RNA sequencing revealed that expression levels of 30 adhesion-like genes were elevated in the elm1Δ strain. Importantly, all these functions were mediated by the kinase activity of Elm1. To our knowledge, this is the first report describing the functional characterization of Elm1 in pathogenic fungi.

Candida glabrata peroxiredoxins, Tsa1 and Tsa2, and sulfiredoxin, Srx1, protect against oxidative damage and are necessary for virulence.

Candida glabrata is an opportunistic fungal pathogen that can cause life-threatening infections in immunocompromised patients. To ensure a successful infection, C. glabrata has evolved a variety of strategies to avoid killing within the host. One of these strategies is the resistance to oxidative stress. Here we show that the sulfiredoxin Srx1 and the peroxiredoxins, Tsa1 and Tsa2, are implicated in the oxidative stress response (OSR) and required for virulence. We analyzed null mutations in SRX1, TSA1 and TSA2 and showed that TSA2 and SRX1 are required to respond to oxidative stress. While TSA1 expression is constitutive, SRX1 and TSA2 are induced in the presence of H2O2 in a process dependent on H2O2 concentration and on both transcription factors Yap1 and Skn7. Msn2 and Msn4 are not necessary for the regulation of SRX1, TSA1 and TSA2. Interestingly, TSA1 and TSA2, which are localized in the cytoplasm, are induced in the presence of neutrophils and required for survival in these phagocytic cells.

Inhibiting Fungal Echinocandin Resistance by Small-Molecule Disruption of Geranylgeranyltransferase Type I Activity.

Echinocandin resistance in Candida is a great concern, as the echinocandin drugs are recommended as first-line therapy for patients with invasive candidiasis. However, therapeutic efforts to thwart echinocandin resistance have been hampered by a lack of fungal specific drug targets. Here, we show that deleting CDC43, the ß subunit of geranylgeranyltransferase type I (GGTase I), confers hypersensitivity to echinocandins, which renders GGTase I a tractable target in combatting echinocandin resistance. The membrane localization of Rho1, which is critical for (1,3)-ß-d-glucan synthase Fks1 activation, is disrupted in the cdc43 mutant, resulting in decreased amounts of glucans in the cell wall, thereby exacerbating the cell wall stress upon caspofungin addition. Guided by this insight, we found that selective chemical inhibition of GGTase I by L-269289 potentiates echinocandin activity and renders echinocandin-resistant Candida albicans responsive to treatment in vitro and in animal models for disseminated infection. Furthermore, L-269289 and echinocandins also act in a synergistic manner for the treatment of Candida tropicalis and Candida parapsilosis Importantly, deletion of CDC43 is lethal in Candida glabrata L-269289 is active on its own to kill C. glabrata, and its fungicidal activity is enhanced when combined with caspofungin. Thus, targeting GGTase I has therapeutic potential to address the clinical challenge of echinocandin-resistant candidiasis.

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.

Novel FKS1 and FKS2 modifications in a high-level echinocandin resistant clinical isolate of Candida glabrata.

Echinocandin resistance in Candida glabrata poses a serious clinical challenge. The underlying resistance mechanism of a pan-echinocandin-resistant C. glabrata isolate (strain L74) was investigated in this study. FKS mutants carrying specific mutations found in L74 were reconstructed by the Alt-R CRISPR-Cas9 system (Fks1 WT/Fks2-E655K, strain CRISPR 31) and site-directed mutagenesis (strain fks1Δ/Fks2-E655K). Sequence analysis of strain L74 revealed a premature stop codon W508stop in FKS1 and an E655K mutation preceding the hotspot 1 region in FKS2. Introduction of the Fks2-E655K mutation in ATCC 2001 (strain CRISPR 31) conferred a modest reduction in susceptibility. However, the same FKS2 mutation in the fks1Δ background (strain fks1Δ/Fks2-E655K) resulted in high levels of resistance to echinocandins. Glucan synthase isolated from L74 was dramatically less sensitive to micafungin (MCF) relative to ATCC 2001. Both FKS1/FKS2 transcript ratios and Fks1/Fks2 protein ratios were significantly lower in L74 and fks1Δ/Fks2-E655K compared to ATCC 2001 and CRISPR 31 (P <0.05). Mice challenged with CRISPR 31 and fks1Δ/Fks2-E655K mutants failed to respond to MCF. In conclusion, the high-level of echinocandin resistance in the clinical isolate of C. glabrata L74 was concluded to result from the combination of null function of Fks1 and the point mutation E655K in Fks2.

Molecular studies of NAD- and NADP-glutamate dehydrogenases decipher the conundrum of yeast-hypha dimorphism in zygomycete Benjaminiella poitrasii.

Benjaminiella poitrasii, a zygomycete, shows glucose- and temperature-dependent yeast (Y)-hypha (H) dimorphic transition. Earlier, we reported the biochemical correlation of relative proportion of NAD- and NADP-glutamate dehydrogenases (GDHs) with Y-H transition. Further, we observed the presence of one NAD-GDH and two form-specific NADP-GDH isoenzymes in B. poitrasii. However, molecular studies are necessary to elucidate the explicit role of GDHs in regulating Y-H reversible transition. Here, we report the isolation and characterization of one NAD (BpNADGDH, 2.643 kb) and two separate genes, BpNADPGDH I (Y-form specific, 1.365 kb) and BpNADPGDH II (H-form specific, 1.368 kb) coding for NADP-GDH isoenzymes in B. poitrasii. The transcriptional profiling during Y-H transition showed higher BpNADPGDH I expression in Y cells while expression of BpNADPGDH II was higher in H cells. Moreover, the yeast-form monomorphic mutant (Y-5) did not show BpNADPGDH II expression under normal dimorphism triggering conditions. Transformation with H-form specific BpNADPGDH II induced the germ tube formation in Y-5, which confirmed the cause-effect relationship between BpNADPGDH genes and morphological outcome in B. poitrasii. Interestingly, expression of H-form specific BpNADPGDH II also induced germ tube formation in human pathogenic, non-dimorphic yeast Candida glabrata, which further corroborated our findings.

Glyoxylate cycle gene ICL1 is essential for the metabolic flexibility and virulence of Candida glabrata.

The human fungal pathogen Candida glabrata appears to utilise unique stealth, evasion and persistence strategies in subverting the onslaught of host immune response during systemic infection. However, macrophages actively deprive the intracellular fungal pathogen of glucose, and therefore alternative carbon sources probably support the growth and survival of engulfed C. glabrata. The present study aimed to investigate the role of the glyoxylate cycle gene ICL1 in alternative carbon utilisation and its importance for the virulence of C. glabrata. The data showed that disruption of ICL1 rendered C. glabrata unable to utilise acetate, ethanol or oleic acid. In addition, C. glabrata icl1∆ cells displayed significantly reduced biofilm growth in the presence of several alternative carbon sources. It was also found that ICL1 is crucial for the survival of C. glabrata in response to macrophage engulfment. Disruption of ICL1 also conferred a severe attenuation in the virulence of C. glabrata in the mouse model of invasive candidiasis. In conclusion, a functional glyoxylate cycle is essential for C. glabrata to utilise certain alternative carbon sources in vitro and to display full virulence in vivo. This reinforces the view that antifungal drugs that target fungal Icl1 have potential for future therapeutic intervention.

Inhibition of recombinant enzyme 3-hydroxy-3-methylglutaryl-CoA reductase from Candida glabrata by α-asarone-based synthetic compounds as antifungal agents.

Due to increasing resistance of Candida species to antifungal drugs, especially azoles, new drugs are needed. The proposed compounds 3 and 4 are analogous to α-asarone (2), a naturally occurring potent inhibitor of HMGR with hypolipidemic and antifungal activity. We used the recombinant enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase of Candida glabrata (CgHMGR) as a model to test the effectiveness of the test compounds. Compounds 3 and 4 demonstrated inhibitory kinetics, having lower IC50 values (42.65 µM and 28.77 µM, respectively) than compound 2 (>100 µM). The docking studies showed better binding energies for compounds 3 and 4 (-5.35 and -6.1 kcal/mol, respectively) than for compound 2 (-4.53 kcal/mol). These findings suggest that the tested compounds are better than their natural analogue. Plaque assays were performed on the C. glabrata strain CBS138 by applying ergosterol or cholesterol to evaluate the possible reversal of the inhibition induced by compounds 2, 3 and 4. Inhibition was easily suppressed in all three cases, recovering the viability of C. glabrata. These results reveal that the CgHMGR model is excellent for testing antifungals. Compound 4 produced the best effect and is herein proposed as a new potent antifungal agent.

Benzenesulfonamides incorporating nitrogenous bases show effective inhibition of ß-carbonic anhydrases from the pathogenic fungi Cryptococcus neoformans, Candida glabrata and Malassezia globosa.

There is an urgent need for new chemotherapic agents to treat human fungal infections due to emerging and spreading globally resistance mechanisms. Among the new targets that have been recently investigated for the development of antifungal drugs there are the metallo-enzymes Carbonic Anhydrases (CAs, EC 4.2.1.1). The inhibition of the ß-CAs identified in many pathogenic fungi leads to an impairment of parasite growth and virulence, which in turn leads to a significant anti-infective effect. Based on antifungal nucleoside antibiotics, the inhibition of the ß-CAs from the resistance-showing fungi Candida glabrata (CgNce103), Cryptococcus neoformans (Can2) and Malasszia globosa (MgCA) with a series of benzenesulfonamides bearing nitrogenous bases, such as uracil and adenine, is here reported. Many such compounds display low nanomolar (<100 nM) inhibitory potency against Can2 and CgNce103, whereas the activity of MgCA is considerably less affected (inhibition constants in the range 138.8-5601.5 nM). The ß-CAs inhibitory data were compared with those against α-class human ubiquitous isoforms. Interesting selective inhibitory activities for the target fungal CAs over hCA I and II were reported, which make nitrogenous base benzenesulfonamides interesting tools and leads for further investigations in search of new antifungal with innovative mechanisms of action.

Vacuolar proton-translocating ATPase is required for antifungal resistance and virulence of Candida glabrata.

Vacuolar proton-translocating ATPase (V-ATPase) is located in fungal vacuolar membranes. It is involved in multiple cellular processes, including the maintenance of intracellular ion homeostasis by maintaining acidic pH within the cell. The importance of V-ATPase in virulence has been demonstrated in several pathogenic fungi, including Candida albicans. However, it remains to be determined in the clinically important fungal pathogen Candida glabrata. Increasing multidrug resistance of C. glabrata is becoming a critical issue in the clinical setting. In the current study, we demonstrated that the plecomacrolide V-ATPase inhibitor bafilomycin B1 exerts a synergistic effect with azole antifungal agents, including fluconazole and voriconazole, against a C. glabrata wild-type strain. Furthermore, the deletion of the VPH2 gene encoding an assembly factor of V-ATPase was sufficient to interfere with V-ATPase function in C. glabrata, resulting in impaired pH homeostasis in the vacuole and increased sensitivity to a variety of environmental stresses, such as alkaline conditions (pH 7.4), ion stress (Na+, Ca2+, Mn2+, and Zn2+ stress), exposure to the calcineurin inhibitor FK506 and antifungal agents (azoles and amphotericin B), and iron limitation. In addition, virulence of C. glabrata Δvph2 mutant in a mouse model of disseminated candidiasis was reduced in comparison with that of the wild-type and VPH2-reconstituted strains. These findings support the notion that V-ATPase is a potential attractive target for the development of effective antifungal strategies.

Azole Resistance Reduces Susceptibility to the Tetrazole Antifungal VT-1161.

Tetrazole antifungals designed to target fungal lanosterol 14α-demethylase (LDM) appear to be effective against a range of fungal pathogens. In addition, a crystal structure of the catalytic domain of Candida albicans LDM in complex with the tetrazole VT-1161 has been obtained. We have addressed concern about artifacts that might arise from crystallizing VT-1161 with truncated recombinant CYP51s and measured the impact on VT-1161 susceptibility of genotypes known to confer azole resistance. A yeast system was used to overexpress recombinant full-length Saccharomyces cerevisiae LDM with a C-terminal hexahistidine tag (ScLDM6×His) for phenotypic analysis and crystallographic studies with VT-1161 or with the widely used triazole drug posaconazole (PCZ). We determined the effect of characterized mutations in LDM on VT-1161 activity and identified drug efflux pumps from fungi, including key fungal pathogens, that efflux VT-1161. The relevance of these yeast-based observations on drug efflux was verified using clinical isolates of C. albicans and Candida glabrata VT-1161 binding elicits a significant conformational difference between the full-length and truncated enzymes not found when posaconazole is bound. Susceptibility to VT-1161 is reduced by ATP-binding cassette (ABC) and major facilitator superfamily (MFS) drug efflux pumps, the overexpression of LDM, and mutations within the drug binding pocket of LDM that affect interaction with the tertiary alcohol of the drug.

Spontaneous Mutational Frequency and FKS Mutation Rates Vary by Echinocandin Agent against Candida glabrata.

Echinocandins are front-line agents for treatment of invasive candidiasis. There are no reported agent-specific differences in Candida mutational frequency of resistance or propensity to develop FKS mutations. The objective of this study was to measure spontaneous and FKS mutation rates among Candida glabrata strains. Twenty bloodstream isolates from patients with or without prior echinocandin exposure were included. Minimum inhibitory concentrations (MICs), minimum fungicidal concentrations (MFCs), and mutation prevention concentrations were higher for caspofungin than for anidulafungin (P < 0.0001) and micafungin (P < 0.0001). Mutational frequencies of resistance at 3× the baseline MIC were highest for caspofungin and lowest for micafungin. A total of 247 isolates were recovered at or above the MFC for caspofungin (n = 159), anidulafungin (n = 74), or micafungin (n = 14). Agent-specific MIC increases were noted for anidulafungin and caspofungin, but not micafungin. Thirty-three percent of isolates harbored hot spot mutations in FKS1 (n = 6) or FKS2 (n = 76). Mutations at the Ser629 (Fks1) or Ser663 (Fks2) loci were more common after selection with anidulafungin or micafungin than with caspofungin (P = 0.003). Four isolates demonstrated >4-fold increases in MICs without FKS hot spot mutations; three of these harbored Fks2 mutations upstream of hot spot 1. The final isolate was FKS1 and FKS2 wild-type, but the 50% inhibitory concentrations of caspofungin and micafungin were increased 2.7- and 8-fold, respectively. In conclusion, micafungin may be superior in vitro to the other agents in limiting the emergence of resistance among C. glabrata Caspofungin exposure may be most likely to promote resistance development. These data provide a foundation for future investigations of newly developed echinocandin agents.

Spatial modulation and cofactor engineering of key pathway enzymes for fumarate production in Candida glabrata.

Fumarate is a naturally occurring organic acid that is an intermediate of the tricarboxylic acid (TCA) cycle and has numerous applications in food, pharmaceutical, and chemical industries. However, microbial fumarate production from renewable feedstock is limited by the intrinsic inefficiency of its synthetic pathway caused by week metabolites transportation and cofactor imbalance. In this study, spatial modulation and cofactor engineering of key pathway enzymes in the reductive TCA pathway were performed for the development of a Candida glabrata strain capable of efficiently producing fumarate. Specifically, DNA-guided scaffold system was first constructed and optimized to modulate pyruvate carboxylase, malate dehydrogenase, and fumarase, increasing the fumarate titer from 0.18 to 11.3 g/L. Then, combinatorially tuning cofactor balance by controlling the expression strengths of adenosine diphosphate-dependent phosphoenolpyruvate carboxykinase and NAD+ -dependent formate dehydrogenase led to a large increase in fumarate production up to 18.5 g/L. Finally, the engineered strain T.G-4G-S(1:1:2) -P(M) -F(H) was able to produce 21.6 g/L fumarate in a 5-L batch bioreactor. This strategy described here, paves the way to develop efficient cell factories for the production of the other industrially useful chemicals.

Fluconazole-induced actin cytoskeleton remodeling requires phosphatidylinositol 3-phosphate 5-kinase in the pathogenic yeast Candida glabrata.

Known azole antifungal resistance mechanisms include mitochondrial dysfunction and overexpression of the sterol biosynthetic target enzyme and multidrug efflux pumps. Here, we identify, through a genetic screen, the vacuolar membrane-resident phosphatidylinositol 3-phosphate 5-kinase (CgFab1) to be a novel determinant of azole tolerance. We demonstrate for the first time that fluconazole promotes actin cytoskeleton reorganization in the emerging, inherently less azole-susceptible fungal pathogen Candida glabrata, and genetic or chemical perturbation of actin structures results in intracellular sterol accumulation and azole susceptibility. Further, CgFAB1 disruption impaired vacuole homeostasis and actin organization, and the F-actin-stabilizing compound jasplakinolide rescued azole toxicity in cytoskeleton defective-mutants including the Cgfab1Δ mutant. In vitro assays revealed that the actin depolymerization factor CgCof1 binds to multiple lipids including phosphatidylinositol 3,5-bisphosphate. Consistently, CgCof1 distribution along with the actin filament-capping protein CgCap2 was altered upon both CgFAB1 disruption and fluconazole exposure. Altogether, these data implicate CgFab1 in azole tolerance through actin network remodeling. Finally, we also show that actin polymerization inhibition rendered fluconazole fully and partially fungicidal in azole-susceptible and azole-resistant C. glabrata clinical isolates, respectively, thereby, underscoring the role of fluconazole-effectuated actin remodeling in azole resistance.

Aspartyl proteases in Candida glabrata are required for suppression of the host innate immune response.

A family of 11 cell surface-associated aspartyl proteases (CgYps1-11), also referred as yapsins, is a key virulence factor in the pathogenic yeast Candida glabrata However, the mechanism by which CgYapsins modulate immune response and facilitate survival in the mammalian host remains to be identified. Here, using RNA-Seq analysis, we report that genes involved in cell wall metabolism are differentially regulated in the Cgyps1-11Δ mutant. Consistently, the mutant contained lower ß-glucan and mannan levels and exhibited increased chitin content in the cell wall. As cell wall components are known to regulate the innate immune response, we next determined the macrophage transcriptional response to C. glabrata infection and observed differential expression of genes implicated in inflammation, chemotaxis, ion transport, and the tumor necrosis factor signaling cascade. Importantly, the Cgyps1-11Δ mutant evoked a different immune response, resulting in an enhanced release of the pro-inflammatory cytokine IL-1ß in THP-1 macrophages. Further, Cgyps1-11Δ-induced IL-1ß production adversely affected intracellular proliferation of co-infected WT cells and depended on activation of spleen tyrosine kinase (Syk) signaling in the host cells. Accordingly, the Syk inhibitor R406 augmented intracellular survival of the Cgyps1-11Δ mutant. Finally, we demonstrate that C. glabrata infection triggers elevated IL-1ß production in mouse organs and that the CgYPS genes are required for organ colonization and dissemination in the murine model of systemic infection. Altogether, our results uncover the basis for macrophage-mediated killing of Cgyps1-11Δ cells and provide the first evidence that aspartyl proteases in C. glabrata are required for suppression of IL-1ß production in macrophages.

Structural analysis of mtEXO mitochondrial RNA degradosome reveals tight coupling of nuclease and helicase components.

Nuclease and helicase activities play pivotal roles in various aspects of RNA processing and degradation. These two activities are often present in multi-subunit complexes from nucleic acid metabolism. In the mitochondrial exoribonuclease complex (mtEXO) both enzymatic activities are tightly coupled making it an excellent minimal system to study helicase-exoribonuclease coordination. mtEXO is composed of Dss1 3'-to-5' exoribonuclease and Suv3 helicase. It is the master regulator of mitochondrial gene expression in yeast. Here, we present the structure of mtEXO and a description of its mechanism of action. The crystal structure of Dss1 reveals domains that are responsible for interactions with Suv3. Importantly, these interactions are compatible with the conformational changes of Suv3 domains during the helicase cycle. We demonstrate that mtEXO is an intimate complex which forms an RNA-binding channel spanning its entire structure, with Suv3 helicase feeding the 3' end of the RNA toward the active site of Dss1.

Efficient biosynthesis of L-phenylglycine by an engineered Escherichia coli with a tunable multi-enzyme-coordinate expression system.

Whole-cell catalysis with co-expression of two or more enzymes in a single host as a simple low-cost biosynthesis method has been widely studied and applied but hardly with regulation of multi-enzyme expression. Here we developed an efficient whole-cell catalyst for biosynthesis of L-phenylglycine (L-Phg) from benzoylformic acid through co-expression of leucine dehydrogenase from Bacillus cereus (BcLeuDH) and NAD+-dependent mutant formate dehydrogenase from Candida boidinii (CbFDHA10C) in Escherichia coli with tunable multi-enzyme-coordinate expression system. By co-expressing one to four copies of CbFDHA10C and optimization of the RBS sequence of BcLeuDH in the expression system, the ratio of BcLeuDH to CbFDH in E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was 28.4 mg L-1 min-1 g-1 dry cell weight for L-Phg production using whole-cell transformation, it's was 3.7 times higher than that of engineered E. coli without enzyme expression regulation. Under optimum conditions (pH 8.0 and 35 °C), 60 g L-1 benzoylformic acid was completely converted to pure chiral L-Phg in 4.5 h with 10 g L-1 dry cells and 50.4 g L-1 ammonium formate, and with enantiomeric excess > 99.9%. This multi-enzyme-coordinate expression system strategy significantly improved L-Phg productivity and demonstrated a novel low-cost method for enantiopure L-Phg production.