Gene mining, codon optimization and analysis of binding mechanism of an aldo-keto reductase with high activity, better substrate specificity and excellent solvent tolerance.

The biosynthesis of chiral alcohols has important value and high attention. Aldo-keto reductases (AKRs) mediated reduction of prochiral carbonyl compounds is an interesting way of synthesizing single enantiomers of chiral alcohols due to the high enantio-, chemo- and regioselectivity of the enzymes. However, relatively little research has been done on characterization and apply of AKRs to asymmetric synthesis of chiral alcohols. In this study, the AKR from Candida tropicalis MYA-3404 (C. tropicalis MYA-3404), was mined and characterized. The AKR shown wider optimum temperature and pH. The AKR exhibited varying degrees of catalytic activity for different substrates, suggesting that the AKR can catalyze a variety of substrates. It is worth mentioning that the AKR could catalytic reduction of keto compounds with benzene rings, such as cetophenone and phenoxyacetone. The AKR exhibited activity on N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP), a key intermediate for biosynthesis of the antidepressant drug duloxetine. Besides, the AKR still has high activity whether in a reaction system containing 10%-30% V/V organic solvent. What's more, the AKR showed the strongest stability in six common organic solvents, DMSO, acetonitrile, ethyl acetate, isopropanol, ethanol, and methanol. And, it retains more that 70% enzyme activity after 6 hours, suggesting that the AKR has strong solvent tolerance. Furthermore, the protein sequences of the AKR and its homology were compared, and a 3D model of the AKR docking with coenzyme NADPH were constructed. And the important catalytic and binding sites were identified to explore the binding mechanism of the enzyme and its coenzyme. These properties, predominant organic solvents resistance and extensive substrate spectrum, of the AKR making it has potential applications in the pharmaceutical field.

Stepwise metabolic engineering of Candida tropicalis for efficient xylitol production from xylose mother liquor.

BACKGROUND: Commercial xylose purification produces xylose mother liquor (XML) as a major byproduct, which has become an inexpensive and abundant carbon source. A portion of this XML has been used to produce low-value-added products such as caramel but the remainder often ends up as an organic pollutant. This has become an issue of industrial concern. In this study, a uracil-deficient Candida tropicalis strain was engineered to efficiently convert XML to the commercially useful product xylitol. RESULTS: The xylitol dehydrogenase gene was deleted to block the conversion of xylitol to xylulose. Then, an NADPH regeneration system was added through heterologous expression of the Yarrowia lipolytica genes encoding 6-phosphate-gluconic acid dehydrogenase and 6-phosphate-glucose dehydrogenase. After process optimization, the engineered strain, C. tropicalis XZX-B4ZG, produced 97.10 g L- 1 xylitol in 120 h from 300 g L- 1 XML in a 5-L fermenter. The xylitol production rate was 0.82 g L- 1 h- 1 and the conversion rate was 92.40 %. CONCLUSIONS: In conclusion, this study performed a combination of metabolic engineering and process optimizing in C. tropicalis to enhance xylitol production from XML. The use of C. tropicalis XZX-B4ZG, therefore, provided a convenient method to transform the industrial by-product XML into the useful material xylitol.

Fe(III)-complex mediated bacterial cell surface immobilization of eGFP and enzymes.

We report a facile and reversible method to immobilize a broad range of His6-tagged proteins on the E. coli cell surface through Fe(iii)-metal complexes. A His6-tagged eGFP and four His6-tagged enzymes were successfully immobilized on the cell surface. Additionally, a hydrogel sheath around E. coli cells was generated by immobilized His6-tagged HRP.

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡.

Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.

Improvement of enzymatic bioxylitol production from sawdust hemicellulose: optimization of parameters.

Enzymatic production of bioxylitol from lignocellulosic biomass (LCB) provides a promising alternative to both chemical and fermentative routes. This study aimed to assess the impacts of catalytic variables on bioxylitol production from wood sawdust using xylose reductase (XR) enzyme and to optimize the bioprocess. Enzyme-based xylitol production was carried out in batch cultivation under various experimental conditions to obtain maximum xylitol yield and productivity. The response surface methodology (RSM) was followed to fine-tune the most significant variables such as reaction time, temperature, and pH, which influence the synthesis of bioxylitol from sawdust hydrolysate and to optimize them. The optimum time, temperature, and pH became were 12.25 h, 35 °C, and 6.5, respectively, with initial xylose 18.8 g/L, NADPH 2.83 g/L, XR 0.027 U/mg, and agitation 100 rpm. The maximum xylitol production was attained at 16.28 g/L with a yield and productivity of 86.6% (w/w) and 1.33 g/L·h, respectively. Optimization of catalytic parameters using sequential strategies resulted in 1.55-fold improvement in overall xylitol production. This study explores a novel strategy for using sawdust hemicellulose in bioxylitol production by enzyme technology.

Increasing Long-Chain Dicarboxylic Acid Production in Candida tropicalis by Engineering Fatty Transporters.

Candida tropicalis can metabolize alkanes or fatty acids to produce long-chain dicarboxylic acids (DCAs). Fatty acid transporters located on the cell or peroxisome membrane may play an important role in this process. Using amino acid sequence homologous alignment, two putative proteins, CtFat1p and CtPxa1p, located on the cell and peroxisome membrane were found, respectively. Moreover, single- and double-knockout homologous recombination technology was used to study ctfat1p and ctpxa1p gene effects on DCA synthesis. In comparison to the wild-type strain, long-chain DCA yield decreased by 65.14%, 88.38% and 56.19% after single and double-copy knockout of ctfat1p genes and double-copy knockout of ctpxa1p genes, respectively, indicating that the knockout of ctfat1p and ctpxa1p genes had a significant effect on the conversion of oils and fats into long-chain DCAs by C. tropicalis. However, the yield of long-chain DCAs increased by 21.90% after single-knockout of the ctpxa1p gene, indicating that the single-knockout of the ctpxa1p gene may reduce fatty acid transport to peroxisome for further oxidation. Moreover, to improve the intracellular transport rate of fatty acids, ctfat1p copy number increased, increasing DCA yield by 30.10%. These results may provide useful information for enhancing the production of long-chain DCAs by C. tropicalis.

Kinome analyses of Candida albicans, C. parapsilosis and C. tropicalis enable novel kinases as therapeutic drug targets in candidiasis.

Candida spp. have attracted considerable attention as they cause serious human diseases in immunocompromised individuals. The genomes of the pathogenic Candida spp. have been sequenced, but systemic characterizations of their kinomes are yet to be reported. As in various eukaryotes, the protein kinases play crucial regulatory roles in pathogenicity of Candida. Increased frequency of antifungal resistance in Candida spp. requires significant attention to explore novel therapeutic molecules for their control. The present in-silico study involves novel bioinformatics strategies to identify the kinase proteins and their potential drug targets with the purpose to combat fungal infections. The study reports 103, 107 and 106 kinase proteins from 3 Candida spp., C. albicans, C. parapsilosis and C. tropicalis, respectively. Moreover, 79 common kinase proteins were identified, of which 54 proteins play essential roles in Candida spp. and 42 proteins were human non-homologues. Among the essential and human non-homologous protein kinases, 9 were found to be common essential human non-homologues, of which 6 are uniquely present in Candida. These 6 protein kinases namely, Hsl1, Npr1, Ptk2, Kin2, Ksp1 and orf19.3854 (CAALFM_CR06040WA) are involved in various molecular and cellular processes regulating virulence or pathogenicity. Further, these 6 kinases are prioritized as potential drug targets and explored for discovering new lead compounds against candidiasis. The drug repurposing approach for these 6 kinases show 13 approved drugs and investigational compounds that might play substantial inhibitory roles during combating candidiasis.

Rapid detection of ERG11 polymorphism associated azole resistance in Candida tropicalis.

Increasing reports of azole resistance in Candida tropicalis, highlight the development of rapid resistance detection techniques. Nonsynonymous mutations in the lanosterol C14 alpha-demethylase (ERG11) gene is one of the predominant mechanisms of azole resistance in C. tropicalis. We evaluated the tetra primer-amplification refractory mutation system-PCR (T-ARMS-PCR), restriction site mutation (RSM), and high-resolution melt (HRM) analysis methods for rapid resistance detection based on ERG11 polymorphism in C. tropicalis. Twelve azole-resistant and 19 susceptible isolates of C. tropicalis were included. DNA sequencing of the isolates was performed to check the ERG11 polymorphism status among resistant and susceptible isolates. Three approaches T-ARMS-PCR, RSM, and HRM were evaluated and validated for the rapid detection of ERG11 mutation. The fluconazole MICs for the 12 resistant and 19 susceptible isolates were 32-256 mg/L and 0.5-1 mg/L, respectively. The resistant isolates showed A339T and C461T mutations in the ERG11 gene. The T-ARMS-PCR and RSM approaches discriminated all the resistant and susceptible isolates, whereas HRM analysis differentiated all except one susceptible isolate. The sensitivity, specificity, analytical sensitivity, time, and cost of analysis suggests that these three methods can be utilized for the rapid detection of ERG11 mutations in C. tropicalis. Additionally, an excellent concordance with DNA sequencing was noted for all three methods. The rapid, sensitive, and inexpensive T-ARMS-PCR, RSM, and HRM approaches are suitable for the detection of azole resistance based on ERG11 polymorphism in C. tropicalis and can be implemented in clinical setups for batter patient management.

Structural Insights into the Interactions of Candidal Enolase with Human Vitronectin, Fibronectin and Plasminogen.

Significant amounts of enolase-a cytosolic enzyme involved in the glycolysis pathway-are exposed on the cell surface of Candida yeast. It has been hypothesized that this exposed enolase form contributes to infection-related phenomena such as fungal adhesion to human tissues, and the activation of fibrinolysis and extracellular matrix degradation. The aim of the present study was to characterize, in structural terms, the protein-protein interactions underlying these moonlighting functions of enolase. The tight binding of human vitronectin, fibronectin and plasminogen by purified C. albicans and C. tropicalis enolases was quantitatively analyzed by surface plasmon resonance measurements, and the dissociation constants of the formed complexes were determined to be in the 10-7-10-8 M range. In contrast, the binding of human proteins by the S.cerevisiae enzyme was much weaker. The chemical cross-linking method was used to map the sites on enolase molecules that come into direct contact with human proteins. An internal motif 235DKAGYKGKVGIAMDVASSEFYKDGK259 in C. albicans enolase was suggested to contribute to the binding of all three human proteins tested. Models for these interactions were developed and revealed the sites on the enolase molecule that bind human proteins, extensively overlap for these ligands, and are well-separated from the catalytic activity center.

The open reading frame 02797 from Candida tropicalis encodes a novel NADH-dependent aldehyde reductase.

Owing to its high-temperature tolerance, robustness, and wide use of carbon sources, Candida tropicalis is considered a good candidate microorganism for bioconversion of lignocellulose to ethanol. It also has the intrinsic ability to in situ detoxify aldehydes derived from lignocellulosic hydrolysis. However, the aldehyde reductases that catalyze this bioconversion in C. tropicalis remain unknown. Herein, we found that the uncharacterized open reading frame (ORF), CTRG_02797, from C. tropicalis encodes a novel and broad substrate-specificity aldehyde reductase that reduces at least seven aldehydes. This enzyme strictly depended on NADH rather than NADPH as the co-factor for catalyzing the reduction reaction. Its highest affinity (Km), maximum velocity (Vmax), catalytic rate constant (Kcat), and catalytic efficiency (Kcat/Km) were observed when reducing acetaldehyde (AA) and its enzyme activity was influenced by different concentrations of salts, metal ions, and chemical protective additives. Protein localization assay demonstrated that Ctrg_02797p was localized in the cytoplasm in C. tropicalis cells, which ensures an effective enzymatic reaction. Finally, Ctrg_02797p was grouped into the cinnamyl alcohol dehydrogenase (CADH) subfamily of the medium-chain dehydrogenase/reductase family. This research provides guidelines for exploring more uncharacterized genes with reduction activity for detoxifying aldehydes.

Exploiting the NADPH pool for xylitol production using recombinant Saccharomyces cerevisiae.

Xylitol is a five-carbon sugar alcohol that has a variety of uses in the food and pharmaceutical industries. In xylose assimilating yeasts, NAD(P)H-dependent xylose reductase (XR) catalyzes the reduction of xylose to xylitol. In the present study, XR with varying cofactor specificities was overexpressed in Saccharomyces cerevisiae to screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH-dependent enzymes (Candida tropicalis XR and S. cerevisiae Gre3p aldose reductase) were expressed, indicating that heterologous enzymes can utilize the intracellular NADPH pool more efficiently than the NADH pool, where they may face competition from native enzymes. This was confirmed by overexpression of a NADH-preferring C. tropicalis XR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of the ZWF1 gene, coding for the first enzyme of the NADPH-generating pentose phosphate pathway, was replaced with the constitutive GPD promoter in a strain expressing C. tropicalis XR. This change led to a ~12% increase in xylitol yield. Deletion of XYL2 and SOR1, whose gene products can use xylitol as substrate, did not further increase xylitol yield. Using wheat stalk hydrolysate as source of xylose, the constructed strain efficiently produced xylitol, demonstrating practical relevance of this approach.

Copper-only superoxide dismutase enzymes and iron starvation stress in Candida fungal pathogens.

Copper (Cu)-only superoxide dismutases (SOD) represent a newly characterized class of extracellular SODs important for virulence of several fungal pathogens. Previous studies of the Cu-only enzyme SOD5 from the opportunistic fungal pathogen Candida albicans have revealed that the active-site structure and Cu binding of SOD5 strongly deviate from those of Cu/Zn-SODs in its animal hosts, making Cu-only SODs a possible target for future antifungal drug design. C. albicans also expresses a Cu-only SOD4 that is highly similar in sequence to SOD5, but is poorly characterized. Here, we compared the biochemical, biophysical, and cell biological properties of C. albicans SOD4 and SOD5. Analyzing the recombinant proteins, we found that, similar to SOD5, Cu-only SOD4 can react with superoxide at rates approaching diffusion limits. Both SODs were monomeric and they exhibited similar binding affinities for their Cu cofactor. In C. albicans cultures, SOD4 and SOD5 were predominantly cell wall proteins. Despite these similarities, the SOD4 and SOD5 genes strongly differed in transcriptional regulation. SOD5 was predominantly induced during hyphal morphogenesis, together with a fungal burst in reactive oxygen species. Conversely, SOD4 expression was specifically up-regulated by iron (Fe) starvation and controlled by the Fe-responsive transcription factor SEF1. Interestingly, Candida tropicalis and the emerging fungal pathogen Candida auris contain a single SOD5-like SOD rather than a pair, and in both fungi, this SOD was induced by Fe starvation. This unexpected link between Fe homeostasis and extracellular Cu-SODs may help many fungi adapt to Fe-limited conditions of their hosts.

pH-Neutralization, Redox-Balanced Process with Coupled Formate Dehydrogenase and Glucose Dehydrogenase Supports Efficient Xylitol Production in Pure Water.

Enzymatic production of xylitol is a promising alternative to the chemical hydrogenation process. However, it encounters problems that are largely due to protein susceptibility to environmental factors. In this study, to develop a robust, practical enzymatic process for xylitol production, a coupled enzyme system consisting of formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and xylose reductase (XR) was constructed, wherein the alkaline product produced by FDH and the acidic product produced by GDH could neutralize each other during cofactor regeneration. After optimization of conditions, a pH-neutralization, redox-balanced process was developed that could be carried out in pure water requiring no pH regulation. As a result, a xylitol production of 273.6 g/L that is much higher than those yet reported was obtained from 2 M xylose in 24 h, with a relatively high productivity of 11.4 g/(L h). The strategy demonstrated here can be adapted for the production of other NADH-consuming products.

Analysis of global antifungal surveillance results reveals predominance of Erg11 Y132F alteration among azole-resistant Candida parapsilosis and Candida tropicalis and country-specific isolate dissemination.

This study evaluated the activity of echinocandins, azoles and amphotericin B against Candida spp. isolates and other yeasts and characterised azole resistance mechanisms in Candida parapsilosis and Candida tropicalis. Invasive Candida spp. isolates (n = 2936) collected in 60 hospitals worldwide during 2016-2017 underwent antifungal susceptibility testing by broth microdilution. Azole-resistant C. parapsilosis and C. tropicalis were submitted to qPCR for ERG11, CDR1 and MDR1, and the whole genome sequence was analysed. Results of non-susceptibility to echinocandins ranged from 0.0-2.3%, being highest in Candida glabrata. More than 99.0% of the Candida albicans isolates were susceptible to both fluconazole and voriconazole. Fluconazole resistance in C. glabrata was 6.5% overall, being highest in the USA (13.0%). Resistance to voriconazole in Candida krusei was only noted in the USA (5.0%). Azoles inhibited 89.1-91.6% of C. parapsilosis isolates, with most resistant isolates noted in Europe (15.1%), including 36 isolates from Italy (three hospitals), of which 34 harboured Erg11 Y132F mutations and overexpressed MDR1. Azole non-wild-type C. tropicalis (7/227) were found in five countries: 3 isolates from Thailand had the same Erg11 Y132F alteration. Fluconazole non-wild-type isolates were noted among 3/77 (3.9%) Candida dubliniensis, 4/17 (23.5%) Candida guilliermondii, 4/47 (8.5%) Candida lusitaniae and other less common yeast species. Echinocandin use has been recommended over fluconazole for invasive Candida infections. However, azoles are still active against the most common Candida spp. and resistance appears to be restricted to certain geographic regions and associated with Erg11 Y132 alterations in C. parapsilosis and C. tropicalis.

Biochemical and structural insights into the cytochrome P450 reductase from Candida tropicalis.

Cytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs). In addition, it can also reduce other proteins and molecules, including cytochrome c, ferricyanide, and different drugs. Although various CPRs have been functionally and structurally characterized, the overall mechanism and its interaction with different redox acceptors remain elusive. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called "uncoupling", whereby NAD(P)H derived electrons are lost due to the reduced intermediates' (FAD and FMN of CPR) interaction with molecular oxygen. Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species (ROS), leading to an inefficient reaction. Here, we present the study of the CPR from Candida tropicalis (CtCPR) lacking the hydrophobic N-terminal part (Δ2-22). The enzyme supports the reduction of cytochrome c and ferricyanide, with an estimated 30% uncoupling during the reactions with cytochrome c. The ROS produced was not influenced by different physicochemical conditions (ionic strength, pH, temperature). The X-ray structures of the enzyme were solved with and without its cofactor, NADPH. Both CtCPR structures exhibited the closed conformation. Comparison with the different solved structures revealed an intricate ionic network responsible for the regulation of the open/closed movement of CtCPR.

Deletion of glycerol-3-phosphate dehydrogenase genes improved 2,3-butanediol production by reducing glycerol production in pyruvate decarboxylase-deficient Saccharomyces cerevisiae.

2,3-Butanediol (2,3-BD) can be produced at high titers by engineered Saccharomyces cerevisiae by abolishing the ethanol biosynthetic pathway and introducing the bacterial butanediol-producing pathway. However, production of 2,3-BD instead of ethanol by engineered S. cerevisiae has resulted in glycerol production because of surplus NADH accumulation caused by a lower degree of reduction (γ = 5.5) of 2,3-BD than that (γ = 6) of ethanol. In order to eliminate glycerol production and resolve redox imbalance during 2,3-BD production, both GPD1 and GPD2 coding for glycerol-3-phosphate dehydrogenases were disrupted after overexpressing NADH oxidase from Lactococcus lactis. As disruption of the GPD genes caused growth defects due to limited supply of C2 compounds, Candida tropicalis PDC1 was additionally introduced to provide a necessary amount of C2 compounds while minimizing ethanol production. The resulting strain (BD5_T2 nox_dGPD1,2_CtPDC1) produced 99.4 g/L of 2,3-BD with 0.5 g/L glycerol accumulation in a batch culture. The fed-batch fermentation led to production of 108.6 g/L 2,3-BD with a negligible amount of glycerol production, resulting in a high BD yield (0.462 g2,3-BD/gglucose) corresponding to 92.4 % of the theoretical yield. These results demonstrate that glycerol-free production of 2,3-BD by engineered yeast is feasible.

Conversion mechanism of enoyl thioesters into acyl thioesters catalyzed by 2-enoyl-thioester reductases from Candida Tropicalis.

Enoyl thioester reductase from Candida tropicalis (Etr1p) catalyzes the NADPH-dependent conversion of enoyl thioesters into acyl thioesters, which are essential in fatty acid and second metabolite biosynthesis. In this paper, we explored the detailed catalytic mechanism of Etr1p by performing QM/MM calculations. Here, we focused on the formation of the covalent ene adduct intermediate and the proton transfer from Tyr79 to the substrate. Our calculation results reveal that the formation of the stable covalent ene adduct follows the Michael addition mechanism rather than the electrocyclic ene reaction. In addition, the ene adduct intermediate can reversibly decompose into the carbanion, and the proton of Tyr79 undertakes a direct electrophilic attack on the substrate to yield the product. In addition, three crystal water molecules do not participate in the catalytic reaction, but they play a crucial role in the hydride transfer and the proton transfer processes by forming a hydrogen bond network. These findings presented here would benefit our understanding of the catalytic mechanism of the NADPH-dependent enzyme.

Mechanistic investigations in ultrasound-assisted xylitol fermentation.

This study has investigated ultrasound-assisted xylitol production through fermentation of dilute acid (pentose-rich) hydrolysate of sugarcane bagasse using free cells of Candida tropicalis. Sonication of fermentation mixture at optimum conditions was carried out in ultrasound bath (37 kHz and 10% duty cycle). Time profiles of substrate and product in control (mechanical shaking) and test (mechanical shaking + sonication) fermentations were fitted to kinetic model using Genetic Algorithm (GA) optimization. Max. xylitol yield of 0.56 g/g and 0.61 g/g of xylose was achieved in control and test fermentations, respectively. The biomass yield also increased marginally (∼17%) with sonication. However, kinetics of fermentation increased drastically (2.5×) with sonication with 2× rise in xylose uptake and utilization by the cells. With comparative analysis of kinetic parameters in control and test experiments, this result was attributed to enhanced permeability of cell membrane that allowed faster diffusion of nutrients, substrates and products across cell membrane, higher enzyme-substrate affinity, dilution of toxic components and reduced inhibition of intracellular enzymes by substrate.

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.

Protein kinase A governs growth and virulence in Candida tropicalis.

Candida tropicalis is one of the most important human fungal pathogens causing superficial infections in locations such as the oral mucosa and genital tract, as well as systemic infections with high mortality. In its sister species Candida albicans, the cyclic AMP/protein kinase A (cAMP/PKA) pathway regulates fungal adhesion and dimorphism, both of which correlate closely with virulence. CaTpk1 and CaTpk2, the catalytic subunits of PKA, not only share redundant functions in hyphal growth, adhesion, and biofilm formation, but also have distinct roles in stress responses and pathogenesis, respectively. However, studies on PKA in the emerging fungal pathogen C. tropicalis are limited. Our results suggest that Tpk1 is involved in cell wall integrity and drug tolerance. The tpk2/tpk2 mutants, which have no protein kinase A activity, have reduced hyphal growth and adhesion. In addition, the tpk1/tpk1 tpk2/tpk2 double deletion mutant demonstrated delayed growth and impaired hyphal formation. In a murine model of systemic infection, both TPK1 and TPK2 were required for full virulence. We further found that EFG1 and HWP1 expression is regulated by PKA, while BCR1, FLO8, GAL4, and RIM101 are upregulated in the tpk1/tpk1 tpk2/tpk2 mutant. This study demonstrates that Tpk1 is involved in drug tolerance and cell wall integrity, while Tpk2 serves as a key regulator in dimorphism and adhesion. Both Tpk1 and Tpk2 are required for growth and full virulence in C. tropicalis.