Metabolic engineering of Candida tropicalis for efficient 1,2,4-butanetriol production.

1,2,4-Butanetriol serves as a precursor in the manufacture of diverse pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. The study involved further modifications to an engineered Candida tropicalis strain, aimed at improving the production efficiency of 1,2,4-butanetriol. Faced with the issue of xylonate accumulation due to the low activity of heterologous xylonate dehydratase, we modulated iron metabolism at the transcriptional level to boost intracellular iron ion availability, thus enhancing the enzyme activity by 2.2-fold. Addressing the NADPH shortfall encountered during 1,2,4-butanetriol biosynthesis, we overexpressed pivotal genes in the NADPH regeneration pathway, achieving a 1,2,4-butanetriol yield of 3.2 g/L. The introduction of calcium carbonate to maintain pH balance led to an increased yield of 4 g/L, marking a 111% improvement over the baseline strain. Finally, the use of corncob hydrolysate as a substrate culminated in 1,2,4-butanetriol production of 3.42 g/L, thereby identifying a novel host for the conversion of corncob hydrolysate to 1,2,4-butanetriol.

Genetic modification of Candida maltosa, a non-pathogenic CTG species, reveals EFG1 function.

Candida maltosa is closely related to important pathogenic Candida species, especially C. tropicalis and C. albicans, but it has been rarely isolated from humans. For this reason, through comparative studies, it could be a powerful model to understand the genetic underpinnings of the pathogenicity of Candida species. Here, we generated a cohesive assembly of the C. maltosa genome and developed genetic engineering tools that will facilitate studying this species at a molecular level. We used a combination of short and long-read sequencing to build a polished genomic draft composed of 14 Mbp, 45 contigs and close to 5700 genes. This assembly represents a substantial improvement from the currently available sequences that are composed of thousands of contigs. Genomic comparison with C. albicans and C. tropicalis revealed a substantial reduction in the total number of genes in C. maltosa. However, gene loss seems not to be associated to the avirulence of this species given that most genes that have been previously associated with pathogenicity were also present in C. maltosa. To be able to edit the genome of C. maltosa we generated a set of triple auxotrophic strains so that gene deletions can be performed similarly to what has been routinely done in pathogenic Candida species. As a proof of concept, we generated gene knockouts of EFG1, a gene that encodes a transcription factor that is essential for filamentation and biofilm formation in C. albicans and C. tropicalis. Characterization of these mutants showed that Efg1 also plays a role in biofilm formation and filamentous growth in C. maltosa, but it seems to be a repressor of filamentation in this species. The genome assembly and auxotrophic mutants developed here are a key step forward to start using C. maltosa for comparative and evolutionary studies at a molecular level.

Optimization of xylanase production by Pichia kudriavzevii and Candida tropicalis isolated from the wood product workshop.

Enzymatic compounds can be found abundantly and provide numerous advantages in microbial organisms. Xylanases are used in various pharmaceutical, food, livestock, poultry, and paper industries. This study aimed to investigate xylanase-producing yeasts, xylose concentration curve and their enzymatic activity under various factors including carbon and nitrogen sources, temperature, and pH. Enzyme activity was evaluated under different conditions before, during, and after purification. The yeast strains were obtained from the wood product workshop and were subsequently cultivated on YPD (yeast extract peptone dextrose) medium. Additionally, the growth curve of the yeast and its molecular identification were conducted. The optimization and design process of xylan isolated from corn wood involved the use of Taguchi software to test different parameters like carbon and nitrogen sources, temperature, and pH, with the goal of determining the most optimal conditions for enzyme production. In addition, the Taguchi method was utilized to conduct a multifactorial optimization of xylanase enzyme activity. The isolated species were partially purified using ammonium sulfate precipitation and dialysis bag techniques. The results indicated that 3 species (8S, 18S, and 16W) after molecular identification based on 18S rRNA gene sequencing were identified as Candida tropicalis SBN-IAUF-1, Candida tropicalis SBN-IAUF-3, and Pichia kudriavzevii SBN-IAUF-2, respectively. The optimal parameters for wheat carbon source and peptone nitrogen source were found at 50 °C and pH 9.0 through single-factor optimization. By using the Taguchi approach, the best combination for highest activity was rice-derived carbon source and peptone nitrogen source at 50 °C and pH 6.0. The best conditions for xylanase enzyme production in single-factor optimization of wheat bran were 2135.6 U/mL, peptone 4475.25 U/mL, temperature 50 °C 1868 U/mL, and pH 9.0 2002.4 U/mL. Among the tested yeast, Candida tropicalis strain SBN-IAUF-1 to the access number MZ816946.1 in NCBI was found to be the best xylanase product. The highest ratio of enzyme production at the end of the delayed phase and the beginning of the logarithmic phase was concluded by comparing the growth ratio of 8S, 16W, and 18S yeasts with the level of enzymatic activity. This is the first report on the production of xylan polymer with a relative purity of 80% in Iran. The extracellular xylanases purified from the yeast species of C. tropicalis were introduced as a desirable biocatalyst due to their high enzymatic activity for the degradation of xylan polymers.

Tandem gene duplications contributed to high-level azole resistance in a rapidly expanding Candida tropicalis population.

Invasive diseases caused by the globally distributed commensal yeast Candida tropicalis are associated with mortality rates of greater than 50%. Notable increases of azole resistance have been observed in this species, particularly within Asia-Pacific regions. Here, we carried out a genetic population study on 1571 global C. tropicalis isolates using multilocus sequence typing (MLST). In addition, whole-genome sequencing (WGS) analysis was conducted on 629 of these strains, comprising 448 clinical invasive strains obtained in this study and 181 genomes sourced from public databases. We found that MLST clade 4 is the predominant azole-resistant clone. WGS analyses demonstrated that dramatically increasing rates of azole resistance are associated with a rapid expansion of cluster AZR, a sublineage of clade 4. Cluster AZR isolates exhibited a distinct high-level azole resistance, which was induced by tandem duplications of the ERG11A395T gene allele. Ty3/gypsy-like retrotransposons were found to be highly enriched in this population. The alarming expansion of C. tropicalis cluster AZR population underscores the urgent need for strategies against growing threats of antifungal resistance.

Development of engineered Candida tropicalis strain for efficient corncob-based xylitol-ethanol biorefinery.

BACKGROUND: Xylitol has a wide range of applications in the pharmaceuticals, cosmetic, food and beverage industry. Microbial xylitol production reduces the risk of contamination and is considered as environment friendly and sustainable compared to the chemical method. In this study, random mutagenesis and genetic engineering approaches were employed to develop Candida tropicalis strains with reduced xylitol dehydrogenase (XDH) activity to eliminate co-substrate requirement for corn cob-based xylitol-ethanol biorefinery. RESULTS: The results suggest that when pure xylose (10% w/v) was fermented in bioreactor, the Ethyl methane sulfonate (EMS) mutated strain (C. tropicalis K2M) showed 9.2% and XYL2 heterozygous (XYL2/xyl2Δ::FRT) strain (C. tropicalis K21D) showed 16% improvement in xylitol production compared to parental strain (C. tropicalis K2). Furthermore, 1.5-fold improvement (88.62 g/L to 132 g/L) in xylitol production was achieved by C. tropicalis K21D after Response Surface Methodology (RSM) and one factor at a time (OFAT) applied for media component optimization. Finally, corncob hydrolysate was tested for xylitol production in biorefinery mode, which leads to the production of 32.6 g/L xylitol from hemicellulosic fraction, 32.0 g/L ethanol from cellulosic fraction and 13.0 g/L animal feed. CONCLUSIONS: This work, for the first time, illustrates the potential of C. tropicalis K21D as a microbial cell factory for efficient production of xylitol and ethanol via an integrated biorefinery framework by utilising lignocellulosic biomass with minimum waste generation.

Clonal aggregation of fluconazole-resistant Candida tropicalis isolated from sterile body fluid specimens from patients in Hefei, China.

Candida tropicalis, a human conditionally pathogenic yeast, is distributed globally, especially in Asia-Pacific. The increasing morbidity and azole resistance of C. tropicalis have made clinical treatment difficult. The correlation between clonality and antifungal susceptibility of clinical C. tropicalis isolates has been reported. To study the putative correlation in C. tropicalis isolated from normally sterile body fluid specimens and explore the distinct clonal complex (CC) in Hefei, 256 clinical C. tropicalis isolates were collected from four teaching hospitals during 2016-2019, of which 30 were fluconazole-resistant (FR). Genetic profiles of 63 isolates, including 30 FR isolates and 33 fluconazole-susceptible (FS) isolates, were characterized using multilocus sequence typing (MLST). Phylogenetic analysis of the data was conducted using UPGMA (unweighted pair group method with arithmetic averages) and the minimum spanning tree algorithm. MLST clonal complexes (CCs) were analyzed using the goeBURST package. Among 35 differentiated diploid sequence types (DSTs), 16 DSTs and 1 genotype were identified as novel. A total of 35 DSTs were assigned to five major CCs based on goeBURST analysis. CC1 (containing DST376, 505, 507, 1221, 1222, 1223, 1226, and 1229) accounted for 86.7% (26/30) of the FR isolates. However, the genetic relationships among the FS isolates were relatively decentralized. The local FR CC1 belongs to a large fluconazole non-susceptible CC8 in global isolates, of which the putative founder genotype was DST225. The putative correlation between MLST types and antifungal susceptibility of clinical C. tropicalis isolates in Hefei showed that DSTs are closely related to FR clones.

A local prevalent FR CC1, accounted for 86.7% of the FR isolates in Hefei, China, which showed that fluconazole resistance is closely related to the genetic background, a finding of great value to local medical treatment and possible reasons for the increase in azole resistance of Candida tropicalis.

Fruits are vehicles of drug-resistant pathogenic Candida tropicalis.

IMPORTANCE: Of 123 identified isolates from the fruit surface, C. tropicalis was the most frequently found species, followed by Meyerozyma caribbica and Candida krusei. All three fluconazole-resistant C. tropicalis were non-susceptible to voriconazole and belonged to the same predominant genotype of azole-resistant C. tropicalis causing candidemia in patients in Taiwan. Our findings provide evidence that fruit should be washed before eaten not only to remove chemicals but also potential drug-resistant pathogenic microbes, especially for immunocompromised individuals. To keep precious treatment options in patients, we not only continuously implement antimicrobial stewardship in hospitals but also reducing/stopping the use of agricultural fungicide classes used in human medicine.

Hotspot mutations and genomic expansion of ERG11 are major mechanisms of azole resistance in environmental and human commensal isolates of Candida tropicalis.

OBJECTIVES: Infections caused by azole-resistant Candida tropicalis strains are increasing in clinical settings. The reason for this epidemical change and the mechanisms of C. tropicalis azole resistance are not fully understood. METHODS: In this study, we performed biological and genomic analyses of 239 C. tropicalis strains, including 115 environmental and 124 human commensal isolates. RESULTS: Most (99.2%) of the isolates had a baseline diploid genome. The strains from both environmental and human niches exhibit similar abilities to survive under stressful conditions and produce secreted aspartic proteases. However, the human commensal isolates exhibited a stronger ability to filament than the environmental strains. We found that 19 environmental isolates (16.5%) and 24 human commensal isolates (19.4%) were resistant to fluconazole. Of the fluconazole-resistant strains, 37 isolates (86.0%) also exhibited cross-resistance to voriconazole. Whole-genome sequencing and phylogenetic analyses revealed that both environmental and commensal isolates were widely distributed in a number of genetic clusters, but the two populations exhibited a close genetic association. The majority of fluconazole-resistant isolates were clustered within a single clade (X). CONCLUSIONS: The combination of hotspot mutations (Y132F and S154F) and genomic expansion of ERG11, which encodes the azole target lanosterol 14-α-demethylase and represents a major target of azole drugs, was a major mechanism for the development of azole resistance. The isolates carrying both hotspot mutations and genomic expansion of ERG11 exhibited cross-resistance to fluconazole and voriconazole. Moreover, the azole-resistant isolates from both the environmental and human commensal niches showed similar genotypes.

Current advances in Candida tropicalis: Yeast overview and biotechnological applications.

Candida tropicalis is a nonconventional yeast with medical and industrial significance, belonging to the CTG clade. Recent advancements in whole-genome sequencing and genetic analysis revealed its close relation to other unconventional yeasts of biotechnological importance. C. tropicalis is known for its immense potential in synthesizing various valuable biomolecules such as ethanol, xylitol, biosurfactants, lipids, enzymes, α,ω-dicarboxylic acids, single-cell proteins, and more, making it an attractive target for biotechnological applications. This review provides an update on C. tropicalis biological characteristics and its efficiency in producing a diverse range of biomolecules with industrial significance from various feedstocks. The information presented in this review contributes to a better understanding of C. tropicalis and highlights its potential for biotechnological applications and market viability.

Expression of ERG11, ERG3, MDR1 and CDR1 genes in Candida tropicalis / Expresión de los genes ERG11, ERG3, MDR1 y CDR1 en Candida tropicalis

INTRODUCTION: Drug resistance to azoles is a growing problem in the Candida genus. OBJECTIVE: To analyze molecularly the genes responsible for fluconazole resistance in Candida tropicalis strains. MATERIALS AND METHODS: Nineteen strains, with and without exposure to fluconazole, were selected for this study. The expression of MDR1, CDR1, ERG11, and ERG3 genes was analyzed in sensitive, dose-dependent sensitive, and resistant strains exposed to different concentrations of the antifungal drug. RESULTS: MDR1, ERG11 and ERG3 genes were significantly overexpressed in the different sensitivity groups. CDR1 gene expression was not statistically significant among the studied groups. Seven of the eight fluconazole-resistant strains showed overexpression of one or more of the analyzed genes. In some dose-dependent sensitive strains, we found overexpression of CDR1, ERG11, and ERG3. CONCLUSION: The frequency of overexpression of ERG11 and ERG3 genes indicates that they are related to resistance. However, the finding of dose-dependent resistant/sensitive strains without overexpression of these genes suggests that they are not exclusive to this phenomenon. More basic research is needed to study other potentially involved genes in the resistance mechanism to fluconazole.

Introducción: La farmacorresistencia a los azoles es un problema creciente en el género Candida. Objetivo: Analizar molecularmente los genes responsables de la resistencia a fluconazol en cepas de Candida tropicalis. Materiales y métodos: Para este estudio, se seleccionaron 19 cepas, con exposición a fluconazol y sin ella. Se analizó la expresión de los genes MDR1, CDR1, ERG11 y ERG3 en cepas sensibles, sensibles dependiente de la dosis, y resistentes, previamente expuestas a diferentes concentraciones del fármaco antifúngico. Resultados: Se encontró que los genes MDR1, ERG11 y ERG3 estaban significativamente sobreexpresados en los diferentes grupos de sensibilidad. La expresión del gen CDR1 no fue estadísticamente significativa entre los grupos estudiados. Siete de las ocho cepas resistentes a fluconazol mostraron sobreexpresión de uno o más de los genes analizados. En algunas cepas sensibles dependientes de la dosis, se encontró sobreexpresión de CDR1, ERG11 y ERG3. Conclusión: La sobreexpresión de los genes ERG11 y ERG3 indica que están relacionados con la resistencia de las cepas de Candida. Sin embargo, el hallazgo de cepas resistentes o sensibles según la dosis, sin sobreexpresión de estos genes, sugiere que pueden existir otros genes involucrados en este fenómeno. Se necesitan más investigaciones básicas que contribuyan al estudio de otros genes potencialmente involucrados en el mecanismo de resistencia al fluconazol.

Rice Big Grain1 enhances biomass and plant growth-promoting traits in rhizospheric yeast Candida tropicalis.

The Big Grain1 (BG1) gene of rice (Oryza sativa L.) is reported to increase the yield of rice crops; however, its molecular mechanism is largely concealed. To explore its functional prospects, we have taken a structure-function-based approach. In silico analyses suggest OsBG1 is a DNA- and phytohormone-binding protein. Heterologous expression of OsBG1 with galactose-inducible promoter GAL1p in the rhizospheric yeast Candida tropicalis SY005 revealed 7.9- and 1.5-fold higher expression of the gene at 12 and 24 h, respectively, compared to the expression at 36 h post-galactose induction. Functional activity of the induced OsBG1 in engineered yeast increased cell density, specific growth rate, and biomass by 28.5%, 29.8%, and 14.1%, respectively, and decreased the generation time by 21.25%. Flow cytometry-based cell cycle analysis of OsBG1-expressing yeast cells exhibited an increase in the cells of the G2/M population by 15.8% after 12 h of post-galactose induction. The gene expression study of yeast transformants disclosed that OsBG1 regulates cell division by upregulating the expression of the endogenous gene cyclin B1 (CtCYB1) by 1.3- and 1.9-folds at 10 and 12 h, respectively, compared to the control, and is positively influenced by the phytohormone indole acetic acid (IAA). Further, the study revealed that OsBG1 significantly increases biofilm formation, stress tolerance, and IAA production in C. tropicalis SY005, implying its prospective role in enhancing plant growth-promoting traits in microbes. OsBG1-expressing rhizospheric yeast cells significantly improved the germination and growth parameters of the bio-inoculated rice seeds. Altogether, this study suggests OsBG1 can be employed to genetically improve suitable bio-inoculants for their plant growth-promoting traits to augment crop productivity. KEY POINTS: • In silico analyses suggested OsBG1 is a phytohormone-binding transcription factor. • OsBG1 enhanced growth in rhizospheric Candida tropicalis by upregulating CtCYB1. • OsBG1 improved plant growth-promoting traits of the rhizospheric yeast C. tropicalis.

Analysis of CDR1 and MDR1 Gene Expression and ERG11 Substitutions in Clinical Candida tropicalis Isolates from Alexandria, Egypt.

INTRODUCTION: Candida tropicalis is a common non-albicans Candida (NAC) species that causes numerous fungal infections. Increasing antifungal resistance to azoles in NAC is becoming a major health problem worldwide; however, in Egypt, almost no data is available regarding fluconazole resistance mechanisms in C. tropicalis. The current study aims to investigate two possible important molecular mechanisms involved in fluconazole resistance in C. tropicalis isolates. MATERIALS: Fifty-four clinical C. tropicalis isolates were included. Identification and antifungal susceptibility profiles of the isolates were carried out using the VITEK 2 compact system. The molecular investigation of fluconazole resistance included the expression of the CDR1 and MDR1 genes by quantitative real-time RT-PCR as well as the sequence analysis of the ERG11 gene. RESULTS: Antifungal susceptibility testing identified 30 fluconazole-non-susceptible isolates. Statistically, CDR1 gene expression in fluconazole-non-susceptible isolates was significantly higher than that in fluconazole-susceptible isolates, with MDR1 gene expression levels that were similar in both non-susceptible and susceptible isolates. Sequence analysis of the ERG11 gene of 26 fluconazole-resistant isolates identified two missense mutations: A395T (Y132F) and G1390A (G464S). CONCLUSIONS: This study has highlighted the role of overexpression of the CDR1 gene and ERG11 gene mutations in fluconazole non-susceptibility. Further studies in Egypt are required to investigate other possible molecular mechanisms involved in azole resistance.

We need to talk about Candida tropicalis: Virulence factors and survival mechanisms.

Candida tropicalis is a notable species of the Candida genus representing an impressive epidemiology in tropical regions, especially in South America and Asia, where India already presents the species as the first in Candida epidemiology. Candida tropicalis has also shown a worrying antifungal resistance profile in recent years. It is essential to highlight that each pathogenic species of the Candida genus has a particular biology; however, Candida virulence factors are almost entirely based on studies with C. albicans. The intrinsic resistance of C. krusei to some azoles, the intrinsic osmotolerance of C. tropicalis, and the multidrug resistance of C. auris are just a few examples of how the biology of each Candida species is unique. Despite being a phylogenetically close species, C. tropicalis can support 15% NaCl, antagonistically metabolize and signal N-acetylglucosamine, encode 16 reported ALS genes, and other specificities discussed here compared to C. albicans. It is essential to clarify the details of the C. tropicalis infectious process, including identifying the participating secreted enzyme(s), the factors responsible for tissue damage, and the mechanisms underlying the morphogenesis and tolerance signaling pathways. In this review, we thoroughly assembled what is known about the main virulence factors of C. tropicalis, highlighting the missing pieces to stimulate further research with C. tropicalis and other non-Candida albicans species.

Genotyping and susceptibility testing uncovers large azole-resistant Candida tropicalis clade in Alexandria, Egypt.

OBJECTIVES: Candida tropicalis is an emerging medically relevant Candida species. The yeast primarily causes opportunistic infections in intensive care units and is highly prevalent in tropical countries. The genetic diversity within this species is high, and nosocomial transmission has been reported. C. tropicalis genotyping of isolates from low- and middle-income countries is underrepresented when compared with that from high-income countries. Also, in Egypt, only limited genotyping has been conducted for C. tropicalis isolates, while antifungal resistance seems to increase, especially against azoles. METHODS: Antifungal susceptibility testing was performed on 64 C. tropicalis isolates from ICU patients collected from multiple hospitals in Alexandria, Egypt. Genotyping by means of short tandem repeat (STR) and whole genome sequencing (WGS) single nucleotide polymorphism (SNP) analysis was performed. RESULTS: Using antifungal susceptibility testing, fluconazole resistance was observed in 24 isolates (38%), of which 23 harboured an ERG11 G464S substitution, previously shown to cause resistance in Candida albicans. STR genotyping showed that these 23 isolates were related, forming a distinct resistant clade. WGS SNP analysis subsequently confirmed this genetic relationship, although isolates within this clade differed in at least 429 SNPs, suggesting that these were independently introduced. CONCLUSION: Overall, STR and WGS SNP analysis of this collection indicates limited C. tropicalis nosocomial transmission in Alexandria, while the presence of this large azole-resistant C. tropicalis clade within this city hampers the treatment of intensive care unit patients.

Convergent and divergent roles of the glucose-responsive kinase SNF4 in Candida tropicalis.

The sucrose non-fermenting 1 (SNF1) complex is a heterotrimeric protein kinase complex that is an ortholog of the mammalian AMPK complex and is evolutionally conserved in most eukaryotes. This complex contains a catalytic subunit (Snf1), a regulatory subunit (Snf4) and a scaffolding subunit (Sip1/Sip2/Gal73) in budding yeast. Although the function of AMPK has been well studied in Saccharomyces cerevisiae and Candida albicans, the role of AMPK in Candida tropicalis has never been investigated. In this study, we focused on SNF4 in C. tropicalis as this fungus cannot produce a snf1Δ mutant. We demonstrated that C. tropicalis SNF4 shares similar roles in glucose derepression and is necessary for cell wall integrity and virulence. The expression of both SNF1 and SNF4 was significantly induced when glucose was limited. Furthermore, snf4Δ strains exhibited high sensitivity to many surface-perturbing agents because the strains contained lower levels of glucan, chitin and mannan. Interestingly, in contrast to C. albicans sak1Δ and snf4Δ, C. tropicalis snf4Δ exhibited phenotypes for cell aggregation and pseudohypha production. These data indicate that SNF4 performs convergent and divergent roles in C. tropicalis and possibly other unknown roles in the C. tropicalis SNF1-SNF4 AMPK pathway.

Candida tropicalis oligopeptide transporters assist in the transmembrane transport of the antimicrobial peptide CGA-N9.

Candida tropicalis is often reported as the second or third most common pathogen causing fungal infections. Antimicrobial peptides (AMPs) have attracted increasing attention for their broad-spectrum antimicrobial properties and low cytotoxicity. Our previous studies have shown that CGA-N9, a non-membrane-rupturing AMP, crosses the cell membrane to exert anticandidal activity. We speculate that there are some related transporters that assist in the transmembrane transport of CGA-N9. In this study, the relationship between CGA-N9 lethality kinetics and its real-time transmembrane amount in C. tropicalis cells was investigated. The results demonstrated that there was a positive correlation between its candicidal activity and transmembrane amount. A total of 12 oligopeptide transporter (OPT) coding sequences (CDSs) were cloned from C. tropicalis by using the conservative OPT gene sequences of Candida spp. to design primers and were named C. tropicalis OPTs (CtOPTs). The results of RT‒qPCR demonstrated that the expression levels of CtOPT1, CtOPT9 and CtOPT12 were correlated with the CGA-N9 transmembrane amount in a time-dependent manner. The results of molecular docking demonstrated that CtOPT1, CtOPT9 and CtOPT12 interact strongly with CGA-N9. Therefore, CtOPT1, CtOPT9 and CtOPT12 were predicted to assist in the transmembrane transport of the AMP CGA-N9.

Adhesion and biofilm formation by the opportunistic pathogen Candida tropicalis: what do we know?

Candida tropicalis is among the most important Candida species in terms of epidemiology, virulence and resistance. Considering the increase in C. tropicalis incidence and high rates of mortality associated with this species, knowledge of its adhesion and biofilm formation abilities is needed. These traits determine the persistence and survival of yeast on different indwelling medical devices and host sites. C. tropicalis is among the most adherent Candida species, and it has been described as a strong biofilm producer. Environmental factors, phenotypic switching and quorum sensing molecules can affect adhesion and biofilm growth. C. tropicalis can form sexual biofilms, which are promoted by mating pheromones. C. tropicalis biofilms are regulated by a wide and complex network of genes and signaling pathways that are currently poorly understood. Morphological studies showed improved biofilm architecture, which was related to the expression of several hypha-specific genes. Based on recent updates, research is still needed to increase our knowledge on the genetic network of adhesion and biofilm formation by C. tropicalis, as well as the protein diversity that mediates interactions with inert materials and biological surfaces. Here, we have reviewed the main aspects related to adhesion and biofilm formation in C. tropicalis and summarized current knowledge on the significance of these virulence factors in this opportunistic species.

Deciphering Colonies of Phenotypic Switching-Derived Morphotypes of the Pathogenic Yeast Candida tropicalis.

BACKGROUND: Phenotypic switching generates fungal colonies with altered morphology and allows pathogens to adapt to changing environments. OBJECTIVE: This study investigated the structure and genetic factors of switched morphotypes colonies in Candida tropicalis. METHODS: Morphotypes of C. tropicalis comprised the clinical strain 49.07 that exhibited smooth colony phenotype and switched (crepe and rough) morphotypes that showed colonies with marked structural variations, including wrinkled surface, depressions areas, and irregular edges (structured morphology). The morphotypes were analyzed for the presence and distribution of the extracellular matrix (ECM) at the ultrastructural level-SEM. The composition of the ECM and the percentage of hyphae in colonies were evaluated. The expression of EFG1 (Enhanced filamentous growth protein 1), WOR1 (White-opaque regulator 1), and BCR1 (Biofilm and cell wall regulator 1) in the morphotypes was measured by RT-qPCR. RESULTS: Colonies of the switched variants exhibited distinct arrangements of ECM compared to the smooth phenotype (clinical strain). In addition, rough variant colonies showed higher amounts of total carbohydrates and proteins in ECM (p < 0.05). Switched (crepe and rough) colonies exhibited a higher percentage of hyphae throughout their development (p < 0.05). The mRNA expression levels of EFG1, WOR1, and BCR1 in the rough morphotype were significantly higher than they were in the smooth morphotype. In addition, there was a positive correlation between the expression of these genes and filamentation (hyphae formation) of the rough morphotype (r2 > 0.9472, p < 0.05). CONCLUSION: Structural variations in switched morphotypes colonies of C. tropicalis seem to be associated with increased hyphae growth and the amount and distribution of ECM. Switched colonies have distinct expressions of the EFG1, WOR1, and BCR1 master regulators genes.

Biodegradation of phenol by Candida tropicalis sp.: Kinetics, identification of putative genes and reconstruction of catabolic pathways by genomic and transcriptomic characteristics.

Candida tropicalis sp. was isolated with predominant biodegradation capability to phenol compounds, even with high concentration or in acid environment. The biodegradation of phenol was evaluated at the following concentrations 10-1750 mg L-1, the strain exhibited well biodegradation efficiency. The maximum specific growth rate was 0.660 h-1 and the specific biodegradation rates was 0.47 mg (phenol) [(mg (VSS) h]-1. Differentially expressed genes were screened out, and results revealed a complete process of energy and carbon metabolism. The genes' arrangements and phylogenetic information showed the unique genetic characteristics of the strain. Catabolic pathways were reconstructed and some key phenol-degrading genes were obviously upregulated, including pheA, catA, OXCT and fadA. A notable detail that CMBL encoding carboxymethylenebutenolidase was speculated to be involved in a shortened pathway of phenol biodegradation, thereby contributing to the reconstruction of the novel phenol catabolic pathway through the hydrolases of dienelactone. Finally, key enzymes were verified by the analysis of specific activity.

Rapid Detection of Candida tropicalis in Clinical Samples From Different Sources Using RPA-LFS.

Candida tropicalis is one of the few Candida species besides Candida albicans that is able to produce true hyphae. At present, the commonly used clinical methods for the identification of this organism are traditional fungal culture, CTB staining, and color development. Polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) are also used to identify this fungus. Since the course of C. tropicalis infection progresses rapidly, there is an urgent need for rapid, sensitive, real-time field assays to meet the needs of clinical diagnosis. Recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) can rapidly amplify and visualize target genes within 20 min, and by pre-processing samples from different sources, the entire process can be controlled within 30 min. In this study, RPA-LFS was used to amplify the internal transcribed spacer-2 (ITS2) gene of C. tropicalis, and primer-probe design was optimized by introducing base mismatches to obtain a specific and sensitive primer-probe combination for clinical sample detection. LFS assay for 37 common clinical pathogens was performed, sensitivity and specificity of the detection system was determined, reaction temperature and time were optimized, and 191 actual clinical samples collected from different sources were tested to evaluate the detection performance of the established RPA-LFS system to provide a reliable molecular diagnostic method for the detection of C. tropicalis, the results show that the RPA-LFS system can specifically detect C. tropicalis without cross-reacting with other fungi or bacterial, with a sensitivity of 9.94 CFU/µL, without interference from genomic DNA of other species, at an optimal reaction temperature of 39°C, and the whole reaction process can be controlled within 20 min, and to meet the clinical need for rapid, sensitive, real-time, and portable field testing.