Discovering the chlamydospore regulatory network in Candida albicans

A normal resident of healthy humans and warm-blooded animals, C. albicans is a commensal fungus that is also among the most common opportunistic pathogens of humans. C. albicans forms unique morphological structures called chlamydospores, which are large, spherical, thick-walled structures formed at the ends of hyphae that have unknown biological function. My goal is to discover the regulatory network controlling chlamydospore formation in C. albicans. By determining this network, we can gain insight into the biological roles of chlamydospores in the C. albicans lifestyle, better understand C. albicans morphological transitions, and determine the selective advantage (if any) provided by chlamydospores to this pathogenic fungus. To determine this regulatory network, I have screened a library of 211 C. albicans transcription factor (TF) homozygous deletion mutants to assay for their abilities to form chlamydospores under standard chlamydospore-inducing growth conditions. I have identified seven TF mutants that fail to produce any chlamydospores andthree TF mutants that produce high levels of chlamydospores relative to WT. To characterize the transcriptional changes occurring during chlamydospore formation, I have performed RNA sequencing (RNA-seq) on these identified regulator mutants to uncover the differentially regulated target genes of each chlamydospore regulator. I will use genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq) on epitope-tagged versions of these regulators to determine which genes are directly under the control of each TF. RNA-seq coupled with ChIP-seq will allow me to determine the regulatory network controlling chlamydospore formation in C. albicans.

Non-Candida albicans Candida Species: Virulence Factors and Species Identification in India

Background and Purpose: The predominant cause of candidiasis was Candida albicans which has recently changed to non-Candida albicans Candida (NCAC) (i.e., Candida spp. other than the C. albicans). The NCAC spp., earlier considered non-pathogenic or minimally virulent, are now considered a primary cause of morbidity and mortality in immunocompromised individuals. Given the NCAC spp.has become more common in clinical cases, this study aimed to determine the prevalence of NCAC spp. in different clinical specimens and assess a few of their virulence factors. Materials and Methods: Routine samples for bacterial culture and sensitivity that showed colony characteristics, like Candida on Blood Agar and microscopic features resembling Candida spp., were processed further. Candida isolates underwent tests for chlamydospore formation and biochemical tests, including sugar fermentation and sugar assimilation tests. These were grown at 42oC, and their colony color was identified using HiCrome™ Candida Differential Agar (HiMedia Laboratories Pvt. Ltd., Mumbai,India), HiCandidaTM Identification Kit (HiMedia Laboratories Pvt. Ltd., Mumbai, India),and VITEK-2® Compact (Biomérieux, France). Virulence factors, such as adherence to buccal epithelial cells (ABEC), biofilm formation, hemolytic activity, and production of coagulase enzyme were also tested. Results: Mean age of the patients was 38.46 years with a male-female ratio of 1.36:1. In total, 137 Candida isolates were recovered; 45.3%, 19.7%, and 13.9% of the isolates were isolated from urine, vaginal swabs, and oropharyngeal swabs, respectively. Moreover, 55(40.1%) isolates were those of C. albicans and 82 (59.9%) isolates belonged to NCAC spp.,with C. tropicalis (23.4%) contributing highest among NCAC species. Furthermore, C. albicans (3; 50%) was the most common spp. in cases of candidemia. Haemolysin production (85.5%) and ABEC (78.2%) were the major virulence factors in C. albicans. C.tropicalis (59.4%) and C. dubliniensis (50%) showed maximum ABEC. Biofilm forming capacity was higher in C. tropicalis (78.1%) than C. albicans (67%). Conclusion: Results of this study suggest varied prevalence and virulence based on geographical locations, even within a subcontinent. It clearly indicates the emergence of the NCAC spp. and their predominance in different body fluids. Identification of Candida to the spp. level should become a routine in all laboratories.

Cunninghamella saisamornae (Cunninghamellaceae, Mucorales), a new soil fungus from northern Thailand

A new endophytic fungus, described herein as Cunninghamella saisamornae, was isolated from soil collected from Chiang Mai Province, Thailand. This species was distinguished from the previously described Cunninghamella species by the presence of a chlamydospore formation, the lack of a zygospore stage and a maximum growth temperature of 45°C. Multi-gene phylogenetic analysis of internal transcribed spacers (ITS) and large subunit (LSU) of the nuclear ribosomal DNA (rDNA), along with the translation elongation factor 1-alpha (tef-1) genes, support the finding that C. saisamornae is distinct from other species within the genus Cunninghamella. A full description, color photographs, illustrations and a phylogenetic tree showing the position of C. saisamornae are provided.

Transcriptome Dynamics Underlying Chlamydospore Formation in Trichoderma virens GV29-8

Trichoderma spp. are widely used biocontrol agents which are antagonistic to a variety of plant pathogens. Chlamydospores are a type of propagules produced by many fungi that have thick walls and are highly resistant to adverse environmental conditions. Chlamydospore preparations of Trichoderma spp. can withstand various storage conditions, have a longer shelf life than conidial preparations and have better application potential. However, large-scale production of chlamydospores has proven difficult. To understand the molecular mechanisms governing chlamydospore formation (CF) in Trichoderma fungi, we performed a comprehensive analysis of transcriptome dynamics during CF across 8 different developmental time points, which were divided into 4 stages according to PCA analysis: the mycelium growth stage (S1), early and middle stage of CF (S2), flourishing stage of CF (S3), and late stage of CF and mycelia initial autolysis (S4). 2864, 3206, and 3630 DEGs were screened from S2 vs S1, S3 vs S2, and S4 vs S3, respectively. We then identified the pathways and genes that play important roles in each stage of CF by GO, KEGG, STC and WGCNA analysis. The results showed that DEGs in the S2 vs S1 were mainly enriched in organonitrogen compound metabolism, those in S3 vs S2 were mainly involved in secondary metabolite, cell cycle, and N-glycan biosynthesis, and DEGs in S4 vs S3 were mainly involved in lipid, glycogen, and chitin metabolic processes. We speculated that mycelial assimilation and absorption of exogenous nitrogen in the early growth stage (S1), resulted in subsequent nitrogen deficiency (S2). At the same time, secondary metabolites and active oxygen free radicals released during mycelial growth produced an adverse growth environment. The resulting nitrogen-deficient and toxin enriched medium may stimulate cell differentiation by initiating cell cycle regulation to induce morphological transformation of mycelia into chlamydospores. High expression of genes relating to glycogen, lipid, mannan, and chitin synthetic metabolic pathways during the flourishing (S3) and late stages (S4) of CF may be conducive to energy storage and cell wall construction in chlamydospores. For further verifying the functions of the amino sugar and nucleotide sugar metabolism (tre00520) pathway in the CF of T. virens GV29-8 strain, the chitin synthase gene (TRIVIDRAFT_90152), one key gene of the pathway, was deleted and resulted in the dysplasia of mycelia and an incapability to form normal chlamydospores, which illustrated the pathway affecting the CF of T. virens GV29-8 strain. Our results provide a new perspective for understanding the genetics of biochemical pathways involved in CF of Trichoderma spp.

Degenerated Virulence and Irregular Development of Fusarium oxysporum f. sp. niveum Induced by Successive Subculture

Successive cultivation of fungi on artificial media has been reported to cause the sectorization, which leads to degeneration of developmental phenotype, and virulence. Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt, forms degenerated sectors after successive cultivation. In the present research, we demonstrated that subculture with aged mycelia increased the incidence of degenerations. To further investigate the differences between the Fon wild type (sporodochial type, ST) and variants (MT: mycelial type and PT: pionnotal type), developmental phenotypes and pathogenicity to watermelon were examined. Results in variants (PT2, PT3, PT11, and MT6) were different from ST with mycelia growth, conidia production and chlamydospore formation. Virulence of degenerated variants on susceptible watermelon Grand Baby (GB) cultivar was determined after inoculation with Fon variants and Fon ST. In root dipping methods, Fon variants showed no significant differences in disease progress compared with ST. Fon variants showed a significant decrease in disease progression compared with ST through infested soil inoculation. The contrasting results of two inoculation methods suggest that the degenerative changes due to repeated successive cultivation may lead to the loss of pathogen virulence-related factors of the early stage of Fon infection process. Therefore, cell wall-degrading enzymes (CWDEs; cellulase, pectinase, and xylanase) activities of different variants were analyzed. All Fon degenerated variants demonstrated significant decreases of CWDEs activities compared with ST. Additionally, transcript levels of 9 virulence-related genes (fmk1, fgb1, pacC, xlnR, pl1, rho1, gas1, wc1, and fow1) were assessed in normal state. The degenerated variants demonstrated a significantly low level of tested virulence-related gene transcripts except for fmk1, xlnR, and fow1. In summary, the degeneration of Fon is triggered with successive subculture through aged mycelia. The degeneration showed significant impacts on virulence to watermelon, which was correlated with the reduction of CWDEs activities and declining expression of a set of virulence-related genes.

Whole Transcriptome Analysis Provides Insights Into the Molecular Mechanisms of Chlamydospore-Like Cell Formation in Phanerochaete chrysosporium

Phanerochaete chrysosporium is a white rot fungus naturally isolated from hardwoods and widely used in environmental pollution control because it produces extracellular peroxidases. It forms chlamydospores during nitrogen starvation, which naturally occurs in the habitat of P. chrysosporium. Chlamydospores protect fungi against many stresses; the molecular basis underlying chlamydospore formation in basidiomycetes is poorly explored. Chlamydospores in P. chrysosporium have a different cell wall compared with hyphae, as confirmed by cell wall digestion and microscopy. Furthermore, this study investigated the transcriptome of P. chrysosporium in different life stages, including conidium, hypha, and chlamydospore formation, through RNA sequencing. A total of 2215 differentially expressed genes were identified during these processes. The expression patterns of genes involved in several molecular events critical for chlamydospore formation, including starch and sucrose metabolism, phosphatase and kinase, and transcription factors, were determined. This study serves as a basis for further investigating the function of chlamydospore formation in the biotechnologically relevant fungus P. chrysosporium.

Degenerated virulence and irregular development of Fusarium oxysporum f. sp. niveum induced by successive subculture

Successive cultivation of fungus has been reported to cause the sectorization, which leads to degeneration of developmental phenotype, and virulence. Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt, demonstrated that successive cultivation formed the degenerated sectors. In the present research, we demonstrated that subculture with aged mycelium increased the incidence of degenerations. To further investigate the differences between the Fon wild type (sporodochial type, ST) and variants (MT: mycelium type and PT: pionnotal type), developmental phenotypes and pathogenicity to watermelon were examined. Results have shown that degeneration variants (PT2, PT3, PT11 and MT6) were different from ST with mycelium growth, conidia production and chlamydospore formation. Virulence of degenerated variants on susceptible watermelon Grand Baby (GB) cultivar was determined after inoculated with Fon variants and Fon ST. In root dipping methods, all Fon variants showed slightly increased disease severity than ST. Conversely, all Fon variants showed a significant decrease in disease progression compared with ST through infested soil inoculation. The contrary results of two inoculation methods suggest that the changes of successive cultural degeneration may lead to the loss of pathogen virulence-related factors of the early stage of Fon infection process. Therefore, Cell wall-degrading enzymes (CWDEs; cellulase, pectinase, and xylanase) activities of different variants were analysed. All Fon degenerated variants demonstrated significantly decreased of CWDEs activities compared with ST. Additionally, transcripts level of 9 virulence-related genes (fmk1, fgb1, pacC, xlnR, pl1, rho1, gas1, wc1 and fow1) were assessed in normal state. The degenerated variants demonstrated a significantly low level of tested virulence-related genes transcripts except for fmk1, xlnR and fow1. In summary, the degeneration of Fon is triggered with successive subculture through aged mycelium. The degeneration showed significant impacts on virulence to watermelon, which caused by the reduction of CWDEs activities and declining expression of a set of virulence-related genes.

The Polyketide Synthase-Encoding Gene Crpks is Involved in Clonostachys Rosea Chlamydospore Formation

Clonostachys rosea is an excellent agent for biocontrol of numerous plant fungal diseases. Polyketide synthases (PKSs) are widely distributed in plants and microorganisms and synthesize various types of polyketides. In this study, a type I PKS-encoding gene, crpks, was cloned and identified from the C. rosea 67-1 genome, and the biological function was investigated through gene knockout. The results showed that crpks deletion did not affect C. rosea morphology, ability for parasitism of sclerotia and the capacity for biocontrol of soybean Sclerotinia white mold, but had a marked influence on the chlamydospore formation ability of C. rosea. After cultivation for 48 and 72 h, chlamydospore production by ∆crpks was increased by 70.1% and 47.6%, respectively, compared to that of the wild-type strain. These data indicate that crpks is involved in C. rosea chlamydospore formation and provide useful insights into the molecular mechanisms of chlamydospore formation in C. rosea.