Polymorphism Analysis of Virulence-Related Genes Among Candida tropicalis Isolates

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Polymorphism Analysis of Virulence-Related Genes Among Candida tropicalis Isolates

Introduction
Candida species represent a significant cause of bloodstream infections globally, with Candida tropicalis emerging as a critical pathogen in immunocompromised populations, including oncology patients and individuals with prolonged hospital stays. Known for its high mortality rates, C. tropicalis exhibits virulence traits such as adhesion, biofilm formation, yeast-to-hyphal transition, and secretion of hydrolytic enzymes. Despite its clinical relevance, comprehensive genomic studies on C. tropicalis virulence genes remain limited compared to C. albicans. This study focuses on analyzing polymorphisms in key virulence-associated genes—ALS2, LIP1, LIP4, and SAPT1-4—across 68 clinical isolates, aiming to elucidate their genetic diversity and phenotypic correlations.

Genetic Polymorphisms in Virulence-Related Genes
The study sequenced the entire lengths of ALS2, LIP1, LIP4, and SAPT1-4 genes from 68 C. tropicalis isolates collected between 2013 and 2017. Single nucleotide polymorphisms (SNPs) and insertion-deletion (indel) variations were systematically cataloged.

  1. Lipase Genes (LIP1 and LIP4)

    • LIP1 (1,398 bp) exhibited high genetic variability with 73 SNPs, while LIP4 (1,392 bp) showed 24 SNPs. Despite these variations, no lipase activity was detected in any isolate, suggesting potential post-transcriptional regulation or functional redundancy.
    • Phylogenetic analysis grouped LIP1 into 66 distinct genotypes and LIP4 into 36 genotypes, highlighting significant intra-species diversity.

  2. Secretory Aspartyl Proteinase Genes (SAPT1-4)

    • SAPT1 (1,185 bp), SAPT2 (1,820 bp), SAPT3 (1,200 bp), and SAPT4 (1,920 bp) displayed 17, 16, 13, and 180 SNPs, respectively. SAPT4 stood out as the most polymorphic, correlating with its potential role in host tissue invasion.
    • Conserved regions across SAPT1-3 contrasted with SAPT4‘s hypervariability. All isolates secreted aspartyl proteases, but no direct correlation between SNP density and enzymatic activity was observed.

  3. Agglutinin-Like Sequence Gene (ALS2)

    • ALS2 (4,071 bp) showed striking structural variations, including 209 SNPs and large fragment indels. Four deletion hotspots (1482–1589, 1697–1925, 1962–2072, and 2073–2272 bp) and two insertion regions (1731–1841 and 2163–2273 bp) were identified.
    • Isolates with deletions in regions 1697–1925 and 2073–2272 bp exhibited reduced adhesion and biofilm formation on polymethylpentene (PMP) surfaces. Bloodstream isolate FXCT01, which harbored deletions across all four sites, demonstrated the lowest adhesive capacity.

Phenotypic Characterization of Virulence Traits
The study evaluated adhesion, biofilm formation, and hydrolytic enzyme activity (aspartyl protease, phospholipase, and hemolysin) across all isolates.

  1. Adhesion and Biofilm Formation

    • Adhesion assays on abiotic (polystyrene) and biotic (human urinary bladder epithelial cells) surfaces revealed strain-dependent variability. Isolate ZRCT47 exhibited the strongest biofilm formation on PMP, as confirmed by crystal violet assays.
    • Deletions in ALS2 correlated with diminished adhesion, underscoring the gene’s role in mediating host-pathogen interactions. For instance, isolates with partial ALS2 deletions showed intermediate adhesive capabilities compared to FXCT01.

  2. Enzymatic Activity

    • All isolates secreted aspartyl proteases and displayed hemolytic activity, with ZRCT28 and ZRCT47 showing exceptionally high protease and hemolysin production, respectively. However, phospholipase activity was absent in all strains.
    • Despite extensive SNP variations in SAPT1-4, enzymatic activity levels (low, medium, high) did not directly align with specific genetic markers, suggesting environmental or regulatory influences.

Phylogenetic and Evolutionary Insights
Phylogenetic trees constructed from concatenated sequences of ALS2, LIP, and SAPT gene families revealed distinct evolutionary patterns:

  • ALS2: High heterogeneity divided isolates into 60 genotypes, with indel-rich strains forming separate clades.
  • LIP Genes: Despite SNP diversity, LIP1 and LIP4 exhibited conserved evolutionary trajectories, indicating purifying selection.
  • SAPT Genes: SAPT4’s hypervariability contrasted with the relative conservation of SAPT1-3, suggesting divergent functional roles.

Clinical Implications and Mechanistic Insights
The study links genetic polymorphisms to virulence attenuation, particularly for ALS2. Large deletions in this gene compromised adhesion, a critical step in colonization and biofilm development. This aligns with clinical observations of reduced pathogenicity in strains with truncated ALS proteins. The absence of lipase activity despite LIP1/LIP4 expression implies compensatory mechanisms or alternative virulence strategies in C. tropicalis.

Conclusion
This comprehensive analysis of C. tropicalis virulence genes provides a foundation for understanding genotype-phenotype relationships in this understudied pathogen. The identification of ALS2 as a key determinant of adhesion highlights its potential as a therapeutic target. Future functional studies should explore regulatory networks governing SAPT4 expression and the mechanistic basis of lipase inactivity. These insights pave the way for improved diagnostic and therapeutic strategies against candidiasis.

doi.org/10.1097/CM9.0000000000000069

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