Genetic and behavioral adaptation ofCandida parapsilosisto the microbiome of hospitalized infants revealed byin situgenomics, transcriptomics and proteomics

Abstract

Candida parapsilosis is a common cause of invasive candidiasis, especially in newborn infants, and infections have been increasing over the past two decades. C. parapsilosis has been primarily studied in pure culture, leaving gaps in understanding of its function in microbiome context. Here, we reconstructed five unique C. parapsilosis genomes from premature infant fecal samples and analyzed their genome structure, population diversity and in situ activity relative to reference strains in pure culture. All five genomes contain hotspots of single nucleotide variants, some of which are shared by strains from multiple hospitals. A subset of environmental and hospital-derived genomes share variants within these hotspots suggesting derivation of that region from a common ancestor. Four of the newly reconstructed C. parapsilosis genomes have four to sixteen copies of the gene RTA3, which encodes a lipid translocase and is implicated in antifungal resistance, potentially indicating adaptation to hospital antifungal use. Time course metatranscriptomics and metaproteomics on fecal samples from a premature infant with a C. parapsilosis blood infection revealed highly variable in situ expression patterns that are distinct from those of similar strains in pure cultures. For example, biofilm formation genes were relatively less expressed in situ, whereas genes linked to oxygen utilization were more highly expressed, indicative of growth in a relatively aerobic environment. In gut microbiome samples, C. parapsilosis coexisted with Enterococcus faecalis that shifted in relative abundance over time, accompanied by changes in bacterial and fungal gene expression and proteome composition. The results reveal potentially medically relevant differences in Candida function in gut vs. laboratory environments, and constrain evolutionary processes that could contribute to hospital strain persistence and transfer into premature infant microbiomes. DECLARATIONS Ethics approval and consent to participateThis study was reviewed and approved by the University of Pittsburgh Institutional Review Board (IRB PRO12100487 and PRO10090089).

Resistance to miltefosine results from amplification of the RTA3 floppase or inactivation of flippases in Candida parapsilosis

“…In 2020, we found that the RTA3 gene has undergone amplification in 23 clinical isolates of Candida parapsilosis, resulting in increased copy number of 24-33x [42]. Variable copy numbers of RTA3 were also found by West et al [43] in four isolates. Here, by genome sequencing, we report that RTA3 amplification is very common in C. parapsilosis.…”

“…We also found that there have been multiple independent events of amplification, with at least 16 unique endpoints. West et al (2021) previously described RTA3 amplifications in 4 C. parapsilosis isolates, including one from the New York subway [43]. Those amplifications also have different endpoints, but because the data comes from metagenomics analyses, we cannot determine if their endpoints differ from the 16 CNVs we describe.…”

“…We previously showed that the RTA3 gene, encoding a putative floppase, had undergone extensive copy number amplification in 23 C. parapsilosis isolates [42]. This was also observed in a small number of isolates by West et al [43]. We therefore characterized copy number variants (CNVs) at the RTA3 locus in all sequenced isolates.…”

“…However, susceptibility to fluconazole was not affected by deleting RTA3 (Fig 3C). Increasing the RTA3 copy number correlates with increased expression, as shown by West et al [43]. However we wondered whether amplifying the upstream region (CNV-B) has the same effect as amplifying the entire open reading frame.…”

CRISPR-Cas9 editing induces Loss of Heterozygosity in the pathogenic yeast Candida parapsilosis

“…CPAR2_301940 ). C. parapsilosis is on average less heterozygous than C. albicans (0.1-0.4 SNP/kb versus 3 SNP/kb, respectively) (34, 35). To maximize the chances of detecting LOH, we selected strains in which the target gene was followed by regions containing at least some heterozygous sites.…”