PCR‐mediated gene modification strategy for construction of fluorescent protein fusions in <i>Candida parapsilosis</i>

Gonia, Sara; Larson, Britta; Gale, Cheryl A.

doi:10.1002/yea.3141

Cited by 5 publications

(10 citation statements)

References 24 publications

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“…The introduction of eGFP by Cormack et al (103) facilitated the tagging and localization of mitochondrial carriers, mitochondrial telomere binding proteins, metabolic enzymes, as well as plasma membrane transporters in C. parapsilosis (99,100,(111)(112)(113). Constructs suitable for PCR-mediated C-terminal tagging of target genes with green, yellow, or mCherry fluorescent proteins using NAT1 as the selectable marker are also available (114). GFP-labeled strains of C. parapsilosis have already been used to study phagocytosis of macrophages (115).…”

Section: Genetic Toolboxmentioning

confidence: 99%

Candida parapsilosis: from Genes to the Bedside

et al. 2019

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SUMMARY Patients with suppressed immunity are at the highest risk for hospital-acquired infections. Among these, invasive candidiasis is the most prevalent systemic fungal nosocomial infection. Over recent decades, the combined prevalence of non-albicans Candida species outranked Candida albicans infections in several geographical regions worldwide, highlighting the need to understand their pathobiology in order to develop effective treatment and to prevent future outbreaks. Candida parapsilosis is the second or third most frequently isolated Candida species from patients. Besides being highly prevalent, its biology differs markedly from that of C. albicans, which may be associated with C. parapsilosis’ increased incidence. Differences in virulence, regulatory and antifungal drug resistance mechanisms, and the patient groups at risk indicate that conclusions drawn from C. albicans pathobiology cannot be simply extrapolated to C. parapsilosis. Such species-specific characteristics may also influence their recognition and elimination by the host and the efficacy of antifungal drugs. Due to the availability of high-throughput, state-of-the-art experimental tools and molecular genetic methods adapted to C. parapsilosis, genome and transcriptome studies are now available that greatly contribute to our understanding of what makes this species a threat. In this review, we summarize 10 years of findings on C. parapsilosis pathogenesis, including the species’ genetic properties, transcriptome studies, host responses, and molecular mechanisms of virulence. Antifungal susceptibility studies and clinician perspectives are discussed. We also present regional incidence reports in order to provide an updated worldwide epidemiology summary.

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Section: Genetic Toolboxmentioning

confidence: 99%

Candida parapsilosis: from Genes to the Bedside

et al. 2019

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“…Candida albicans penetration of pIECs was determined as described previously (14, 15, 24). In general, the yeast morphologic form is non-invasive whereas the hyphal form is capable of penetration with respect to H4 pIECs (14, 15).…”

Section: Methodsmentioning

confidence: 99%

“…In general, the yeast morphologic form is non-invasive whereas the hyphal form is capable of penetration with respect to H4 pIECs (14, 15). Hyphal cells were determined to be invading or not invading pIECs after 3 h of infection with C. albicans , in the presence or absence of C. parapsilosis or C. parapsilosis supernatant, using an immunocytochemical method.…”

Section: Methodsmentioning

confidence: 99%

“…The ability to undergo hyphal morphogenesis is associated with the ability of C. albicans to invade and damage various human epithelial and endothelial tissues (12, 13). In particular, our laboratory has shown that C. albicans hyphae, but not yeast forms, cause significant invasion and damage of premature intestinal epithelial cells (pIECs) (14, 15). Other Candida species that do not form hyphae, including C. parapsilosis , are essentially inert with respect to this epithelial cell line (14).…”

Section: Introductionmentioning

confidence: 99%

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Candida parapsilosis Protects Premature Intestinal Epithelial Cells from Invasion and Damage by Candida albicans

et al. 2017

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Candida is a leading cause of late-onset sepsis in premature infants and is thought to invade the host via immature or damaged epithelial barriers. We previously showed that the hyphal form of Candida albicans invades and causes damage to premature intestinal epithelial cells (pIECs), whereas the non-hyphal Candida parapsilosis, also a fungal pathogen of neonates, has less invasion and damage abilities. In this study, we investigated the potential for C. parapsilosis to modulate pathogenic interactions of C. albicans with the premature intestine. While a mixed infection with two fungal pathogens may be expected to result in additive or synergistic damage to pIECs, we instead found that C. parapsilosis was able to protect pIECs from invasion and damage by C. albicans. C. albicans-induced pIEC damage was reduced to a similar extent by multiple different C. parapsilosis strains, but strains differed in their ability to inhibit C. albicans invasion of pIECs, with the inhibitory activity correlating with their adhesiveness for C. albicans and epithelial cells. C. parapsilosis cell-free culture fractions were also able to significantly reduce C. albicans adhesion and damage to pIECs. Furthermore, coadministration of C. parapsilosis cell-free fractions with C. albicans was associated with decreased infection and mortality in zebrafish. These results indicate that C. parapsilosis is able to reduce invasion, damage, and virulence functions of C. albicans. Additionally, the results with cellular and cell-free fractions of yeast cultures suggest that inhibition of pathogenic interactions between C. albicans and host cells by C. parapsilosis occurs via secreted molecules as well as by physical contact with the C. parapsilosis cell surface. We propose that non-invasive commensals can be used to inhibit virulence features of pathogens and deserve further study as a non-pharmacological strategy to protect the fragile epithelial barriers of premature infants.

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“…Plasmids containing FP sequences, optimized for expression in Candida albicans and that can be used in the PCR-mediated gene modification strategy, have been previously constructed 2,3,4,5 . Plasmids contain FP transformation "cassettes": a FP sequence linked to a nutritional marker gene that facilitates the transformation of C. albicans and C. parapsilosis 2,3,4,5,6,7 . Currently available plasmids contain a variety of selectable nutritional marker genes (URA3, HIS1, ARG4) for transformation of auxotrophic strains as well as a dominant drug resistance marker (NAT1), which facilitates transformation of clinical strains lacking auxotrophies.…”

Section: Introductionmentioning

confidence: 99%

Generation of Fluorescent Protein Fusions in <em>Candida</em> Species

Gonia

Berman

Gale

2017

JoVE

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Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

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PCR‐mediated gene modification strategy for construction of fluorescent protein fusions in Candida parapsilosis

Cited by 5 publications

References 24 publications

Candida parapsilosis: from Genes to the Bedside

Candida parapsilosis: from Genes to the Bedside

Candida parapsilosis Protects Premature Intestinal Epithelial Cells from Invasion and Damage by Candida albicans

Generation of Fluorescent Protein Fusions in <em>Candida</em> Species

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