Attila Gácser

Candida parapsilosis, an Emerging Fungal Pathogen

SUMMARY Candida parapsilosis is an emerging major human pathogen that has dramatically increased in significance and prevalence over the past 2 decades, such that C. parapsilosis is now one of the leading causes of invasive candidal disease. Individuals at the highest risk for severe infection include neonates and patients in intensive care units. C. parapsilosis infections are especially associated with hyperalimentation solutions, prosthetic devices, and indwelling catheters, as well as the nosocomial spread of disease through the hands of health care workers. Factors involved in disease pathogenesis include the secretion of hydrolytic enzymes, adhesion to prosthetics, and biofilm formation. New molecular genetic tools are providing additional and much-needed information regarding C. parapsilosis virulence. The emerging information will provide a deeper understanding of C. parapsilosis pathogenesis and facilitate the development of new therapeutic approaches for treating C. parapsilosis infections.

Targeted gene deletion in Candida parapsilosis demonstrates the role of secreted lipase in virulence

Candida parapsilosis is a major cause of human disease, yet little is known about the pathogens virulence. We have developed an efficient gene deletion system for C. parapsilosis based on the repeated use of the dominant nourseothricin resistance marker (caSAT1) and its subsequent deletion by FLP-mediated, site-specific recombination. Using this technique, we deleted the lipase locus in the C. parapsilosis genome consisting of adjacent genes CpLIP1 and CpLIP2. Additionally we reconstructed the CpLIP2 gene, which restored lipase activity. Lipolytic activity was absent in the null mutants, whereas the WT, heterozygous, and reconstructed mutants showed similar lipase production. Biofilm formation was inhibited with lipase-negative mutants and their growth was significantly reduced in lipid-rich media. The knockout mutants were more efficiently ingested and killed by J774.16 and RAW 264.7 macrophage-like cells. Additionally, the lipase-negative mutants were significantly less virulent in infection models that involve inoculation of reconstituted human oral epithelium or murine intraperitoneal challenge. These studies represent what we believe to be the first targeted disruption of a gene in C. parapsilosis and show that C. parapsilosis-secreted lipase is involved in disease pathogenesis. This efficient system for targeted gene deletion holds great promise for rapidly enhancing our knowledge of the biology and virulence of this increasingly common invasive fungal pathogen.

Expression analysis of the Candida albicans lipase gene family during experimental infections and in patient samples

Secreted lipases of Candida albicans are encoded by a gene family with at least 10 members (LIP1-LIP10). The expression pattern of this multigene family was investigated using reverse transcription polymerase chain reaction in experimental infections and in samples of patients suffering from oral candidosis. The findings illustrate that individual lipase genes are differentially regulated in a mouse model of systemic candidosis with some members showing sustained expression and others being transiently expressed or even silent. The lipase gene expression profile depended on the stage of infection rather than on the organ localization. This temporal regulation of lipase gene expression was also detected in an experimental model of oral candidosis. Furthermore, the expression of candidal lipase genes in human specimens is shown for the first time.

Candida parapsilosis is a significant neonatal pathogen a systematic review and meta-analysis

Background: Candida is the third most common cause of late-onset neonatal sepsis in infants born at <1500 g. Candida parapsilosis infections are increasingly reported in preterm neonates in association with indwelling catheters. Methods: We systematically reviewed neonatal literature and synthesized data pertaining to percentage of C. parapsilosis infections and mortality by meta-analyses. We also reviewed risk factors, virulence determinants, antimicrobial susceptibility patterns and outlined clinical management strategies. Results: C. parapsilosis infections comprised 33.47% (95% confidence interval [CI]: 30.02, 37.31) of all neonatal Candida infections. C. parapsilosis rates were similar in studies performed before the year 2000, 33.53% (95% CI: 30.06, 37.40) (28 studies), to those after 2000, 27.00% (95% CI: 8.25, 88.37) (8 studies). The mortality due to neonatal C. parapsilosis infections was 10.02% (95% CI: 7.66, 13.12). Geographical variations in C. parapsilosis infections included a low incidence in Europe and higher incidence in North America and Australia. Biofilm formation was a significant virulence determinant and predominant risk factors for C. parapsilosis infections were prematurity, prior colonization and catheterization. Amphotericin B remains the antifungal drug of choice and combination therapy with caspofungin or other echinocandins may be considered in resistant cases. Conclusion: C. parapsilosis is a significant neonatal pathogen, comprises a third of all Candida infections and is associated with 10% mortality. Availability of tools for genetic manipulation of this organism will identify virulence determinants and organism characteristics that may explain predilection for preterm neonates. Strategies to prevent horizontal transmission in the neonatal unit are paramount in decreasing infection rates.

The PD-1/PD-L costimulatory pathway critically affects host resistance to the pathogenic fungus Histoplasma capsulatum

The PD-1 costimulatory receptor inhibits T cell receptor signaling upon interacting with its ligands PD-L1 and PD-L2. The PD-1/PD-L pathway is critical in maintaining self-tolerance. In this study, we examined the role of PD-1 in a mouse model of acute infection with Histoplasma capsulatum, a major human pathogenic fungus. In a lethal model of histoplasmosis, all PD-1-deficient mice survived infection, whereas the wild-type mice died with disseminated disease. PD-L expression on macrophages and splenocytes was up-regulated during infection, and macrophages from infected mice inhibited in vitro T cell activation. Of interest, antibody blocking of PD-1 significantly increased survival of lethally infected wild-type mice. Thus, our studies extend the role of the PD-1/PD-L pathway in regulating antimicrobial immunity to fungal pathogens. The results show that the PD-1/PD-L pathway has a key role in the regulation of antifungal immunity, and suggest that manipulation of this pathway represents a strategy of immunotherapy for histoplasmosis.

Lipase 8 Affects the Pathogenesis of Candida albicans

ABSTRACT The production of lipases can affect microbial fitness and virulence. We examined the role of the lipase 8 (LIP8) gene in the virulence of Candida albicans by constructing Δlip8 strains by the URA-blaster disruption method. Reverse transcription-PCR experiments demonstrated the absence of LIP8 expression in the homozygous knockout mutants. Reconstituted strains and overexpression mutants were generated by introducing a LIP8 open reading frame under control of a constitutive actin promoter. Knockout mutants produced more mycelium, particularly at higher temperatures and pH ≥7. Diminished LIP8 expression resulted in reduced growth in lipid-containing media. Mutants deficient in the LIP8 gene were significantly less virulent in a murine intravenous infection model. The results clearly indicate that Lip8p is an important virulence factor of C. albicans.

Latent homology and convergent regulatory evolution underlies the repeated emergence of yeasts

Convergent evolution is common throughout the tree of life, but the molecular mechanisms causing similar phenotypes to appear repeatedly are obscure. Yeasts have arisen in multiple fungal clades, but the genetic causes and consequences of their evolutionary origins are unknown. Here we show that the potential to develop yeast forms arose early in fungal evolution and became dominant independently in multiple clades, most likely via parallel diversification of Zn-cluster transcription factors, a fungal-specific family involved in regulating yeast-filamentous switches. Our results imply that convergent evolution can happen by the repeated deployment of a conserved genetic toolkit for the same function in distinct clades via regulatory evolution. We suggest that this mechanism might be a common source of evolutionary convergence even at large time scales.

Characterization of virulence properties in the C. parapsilosis sensu lato species.

The C. parapsilosis sensu lato group involves three closely related species, C. parapsilosis sensu stricto, C . orthopsilosis and C . metapsilosis . Although their overall clinical importance is dramatically increasing, there are few studies regarding the virulence properties of the species of the psilosis complex. In this study, we tested 63 C. parapsilosis sensu stricto, 12 C . metapsilosis and 18 C . orthopsilosis isolates for the ability to produce extracellular proteases, secrete lipases and form pseudohyphae. Significant differences were noted between species, with the C . metapsilosis strains failing to secrete lipase or to produce pseudohyphae. Nine different clinical isolates each of C. parapsilosis sensu stricto, C . orthopsilosis and C . metapsilosis were co-cultured with immortalized murine or primary human macrophages. C. parapsilosis sensu stricto isolates showed a significantly higher resistance to killing by primary human macrophages compared to C . orthopsilosis and C . metapsilosis isolates. In contrast, the killing of isolates by J774.2 mouse macrophages did not differ significantly between species. However, C. parapsilosis sensu stricto isolates induced the most damage to murine and human macrophages, and C . metapsilosis strains were the least toxic. Furthermore, strains that produced lipase or pseudohyphae were most resistant to macrophage-mediated killing and produced the most cellular damage. Finally, we used 9 isolates of each of the C. parapsilosis sensus lato species to examine their impact on the survival of Galleria mellonella larvae. The mortality rate of G . mellonella larvae infected with C . metapsilosis isolates was significantly lower than those infected with C. parapsilosis sensu stricto or C . orthopsilosis strains. Taken together, our findings demonstrate that C . metapsilosis is indeed the least virulent member of the psilosis group, and also highlight the importance of pseudohyphae and secreted lipases during fungal-host interactions.

Comparative Phenotypic Analysis of the Major Fungal Pathogens Candida parapsilosis and Candida albicans

Candida parapsilosis and Candida albicans are human fungal pathogens that belong to the CTG clade in the Saccharomycotina. In contrast to C. albicans, relatively little is known about the virulence properties of C. parapsilosis, a pathogen particularly associated with infections of premature neonates. We describe here the construction of C. parapsilosis strains carrying double allele deletions of 100 transcription factors, protein kinases and species-specific genes. Two independent deletions were constructed for each target gene. Growth in >40 conditions was tested, including carbon source, temperature, and the presence of antifungal drugs. The phenotypes were compared to C. albicans strains with deletions of orthologous transcription factors. We found that many phenotypes are shared between the two species, such as the role of Upc2 as a regulator of azole resistance, and of CAP1 in the oxidative stress response. Others are unique to one species. For example, Cph2 plays a role in the hypoxic response in C. parapsilosis but not in C. albicans. We found extensive divergence between the biofilm regulators of the two species. We identified seven transcription factors and one protein kinase that are required for biofilm development in C. parapsilosis. Only three (Efg1, Bcr1 and Ace2) have similar effects on C. albicans biofilms, whereas Cph2, Czf1, Gzf3 and Ume6 have major roles in C. parapsilosis only. Two transcription factors (Brg1 and Tec1) with well-characterized roles in biofilm formation in C. albicans do not have the same function in C. parapsilosis. We also compared the transcription profile of C. parapsilosis and C. albicans biofilms. Our analysis suggests the processes shared between the two species are predominantly metabolic, and that Cph2 and Bcr1 are major biofilm regulators in C. parapsilosis.

Genome comparison of Candida orthopsilosis clinical strains reveals the existence of hybrids between two distinct subspecies

The Candida parapsilosis species complex comprises a group of emerging human pathogens of varying virulence. This complex was recently subdivided into three different species: C. parapsilosis sensu stricto, C. metapsilosis, and C. orthopsilosis. Within the latter, at least two clearly distinct subspecies seem to be present among clinical isolates (Type 1 and Type 2). To gain insight into the genomic differences between these subspecies, we undertook the sequencing of a clinical isolate classified as Type 1 and compared it with the available sequence of a Type 2 clinical strain. Unexpectedly, the analysis of the newly sequenced strain revealed a highly heterozygous genome, which we show to be the consequence of a hybridization event between both identified subspecies. This implicitly suggests that C. orthopsilosis is able to mate, a so-far unanswered question. The resulting hybrid shows a chimeric genome that maintains a similar gene dosage from both parental lineages and displays ongoing loss of heterozygosity. Several of the differences found between the gene content in both strains relate to virulent-related families, with the hybrid strain presenting a higher copy number of genes coding for efflux pumps or secreted lipases. Remarkably, two clinical strains isolated from distant geographical locations (Texas and Singapore) are descendants of the same hybrid line, raising the intriguing possibility of a relationship between the hybridization event and the global spread of a virulent clone.