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1.  Kinetic studies of Candida parapsilosis phagocytosis by macrophages and detection of intracellular survival mechanisms 
Even though the number of Candida infections due to non-albicans species like C. parapsilosis has been increasing, little is known about their pathomechanisms. Certain aspects of C. parapsilosis and host interactions have already been investigated; however we lack information about the innate cellular responses toward this species. The aim of our project was to dissect and compare the phagocytosis of C. parapsilosis to C. albicans and to another Candida species C. glabrata by murine and human macrophages by live cell video microscopy. We broke down the phagocytic process into three stages: macrophage migration, engulfment of fungal cells and host cell killing after the uptake. Our results showed increased macrophage migration toward C. parapsilosis and we observed differences during the engulfment processes when comparing the three species. The engulfment time of C. parapsilosis was comparable to that of C. albicans regardless of the pseudohypha length and spatial orientation relative to phagocytes, while the rate of host cell killing and the overall uptake regarding C. parapsilosis showed similarities mainly with C. glabrata. Furthermore, we observed difference between human and murine phagocytes in the uptake of C. parapsilosis. UV-treatment of fungal cells had varied effects on phagocytosis dependent upon which Candida strain was used. Besides statistical analysis, live cell imaging videos showed that this species similarly to the other two also has the ability to survive in host cells via the following mechanisms: yeast replication, and pseudohypha growth inside of phagocytes, exocytosis of fungal cells and also abortion of host cell mitosis following the uptake. According to our knowledge this is the first study that provides a thorough examination of C. parapsilosis phagocytosis and reports intracellular survival mechanisms associated with this species.
doi:10.3389/fmicb.2014.00633
PMCID: PMC4238376  PMID: 25477874
video microscopy; phagocytic stages; Candida parapsilosis; intracellular survival mechanisms; pseudohypha uptake
2.  Comparative Phenotypic Analysis of the Major Fungal Pathogens Candida parapsilosis and Candida albicans 
PLoS Pathogens  2014;10(9):e1004365.
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.
Author Summary
Candida species are among the most common causes of fungal infection worldwide. Infections can be both community-based and hospital-acquired, and are particularly associated with immunocompromised individuals. Candida albicans is the most commonly isolated species and is the best studied. However, other species are becoming of increasing concern. Candida parapsilosis causes outbreaks of infection in neonatal wards, and is one of the few Candida species that is transferred from the hands of healthcare workers. C. parapsilosis, like C. albicans, grows as biofilms (cell communities) on the surfaces of indwelling medical devices like feeding tubes. We describe here the construction of a set of tools that allow us to characterize the virulence properties of C. parapsilosis, and in particular its ability to grow as biofilms. We find that some of the regulatory mechanisms are shared with C. albicans, but others are unique to each species. Our tools, based on selectively deleting regulatory genes, will provide a major resource to the fungal research community.
doi:10.1371/journal.ppat.1004365
PMCID: PMC4169492  PMID: 25233198
3.  Candida albicans and Candida parapsilosis Induce Different T-Cell Responses in Human Peripheral Blood Mononuclear Cells 
The Journal of Infectious Diseases  2013;208(4):690-698.
Candida parapsilosis is the third most frequent cause of candidemia. Despite its clinical importance, little is known about the human immunological response to C. parapsilosis. In this study, we compared the cytokine responses evoked by Candida albicans and C. parapsilosis. C. parapsilosis–stimulated human peripheral blood mononuclear cells (PBMCs) produced similar quantities of tumor necrosis factor α and interleukin 6 and slightly lower amounts of interleukin 1β, compared with C. albicans–stimulated cells. PBMCs stimulated with C. parapsilosis displayed a skewed T-helper cell response, producing more interleukin 10 and less interferon γ than cells stimulated with C. albicans. Notably, C. parapsilosis induced much less interleukin 17 and interleukin 22 production as compared to C. albicans. Inhibition of the 3 classical mitogen-activated protein kinases (p38 kinase, ERK, and JNK) revealed kinase-dependent differences in reductions in cytokine production by the 2 Candida species. Decreased cytokine production after inhibition of dectin 1 revealed that this receptor plays a major role in the recognition of both C. albicans and C. parapsilosis. These data improve understanding of the immune response triggered by C. parapsilosis, a first step for the future design of immunotherapeutic strategies for these infections.
doi:10.1093/infdis/jit188
PMCID: PMC3719900  PMID: 23661798
Candida parapsilosis; human PBMC; T cell response
4.  Genome Comparison of Candida orthopsilosis Clinical Strains Reveals the Existence of Hybrids between Two Distinct Subspecies 
Genome Biology and Evolution  2014;6(5):1069-1078.
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.
doi:10.1093/gbe/evu082
PMCID: PMC4040990  PMID: 24747362
Candida orthopsilosis; hybridization; pathogens; genome sequencing; fungi
5.  Unexpected Genomic Variability in Clinical and Environmental Strains of the Pathogenic Yeast Candida parapsilosis 
Genome Biology and Evolution  2013;5(12):2382-2392.
Invasive candidiasis is the most commonly reported invasive fungal infection worldwide. Although Candida albicans remains the main cause, the incidence of emerging Candida species, such as C. parapsilosis is increasing. It has been postulated that C. parapsilosis clinical isolates result from a recent global expansion of a virulent clone. However, the availability of a single genome for this species has so far prevented testing this hypothesis at genomic scales. We present here the sequence of three additional strains from clinical and environmental samples. Our analyses reveal unexpected patterns of genomic variation, shared among distant strains, that argue against the clonal expansion hypothesis. All strains carry independent expansions involving an arsenite transporter homolog, pointing to the existence of directional selection in the environment, and independent origins of the two clinical isolates. Furthermore, we report the first evidence for the existence of recombination in this species. Altogether, our results shed new light onto the dynamics of genome evolution in C. parapsilosis.
doi:10.1093/gbe/evt185
PMCID: PMC3879973  PMID: 24259314
genome comparison; recombination; pathogens; Candida
6.  Characterization of Virulence Properties in the C. parapsilosis Sensu Lato Species 
PLoS ONE  2013;8(7):e68704.
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 Galleriamellonella 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.
doi:10.1371/journal.pone.0068704
PMCID: PMC3706360  PMID: 23874732
7.  In vitro interactions of Candida parapsilosis wild type and lipase deficient mutants with human monocyte derived dendritic cells 
BMC Microbiology  2011;11:122.
Background
Candida parapsilosis typically is a commensal of human skin. However, when host immune defense is compromised or the normal microflora balance is disrupted, C. parapsilosis transforms itself into an opportunistic pathogen. Candida-derived lipase has been identified as potential virulence factor. Even though cellular components of the innate immune response, such as dendritic cells, represent the first line of defense against invading pathogens, little is known about the interaction of these cells with invading C. parapsilosis. Thus, the aim of our study was to assess the function of dendritic cells in fighting C. parapsilosis and to determine the role that C. parapsilosis-derived lipase plays in the interaction with dendritic cells.
Results
Monocyte-derived immature and mature dendritic cells (iDCs and mDCs, respectively) co-cultured with live wild type or lipase deficient C. parapsilosis strains were studied to determine the phagocytic capacity and killing efficiency of host cells. We determined that both iDCs and mDCs efficiently phagocytosed and killed C. parapsilosis, furthermore our results show that the phagocytic and fungicidal activities of both iDCs and mDCs are more potent for lipase deficient compared to wild type yeast cells. In addition, the lipase deficient C. parapsilosis cells induce higher gene expression and protein secretion of proinflammatory cytokines and chemokines in both DC types relative to the effect of co-culture with wild type yeast cells.
Conclusions
Our results show that DCs are activated by exposure to C. parapsilosis, as shown by increased phagocytosis, killing and proinflammatory protein secretion. Moreover, these data strongly suggest that C. parapsilosis derived lipase has a protective role during yeast:DC interactions, since lipase production in wt yeast cells decreased the phagocytic capacity and killing efficiency of host cells and downregulated the expression of host effector molecules.
doi:10.1186/1471-2180-11-122
PMCID: PMC3148963  PMID: 21619700
Candida; dendritic cell; innate immunity; secreted lipase
8.  Acetylsalicylic acid (aspirin) reduces damage to reconstituted human tissues infected with Candida species by inhibiting extracellular fungal lipases 
Microbes and infection / Institut Pasteur  2009;11(14-15):1131-1139.
A reconstituted human tissue model was used to mimic Candida albicans and Candida parapsilosis infection in order to investigate the protective effects of acetylsalicylic acid (aspirin, ASA). We found that therapeutic concentrations of ASA reduced tissue damage in the in vitro infection model. We further evaluated the lipase inhibitory effects of ASA by investigating the growth of C. albicans, C. parapsilosis and C. parapsilosis lipase negative (Δcplip1-2/Δcplip1-2) mutants in a lipid rich minimal medium supplemented with olive oil and found that a therapeutic concentration of ASA inhibited the growth of wild type fungi. The lipase inhibitors quinine and ebelactone B were also shown to reduce growth and protect against tissue damage from Candida species, respectively. A lipolytic activity assay also showed that therapeutic concentrations of ASA inhibited C. antarctica and C. cylindracea purified lipases obtained through a commercial kit. The relationship between ASA and lipase was characterized through a computed structural model of the Lipase-2 protein from C. parapsilosis in complex with ASA. The results suggest that development of inhibitors of fungal lipases could result in broad-spectrum therapeutics, especially since fungal lipases are not homologous to their human analogues.
doi:10.1016/j.micinf.2009.08.007
PMCID: PMC2787780  PMID: 19703582
Candida; Acetylsalicylic Acid; Lipases; Human Fungal Pathogen; Yeast; Inhibition; Pathogenesis
9.  Candida parapsilosis, an Emerging Fungal Pathogen 
Clinical Microbiology Reviews  2008;21(4):606-625.
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.
doi:10.1128/CMR.00013-08
PMCID: PMC2570155  PMID: 18854483
10.  A Monoclonal Antibody to Histoplasma capsulatum Alters the Intracellular Fate of the Fungus in Murine Macrophages▿ †  
Eukaryotic Cell  2008;7(7):1109-1117.
Monoclonal antibodies (MAbs) to a cell surface histone on Histoplasma capsulatum modify murine infection and decrease the growth of H. capsulatum within macrophages. Without the MAbs, H. capsulatum survives within macrophages by modifying the intraphagosomal environment. In the present study, we aimed to analyze the affects of a MAb on macrophage phagosomes. Using transmission electron and fluorescence microscopy, we showed that phagosome activation and maturation are significantly greater when H. capsulatum yeast are opsonized with MAb. The MAb reduced the ability of the organism to regulate the phagosomal pH. Additionally, increased antigen processing and reduced negative costimulation occur in macrophages that phagocytose yeast cells opsonized with MAb, resulting in more-efficient T-cell activation. The MAb alters the intracellular fate of H. capsulatum by affecting the ability of the fungus to regulate the milieu of the phagosome.
doi:10.1128/EC.00036-08
PMCID: PMC2446677  PMID: 18487350
11.  Voriconazole Inhibits Melanization in Cryptococcus neoformans▿  
Antimicrobial Agents and Chemotherapy  2007;51(12):4396-4400.
Voriconazole is a triazole antifungal drug that inhibits ergosterol synthesis and has broad activity against yeast and molds. While studying the interaction of voriconazole and Cryptococcus neoformans, we noted that cells grown in the presence of subinhibitory concentrations of voriconazole reduced melanin pigmentation. We investigated this effect systematically by assessing melanin production in the presence of voriconazole, amphotericin B, caspofungin, itraconazole, and fluconazole. Only voriconazole impeded the formation of melanin at subinhibitory concentrations. Voriconazole did not affect the autopolymerization of l-dopa, and 0.5 MIC of voriconazole did affect the gene expression of C. neoformans. However, voriconazole inhibited the capacity of laccase to catalyze the formation of melanin. Hence, voriconazole affects melanization in C. neoformans by interacting directly with laccase, which may increase the efficacy of this potent antifungal against certain pigmented fungi.
doi:10.1128/AAC.00376-07
PMCID: PMC2167997  PMID: 17923488
12.  Correction: Methamphetamine Inhibits Antigen Processing, Presentation, and Phagocytosis 
PLoS Pathogens  2008;4(3):10.1371/annotation/bd02ad26-a081-4c61-88c2-ebda285b8bca.
doi:10.1371/annotation/bd02ad26-a081-4c61-88c2-ebda285b8bca
PMCID: PMC2637120
13.  Methamphetamine Inhibits Antigen Processing, Presentation, and Phagocytosis 
PLoS Pathogens  2008;4(2):e28.
Methamphetamine (Meth) is abused by over 35 million people worldwide. Chronic Meth abuse may be particularly devastating in individuals who engage in unprotected sex with multiple partners because it is associated with a 2-fold higher risk for obtaining HIV and associated secondary infections. We report the first specific evidence that Meth at pharmacological concentrations exerts a direct immunosuppressive effect on dendritic cells and macrophages. As a weak base, Meth collapses the pH gradient across acidic organelles, including lysosomes and associated autophagic organelles. This in turn inhibits receptor-mediated phagocytosis of antibody-coated particles, MHC class II antigen processing by the endosomal–lysosomal pathway, and antigen presentation to splenic T cells by dendritic cells. More importantly Meth facilitates intracellular replication and inhibits intracellular killing of Candida albicans and Cryptococcus neoformans, two major AIDS-related pathogens. Meth exerts previously unreported direct immunosuppressive effects that contribute to increased risk of infection and exacerbate AIDS pathology.
Author Summary
There is a new population of HIV+ men who are developing AIDS over months instead of years as typical. It has recently become popular among gay and bisexual men to consume very high levels of Meth. Unsafe sex together with Meth abuse has been suspected to lead to rapid disease progression. While studies show exacerbated AIDS symptoms and disease progression in HIV+ Meth abusers, the molecular mechanism is yet unknown. It was postulated, yet unproven, that the rapid disease progression might be due to a mutant “superstrain” of HIV that was extremely virulent. It was also assumed that the effects of the drug on behavior may lead to unsafe sex, although this would not explain the more rapid time course of the disease. We now demonstrate the first direct evidence that Meth is an immunosuppressive agent, and that the molecular mechanism of this immunosuppression is due to the collapse of acidic organelle pH in cells of the immune system, inhibiting the functions of antigen presentation, as well as phagocytosis. These effects compromise the immune response to opportunistic infections and HIV. These findings could have a major impact on public health, as there are over 35 million Meth abusers worldwide
doi:10.1371/journal.ppat.0040028
PMCID: PMC2242831  PMID: 18282092
14.  Lipase 8 Affects the Pathogenesis of Candida albicans▿  
Infection and Immunity  2007;75(10):4710-4718.
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.
doi:10.1128/IAI.00372-07
PMCID: PMC2044512  PMID: 17646357
15.  Targeted gene deletion in Candida parapsilosis demonstrates the role of secreted lipase in virulence  
The Journal of Clinical Investigation  2007;117(10):3049-3058.
Candida parapsilosis is a major cause of human disease, yet little is known about the pathogen’s 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.
doi:10.1172/JCI32294
PMCID: PMC1974868  PMID: 17853941
16.  The APSES transcription factor Efg1 is a global regulator that controls morphogenesis and biofilm formation in Candida parapsilosis 
Molecular Microbiology  2013;90(1):36-53.
Efg1 (a member of the APSES family) is an important regulator of hyphal growth and of the white-to-opaque transition in Candida albicans and very closely related species. We show that in Candida parapsilosis Efg1 is a major regulator of a different morphological switch at the colony level, from a concentric to smooth morphology. The rate of switching is at least 20-fold increased in an efg1 knockout relative to wild type. Efg1 deletion strains also have reduced biofilm formation, attenuated virulence in an insect model, and increased sensitivity to SDS and caspofungin. Biofilm reduction is more dramatic in in vitro than in in vivo models. An Efg1 paralogue (Efh1) is restricted to Candida species, and does not regulate concentric-smooth phenotype switching, biofilm formation or stress response. We used ChIP-seq to identify the Efg1 regulon. A total of 931 promoter regions bound by Efg1 are highly enriched for transcription factors and regulatory proteins. Efg1 also binds to its own promoter, and negatively regulates its expression. Efg1 targets are enriched in binding sites for 93 additional transcription factors, including Ndt80. Our analysis suggests that Efg1 has an ancient role as regulator of development in fungi, and is central to several regulatory networks.
doi:10.1111/mmi.12345
PMCID: PMC3912905  PMID: 23895281

Results 1-16 (16)