pepstatin and Candidiasis

The opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera) are notable for their intrinsic resistance to different antifungal classes. Little is known about the virulence attributes in this emerging fungal complex. However, it is well-recognized that enzymes play important roles in virulence/pathogenesis of candidiasis. Herein, we aimed to identify aspartyl-type peptidases in 12 clinical isolates belonging to the C. haemulonii complex. All isolates were able to grow in a chemically defined medium containing albumin as the sole nitrogen source, and a considerable consumption of this protein occurred after 72-96 h. C. haemulonii var. vulnera isolates showed the lowest albumin degradation capability and the poorest growth rate. The measurement of secreted aspartyl peptidase (Sap) activity, using the cathepsin D fluorogenic substrate, varied from 91.6 to 413.3 arbitrary units and the classic aspartyl peptidase inhibitor, pepstatin A, significantly blocked the Sap released by C. haemulonii complex. No differences were observed in the Sap activity among the three fungal species. Flow cytometry, using a polyclonal antibody against Sap1-3 of C. albicans, detected homologous proteins at the surface of C. haemulonii complex (anti-Sap1-3-labeled cells ranged from 24.6 to 79.1%). Additionally, the immunoblotting assay, conducted with the same Sap1-3 antibody, recognized a protein of ∼50 kDa in all fungal isolates. A glimpse in the genome of these fungi revealed several potential proteins containing Sap1-3-like conserved domain. Altogether, our results demonstrated the potential of C. haemulonii species complex to produce Saps, an important virulence factor of Candida spp.

Topics: Antifungal Agents; Candida; Candida albicans; Candidiasis; Dipeptidases; Drug Resistance, Multiple; Humans; Pepstatins; Protease Inhibitors; Sequence Analysis, Protein

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.

Topics: Animals; Aspartic Acid Proteases; beta-Glucans; Biofilms; Candida albicans; Candidiasis; Cell Membrane; Culture Media; Environment; Enzyme Activation; Fungal Proteins; Hyphae; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 3; Models, Biological; Mouth Diseases; Mutation; Pepstatins; Pharyngeal Diseases; Phosphorylation; Plankton; Proteolysis

The screening of a library of small molecule peptidomimetics toward secreted aspartic proteinase-2 (SAP2) of Candida albicans allowed us to identify two compounds that showed in vitro inhibitory potency comparable to pepstatin A. In an experimental model of vaginal candidiasis, the two candidate compounds were as active as a therapeutic dose of fluconazole. Importantly, this activity was fully preserved when the challenger was a fluconazole-resistant strain of the fungus. Altogether, our data demonstrate SAP2 as a valid C. albicans target for the development of new drugs against this important human pathogen.

Topics: Animals; Antifungal Agents; Aspartic Acid Endopeptidases; Candida albicans; Candidiasis; Dose-Response Relationship, Drug; Drug Resistance, Fungal; Enzyme Inhibitors; Female; Fluconazole; Fungal Proteins; Humans; Models, Chemical; Molecular Structure; Pepstatins; Peptide Library; Peptides, Cyclic; Rats; Rats, Wistar; Structure-Activity Relationship; Virulence

Oral candidosis is a common problem in immunocompromised patients, and whilst Candida albicans is regarded as the principal cause of infection, other non-Candida albicans Candida (NCAC) species are increasingly being recognized as human pathogens. Relatively little is known about the virulence factors associated with NCAC species, and the aim of this study was to use a reconstituted human oral epithelium (RHOE) to examine epithelial infection withCandida parapsilosis. Strains originating from the oral and vaginal mucosa and from the urinary tract were all shown to colonize RHOE in a strain-dependent manner. Strain differences were found in the colonizing morphology and in the extent of invasion of the RHOE. Low invasion of RHOE was detected for strains after 12 h, whereas extensive tissue damage was evident after 24 h when assessed using histological examination and lactate dehydrogenase activity determination. Tissue damage was reduced in the presence of pepstatin A, although C. parapsilosis invasion of the tissue was not inhibited. Real-time polymerase chain reaction of secreted aspartyl proteinase (SAP) genes (SAPP1-3) showed that expression was strain dependent, with an increased expression generally occurring for Candida infecting RHOE compared with planktonic equivalents. In summary, C. parapsilosis was not highly invasive of RHOE but did induce significant tissue damage, which could relate to specific SAPgene expression.

Topics: Aspartic Acid Endopeptidases; Candida; Candidiasis; Candidiasis, Oral; Candidiasis, Vulvovaginal; Epithelium; Female; Fungal Proteins; Humans; L-Lactate Dehydrogenase; Microscopy, Confocal; Microscopy, Electron, Scanning; Mouth Mucosa; Pepstatins; Polymerase Chain Reaction; Protease Inhibitors; Time Factors; Urinary Tract Infections; Virulence

Candida parapsilosis is an increasingly important human pathogen. To study the interactions of C. parapsilosis with human tissues, we evaluated the effects of the CBS 604 type strain and three different clinical isolates on reconstituted human oral epithelial and epidermal tissues. The newly described species Candida orthopsilosis and Candida metapsilosis were also examined in these models. Microscopy of reconstituted tissues infected with yeast cells revealed severe attenuation, morphological changes and cellular damage. C. orthopsilosis caused damage similar to C. parapsilosis isolates, whereas C. metapsilosis was less virulent. To further quantitate tissue damage, we measured lactate dehydrogenase (LDH) in the culture supernatant. The relative LDH measurements correlated with our histopathological observations. We also examined the effect of the lipase inhibitor Ebelactone B and proteinase inhibitor Pepstatin A, to establish the utility of this model for studying factors of C. parapsilosis virulence. Both Ebelactone B and Pepstatin A reduced the destruction of epidermal and epithelial tissues. Our data show that reconstituted human tissues are extremely useful for modeling host interactions with C. parapsilosis and for studying fungal virulence factors.

Topics: Candida; Candidiasis; Epidermis; Epithelium; Humans; Lactate Dehydrogenases; Lactones; Pepstatins; Species Specificity; Tissue Engineering; Virulence

Non-albicans Candida species cause 35-65% of all candidemias in the general population, especially in immunosuppressed individuals. Here, we describe a case of a 19-year-old HIV-infected man with pneumonia due to a yeast-like organism. This clinical yeast isolate was identified as Candida guilliermondii through mycological tests. C. guilliermondii was cultivated in brain heart infusion medium for 48 h at 37 degrees C. After sequential centrifugation and concentration steps, the free-cell culture supernatant was obtained and extracellular proteolytic activity was assayed firstly using gelatin-SDS-PAGE. A 50 kDa proteolytic enzyme was detected with activity at physiological pH. This activity was completely blocked by 10 mM phenylmethylsulphonyl fluoride (PMSF), a serine proteinase inhibitor, suggesting that this extracellular proteinase belongs to the serine proteinase class. E-64, a strong cysteine proteinase inhibitor, and pepstatin A, a specific aspartic proteolytic inhibitor, did not interfere with the 50 kDa proteinase. Conversely, a zinc-metalloproteinase inhibitor (1,10-phenanthroline) restrained the proteinase activity released by C. guilliermondii by approximately 50%. Proteinases are a well-known class of enzymes that participate in a vast context of yeast-host interactions. In an effort to establish a functional implication for this extracellular serine-type enzyme, we investigated its capacity to hydrolyze some serum proteins and extracellular matrix components. We demonstrated that the 50 kDa exocellular serine proteinase cleaved human serum albumin, non-immune human immunoglobulin G, human fibronectin and human placental laminin, generating low molecular mass polypeptides. Collectively, these results showed for the first time the ability of an extracellular proteolytic enzyme other than aspartic-type proteinases in destroying a broad spectrum of relevant host proteins by a clinical species of non-albicans Candida.

Topics: Adult; AIDS-Related Opportunistic Infections; Blood Proteins; Brazil; Candida; Candidiasis; Enzyme Inhibitors; Extracellular Matrix Proteins; Gelatin; Humans; Hydrogen-Ion Concentration; Leucine; Lung Diseases, Fungal; Male; Molecular Weight; Pepstatins; Phenanthrolines; Phenylmethylsulfonyl Fluoride; Pneumonia; Serine Endopeptidases

Secreted aspartic proteinases (Saps) encoded by 10 genes of Candida albicans are important virulence factors for different types of candidiasis. Distinct SAP genes have previously been shown to contribute to tissue damage in a model of oral candidiasis. In this study a progressive SAP expression in the order SAP1 and SAP2 > SAP8 > SAP6 > SAP3 was observed in an in vitro model of cutaneous candidiasis based on reconstituted human epidermis. Transcripts of SAP1 and SAP2 were detected during initial invasion of the stratum corneum by C. albicans. Deeper, extensive penetration of the corneal layer was accompanied by additional SAP8 mRNA. SAP6 expression occurred concomitantly with germ tube formation and extensive hyphal growth in the strata granulosum, spinosum, and basale. Ultrastructural studies using specific polyclonal antibodies directed against the gene products of SAP1-3 and SAP4-6 revealed predominant expression of Sap1-3. The protective effect of the aspartic proteinase inhibitor pepstatin A during infection of the epidermis and an attenuated virulence phenotype of SAP-deficient mutants suggest that the observed SAP expression correlates with tissue damage in the skin.

Topics: Aspartic Acid Endopeptidases; Candida albicans; Candidiasis; Epidermis; Humans; Microscopy, Immunoelectron; Mutation; Pepstatins; Protease Inhibitors

A murine model of disseminated candidiasis involving intranasal challenge with Candida albicans was developed and used to explore the role of C. albicans aspartic proteases as virulence factors during early dissemination. Pretreatment of neutropenic mice with the aspartic protease inhibitor pepstatin A by intraperitoneal injection afforded strong dose-dependent protection against a subsequent lethal intranasal dose of an aspartic protease-producing strain (ATCC 32354) of C. albicans. Administration of 0.6 mg of pepstatin A kg of body weight(-1) prior to challenge and on days 1 to 4 postchallenge resulted in 100% survival at day 15 postchallenge, whereas 100% of animals receiving saline had died by day 6. This effect was comparable to the dose-dependent protection obtained with amphotericin B, which resulted in 100% survival when administered at 0.1 mg kg(-1). The reduction in mortality afforded by pepstatin A correlated with its dose-dependent blockade of C. albicans numbers in the lungs, liver, and kidneys. By sharp contrast, no protection by pepstatin A was observed in mice challenged intravenously, and protection was markedly attenuated in mice given pepstatin A after intranasal challenge only. These data show the utility of pepstatin A in the prophylaxis of disseminated Candida infections and suggest that Candida aspartic proteases play an essential role early in dissemination.

Topics: Administration, Intranasal; Amphotericin B; Animals; Aspartic Acid Endopeptidases; Candida albicans; Candidiasis; Disease Models, Animal; Epithelium; Female; Mice; Pepstatins

The intravenous injection of 10(6) to 10(7) Candida albicans cells revealed to be a reliable model in mice produce infections of the kidney. Higher germ contents could be yielded in the kidney after the application of protease positive Candida-strains as compared to protease negative ones. Additionally, after the infection with protease positive strains a proteolytic activity could be found in the kidney homogenates in vitro on casein plates. The modulation of this Candida infection by pepstatin A, an inhibitor of extracellular yeast proteases in vitro, has also been studied in the same mice model in vivo. The growth rate of Candida albicans has been measured in the left kidney by counting the germ content as described by Haenel. Infections could be reduced after single doses of 120 micrograms pepstatin A contrary to 60 micrograms pepstatin A. The same was with three doses of 180 micrograms at 24 h intervals. This protease inhibitory effect could also be found in kidney homogenates in vitro on casein plates and lasted until 48 h post injection. On the basis of this positive effects on the Candida infection in mice pepstatin A should be considered as an adjuvans in the therapy of severe yeast infections.

Topics: Animals; Candida albicans; Candidiasis; Dose-Response Relationship, Drug; Kidney; Mice; Oligopeptides; Pepstatins

Adherence of blastoconidia to epidermal corneocytes is an early event in Candida colonization and infection of the skin. Pathogenic species adhere more avidly than nonpathogenic species, transform to hyphal growth, and invade the stratum corneum of the skin. Adherence was studied by scanning electron microscopy of experimental murine cutaneous Candida infections, using six species of Candida. Candida albicans and C. stellatoidea blastoconidia, applied to newborn mouse skin, adhered to the stratum corneum in greater numbers than other species tested, acquired fibrils and strands of amorphous mucinlike material ("cohesin") between spores and the corneocyte cell surface, formed cavitations in the corneocyte surface, and invaded the corneocyte envelope by hyphal growth at sites distant to the point of blastoconidia attachment. Other species showed little or no adherence, colonization, or cavitation of the corneocyte surface, except C. tropicalis, which showed intermediate results. Pepstatin, an inhibitor of Candida acid proteinase, did not alter adherence or cohesion formation, but inhibited formation of corneocyte cavitations about adherent blastoconidia, suggesting that this enzyme may facilitate adherence/invasion events on skin. Depletion of surface lipids did not alter the formation of cohesin material or the adherence process. Adherence and invasion of epithelium by pathogenic Candida species include the interaction of blastoconidia with an epithelial surface cohesin material that participates in the adherence process. Candida acid proteinase, a keratinolytic enzyme, may participate in the cavitation process of the corneocyte surface by C. albicans.

Topics: Animals; Aspartic Acid Endopeptidases; Candida; Candidiasis; Cell Adhesion; Endopeptidases; Epidermis; Mice; Microscopy, Electron, Scanning; Pepstatins; Time Factors