Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

BACKGROUND: Candida albicans, a commensal organism, is a part of the normal flora of healthy individuals. However, once the host immunity is compromised, C. albicans opportunistically causes recurrent superficial or fatal systemic candidiasis. Secreted aspartic proteases (Sap), encoded by 10 types of SAP genes, have been suggested to contribute to various virulence processes. Thus, it is important to elucidate their biochemical properties for better understanding of the molecular mechanisms that how Sap isozymes damage host tissues. METHODOLOGY/PRINCIPAL FINDINGS: The SAP7 gene was cloned from C. albicans SC5314 and heterogeneously produced by Pichia pastoris. Measurement of Sap7 proteolytic activity using the FRETS-25Ala library showed that Sap7 was a pepstatin A-insensitive protease. To understand why Sap7 was insensitive to pepstatin A, alanine substitution mutants of Sap7 were constructed. We found that M242A and T467A mutants had normal proteolytic activity and sensitivity to pepstatin A. M242 and T467 were located in close proximity to the entrance to an active site, and alanine substitution at these positions widened the entrance. Our results suggest that this alteration might allow increased accessibility of pepstatin A to the active site. This inference was supported by the observation that the T467A mutant has stronger proteolytic activity than the wild type. CONCLUSIONS/SIGNIFICANCE: We found that Sap7 was a pepstatin A-insensitive protease, and that M242 and T467 restricted the accessibility of pepstatin A to the active site. This finding will lead to the development of a novel protease inhibitor beyond pepstatin A. Such a novel inhibitor will be an important research tool as well as pharmaceutical agent for patients suffering from candidiasis

Effect of surface roughness of biomaterials on Staphylococcus epidermidis adhesion

Background: Implant-related infections are caused by adhesion of bacteria to the surface of biomaterials. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis (ATCC35984) to adhere to the surface of solid biomaterials at different levels of roughness below 30 nm Ra and investigated the minimum level of roughness required to promote bacterial adhesion on five kinds of biomaterials: oxidized zirconium-niobium alloy (Oxinium), cobalt-chromium-molybdenum alloy (Co-Cr-Mo), titanium alloy (Ti-6Al-4 V), commercially pure titanium (Cp-Ti) and stainless steel (SUS316L), samples of which were categorized into a fine group and a coarse group according to surface roughness. The test specimens were physically analyzed and the viable bacterial density of the adhered bacteria was quantitatively determined (n = 20).Results: The amount of bacteria that adhered to the biomaterials in the coarse group was higher than those in the fine group. Oxinium, Ti-6Al-4 V and SUS316L in particular demonstrated statistically significant differences between the two groups (P < 0.05). Of the materials, the Co-Cr-Mo specimens exhibited significantly lower amounts of adhered bacteria than the Ti-6Al-4 V, Cp-Ti and SUS316L specimens in the fine group. Similarly, the Co-Cr-Mo specimens in the coarse group exhibited significantly lower values than the other four materials.Conclusions: These results suggest that minimum level of roughness affecting initial bacterial adherence activity differs according to the type of biomaterial used, and that even a surface roughness of below 30 nm Ra in Oxinium, Ti-6Al-4 V and SUS316L can promote bacterial adhesion. Relative hydrophobic Co-Cr-Mo surfaces were less susceptible to bacterial adherence

Early Staphylococcal Biofilm Formation on Solid Orthopaedic Implant Materials: In Vitro Study

Biofilms forming on the surface of biomaterials can cause intractable implant-related infections. Bacterial adherence and early biofilm formation are influenced by the type of biomaterial used and the physical characteristics of implant surface. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis, the main pathogen in implant-related infections, to form biofilms on the surface of the solid orthopaedic biomaterials, oxidized zirconium-niobium alloy, cobalt-chromium-molybdenum alloy (Co-Cr-Mo), titanium alloy (Ti-6Al-4V), commercially pure titanium (cp-Ti) and stainless steel. A bacterial suspension of Staphylococcus epidermidis strain RP62A (ATCC35984) was added to the surface of specimens and incubated. The stained biofilms were imaged with a digital optical microscope and the biofilm coverage rate (BCR) was calculated. The total amount of biofilm was determined with the crystal violet assay and the number of viable cells in the biofilm was counted using the plate count method. The BCR of all the biomaterials rose in proportion to culture duration. After culturing for 2-4 hours, the BCR was similar for all materials. However, after culturing for 6 hours, the BCR for Co-Cr-Mo alloy was significantly lower than for Ti-6Al-4V, cp-Ti and stainless steel (P0.05). These results suggest that surface properties, such as hydrophobicity or the low surface free energy of Co-Cr-Mo, may have some influence in inhibiting or delaying the two-dimensional expansion of biofilm on surfaces with a similar degree of smoothness