Wataru Aoki

Characterization of Antimicrobial Peptides toward the Development of Novel Antibiotics

Antimicrobial agents have eradicated many infectious diseases and significantly improved our living environment. However, abuse of antimicrobial agents has accelerated the emergence of multidrug-resistant microorganisms, and there is an urgent need for novel antibiotics. Antimicrobial peptides (AMPs) have attracted attention as a novel class of antimicrobial agents because AMPs efficiently kill a wide range of species, including bacteria, fungi, and viruses, via a novel mechanism of action. In addition, they are effective against pathogens that are resistant to almost all conventional antibiotics. AMPs have promising properties; they directly disrupt the functions of cellular membranes and nucleic acids, and the rate of appearance of AMP-resistant strains is very low. However, as pharmaceuticals, AMPs exhibit unfavorable properties, such as instability, hemolytic activity, high cost of production, salt sensitivity, and a broad spectrum of activity. Therefore, it is vital to improve these properties to develop novel AMP treatments. Here, we have reviewed the basic biochemical properties of AMPs and the recent strategies used to modulate these properties of AMPs to enhance their safety.

Comprehensive characterization of secreted aspartic proteases encoded by a virulence gene family in Candida albicans

Candida albicans is a commensal organism, but causes life-threatening infections in immunocompromised patients. Certain factors such as yeast-hyphae transition and hydrolytic enzymes are suggested as virulence attributes of C. albicans. Among them, 10 types of secreted aspartic protease (SAP) genes have received particular attention as a major virulence gene family. However, their full functional repertoire, including its biochemical properties, remains to be elucidated. Hence, we purified all Sap isozymes using Pichia pastoris and comprehensively determined and compared their biochemical properties. While optimum pH of Sap7 was 6.5 and that of Sap8 was 2.5, presence of other Sap isozymes functioning within a broad range of optimum pH could allow C. albicans to survive and cause infections in various tissues. The substrate specificities of Sap isozymes were analysed by using FRETS-25Xaa libraries. Sap7 and Sap10 showed high substrate specificity, while other Sap isozymes had broad substrate specificities. Principal component analysis revealed that the 10 Sap isozymes were clustered into 3 distinct groups in terms of their substrate specificities. Interestingly, Sap4-6, which are coproduced in the hyphal form, were clustered as the same group, indicating that they may target similar host proteins. These results will lead to further understanding of C. albicans pathogenicity.

Inactivation of the Antifungal and Immunomodulatory Properties of Human Cathelicidin LL-37 by Aspartic Proteases Produced by the Pathogenic Yeast Candida albicans

ABSTRACT Constant cross talk between Candida albicans yeast cells and their human host determines the outcome of fungal colonization and, eventually, the progress of infectious disease (candidiasis). An effective weapon used by C. albicans to cope with the host defense system is the release of 10 distinct secreted aspartic proteases (SAPs). Here, we validate a hypothesis that neutrophils and epithelial cells use the antimicrobial peptide LL-37 to inactivate C. albicans at sites of candidal infection and that C. albicans uses SAPs to effectively degrade LL-37. LL-37 is cleaved into multiple products by SAP1 to -4, SAP8, and SAP9, and this proteolytic processing is correlated with the gradual decrease in the antifungal activity of LL-37. Moreover, a major intermediate of LL-37 cleavage—the LL-25 peptide—is antifungal but devoid of the immunomodulatory properties of LL-37. In contrast to LL-37, LL-25 did not affect the generation of reactive oxygen species by neutrophils upon treatment with phorbol esters. Stimulating neutrophils with LL-25 (rather than LL-37) significantly decreased calcium flux and interleukin-8 production, resulting in lower chemotactic activity of the peptide against neutrophils, which may decrease the recruitment of neutrophils to infection foci. LL-25 also lost the function of LL-37 as an inhibitor of neutrophil apoptosis, thereby reducing the life span of these defense cells. This study indicates that C. albicans can effectively use aspartic proteases to destroy the antimicrobial and immunomodulatory properties of LL-37, thus enabling the pathogen to survive and propagate.

Next generation of antimicrobial peptides as molecular targeted medicines.

Antibiotics have significantly improved our living environments. However, overuse of antibiotics has led to the emergence of multi-drug resistant microorganisms, and the subsequent constant demand for the exploration of novel antibiotics. To this end, antimicrobial peptides (AMPs) have attracted much attention as a novel class of antibiotics. AMPs have strong antimicrobial activity against a wide-range of species, including gram-positive and gram-negative bacteria, fungi, and viruses. In addition, they are also effective against pathogenic organisms that are resistant to conventional drugs. Despite their great potential, the hemolytic activity and a highly broad spectrum of activity of AMPs dictate the need for amendments to develop safe pharmaceuticals. The human body contains commensal microflora as an integral part of complex mucosal surfaces that offers protection against pathogenic organisms. Administration of antibiotics with broad spectra of activity disrupts the indigenous microflora and increases the risks of diarrhea and other fatal infections. Therefore, it is difficult, but vital, to develop treatments capable of rapidly eliminating pathogenic organisms while maintaining the commensal microbiota. As such, novel pharmaceuticals, safe designer AMPs have been heavily researched. In this article, we review recent attempts to spatially and temporally regulate AMPs to enhance the quality-of-life of patients.

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.

Time-course proteomic profile of Candida albicans during adaptation to a fetal serum

Candida albicans is a commensal organism; however, it causes fatal diseases if the host immunity is compromised. The mortality rate is very high due to the lack of effective treatment, leading to ceaseless demand for novel pharmaceuticals. In this study, time-course proteomics of C. albicans during adaptation to fetal bovine serum (FBS) was described. Time-course proteomics is a promising way to understand the exact process of going adaptation in dynamically changing environments. Candida albicans was cultivated in yeast nitrogen base (YNB) ± FBS media, and we identified 1418 proteins in the endpoint samples incubated for 0 or 60 min by a LC-MS/MS system with a long monolithic silica capillary column. Next, we carried out time-course proteomics of the YNB + FBS samples to identify top-priority proteins for adaption to FBS. We identified 16 proteins as nascent/newly synthesized proteins, and they were recognized as candidates of important virulent factors. Gene ontology analysis revealed that transport-related proteins were enriched in the 16 proteins, indicating that C. albicans probably put priority in time on the acquisition of essential elements. Time-course proteomics of C. albicans revealed the order of priority to adapt to FBS. Depicting time-course dynamics will lead to profound understandings of virulence of C. albicans.

Profiling of adhesive properties of the agglutinin-like sequence (ALS) protein family, a virulent attribute of Candida albicans.

Candida albicans is normally present in nearly all humans but can cause fatal diseases in immunocompromised patients. The agglutinin-like sequence (ALS) gene family of C. albicans has been suggested to be important for biofilm formation on medical devices. Here, we cloned all ALS genes and determined the binding properties of their gene products by cell surface engineering of Saccharomyces cerevisiae. Most of the ALS homologues had the ability to bind polypropylene, borosilicate glass and polyvinyl chloride, which are often used as materials in medical devices. However, ALS homologues were not able to bind polyurethane, polymethyl methacrylate, polytetrafluoroethylene or titanium. These findings will aid in the development of biofilm-resistant medical devices.

Secreted aspartic peptidases of Candida albicans liberate bactericidal hemocidins from human hemoglobin.

Secreted aspartic peptidases (Saps) are a group of ten acidic hydrolases considered as key virulence factors of Candida albicans. These enzymes supply the fungus with nutrient amino acids as well as are able to degrade the selected hosts proteins involved in the immune defense. Our previous studies showed that the human menstrual discharge is exceptionally rich in bactericidal hemoglobin (Hb) fragments - hemocidins. However, to date, the genesis of such peptides is unclear. The presented study demonstrates that the action of C. albicans isozymes Sap1-Sap6, Sap8 and Sap9, but not Sap7 and Sap10, toward human hemoglobin leads to limited proteolysis of this protein and generates a variety of antimicrobial hemocidins. We have identified these peptides and checked their activity against selected microorganisms representative for human vagina. We have also demonstrated that the process of Hb hydrolysis is most effective at pH 4.0, characteristic for vagina, and the liberated peptides showed pronounced killing activity toward Lactobacillus acidophilus, and to a lower degree, Escherichia coli. However, only a very weak activity toward Staphylococcus aureus and C. albicans was noticed. These findings provide interesting new insights into pathophysiology of human vaginal candidiasis and suggest that C. albicans may be able to compete with the other microorganisms of the same physiological niche using the microbicidal peptides generated from the host protein.

Elucidation of potentially virulent factors of Candida albicans during serum adaptation by using quantitative time-course proteomics.

UNLABELLED Candida albicans is an opportunistic pathogen that causes fatal disease if the host immunity is compromised. The mortality rate of systemic candidiasis is very high; hence, there is a ceaseless demand for novel pharmaceuticals. In this study, quantitative time-course proteomics of C. albicans during adaptation to fetal bovine serum (FBS) is described. Survival in blood is essential for virulence of C. albicans, and a detailed analysis is required. We cultivated C. albicans in FBS for 0-180min, and determined quantitative time-course variations of 1024 proteins in the cultured cells by using a LC-MS/MS system with a long monolithic silica capillary column. Clustering analysis identified FBS-induced proteins associated with detoxification of oxidative species, high-affinity glucose transport, citrate cycle, oxidative phosphorylation, and iron acquisition. Furthermore, we identified possible virulence factors such as orf19.4914.1 (named Blood-induced peptide 1, Blp1). Heterologous expression of BLP1 in Saccharomyces cerevisiae shortened the lag phase and resulted in a pleiotropic stress-tolerance phenotype, indicating a possible role for quick adaptation to a stressful environment. While further experiments are necessary to prove virulence of the identified factors, systematic identification of candidate virulence proteins in this study will lead to profound understanding of virulence of C. albicans. BIOLOGICAL SIGNIFICANCE This paper describes time-course proteomics of C. albicans during adaptation to serum, which is an essential process for fatal systemic candidiasis. Using a LC-MS/MS system with a monolithic silica capillary column, we have successfully characterized time-course variations of 1024 proteins. Among them, orf19.4914.1 (Blp1) was identified as a novel pleiotropic stress-tolerance peptide, which could have an important role for virulence of C. albicans.

High-throughput screening of improved protease inhibitors using a yeast cell surface display system and a yeast cell chip.

Protease-targeted inhibitors have been promising pharmaceuticals. Here, we combined a yeast cell surface display system with a yeast cell chip for the high-throughput screening of protease inhibitors, and succeeded in improving the activity of a protease inhibitor.