Antimicrobial peptide LL-37 disrupts plasma membrane and calcium homeostasis in Candida albicans via the Rim101 pathway.

IMPORTANCE: Candida albicans is a major human fungal pathogen, and antimicrobial peptides are key components of innate immunity. Studying the interplay between C. albicans and human antimicrobial peptides would enhance a better understanding of pathogen-host interactions. Moreover, potential applications of antimicrobial peptides in antifungal therapy have aroused great interest. This work explores new mechanisms of LL-37 against C. albicans and reveals the complex connection among calcium homeostasis, oxidative stress, signaling, and possibly organelle interaction. Notably, these findings support the possible use of antimicrobial peptides to prevent and treat fungal infections.

Fruits are vehicles of drug-resistant pathogenic Candida tropicalis.

IMPORTANCE: Of 123 identified isolates from the fruit surface, C. tropicalis was the most frequently found species, followed by Meyerozyma caribbica and Candida krusei. All three fluconazole-resistant C. tropicalis were non-susceptible to voriconazole and belonged to the same predominant genotype of azole-resistant C. tropicalis causing candidemia in patients in Taiwan. Our findings provide evidence that fruit should be washed before eaten not only to remove chemicals but also potential drug-resistant pathogenic microbes, especially for immunocompromised individuals. To keep precious treatment options in patients, we not only continuously implement antimicrobial stewardship in hospitals but also reducing/stopping the use of agricultural fungicide classes used in human medicine.

Associations of Rap1 with Cell Wall Integrity, Biofilm Formation, and Virulence in Candida albicans.

Rap1 (repressor activator protein 1) is a multifunctional protein, playing important roles in telomeric and nontelomeric functions in many eukaryotes. Candida albicans Rap1 has been previously shown to be involved in telomeric regulation, but its other functions are still mostly unknown. In this study, we found that the deletion of the RAP1 gene altered cell wall properties, composition, and gene expression. In addition, deletion of RAP1 affected C. albicans biofilm formation and modulated phagocytosis and cytokine release by host immune cells. Finally, the RAP1 gene deletion mutant showed attenuation of C. albicans virulence in a Galleria mellonella infection model. Therefore, these findings provide new insights into Rap1 functions that are particularly relevant to pathogenesis and virulence of C. albicans. IMPORTANCE C. albicans is an important fungal pathogen of humans. The cell wall is the outermost layer of C. albicans and is important for commensalism and infection by this pathogen. Moreover, the cell wall is also an important target for antifungals. Studies of how C. albicans maintains its cell wall integrity are critical for a better understanding of fungal pathogenesis and virulence. This work focuses on exploring unknown functions of C. albicans Rap1 and reveals its contribution to cell wall integrity, biofilm formation, and virulence. Notably, these findings will also improve our general understanding of complex machinery to control pathogenesis and virulence of fungal pathogens.

The Role of Sfp1 in Candida albicans Cell Wall Maintenance.

The cell wall is the first interface for Candida albicans interaction with the surrounding environment and the host cells. Therefore, maintenance of cell wall integrity (CWI) is crucial for C. albicans survival and host-pathogen interaction. In response to environmental stresses, C. albicans undergoes cell wall remodeling controlled by multiple signaling pathways and transcription regulators. Here, we explored the role of the transcription factor Sfp1 in CWI. A deletion of the SFP1 gene not only caused changes in cell wall properties, cell wall composition and structure but also modulated expression of cell wall biosynthesis and remodeling genes. In addition, Cas5 is a known transcription regulator for C. albicans CWI and cell wall stress response. Interestingly, our results indicated that Sfp1 negatively controls the CAS5 gene expression by binding to its promoter element. Together, this study provides new insights into the regulation of C. albicans CWI and stress response.

Causative links between ER stress and oxidative damage in a yeast model of human N88S seipinopathy.

Seipin is encoded by the gene Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) and FLD1/SEI1 in yeast. The gain-of-function N88S mutation in the BSCL2 gene was identified in a cohort of autosomal dominant motor neuron diseases (MNDs) collectively known as seipinopathies. Previous work has shown that this mutation disrupts N-glycosylation, leading to the formation of inclusion bodies (IBs) and contributing to severe Endoplasmic Reticulum (ER) stress and cell death. In this work, we established a humanized yeast model of N88S seipinopathy that recapitulated the formation of IBs and activation of the unfolded protein response (UPR) observed in mammalian systems. Autophagy and the Hrd1-mediated endoplasmic reticulum-associated degradation (ERAD) were fully functional in cells expressing mutant homomers and WT-mutant heteromers of seipin, discarding the possibility that mutant seipin accumulate due to impaired protein quality control systems. Importantly, the N88S seipin form IBs that appear to induce changes in ER morphology, in association with Kar2 chaperone and the Hsp104 disaggregase. For the first time, we have determined that N88S homo-oligomers expressing cells present reduced viability, decreased antioxidant activity and increased oxidative damage associated with loss of mitochondrial membrane potential, higher reactive oxygen species (ROS) levels and lipid peroxidation. This was correlated with the activation of oxidative stress sensor Yap1. Moreover, activation of ERAD and UPR quality control mechanisms were essential for proper cell growth, and crucial to prevent excessive accumulation of ROS in cells expressing N88S homomers solely. Overall, this study provides new insights into the molecular underpinnings of these rare diseases and offers novel targets for potential pharmacological intervention.

Helical structure motifs made searchable for functional peptide design.

The systematic design of functional peptides has technological and therapeutic applications. However, there is a need for pattern-based search engines that help locate desired functional motifs in primary sequences regardless of their evolutionary conservation. Existing databases such as The Protein Secondary Structure database (PSS) no longer serves the community, while the Dictionary of Protein Secondary Structure (DSSP) annotates the secondary structures when tertiary structures of proteins are provided. Here, we extract 1.7 million helices from the PDB and compile them into a database (Therapeutic Peptide Design database; TP-DB) that allows queries of compounded patterns to facilitate the identification of sequence motifs of helical structures. We show how TP-DB helps us identify a known purification-tag-specific antibody that can be repurposed into a diagnostic kit for Helicobacter pylori. We also show how the database can be used to design a new antimicrobial peptide that shows better Candida albicans clearance and lower hemolysis than its template homologs. Finally, we demonstrate how TP-DB can suggest point mutations in helical peptide blockers to prevent a targeted tumorigenic protein-protein interaction. TP-DB is made available at http://dyn.life.nthu.edu.tw/design/ .

Candida albicans Sfp1 Is Involved in the Cell Wall and Endoplasmic Reticulum Stress Responses Induced by Human Antimicrobial Peptide LL-37.

Candida albicans is a commensal fungus of humans but can cause infections, particularly in immunocompromised individuals, ranging from superficial to life-threatening systemic infections. The cell wall is the outermost layer of C. albicans that interacts with the host environment. Moreover, antimicrobial peptides (AMPs) are important components in innate immunity and play crucial roles in host defense. Our previous studies showed that the human AMP LL-37 binds to the cell wall of C. albicans, alters the cell wall integrity (CWI) and affects cell adhesion of this pathogen. In this study, we aimed to further investigate the molecular mechanisms underlying the C. albicans response to LL-37. We found that LL-37 causes cell wall stress, activates unfolded protein response (UPR) signaling related to the endoplasmic reticulum (ER), induces ER-derived reactive oxygen species and affects protein secretion. Interestingly, the deletion of the SFP1 gene encoding a transcription factor reduced C. albicans susceptibility to LL-37, which is cell wall-associated. Moreover, in the presence of LL-37, deletion of SFP1 attenuated the UPR pathway, upregulated oxidative stress responsive (OSR) genes and affected bovine serum albumin (BSA) degradation by secreted proteases. Therefore, these findings suggested that Sfp1 positively regulates cell wall integrity and ER homeostasis upon treatment with LL-37 and shed light on pathogen-host interactions.

Antimicrobial Activity of the Peptide LfcinB15 against Candida albicans.

Lactoferricin (Lfcin) is an amphipathic, cationic peptide derived from proteolytic cleavage of the N-lobe of lactoferrin (Lf). Lfcin and its derivatives possess broad-spectrum antibacterial and antifungal activities. However, unlike their antibacterial functions, the modes of action of Lfcin and its derivatives against pathogenic fungi are less well understood. In this study, the mechanisms of LfcinB15, a derivative of bovine Lfcin, against Candida albicans were, therefore, extensively investigated. LfcinB15 exhibited inhibitory activity against planktonic cells, biofilm cells, and clinical isolates of C. albicans and non-albicans Candida species. We further demonstrated that LfcinB15 is localized on the cell surface and vacuoles of C. albicans cells. Moreover, LfcinB15 uses several different methods to kill C. albicans, including disturbing the cell membrane, inducing reactive oxygen species (ROS) generation, and causing mitochondrial dysfunction. Finally, the Hog1 and Mkc1 mitogen-activated protein kinases were both activated in C. albicans cells in response to LfcinB15. These findings help us to obtain more insight into the complex mechanisms used by LfcinB15 and other Lfcin-derived peptides to fight fungal pathogens.

Characterization of biofilm production in different strains of Acinetobacter baumannii and the effects of chemical compounds on biofilm formation.

Acinetobacter baumannii, an important emerging pathogen of nosocomial infections, is known for its ability to form biofilms. Biofilm formation increases the survival rate of A. baumannii on dry surfaces and may contribute to its persistence in the hospital environment, which increases the probability of nosocomial infections and outbreaks. This study was undertaken to characterize the biofilm production of different strains of A. baumannii and the effects of chemical compounds, especially antibiotics, on biofilm formation. In this study, no statistically significant relationship was observed between the ability to form a biofilm and the antimicrobial susceptibility of the A. baumannii clinical isolates. Biofilm formation caused by A. baumannii ATCC 17978 after gene knockout of two-component regulatory system gene baeR, efflux pump genes emrA/emrB and outer membrane coding gene ompA revealed that all mutant strains had less biofilm formation than the wild-type strain, which was further supported by the images from scanning electron microscopy and confocal laser scanning microscopy. The addition of amikacin, colistin, LL-37 or tannic acid decreased the biofilm formation ability of A. baumannii. In contrast, the addition of lower subinhibitory concentration tigecycline increased the biofilm formation ability of A. baumannii. Minimum biofilm eradication concentrations of amikacin, imipenem, colistin, and tigecycline were increased obviously for both wild type and multidrug resistant clinical strain A. baumannii VGH2. In conclusion, the biofilm formation ability of A. baumannii varied in different strains, involved many genes and could be influenced by many chemical compounds.

Human Antimicrobial Peptide Hepcidin 25-Induced Apoptosis in Candida albicans.

Hepcidin 25 (hep 25) is a cysteine-rich 25-amino acid antimicrobial peptide containing the amino-terminal Cu(II)/Ni(II)-binding (ATCUN) motif. Upon metal binding, the ATCUN motif is known to be involved in the generation of reactive oxygen species (ROS), especially hydrogen peroxide and hydroxyl radicals, which act against different bacterial species. However, the antifungal activity and its correlation to the Cu(II)-ATCUN complex of Hep 25 are still poorly understood. Here, we found that ROS accumulation plays an important role in the fungicidal activity of hep 25 against Candida albicans. In addition, Annexin V-FITC staining and TUNEL assay results provide clues about the apoptosis induced by hep 25. Moreover, hep 25 also increases the generation of ROS, possibly because of copper binding to the ATCUN motif, which is relevant to its activity against C. albicans. Finally, the C. albicans killing action of hep 25 is an energy- and temperature-dependent process that does not involve targeting the membrane. Taken together, our results provide new insights into the mechanisms of hep 25 against C. albicans cells and the potential use of hep 25 and its derivatives as novel antifungal agents.

The interaction Between Carbohydrates and the Antimicrobial Peptide P-113Tri is Involved in the Killing of Candida albicans.

The emergence of drug resistance to Candida albicans is problematic in the clinical setting. Therefore, developing new antifungal drugs is in high demand. Our previous work indicated that the antimicrobial peptide P-113Tri exhibited higher antifungal activity against planktonic cells, biofilm cells, and clinical isolates of Candida species compared to its parental peptide P-113. In this study, we further investigated the difference between these two peptides in their mechanisms against C. albicans. Microscopic examination showed that P-113 rapidly gained access to C. albicans cells. However, most of the P-113Tri remained on the cell surface. Moreover, using a range of cell wall-defective mutants and competition assays, the results indicated that phosphomannan and N-linked mannan in the cell wall are important for peptide binding to C. albicans cells. Furthermore, the addition of exogenous phosphosugars reduced the efficacy of the peptide, suggesting that negatively charged phosphosugars also contributed to the peptide binding to the cell wall polysaccharides. Finally, using a glycan array, P-113Tri, but not P-113, can bind to other glycans commonly present on other microbial and mammalian cells. Together, these results suggest that P-113 and P-113Tri have fundamental differences in their interaction with C. albicans and candidacidal activities.

Candida albicans Aro1 affects cell wall integrity, biofilm formation and virulence.

BACKGROUND: Candida albicans is an opportunistic pathogen capable of causing life-threatening systemic infections. The C. albicans ARO1 gene encodes an arom multifunctional enzyme, which can possibly catalyze reactions of the shikimate pathway to synthesize aromatic amino acids. However, the functions of C. albicans Aro1 have not been extensively characterized. METHODS: ARO1 knockdown mutant strain was constructed, using a tetracycline-regulated (TR) expression system. Cell growth of the mutant strain was compared with wild type. Effects of the ARO1 gene knockdown on cell wall properties, adhesion to polystyrene and biofilm formation were further investigated. Finally, Galleria mellonella was used as a model host to study the role of ARO1 in virulence of C. albicans. RESULTS: We showed that defective growth in the ARO1 knockdown strain was rescued by supplemental aromatic amino acids. In addition, the ARO1 knockdown strain was easily aggregated and precipitated. The knockdown of ARO1 also caused changes in cell wall properties and compositions and promoted C. albicans cell adhesion to polystyrene and biofilm formation. Finally, the ARO1 knockdown strain showed attenuation of C. albicans virulence. CONCLUSION: This work provides new insights into C. albicans metabolism, cell wall and virulence.

Novel mitochondrial complex I-inhibiting peptides restrain NADH dehydrogenase activity.

The emergence of drug-resistant fungal pathogens is becoming increasingly serious due to overuse of antifungals. Antimicrobial peptides have potent activity against a broad spectrum of pathogens, including fungi, and are considered a potential new class of antifungals. In this study, we examined the activities of the newly designed peptides P-113Du and P-113Tri, together with their parental peptide P-113, against the human fungal pathogen Candida albicans. The results showed that these peptides inhibit mitochondrial complex I, specifically NADH dehydrogenase, of the electron transport chain. Moreover, P-113Du and P-113Tri also block alternative NADH dehydrogenases. Currently, most inhibitors of the mitochondrial complex I are small molecules or artificially-designed antibodies. Here, we demonstrated novel functions of antimicrobial peptides in inhibiting the mitochondrial complex I of C. albicans, providing insight in the development of new antifungal agents.

Rhamnose Binding Protein as an Anti-Bacterial Agent-Targeting Biofilm of Pseudomonas aeruginosa.

More than 80% of infectious bacteria form biofilm, which is a bacterial cell community surrounded by secreted polysaccharides, proteins and glycolipids. Such bacterial superstructure increases resistance to antimicrobials and host defenses. Thus, to control these biofilm-forming pathogenic bacteria requires antimicrobial agents with novel mechanisms or properties. Pseudomonas aeruginosa, a Gram-negative opportunistic nosocomial pathogen, is a model strain to study biofilm development and correlation between biofilm formation and infection. In this study, a recombinant hemolymph plasma lectin (rHPLOE) cloned from Taiwanese Tachypleus tridentatus was expressed in an Escherichia coli system. This rHPLOE was shown to have the following properties: (1) Binding to P. aeruginosa PA14 biofilm through a unique molecular interaction with rhamnose-containing moieties on bacteria, leading to reduction of extracellular di-rhamnolipid (a biofilm regulator); (2) decreasing downstream quorum sensing factors, and inhibiting biofilm formation; (3) dispersing the mature biofilm of P. aeruginosa PA14 to improve the efficacies of antibiotics; (4) reducing P. aeruginosa PA14 cytotoxicity to human lung epithelial cells in vitro and (5) inhibiting P. aeruginosa PA14 infection of zebrafish embryos in vivo. Taken together, rHPLOE serves as an anti-biofilm agent with a novel mechanism of recognizing rhamnose moieties in lipopolysaccharides, di-rhamnolipid and structural polysaccharides (Psl) in biofilms. Thus rHPLOE links glycan-recognition to novel anti-biofilm strategies against pathogenic bacteria.

The Transcription Factor Sfp1 Regulates the Oxidative Stress Response in Candida albicans.

Candida albicans is a commensal that inhabits the skin and mucous membranes of humans. Because of the increasing immunocompromised population and the limited classes of antifungal drugs available, C. albicans has emerged as an important opportunistic pathogen with high mortality rates. During infection and therapy, C. albicans frequently encounters immune cells and antifungal drugs, many of which exert their antimicrobial activity by inducing the production of reactive oxygen species (ROS). Therefore, antioxidative capacity is important for the survival and pathogenesis of C. albicans. In this study, we characterized the roles of the zinc finger transcription factor Sfp1 in the oxidative stress response against C. albicans. A sfp1-deleted mutant was more resistant to oxidants and macrophage killing than wild-type C. albicans and processed an active oxidative stress response with the phosphorylation of the mitogen-activated protein kinase (MAPK) Hog1 and high CAP1 expression. Moreover, the sfp1-deleted mutant exhibited high expression levels of antioxidant genes in response to oxidative stress, resulting in a higher total antioxidant capacity, glutathione content, and glutathione peroxidase and superoxide dismutase enzyme activity than the wild-type C. albicans. Finally, the sfp1-deleted mutant was resistant to macrophage killing and ROS-generating antifungal drugs. Together, our findings provide a new understanding of the complex regulatory machinery in the C. albicans oxidative stress response.

A method to assess influence of different medical tubing on biofilm formation by Acinetobacter baumannii.

In this study, we developed a method to assess influence of different medical tubing on biofilm formation by A. baumannii. The results of biofilm quantification and scanning electron microscopy showed that the biofilm formation susceptibility of different tubing materials was rubber latex > polyvinyl chloride > silicone.

Investigating Common Pathogenic Mechanisms between Homo sapiens and Different Strains of Candida albicans for Drug Design: Systems Biology Approach via Two-Sided NGS Data Identification.

Candida albicans (C. albicans) is the most prevalent fungal species. Although it is a healthy microbiota, genetic and epigenetic alterations in host and pathogen, and microenvironment changes would lead to thrush, vaginal yeast infection, and even hematogenously disseminated infection. Despite the fact that cytotoxicity is well-characterized, few studies discuss the genome-wide genetic and epigenetic molecular mechanisms between host and C. albicans. The aim of this study is to identify drug targets and design a multiple-molecule drug to prevent the infection from C. albicans. To investigate the common and specific pathogenic mechanisms in human oral epithelial OKF6/TERT-2 cells during the C. albicans infection in different strains, systems modeling and big databases mining were used to construct candidate host⁻pathogen genetic and epigenetic interspecies network (GEIN). System identification and system order detection are applied on two-sided next generation sequencing (NGS) data to build real host⁻pathogen cross-talk GEINs. Core host⁻pathogen cross-talk networks (HPCNs) are extracted by principal network projection (PNP) method. By comparing with core HPCNs in different strains of C. albicans, common pathogenic mechanisms were investigated and several drug targets were suggested as follows: orf19.5034 (YBP1) with the ability of anti-ROS; orf19.939 (NAM7), orf19.2087 (SAS2), orf19.1093 (FLO8) and orf19.1854 (HHF22) with high correlation to the hyphae growth and pathogen protein interaction; orf19.5585 (SAP5), orf19.5542 (SAP6) and orf19.4519 (SUV3) with the cause of biofilm formation. Eventually, five corresponding compounds-Tunicamycin, Terbinafine, Cerulenin, Tetracycline and Tetrandrine-with three known drugs could be considered as a potential multiple-molecule drug for therapeutic treatment of C. albicans.

The small GTPase Rhb1 is involved in the cell response to fluconazole in Candida albicans.

Candida albicans is an important fungal pathogen in humans. Rhb1 is a small GTPase of the Ras superfamily and is conserved from yeasts to humans. In C. albicans, Rhb1 regulates the expression of secreted protease 2, low nitrogen-mediated morphogenesis, and biofilm formation. Moreover, our previous studies have indicated that Rhb1 is associated with the target of rapamycin (TOR) signaling pathway. In this study, we further explored the relationship between Rhb1 and drug susceptibility. The RHB1 deletion mutant exhibited reduced fluconazole susceptibility, and this phenotype occurred mainly through the increased gene expression and activity of efflux pumps. In addition, Mrr1 and Tac1 are transcription factors that can activate efflux pump gene expression. However, the RHB1 deletion, RHB1/MRR1 and RHB1/TAC1 double deletion mutants had no significant differences in efflux pump gene expression and fluconazole susceptibility, suggesting that Rhb1-regulated efflux pump genes do not act through Mrr1 and Tac1. We also showed that membrane localization is crucial for Rhb1 activity in response to fluconazole. Finally, Rhb1 was linked not only to the TOR but also to the Mkc1 mitogen-activated protein kinase signaling pathway in response to fluconazole. In sum, this study unveiled a new role of Rhb1 in the regulation of C. albicans drug susceptibility.

Contribution of EmrAB efflux pumps to colistin resistance in Acinetobacter baumannii.

Efflux pumps play an important role in antimicrobial resistance for Acinetobacter baumannii. However, the function of the Emr pump system and the relationship between Emr and drug resistance has not been characterized in A. baumannii. In this study, four possible groups of emr-like genes were found by searching a genome database. Among them, A1S_1772 (emrB) and A1S_1773 (emrA) were demonstrated to be co-transcribed as a single operon. Moreover, during osmotic stress, A1S_1772 showed the largest change in gene expression compared to the other emrB-like genes, and deletion of A1S_1772 (AB ΔemrB) significantly slowed cell growth in 20% sucrose. Using a phenotypic microarray analysis, the AB ΔemrB mutant was more susceptible to colistin and nafcillin, paromomycin, spiramycin, and D,L-serine hydroxmate than the wild type. The spot assay, time kill assay and minimal inhibition concentration determination also indicated that the wild type could tolerate colistin better than the AB ΔemrB mutant. Finally, the increased expression levels of all emrB-like genes, including A1S_0775, A1S_0909, A1S_1772, and A1S_1799, in colistin resistance-induced A. baumannii further supported the possible involvement of the emrB genes in A. baumannii colistin resistance. Together, the Emr pump systems in A. baumannii contribute to adaptation to osmotic stress and resistance to colistin.

Distribution of different efflux pump genes in clinical isolates of multidrug-resistant Acinetobacter baumannii and their correlation with antimicrobial resistance.

BACKGROUND/PURPOSE: Efflux pumps are one of the major mechanisms of antimicrobial resistance in Acinetobacter baumannii. This study aimed to understand the distribution of different types of pump genes in clinical isolates of multidrug-resistant A. baumannii (MDRAB) and to reveal the relationship between their presence and expression with antimicrobial resistance. METHODS: MDRAB isolates were collected from five hospitals in Taiwan. Different categories of pump genes, including adeB, adeJ, macB, abeM, abeS, emrA-like, emrB-like, and craA, were chosen, and their presence in the collected isolates was determined. Three induced resistant strains of A. baumannii ATCC 17978 to tigecycline, imipenem, and amikacin were also included. The expressions of the selected pump genes were determined using quantitative reverse transcription-polymerase chain reaction. RESULTS: Twenty-one MDRAB clinical isolates were obtained from five hospitals. All of the studied pump genes were present in the collected MDRAB isolates except one isolate that lacked the emrA-like gene. The gene expression of these efflux pumps was variable among the strains. The upregulation of the adeB, adeJ, and macB genes was responsible for tigecycline resistance, and the increased abeS expression was strongly related to amikacin resistance. Of all the antibiotics studied, tigecycline was the strongest inducer of gene expression for many efflux pumps in A. baumannii. CONCLUSION: Efflux pump genes are universally present in the collected clinical MDRAB isolates. The upregulation of the adeB, adeJ, macB and abeS genes is more related with antibiotic resistance.