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1.
Nat Commun ; 13(1): 102, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013238

RESUMO

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/ .


Assuntos
Aminoácidos/química , Antineoplásicos/química , Software , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , /farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Bases de Dados de Proteínas , Desenho de Fármacos/métodos , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Relação Estrutura-Atividade
2.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638975

RESUMO

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.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Candida albicans/metabolismo , Parede Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Parede Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Proteínas Fúngicas/genética , Deleção de Genes , Homeostase/efeitos dos fármacos , Homeostase/genética , Interações Hospedeiro-Patógeno , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
3.
J Fungi (Basel) ; 7(7)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209722

RESUMO

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.

4.
PeerJ ; 8: e9020, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32523805

RESUMO

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.

5.
Microorganisms ; 8(4)2020 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-32316661

RESUMO

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.

6.
Microorganisms ; 8(2)2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32098211

RESUMO

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.

7.
J Microbiol Immunol Infect ; 53(1): 115-124, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29807722

RESUMO

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.


Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/genética , Candida albicans/patogenicidade , Parede Celular , Proteínas Fúngicas/genética , Animais , Candida albicans/crescimento & desenvolvimento , Técnicas de Silenciamento de Genes , Larva/microbiologia , Lepidópteros/microbiologia , Mutação , Virulência
8.
Sci Rep ; 9(1): 13694, 2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31548559

RESUMO

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.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Histatinas/farmacologia , NADH Desidrogenase/metabolismo , Candida albicans/metabolismo , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Mar Drugs ; 17(6)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207891

RESUMO

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.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Ramnose/metabolismo , Células A549 , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Escherichia coli/metabolismo , Glicolipídeos/metabolismo , Caranguejos Ferradura/metabolismo , Humanos , Lectinas/metabolismo , Polissacarídeos Bacterianos/metabolismo , Percepção de Quorum/efeitos dos fármacos , Peixe-Zebra
10.
Microorganisms ; 7(5)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091716

RESUMO

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.

11.
J Microbiol Methods ; 160: 84-86, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30928423

RESUMO

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.


Assuntos
Acinetobacter baumannii/fisiologia , Biofilmes , Infecções Relacionadas a Cateter/microbiologia , Humanos , Látex/química , Cloreto de Polivinila/química , Silicones/química
12.
Toxins (Basel) ; 11(2)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30769958

RESUMO

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.


Assuntos
Antifúngicos , Candida albicans/fisiologia , Candidíase/tratamento farmacológico , Interações Hospedeiro-Patógeno , Linhagem Celular , Desenho de Fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biologia de Sistemas
13.
FEMS Yeast Res ; 19(2)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649293

RESUMO

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.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Fluconazol/farmacologia , Regulação Fúngica da Expressão Gênica , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transporte Biológico Ativo , Candida albicans/genética , Farmacorresistência Fúngica , Deleção de Genes , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Proteínas Monoméricas de Ligação ao GTP/deficiência
14.
J Microbiol ; 55(2): 130-136, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28120193

RESUMO

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.


Assuntos
Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Colistina/farmacologia , Farmacorresistência Bacteriana , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/crescimento & desenvolvimento , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos , Óperon , Pressão Osmótica , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Sacarose/farmacologia , Transcrição Genética
15.
J Microbiol Immunol Infect ; 50(2): 224-231, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26055688

RESUMO

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.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Membrana Transportadoras/genética , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacocinética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/análise , Farmacorresistência Bacteriana Múltipla , Genes Bacterianos , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , RNA Bacteriano/análise
16.
J Microbiol Immunol Infect ; 50(6): 863-871, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27089825

RESUMO

BACKGROUND/PURPOSE: Candida albicans is a common fungal pathogen in humans. In healthy individuals, C. albicans represents a harmless commensal organism, but infections can be life threatening in immunocompromised patients. The complete genome sequence of C. albicans is extremely useful for identifying genes that may be potential drug targets and important for pathogenic virulence. However, there are still many uncharacterized genes in the Candida genome database. In this study, we investigated C. albicans Hom6, the functions of which remain undetermined experimentally. METHODS: HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome profiling, and cell adhesion to polystyrene was also determined. RESULTS: C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. CONCLUSION: Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs.


Assuntos
Candida albicans/enzimologia , Candida albicans/genética , Proteínas Fúngicas/biossíntese , Homosserina Desidrogenase/genética , Sequência de Aminoácidos , Antifúngicos/farmacologia , Adesão Celular , Proteínas Fúngicas/genética , Deleção de Genes , Genoma Fúngico , Homosserina Desidrogenase/metabolismo , Humanos , Metionina/biossíntese , Poliestirenos , Alinhamento de Sequência , Treonina/biossíntese
17.
Antimicrob Agents Chemother ; 60(10): 6369-73, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27458227

RESUMO

Two antimicrobial P-113 peptide derivatives, P-113Du and P-113Tri, were investigated in this study. Notably, P-113Du and P-113Tri contained significant fractions of α-helix conformation and were less sensitive to high salt and low pH than P-113. Moreover, compared to P-113, these peptides exhibited increased antifungal activity against planktonic cells, biofilm cells, and clinical isolates of Candida albicans and non-albicans Candida spp. These results suggest that P-113Du and P-113Tri are promising candidates for development as novel antifungal agents.


Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Histatinas/farmacologia , Ácido Ascórbico/farmacologia , Biofilmes/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Histatinas/química , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Plâncton/microbiologia , Conformação Proteica
18.
PLoS One ; 10(10): e0141107, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26484669

RESUMO

Multidrug-resistant Acinetobacter baumannii has recently emerged as an important pathogen in nosocomial infection; thus, effective antimicrobial regimens are urgently needed. Human antimicrobial peptides (AMPs) exhibit multiple functions and antimicrobial activities against bacteria and fungi and are proposed to be potential adjuvant therapeutic agents. This study examined the effect of the human cathelicidin-derived AMP LL-37 on A. baumannii and revealed the underlying mode of action. We found that LL-37 killed A. baumannii efficiently and reduced cell motility and adhesion. The bacteria-killing effect of LL-37 on A. baumannii was more efficient compared to other AMPs, including human ß-defensin 3 (hBD3) and histatin 5 (Hst5). Both flow cytometric analysis and immunofluorescence staining showed that LL-37 bound to A. baumannii cells. Moreover, far-western analysis demonstrated that LL-37 could bind to the A. baumannii OmpA (AbOmpA) protein. An ELISA assay indicated that biotin-labelled LL-37 (BA-LL37) bound to the AbOmpA74-84 peptide in a dose-dependent manner. Using BA-LL37 as a probe, the ~38 kDa OmpA signal was detected in the wild type but the ompA deletion strain did not show the protein, thereby validating the interaction. Finally, we found that the ompA deletion mutant was more sensitive to LL-37 and decreased cell adhesion by 32% compared to the wild type. However, ompA deletion mutant showed a greatly reduced adhesion defect after LL-37 treatment compared to the wild strain. Taken together, this study provides evidence that LL-37 affects A. baumannii through OmpA binding.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Catelicidinas/farmacologia , Regulação Bacteriana da Expressão Gênica , Infecções por Acinetobacter/metabolismo , Infecções por Acinetobacter/microbiologia , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos , Aderência Bacteriana/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Western Blotting , Movimento Celular/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , beta-Defensinas/farmacologia
19.
PLoS One ; 10(7): e0132843, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26161744

RESUMO

Bacterial two-component regulatory systems (TCSs) facilitate changes in gene expression in response to environmental stimuli. TCS BaeR regulons influence tigecycline susceptibility in Acinetobacter baumannii through positively regulating the pump genes adeA and adeB. In this study, we demonstrate that an additional two transport systems, AdeIJK and MacAB-TolC, are also regulated by BaeSR. In the wild type and clinical tigecycline-resistant A. baumannii strains, gene expression of AdeIJK and MacAB-TolC increased after tigecycline induction, implicating their importance to tigecycline resistance in addition to AdeABC. Phenotypic microarray results showed that A. baumannii is vulnerable to certain chemicals, especially tannic acid, after deleting baeR, which was confirmed using the spot assay. The wild-type strain of A. baumannii also exhibited 1.6-fold and 4.4-fold increase in gene expression of adeJ and macB in the medium with 100 µg/mL tannic acid, but the increase was fully inhibited by baeR deletion. An electrophoretic motility shift assay based on an interaction between His-BaeR and the adeA, adeI and macA promoter regions did not demonstrate direct binding. In conclusion, A. baumannii can use the TCS BaeSR in disposing chemicals, such as tannic acid and tigecycline, through regulating the efflux pumps.


Assuntos
Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Minociclina/análogos & derivados , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Testes de Sensibilidade Microbiana , Minociclina/metabolismo , Mutação/genética , Fenótipo , Taninos/farmacologia , Tigeciclina
20.
PLoS One ; 10(6): e0129903, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26087243

RESUMO

Candida albicans is a major human fungal pathogen. One of the important features of C. albicans pathogenicity is the ability to form biofilms on mucosal surfaces and indwelling medical devices. Biofilm formation involves complex processes in C. albicans, including cell adhesion, filamentous growth, extracellular matrix secretion and cell dispersion. In this work, we characterized the role of the transcription factor Sfp1, particularly with respect to its function in the regulation of biofilm formation. The deletion of the SFP1 gene enhanced cell adhesion and biofilm formation in comparison to the wild-type strain. Interestingly, the sfp1-deleted mutant also exhibited an increase in the expression of the ALS1, ALS3 and HWP1 genes, which encode adhesin proteins. In addition, Sfp1 was demonstrated to function downstream of the Rhb1-TOR signaling pathway. Bcr1 and Efg1 are transcription factors that are critical for controlling biofilm formation, and Efg1 is also required for hyphal growth. Deleting either the BCR1 or EFG1 gene in the sfp1-null background led to reduced adhesin gene expression. As a result, the bcr1/sfp1 or efg1/sfp1 double deletion mutants exhibited dramatically reduced biofilm formation. The results indicated that Sfp1 negatively regulates the ALS1, ALS3 and HWP1 adhesin genes and that the repression of these genes is mediated by the inhibition of Bcr1 and Efg1.


Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/fisiologia , Candidíase/microbiologia , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/metabolismo , Candida albicans/genética , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Humanos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética
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