RESUMEN
BACKGROUND: Resonant recognition model-myxoma virus (RRM-MV), a bioactive peptide analogue for myxoma virus MV-T5 protein, was computationally designed by the RRM. In this study, the anticancer effects of RRM-MV were assessed in vitro against four negative control peptides on human skin cancer and normal cells. RESULTS & DISCUSSION: The effects of RRM-MV versus negative control peptides on cells were evaluated by quantitative and qualitative assays. The RRM-MV treatment was able to induce cell death in cancer cells without triggering similar effects on normal cells. However, the negative control peptides produced no toxic effects on skin cancer and normal cells. No effects on human erythrocytes were detected when treated with all peptides. CONCLUSION: It is suggested that the RRM can be applied to design therapeutic anticancer peptides.
Asunto(s)
Péptidos/toxicidad , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Myxoma virus/metabolismo , Péptidos/química , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/patología , Proteínas Virales/químicaRESUMEN
BACKGROUND: Cancer is an international health problem, and the search for effective treatments is still in progress. Peptide therapy is focused on the development of short peptides with strong tumoricidal activity and low toxicity. In this study, we investigated the efficacy of a myxoma virus peptide analogue (RRM-MV) as a candidate for skin cancer therapy. RRM-MV was designed using the Resonant Recognition Model (RRM) and its effect was examined on human skin cancer and normal human skin cells in vitro. METHODS: Cell cultures were treated with various concentrations of the peptides at different incubation intervals. Cellular morphological changes (apoptosis and necrosis) were evaluated using confocal laser scanning microscopy. The cytotoxic effects of RRM-MV on human skin cancer and normal human skin cells were quantitatively determined by cytotoxicity and cell viability assays. The effect on human erythrocytes was also determined using quantitative hemolysis assay. DNA fragmentation assay was performed to detect early apoptotic events in treated cancer cells. Furthermore, to investigate the possible cell signalling pathway targeted by the peptides treatment, the levels of p-Akt expression in skin cancer and normal cells were detected by immunoblotting. RESULTS: Our results indicate that RRM-MV has a dose-dependent toxic effect on cancer cells only up to 18 h. The immunoblotting results indicated that the RRM-MV slightly increased p-Akt expression in melanoma and carcinoma cells, but did not seem to affect p-Akt expression in normal skin cells. CONCLUSIONS: RRM-MV targets and lethally harms cancer cells and leaves normal cells unharmed. It is able to reduce the cancer cell viability, disrupting the LDH activity in cancer cells and can significantly affect cancer progression. Further investigation into other cell signalling pathways is needed in the process leading to the in vivo testing of this peptide to prove its safety as a possible effective treatment for skin cancer.
Asunto(s)
Carcinoma de Células Escamosas , Melanoma , Péptidos , Neoplasias Cutáneas , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/terapia , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Humanos , Técnicas In Vitro , Melanoma/genética , Melanoma/terapia , Myxoma virus/química , Necrosis , Péptidos/administración & dosificación , Péptidos/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/terapiaRESUMEN
BACKGROUND: The Resonant Recognition Model (RRM) is a physico-mathematical model that interprets protein sequence linear information using digital signal processing methods. In this study the RRM concept was employed for structure-function analysis of myxoma virus (MV) proteins and the design of a short bioactive therapeutic peptide with MV-like antitumor/cytotoxic activity. METHODOLOGY/PRINCIPAL FINDINGS: The analogue RRM-MV was designed by RRM as a linear 18 aa 2.3 kDa peptide. The biological activity of this computationally designed peptide analogue against cancer and normal cell lines was investigated. The cellular cytotoxicity effects were confirmed by confocal immunofluorescence microscopy, by measuring the levels of cytoplasmic lactate dehydrogenase (LDH) and by Prestoblue cell viability assay for up to 72 hours in peptide treated and non-treated cell cultures. Our results revealed that RRM-MV induced a significant dose and time-dependent cytotoxic effect on murine and human cancer cell lines. Yet, when normal murine cell lines were similarly treated with RRM-MV, no cytotoxic effects were observed. Furthermore, the non-bioactive RRM designed peptide RRM-C produced negligible cytotoxic effects on these cancer and normal cell lines when used at similar concentrations. The presence/absence of phosphorylated Akt activity in B16F0 mouse melanoma cells was assessed to indicate the possible apoptosis signalling pathway that could be affected by the peptide treatment. So far, Akt activity did not seem to be significantly affected by RRM-MV as is the case for the original viral protein. CONCLUSIONS/SIGNIFICANCE: Our findings indicate the successful application of the RRM concept to design a bioactive peptide analogue (RRM-MV) with cytotoxic effects on tumor cells only. This 2.345 kDa peptide analogue to a 49 kDa viral protein may be suitable to be developed as a potential cancer therapeutic. These results also open a new direction to the rational design of therapeutic agents for future cancer treatment.
Asunto(s)
Myxoma virus/química , Neoplasias/patología , Péptidos/farmacología , Animales , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Biología Computacional , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Microscopía Confocal , Modelos Moleculares , Myxoma virus/efectos de los fármacos , Fosforilación/efectos de los fármacos , Propidio/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Virales/química , Proteínas Virales/farmacologíaRESUMEN
BACKGROUND: Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests. METHODS: Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms. RESULTS: Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration. CONCLUSION: We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.
Asunto(s)
Antibacterianos/farmacología , Bacteriemia/microbiología , Biopelículas/efectos de los fármacos , Recién Nacido de muy Bajo Peso , Infecciones Estafilocócicas/microbiología , Staphylococcus/efectos de los fármacos , Staphylococcus/fisiología , Biopelículas/crecimiento & desarrollo , Inhibidores de Crecimiento/farmacología , Humanos , Recién Nacido , Unidades de Cuidado Intensivo Neonatal , Péptidos y Proteínas de Señalización Intercelular/farmacología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Microscopía Confocal , Microscopía Fluorescente/métodos , Polisacáridos Bacterianos/análisis , Coloración y Etiquetado/métodos , Staphylococcus/aislamiento & purificaciónRESUMEN
OBJECTIVES: (i) To evaluate the role of the adherent growth mode and extracellular polymer substance build-up in biofilm resistance to antibiotics. (ii) To re-assess various mechanisms leading to biofilm resistance to antibiotics. METHODS: We compared the biofilm MICs, biofilm MBCs using the viable count method, biofilm MBCs based on broth recovery methods and minimum biofilm eradication concentrations (MBECs) of antistaphylococcal antibiotics for multilayer biofilms formed by 'biofilm-positive' S. epidermidis strains and monolayer biofilms formed by their 'biofilm-negative' mutants/variants. Bacterial densities and the quantity of persister cells in both multilayer and monolayer biofilms were assessed to evaluate their roles in biofilm resistance. RESULTS: Monolayer and multilayer biofilms presented similar susceptibilities to multiple antibiotics, based on biofilm MIC, broth recovery-based biofilm MBC and MBEC results. Multilayer biofilms demonstrated higher viable count-based MBCs than monolayer biofilms. Both monolayer and multilayer biofilms had very high bacterial densities of approximately 10(11-12) cfu/mL. Persister cells were found in both monolayer and multilayer biofilms, but not in planktonic cultures at log phase. The presence of persister cells in monolayer and multilayer biofilms appeared to be strain and antibiotic dependent. CONCLUSIONS: The adherent growth mode, rather than the ability to build up a typical multilayer biofilm structure, contributes to the high resistance of biofilms to antibiotics, and therefore might be the main virulence factor of coagulase-negative staphylococci (CoNS) with respect to antibiotic resistance. The presence of persister cells in CoNS biofilms plays an important role in antibiotic resistance. Growth at high bacterial densities is another significant factor in biofilm resistance.
Asunto(s)
Antibacterianos/farmacología , Adhesión Bacteriana , Biopelículas/efectos de los fármacos , Biopolímeros/metabolismo , Farmacorresistencia Bacteriana , Staphylococcus epidermidis/efectos de los fármacos , Staphylococcus epidermidis/fisiología , Proteínas Bacterianas , Biopelículas/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Factores de TranscripciónRESUMEN
Coagulase-negative staphylococci (CoNS) are the main causative agents of bacteraemia in infants managed in neonatal intensive care units (NICUs). Intraluminal colonization of long-term central venous catheters by these bacteria and subsequent biofilm formation are the prerequisites of the bloodstream infections acquired in NICUs. The catheter lock technique has been used to treat catheter colonization; however, the optimum choice of antimicrobial agents and their corresponding concentrations and exposure times have not been determined. The effectiveness of catheter lock solutions (CLSs) was assessed by determining the minimal biofilm eradication concentration of antimicrobial agents against CoNS biofilms. Five conventional antibiotics (oxacillin, gentamicin, vancomycin, ciprofloxacin and rifampicin) alone or in combination, as well as ethanol, were evaluated. Ethanol was found to be superior to all of these conventional antibiotics when used as a CLS. A time-kill study and confocal laser scanning microscopy revealed that exposure to 40 % ethanol for 1 h was sufficient to kill CoNS biofilm cells. To our knowledge, this is the first in vitro study to provide solid evidence to support the rationale of using ethanol at low concentrations for a short time as a CLS, instead of using conventional antibiotics at high concentrations for a long period to treat catheter-related bloodstream infections.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Etanol/farmacología , Staphylococcus/efectos de los fármacos , Staphylococcus/enzimología , Antibacterianos/administración & dosificación , Catéteres de Permanencia/microbiología , Quimioterapia Combinada , Etanol/administración & dosificación , Staphylococcus/clasificación , Factores de TiempoRESUMEN
Campylobacter concisus is an opportunistic pathogen commonly found in the human oral cavity. It has also been isolated from clinical sources including gastroenteritis cases. Both secreted and cell-associated hemolytic activities were detected in C. concisus strains isolated from children with gastroenteritis. The secreted hemolytic activity of C. concisus strains was labile and was detected in variable levels from fresh-culture filtrates only. In addition, another secreted hemolysin/cytotoxin with a molecular weight < 10 kDa was detected in a single C. concisus strain (RCH 12). A C. concisus genomic library, constructed from strain RCH 3 in Escherichia coli XL1-Blue, was screened for hemolytic clones. Subcloning and sequence analysis of selected hemolytic clones identified ORFs for genes that enhance hemolytic activity but do not appear to be related to any known hemolysin genes found in Gram-negative bacteria. In a previous study, a stable cell-associated hemolysin was identified as an outer-membrane phospholipase A (OMPLA) encoded by the pldA gene. In this study, we report cloning of the pldA gene of the clinical strain C. concisus RCH 3 and the complementation of phospholipase A activity in an E. coli pldA mutant.
Asunto(s)
Campylobacter/genética , Proteínas Hemolisinas/genética , Hemólisis , Fosfolipasas A/genética , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Campylobacter/aislamiento & purificación , Campylobacter/patogenicidad , Infecciones por Campylobacter/microbiología , Niño , Clonación Molecular , Gastroenteritis/microbiología , Prueba de Complementación Genética , Biblioteca Genómica , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Humanos , Hierro/metabolismo , Datos de Secuencia Molecular , Fosfolipasas A/química , Fosfolipasas A/metabolismo , Fosfolipasas A1/genética , Fosfolipasas A1/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Phospholipase A (PLA) is one of the few enzymes present in the outer membrane of Gram-negative bacteria, and is likely to be involved in the membrane disruption processes that occur during host cell invasion. Both secreted and membrane-bound phospholipase A(2) activities have been described in bacteria, fungi and protozoa. Recently there have been increasing reports on the involvement of PLA in bacterial invasion and pathogenesis. This review highlights the latest findings on PLA as a virulence factor in Gram-negative bacteria.
Asunto(s)
Bacterias Gramnegativas/enzimología , Fosfolipasas A/fisiología , Factores de Virulencia/fisiología , Secuencia de Aminoácidos , Membrana Celular/enzimología , Bacterias Gramnegativas/patogenicidad , Datos de Secuencia Molecular , Fosfolipasas A/química , Fosfolipasas A/genética , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
A membrane-bound, haemolytic phospholipase A(2) (PLA(2)) activity was detected in clinical strains of Campylobacter concisus isolated from children with gastroenteritis. The clinical strains were assigned into two molecular groups (genomospecies) based on PCR amplification of their 23S rDNA. This calcium-dependent, heat-stable, haemolytic PLA(2) activity was detected in strains from both genomospecies. A crude haemolysin extract (CHE) was initially prepared from cellular outer-membrane proteins of these isolates and was further fractionated by ultrafiltration. The haemolytic activity of the extracted fraction (R30) was retained by ultrafiltration using a 30 kDa molecular mass cut-off filter, and was designated haemolysin extract (HE). Both CHE and HE had PLA(2) activity and caused stable vacuolating and cytolytic effects on Chinese hamster ovary cells in tissue culture. Primers for the conserved region of pldA gene (phospholipase A gene) from Campylobacter coli amplified a gene region of 460 bp in all tested isolates, confirming the presence of a homologous PLA gene sequence in C. concisus. The detection of haemolytic PLA(2) activity in C. concisus indicates the presence of a potential virulence factor in this species and supports the hypothesis that C. concisus is a possible opportunistic pathogen.