RESUMEN
Menthol (C(10)H(20)O) possesses antibacterial activity; nevertheless, bacterial adaptation to this compound has never been studied. Here we report that precultivation of enterohemorrhagic Escherichia coli (EHEC) strains in increasing subinhibitory (SI) concentrations of menthol significantly elevates (4- to 16-fold) their resistance to menthol. Concomitant morphological alterations included the appearance of mucoid colonies and reduced biofilm production. Scanning electron microscopy (SEM) examination revealed suppressed curli formation in menthol-adapted cells. Expression of the gene cpsB10 (encoding one of the enzymes responsible for colanic acid production) was elevated in response to SI concentrations of menthol in a laboratory E. coli strain, whereas expression in an rcsC null mutant was reduced, implicating a partial role for the Rcs phosphorelay system in mediating the menthol signal. Adaptation to menthol also reduced expression of the locus of enterocyte effacement-encoded regulator (Ler). This reduction, together with reduced curli and biofilm formation and elevated mucoidity, suggests a general reduction in bacterial virulence following adaptation to menthol. Our results thus suggest menthol as a potential lead in the recently emerging alternative strategy of targeting bacterial virulence factors to develop new types of anti-infective agents.
Asunto(s)
Adaptación Biológica/efectos de los fármacos , Biopelículas/efectos de los fármacos , Farmacorresistencia Bacteriana/fisiología , Escherichia coli Enterohemorrágica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Mentol/farmacología , Farmacorresistencia Bacteriana/genética , Escherichia coli Enterohemorrágica/patogenicidad , Escherichia coli Enterohemorrágica/ultraestructura , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Virulencia , beta-GalactosidasaRESUMEN
We previously found that a short exposure of Staphylococcus aureus to subinhibitory (SI) doses of epigallocatechin gallate (EGCG) results in increased cell wall thickness, adaptation, and enhanced tolerance to cell-wall-targeted antibiotics. In this study, the response to EGCG of sigB and vraSR transcription factor mutants was characterized. We show that in contrast to the results observed for wild-type (WT) strains, an S. aureus 315 vraSR null mutant exposed to SI doses of EGCG did not exhibit increased tolerance to EGCG and oxacillin. A diminished increase in tolerance to ampicillin (from 16-fold to 4-fold) and no change in the magnitude of resistance to vancomycin were observed. Preexposure to EGCG enhanced the tolerance of wild-type and sigB null mutant cells to lysostaphin, but this enhancement was much weaker in the vraSR null mutant. Marked upregulation (about 60-fold) of vraR and upregulation of the peptidoglycan biosynthesis-associated genes murA, murF, and pbp2 (2-, 5-, and 6-fold, respectively) in response to SI doses of EGCG were determined by quantitative reverse transcription-PCR (qRT-PCR). EGCG also induced the promoter of sas016 (encoding a cell wall stress protein of unknown function which is not induced in vraSR null mutants) in a concentration-dependent manner, showing kinetics comparable to those of cell-wall-targeting antibiotics. Taken together, our results suggest that the two-component VraSR system is involved in modulating the cell response to SI doses of EGCG.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Catequina/análogos & derivados , Pared Celular/efectos de los fármacos , Proteínas de Unión al ADN/biosíntesis , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Transducción de Señal , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Catequina/metabolismo , Pared Celular/metabolismo , Proteínas de Unión al ADN/genética , Tolerancia a Medicamentos , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Lisostafina/metabolismo , Oxacilina/metabolismo , Oxacilina/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Staphylococcus aureus/metabolismoRESUMEN
The armadillo-family protein, p120 catenin (p120), binds to the juxtamembrane domain of classical cadherins and increases cell-cell junction stability. Overexpression of p120 modulates the activity of Rho family GTPases and augments cell migratory ability. Here we show that down-regulation of p120 in epithelial MCF-7 cells by siRNA leads to a striking decrease in lamellipodial persistence and focal adhesion formation. Similar alterations in lamellipodial activity were observed in MCF-7 cells treated with siRNA to cortactin, an activator of Arp2/3-dependent actin polymerization. We found that, in many cell types, p120 is colocalized with cortactin-containing actin structures not only at cell-cell junctions, but also at extrajunctional sites including membrane ruffles and actin-rich halos around endocytotic vesicles. p120 depletion led to dramatic loss of cortactin and its partner, Arp3, from the cell leading edges. Cortactin and p120 are shown to directly interact with each other via the cortactin N-terminal region. We propose that the mechanism underlying p120 functions at the leading edge involves its cooperation with cortactin.
Asunto(s)
Proteína 3 Relacionada con la Actina/metabolismo , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/fisiología , Adhesión Celular , Cortactina/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiología , Seudópodos/metabolismo , Cateninas , Línea Celular Tumoral , Células Epiteliales , Adhesiones Focales , Humanos , Unión Proteica , Catenina deltaRESUMEN
Epigallocathechin gallate (EGCG) possesses many beneficial properties, such as anticarcinogenicity, antiatherogenicity, as well as antioxidant and antibacterial activities. However, the bacterial response to sublethal concentrations of EGCG has not been studied. Here we investigated whether short exposure of staphylococci strains to sublethal doses of EGCG can lead to adaptation and cross-resistance. Two-hour exposure of five strains to 20 microg/ml of EGCG did not affect the growth rate but significantly elevated the resistance towards antibiotics targeting the bacterial cell wall. The magnitude of cross-resistance towards such antibiotics varied with the staphylococci strain, with Staphylococcus aureus Newman exhibiting the highest magnitude of cross-resistance, showing a 2, 4 and 8-fold increase in resistance towards vancomycin, oxacillin and ampicillin respectively. All EGCG-adapted strains were also more heat tolerant than their control counterparts as derived from the Weibull model. Adaptation to EGCG led to a moderate increase in heat resistance of the adapted strains S. epidermis ATCC 12228, S. aureus Newman, and S. aureus ATCC 29213, and an extremely pronounced increase for S. aureus ATCC 6538 and S. aureus RN4220. The shape of the survival curve also varied with the staphylococci strain. Transmission electron microscopy (TEM) analysis revealed suppressed separation of daughter cells in cultures exposed to EGCG, as evidenced by the pseudomulticellular appearance and by more than 2-fold increase in cell wall thickness. These observations raise concerns over the potential of EGCG utilization in therapy in that it may contribute to the development and enhancement of microbial resistance mechanisms.