RESUMO
Chronic, biofilm-based bacterial infections are exceptionally difficult to eradicate due to the high degree of antibiotic recalcitrance exhibited by cells in biofilm communities. In the opportunistic pathogen Pseudomonas aeruginosa, biofilm recalcitrance is multifactorial and arises in part from the preferential expression of resistance genes in biofilms compared to exponential-phase planktonic cells. One such mechanism involves ndvB, which we have previously shown to be expressed specifically in biofilms. In this study, we investigated the regulatory basis of this lifestyle-specific expression by developing an unstable green fluorescent protein (GFP) transcriptional reporter to observe the expression pattern of ndvB We found that in addition to its expression in biofilms, ndvB was upregulated in planktonic cells as they enter stationary phase. The transcription of ndvB in both growth phases was shown to be dependent on the stationary-phase sigma factor RpoS, and mutation of a putative RpoS binding site in the ndvB promoter abolished the activity of the promoter in stationary-phase cells. Overall, we have expanded our understanding of the temporal expression of ndvB in P. aeruginosa and have uncovered a regulatory basis for its growth phase-dependent expression.IMPORTANCE Bacterial biofilms are more resistant to antibiotics than free-living planktonic cells, and understanding the mechanistic basis of this resistance can inform treatments of biofilm-based infections. In addition to chemical and structural barriers that can inhibit antibiotic entry, the upregulation of specific genes in biofilms contributes to the resistance. We investigated this biofilm-specific gene induction by examining expression patterns of ndvB, a gene involved in biofilm resistance of the opportunistic pathogen Pseudomonas aeruginosa We characterized ndvB expression in planktonic and biofilm growth conditions with an unstable green fluorescent protein (GFP) reporter and found that the expression of ndvB in biofilms is dependent on the stationary-phase sigma factor RpoS. Overall, our results support the physiological similarity between biofilms and stationary-phase cells and suggest that the induction of some stationary-phase genes in biofilms may contribute to their increased antibiotic resistance.
Assuntos
Proteínas de Bactérias/genética , Biofilmes , Resistência Microbiana a Medicamentos/genética , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/fisiologia , Fator sigma/genética , Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/genética , Fator sigma/metabolismoRESUMO
Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that infects immunocompromised and cystic fibrosis patients. Treatment is difficult due to antibiotic resistance, and new antimicrobials are needed to treat infections. The alternative sigma factor 54 (σ54, RpoN), regulates many virulence-associated genes. Thus, we evaluated inhibition of virulence in P. aeruginosa by a designed peptide (RpoN molecular roadblock, RpoN*) which binds specifically to RpoN consensus promoters. We expected that RpoN* binding to its consensus promoter sites would repress gene expression and thus virulence by blocking RpoN and/or other transcription factors. RpoN* reduced transcription of approximately 700 genes as determined by microarray analysis, including genes related to virulence. RpoN* expression significantly reduced motility, protease secretion, pyocyanin and pyoverdine production, rhamnolipid production, and biofilm formation. Given the effectiveness of RpoN* in vitro, we explored its effects in a Caenorhabditis elegans-P. aeruginosa infection model. Expression of RpoN* protected C. elegans in a paralytic killing assay, whereas worms succumbed to paralysis and death in its absence. In a slow killing assay, which mimics establishment and proliferation of an infection, C. elegans survival was prolonged when RpoN* was expressed. Thus, blocking RpoN consensus promoter sites is an effective strategy for abrogation of P. aeruginosa virulence.
Assuntos
Peptídeos/genética , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/genética , RNA Polimerase Sigma 54/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiologia , Movimento Celular/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/biossíntese , Glicolipídeos/genética , Humanos , Terapia de Alvo Molecular , Peptídeos/administração & dosagem , Ligação Proteica , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/patogenicidade , RNA Polimerase Sigma 54/administração & dosagem , RNA Polimerase Sigma 54/antagonistas & inibidores , Virulência/genéticaRESUMO
The increasing number of multidrug resistant bacteria has revitalized interest in seeking alternative sources for controlling bacterial infection. Silver nanoparticles (AgNPs), are amongst the most promising candidates due to their wide microbial spectrum of action. In this work, we report on the safety and efficacy of the incorporation of collagen coated AgNPs into collagen hydrogels for tissue engineering. The resulting hybrid materials at [AgNPs] < 0.4 µM retained the mechanical properties and biocompatibility for primary human skin fibroblasts and keratinocytes of collagen hydrogels; they also displayed remarkable anti-infective properties against S. aureus, S. epidermidis, E. coli and P. aeruginosa at considerably lower concentrations than silver nitrate. Further, subcutaneous implants of materials containing 0.2 µM AgNPs in mice showed a reduction in the levels of IL-6 and other inflammation markers (CCL24, sTNFR-2, and TIMP1). Finally, an analysis of silver contents in implanted mice showed that silver accumulation primarily occurred within the tissue surrounding the implant.
