RESUMEN
Exposure of wild-type (WT) Pseudomonas aeruginosa PAO1 to ZnCl2 (Zn) yielded a concentration-dependent increase in depolarization of the cytoplasmic membrane (CM), an indication that this metal is membrane-damaging. Consistent with this, Zn activated the AmgRS envelope stress-responsive two-component system (TCS) that was previously shown to be activated by and to protect P. aeruginosa from the membrane-damaging effects of aminoglycoside (AG) antibiotics. A mutant lacking amgR showed enhanced Zn-promoted CM perturbation and was Zn-sensitive, an indication that the TCS protected cells from the CM-damaging effects of this metal. In agreement with this, a mutant carrying an AmgRS-activating amgS mutation was less susceptible to Zn-promoted CM perturbation and more tolerant of elevated levels of Zn than WT. AG activation of AmgRS is known to drive expression of the AG resistance-promoting mexXY multidrug efflux operon, and while Zn similarly induced mexXY expression this was independent of AmgRS and reliant on a second TCS implicated in mexXY regulation, ParRS. MexXY did not, however, contribute to Zn resistance or protection from Zn-promoted CM damage. Despite its activation of AmgRS and induction of mexXY, Zn had a minimal impact on the AG resistance of WT P. aeruginosa although, given that Zn-tolerant AmgRS-activated amgS mutant strains are AG resistant, there is still the prospect of this metal promoting AG resistance development in this organism.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Zinc/metabolismo , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple , Regulación Bacteriana de la Expresión Génica , Pruebas de Sensibilidad Microbiana , Operón , Pseudomonas aeruginosa/genéticaRESUMEN
Exposure of P. aeruginosa to the aminoglycoside (AG) paromomycin (PAR) induced expression of the PA3720-armR locus and the mexAB-oprM multidrug efflux operon that AmgR controls, although PAR induction of mexAB-oprM was independent of armR. Multiple AGs promoted mexAB-oprM expression and this was lost in the absence of the amgRS locus encoding an aminoglycoside-activated envelope stress-responsive 2-component system (TCS). Purified AmgR bound to the mexAB-oprM promoter region consistent with this response regulator directly regulating expression of the efflux operon. The thiol-active reagent, diamide, which, like AGs, promotes protein aggregation and cytoplasmic membrane damage also promoted AmgRS-dependent mexAB-oprM expression, a clear indication that the MexAB-OprM efflux system is recruited in response to membrane perturbation and/or circumstances that lead to this. Despite the AG and diamide induction of mexAB-oprM, however, MexAB-OprM does not appear to contribute to resistance to these agents.
Asunto(s)
Aminoglicósidos/farmacología , Proteínas Bacterianas/genética , Resistencia a Múltiples Medicamentos/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Operón/genética , Pseudomonas aeruginosa/efectos de los fármacos , Estrés Fisiológico/genética , Proteínas Bacterianas/metabolismo , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Operón/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiología , Estrés Fisiológico/efectos de los fármacosRESUMEN
Pseudomonas aeruginosa utilizes the Type II secretion system (T2SS) to translocate a wide range of large, structured protein virulence factors through the periplasm to the extracellular environment for infection. In the T2SS, five pseudopilins assemble into the pseudopilus that acts as a piston to extrude exoproteins out of cells. Through structure determination of the pseudopilin complexes of XcpVWX and XcpVW and function analysis, we have confirmed that two minor pseudopilins, XcpV and XcpW, constitute a core complex indispensable to the pseudopilus tip. The absence of either XcpV or -W resulted in the non-functional T2SS. Our small-angle X-ray scattering experiment for the first time revealed the architecture of the entire pseudopilus tip and established the working model. Based on the interaction interface of complexes, we have developed inhibitory peptides. The structure-based peptides not only disrupted of the XcpVW core complex and the entire pseudopilus tip in vitro but also inhibited the T2SS in vivo. More importantly, these peptides effectively reduced the virulence of P. aeruginosa towards Caenorhabditis elegans.
Asunto(s)
Proteínas Bacterianas/química , Caenorhabditis elegans/crecimiento & desarrollo , Fimbrias Bacterianas/metabolismo , Proteínas de la Membrana/química , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreción Tipo II/química , Animales , Proteínas Bacterianas/metabolismo , Caenorhabditis elegans/microbiología , Cristalografía por Rayos X , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Conformación Proteica , Infecciones por Pseudomonas/metabolismo , Sistemas de Secreción Tipo II/metabolismo , VirulenciaRESUMEN
Objectives: To assess the ability of meropenem to potentiate aminoglycoside (AG) activity against laboratory and AG-resistant cystic fibrosis (CF) isolates of Pseudomonas aeruginosa and to elucidate its mechanism of action. Methods: AG resistance gene deletions were engineered into P. aeruginosa laboratory and CF isolates using standard gene replacement technology. Susceptibility to AGs ± meropenem (at ½ MIC) was assessed using a serial 2-fold dilution assay. mexXY expression and MexXY-OprM efflux activity were quantified using quantitative PCR and an ethidium bromide accumulation assay, respectively. Results: A screen for agents that rendered WT P. aeruginosa susceptible to a sub-MIC concentration of the AG paromomycin identified the carbapenem meropenem, which potentiated several additional AGs. Meropenem potentiation of AG activity was largely lost in a mutant lacking the MexXY-OprM multidrug efflux system, an indication that it was targeting this efflux system in enhancing P. aeruginosa susceptibility to AGs. Meropenem failed to block AG induction of mexXY expression or MexXY-OprM efflux activity, suggesting that it may be interfering with some MexXY-dependent process linked to AG susceptibility. Meropenem potentiated AG activity versus AG-resistant CF isolates, enhancing susceptibility to at least one AG in all isolates and susceptibility to all tested AGs in 50% of the isolates. Notably, meropenem potentiation of AG activity was linked to MexXY in some but not all CF isolates in which this was examined. Conclusions: Meropenem potentiates AG activity against laboratory and CF strains of P. aeruginosa, both dependent on and independent of MexXY, highlighting the complexity of AG resistance in this organism.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Sinergismo Farmacológico , Proteínas de Transporte de Membrana/metabolismo , Meropenem/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVES: Rifampicin potentiates the activity of aminoglycosides (AGs) versus Pseudomonas aeruginosa by targeting the AmgRS two-component system. In this study we examine the impact of rifampicin on the AG susceptibility of cystic fibrosis (CF) lung isolates of P. aeruginosa and the contribution of AmgRS to AG resistance in these isolates. METHODS: amgR deletion derivatives of clinical isolates were constructed using standard gene replacement technology. Susceptibility to AGs ± rifampicin (at ½ MIC) was assessed using a serial 2-fold dilution assay. RESULTS: Rifampicin showed a variable ability to potentiate AG activity versus the CF isolates, enhancing AG susceptibility between 2- and 128-fold. Most strains showed potentiation for at least two AGs, with only a few strains showing no AG potentiation by rifampicin. Notably, loss of amgR increased AG susceptibility although rifampicin potentiation of AG activity was still observed in the ΔamgR derivatives. CONCLUSIONS: AmgRS contributes to AG resistance in CF isolates of P. aeruginosa and rifampicin shows a variable ability to potentiate AG activity against these, highlighting the complexity of AG resistance in such isolates.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Sinergismo Farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Rifampin/farmacología , Fibrosis Quística/complicaciones , Técnicas de Inactivación de Genes , Genes Bacterianos , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Rifampin/metabolismoRESUMEN
Environmental influences on antibiotic activity and resistance can wreak havoc with in vivo antibiotic efficacy and, ultimately, antimicrobial chemotherapy. In nature, bacteria encounter a variety of metal ions, particularly copper (Cu) and zinc (Zn), as contaminants in soil and water, as feed additives in agriculture, as clinically-used antimicrobials, and as components of human antibacterial responses. Importantly, there is a growing body of evidence for Cu/Zn driving antibiotic resistance development in metal-exposed bacteria, owing to metal selection of genetic elements harbouring both metal and antibiotic resistance genes, and metal recruitment of antibiotic resistance mechanisms. Many classes of antibiotics also form complexes with metal cations, including Cu and Zn, and this can hinder (or enhance) antibiotic activity. This review highlights the ways in which Cu/Zn influence antibiotic resistance development and antibiotic activity, and in so doing impact in vivo antibiotic efficacy.
Asunto(s)
Antibacterianos/química , Cobre/química , Cementos de Resina/análisis , Zinc/química , Animales , Farmacorresistencia Bacteriana Múltiple/fisiología , Ambiente , Humanos , Contaminantes del Suelo/análisisRESUMEN
A screen for agents that potentiated the activity of paromomycin (PAR), a 4,5-linked aminoglycoside (AG), against wild-type Pseudomonas aeruginosa identified the RNA polymerase inhibitor rifampin (RIF). RIF potentiated additional 4,5-linked AGs, such as neomycin and ribostamycin, but not the clinically important 4,6-linked AGs amikacin and gentamicin. Potentiation was absent in a mutant lacking the AmgRS envelope stress response two-component system (TCS), which protects the organism from AG-generated membrane-damaging aberrant polypeptides and, thus, promotes AG resistance, an indication that RIF was acting via this TCS in potentiating 4,5-linked AG activity. Potentiation was also absent in a RIF-resistant RNA polymerase mutant, consistent with its potentiation of AG activity being dependent on RNA polymerase perturbation. PAR-inducible expression of the AmgRS-dependent genes htpX and yccA was reduced by RIF, suggesting that AG activation of this TCS was compromised by this agent. Still, RIF did not compromise the membrane-protective activity of AmgRS, an indication that it impacted some other function of this TCS. RIF potentiated the activities of 4,5-linked AGs against several AG-resistant clinical isolates, in two cases also potentiating the activity of the 4,6-linked AGs. These cases were, in one instance, explained by an observed AmgRS-dependent expression of the MexXY multidrug efflux system, which accommodates a range of AGs, with RIF targeting of AmgRS undermining mexXY expression and its promotion of resistance to 4,5- and 4,6-linked AGs. Given this link between AmgRS, MexXY expression, and pan-AG resistance in P. aeruginosa, RIF might be a useful adjuvant in the AG treatment of P. aeruginosa infections.
Asunto(s)
Antibacterianos/farmacología , Paromomicina/farmacología , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Rifampin/farmacología , Amicacina/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Sinergismo Farmacológico , Regulación Bacteriana de la Expresión Génica , Gentamicinas/farmacología , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Ribostamicina/farmacología , Estrés Fisiológico/genéticaRESUMEN
The ribosome-targeting antimicrobial, spectinomycin (SPC), strongly induced the mexXY genes of the MexXY-OprM multidrug efflux system in Pseudomonas aeruginosa and increased susceptibility to the polycationic antimicrobials polymyxin B and polymyxin E, concomitant with a decrease in expression of the polymyxin resistance-promoting lipopolysaccharide (LPS) modification loci, arnBCADTEF and PA4773-74. Consistent with the SPC-promoted reduction in arn and PA4773-74 expression being linked to mexXY, expression of these LPS modification loci was moderated in a mutant constitutively expressing mexXY and enhanced in a mutant lacking the efflux genes. Still, the SPC-mediated increase in polymyxin susceptibility was retained in mutants lacking arnB and/or PA4773-74, an indication that their reduced expression in SPC-treated cells does not explain the enhanced polymyxin susceptibility. That the polymyxin susceptibility of a mutant strain lacking mexXY was unaffected by SPC exposure, however, was an indication that the unknown polymyxin resistance 'mechanism' is also influenced by the MexXY status of the cell. In agreement with SPC and MexXY influencing polymyxin susceptibility as a result of changes in the LPS target of these agents, SPC treatment yielded a decline in common polysaccharide antigen (CPA) synthesis in wild-type P. aeruginosa but not in the ΔmexXY mutant. A mutant lacking CPA still showed the SPC-mediated decline in polymyxin MICs, however, indicating that the loss of CPA did not explain the SPC-mediated MexXY-dependent increase in polymyxin susceptibility. It is possible, therefore, that some additional change in LPS promoted by SPC-induced mexXY expression impacted CPA synthesis or its incorporation into LPS and that this was responsible for the observed changes in polymyxin susceptibility.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Colistina/farmacología , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Polimixina B/farmacología , Pseudomonas aeruginosa/genética , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Eliminación de Gen , Sitios Genéticos , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/genética , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Espectinomicina/farmacologíaRESUMEN
mexCD-oprJ is an envelope stress-inducible multidrug efflux operon of Pseudomonas aeruginosa. A gene encoding a homologue of the NfxB repressor of this operon, PA4596, occurs downstream of oprJ and was proposed as a second repressor of this efflux operon. Inactivation of this gene had no impact on mexCD-oprJ expression in cells not exposed to envelope stress although its loss under envelope stress conditions yielded a > 10-fold increase in mexCD-oprJ expression. Consistent with PA4596 functioning as a mexCD-oprJ repressor, the purified protein was able to bind to a DNA fragment carrying the mexCD-oprJ promoter region. Expression of PA4596 was induced under conditions of envelope stress dependent on the AlgU envelope stress sigma factor, consistent with PA4596 operating under envelope stress conditions where it possibly serves to moderate envelope stress-inducible mexCD-oprJ expression. nfxB mutants showed elevated PA4596 expression and purified NfxB bound to DNA encompassing the PA4596 upstream region, an indication that NfxB functions as a repressor of PA4596 expression. Elimination of PA4596 in P. aeruginosa lacking nfxB and hyperexpressing mexCD-oprJ had no additional impact on mexCD-oprJ expression, regardless of the presence of envelope stress, suggesting that PA4596 repressor activity may be dependent on NfxB. This envelope stress-regulated repressor of mexCD-oprJ has been renamed esrC.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Operón , Pseudomonas aeruginosa/genética , Proteínas Represoras/metabolismo , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/metabolismo , Farmacorresistencia Bacteriana Múltiple , Proteínas de Transporte de Membrana/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Represoras/genética , Estrés Fisiológico/genética , Factores de Transcripción/metabolismoRESUMEN
AmgRS is an envelope stress-responsive two-component system and aminoglycoside resistance determinant in Pseudomonas aeruginosa that is proposed to protect cells from membrane damage caused by aminoglycoside-generated mistranslated polypeptides. Consistent with this, a ΔamgR strain showed increased aminoglycoside-promoted membrane damage, damage that was largely absent in AmgRS-activated amgS-mutant strains. Intriguingly, one such mutation, V121G, while providing for enhanced resistance to aminoglycosides, rendered P. aeruginosa susceptible to several ribosome-targeting nonaminoglycoside antimicrobials that are inducers and presumed substrates of the MexXY-OprM multidrug efflux system. Surprisingly, the amgSV 121G mutation increased mexXY expression threefold, suggesting that export of these nonaminoglycosides was compromised in the amgSV 121G mutant. Nonetheless, a link was established between AmgRS activation and mexXY expression and this was confirmed in studies showing that aminoglycoside-promoted mexXY expression is dependent on AmgRS. While nonaminoglycosides also induced mexXY expression, this was not AmgRS-dependent, consistent with these agents not generating mistranslated polypeptides and not activating AmgRS. The aminoglycoside inducibility of mexXY was abrogated in a mutant lacking the AmgRS target genes htpX and PA5528, encoding a presumed cytoplasmic membrane-associated protease and a membrane protein of unknown function, respectively. Thus, aminoglycoside induction of mexXY is a response to membrane damage and activation of the AmgRS two-component system.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Genes MDR , Operón , Pseudomonas aeruginosa/genética , Aminoglicósidos/farmacología , Farmacorresistencia Bacteriana/genética , Mutación , Transporte de Proteínas , Pseudomonas aeruginosa/efectos de los fármacos , Estrés Fisiológico/genéticaRESUMEN
Pseudomonas aeruginosa is a notoriously antimicrobial-resistant organism that is increasingly refractory to antimicrobial chemotherapy. While the usual array of acquired resistance mechanisms contribute to resistance development in this organism a multitude of endogenous genes also play a role. These include a variety of multidrug efflux loci that contribute to both intrinsic and acquired antimicrobial resistance. Despite their roles in resistance, however, it is clear that these efflux systems function in more than just antimicrobial efflux. Indeed, recent data indicate that they are recruited in response to environmental stress and, therefore, function as components of the organism's stress responses. In fact, a number of endogenous resistance-promoting genes are linked to environmental stress, functioning as part of known stress responses or recruited in response to a variety of environmental stress stimuli. Stress responses are, thus, important determinants of antimicrobial resistance in P. aeruginosa. As such, they represent possible therapeutic targets in countering antimicrobial resistance in this organism.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple , Proteínas de Transporte de Membrana/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiología , Estrés Fisiológico , Antibacterianos/metabolismo , Proteínas Bacterianas/genética , Genes Bacterianos , Genes MDR , Respuesta al Choque Térmico/genética , Proteínas de Transporte de Membrana/genética , Estrés Oxidativo , Pseudomonas aeruginosa/genéticaRESUMEN
The resistance-nodulation-division (RND) family multidrug efflux system MexXY-OprM is a major determinant of aminoglycoside resistance in Pseudomonas aeruginosa, although the details of aminoglycoside recognition and export by MexY, the substrate-binding RND component of this efflux system, have not been elucidated. To identify regions/residues of MexY important for aminoglycoside resistance, plasmid-borne mexY was mutagenized and mutations that impaired MexY-promoted aminoglycoside (streptomycin) resistance were identified in a ΔmexY strain of P. aeruginosa. Sixty-one streptomycin-sensitive mexY mutants were recovered; among these, 7 unique mutations that yielded wild-type levels of MexY expression were identified. These mutations compromised resistance to additional aminoglycosides and to other antimicrobials and occurred in both the transmembrane and periplasmic regions of the protein. Mapping of the mutated residues onto a 3-dimensional structure of MexY modeled on Escherichia coli AcrB revealed that these tended to occur in regions implicated in general pump operation (transmembrane domain) and MexY trimer assembly (docking domain) and, thus, did not provide insights into aminoglycoside recognition. A region corresponding to a proximal binding pocket connected to a periplasm-linked cleft, part of a drug export pathway of AcrB, was identified in MexY and proposed to play a role in aminoglycoside recognition. To test this, selected residues (K79, D133, and Y613) within this pocket were mutagenized and the impact on aminoglycoside resistance was assessed. Mutations of D133 and Y613 compromised aminoglycoside resistance, while, surprisingly, the K79 mutation enhanced aminoglycoside resistance, confirming a role for this putative proximal binding pocket in aminoglycoside recognition and export. IMPORTANCE Bacterial RND pumps do not typically accommodate highly hydrophilic agents such as aminoglycosides, and it is unclear how those, such as MexY, which accommodate these unique substrates, do so. The results presented here indicate that aminoglycosides are likely not captured and exported by this RND pump component in a unique manner but rather utilize a previously defined export pathway that involves a proximal drug-binding pocket that is also implicated in the export of nonaminoglycosides. The observation, too, that a mutation in this pocket enhances MexY-mediated aminoglycoside resistance (K79A), an indication that it is not optimally designed to accommodate these agents, lends further support to earlier proposals that antimicrobials are not the intended pump substrates.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Sitios de Unión , Análisis Mutacional de ADN , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión ProteicaRESUMEN
The mexCD-oprJ multidrug efflux operon of Pseudomonas aeruginosa is regulated by the NfxB repressor. Two forms of NfxB have been reported [Shiba et al. (1995). J Bacteriol 177, 5872) although mutagenesis studies here confirm that the larger protein (199 amino acids, 22.4 kDa) is the functional repressor. NfxB binds upstream of the mexCD-oprJ transcription initiation site to a region containing two inverted repeats, both of which are required for binding. Two-hybrid assays confirmed that NfxB is a multimer, with the C-terminal two-thirds of the repressor required for multimerization. Random mutagenesis identified several mutations within the C-terminal region of NfxB required for multimerization, all of which mapped to a three-helix subdomain of the C-terminal region in a structural model of the repressor, which may thus represent the multimerization domain. These mutations compromised NfxB binding to its target DNA in electromobility shift assays, and their introduction into the chromosome of P. aeruginosa enhanced mexCD-oprJ expression and promoted multidrug resistance, consistent with the functional NfxB repressor being a multimer. Site-directed and spontaneous nfxB mutants showing increased mexCD-oprJ expression and multidrug resistance were also recovered, with mutations mapping to the three-helix subdomain again impacting multimerization and DNA binding. Mutations mapping to the N-terminal helix-turn-helix motif implicated in DNA binding did not impact multimerization although they did render the repressor insoluble and unsuitable for mobility shift assays. Size exclusion column chromatography demonstrated that wild-type NfxB forms tetramers in solution, although a mutant form of the repressor carrying a G192D substitution near the C terminus of the protein and compromised for DNA binding and repressor activity forms dimers. These results suggest that NfxB operates as a tetramer (dimer of dimers) and that the C terminus of the protein serves as a tetramerization domain.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana/biosíntesis , Proteínas de Transporte de Membrana/biosíntesis , Operón , Pseudomonas aeruginosa/genética , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Cromatografía en Gel , Análisis Mutacional de ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Pseudomonas aeruginosa/fisiología , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The amgRS operon encodes a presumed membrane stress-responsive two-component system linked to intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Genome sequencing of a lab isolate showing modest pan-aminoglycoside resistance, strain K2979, revealed a number of mutations, including a substitution in amgS that produced an R182C change in the AmgS sensor kinase product of this gene. Introduction of this mutation into an otherwise wild-type strain recapitulated the resistance phenotype, while correcting the mutation in the resistant mutant abrogated the resistant phenotype, confirming that the amgS mutation is responsible for the aminoglycoside resistance of strain K2979. The amgSR182 mutation promoted an AmgR-dependent, 2- to 3-fold increase in expression of the AmgRS target genes htpX and PA5528, mirroring the impact of aminoglycoside exposure of wild-type cells on htpX and PA5528 expression. This suggests that amgSR182 is a gain-of-function mutation that activates AmgS and the AmgRS two-component system in promoting modest resistance to aminoglycosides. Screening of several pan-aminoglycoside-resistant clinical isolates of P. aeruginosa revealed three that showed elevated htpX and PA5528 expression and harbored single amino acid-altering mutations in amgS (V121G or D106N) and no mutations in amgR. Introduction of the amgSV121G mutation into wild-type P. aeruginosa generated a resistance phenotype reminiscent of the amgSR182 mutant and produced a 2- to 3-fold increase in htpX and PA5528 expression, confirming that it, too, is a gain-of-function aminoglycoside resistance-promoting mutation. These results highlight the contribution of amgS mutations and activation of the AmgRS two-component system to acquired aminoglycoside resistance in lab and clinical isolates of P. aeruginosa.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/efectos de los fármacos , Genoma Bacteriano , Mutación , Pseudomonas aeruginosa/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Humanos , Operón , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/metabolismoRESUMEN
Expression of the mexXY multidrug efflux operon in wild type Pseudomonas aeruginosa is substantially enhanced by the ribosome-targeting antimicrobial spectinomycin (18-fold) and this is wholly dependent upon the product of the PA5471 gene. In a mutant strain lacking the mexZ gene encoding a repressor of mexXY gene expression, expression of the efflux operon increases modestly (5-fold) and is still responsive (18-fold) to spectinomycin. Spectinomycin induction of mexXY expression in the mexZ mutant is, however, independent of PA5471 suggesting that PA5471 functions as an anti-repressor (dubbed ArmZ for anti-repressor MexZ) that serves only to modulate MexZ's repressor activity, with additional gene(s)/gene product(s) providing for the bulk of the antimicrobial-inducible mexXY expression. Consistent with PA5471/ArmZ functioning as a MexZ anti-repressor, an interaction between MexZ and ArmZ was confirmed using a bacterial 2-hybrid assay. Mutations compromising this interaction (P68S, G76S, R216C, R221W, R221Q, G231D and G252S) were identified and localized to one region of an ArmZ structural model that may represent a MexZ-interacting domain. Introduction of representative mutations into the chromosome of P. aeruginosa reduced (P68S, G76S) or obviated (R216C, R2211W) antimicrobial induction of mexXY gene expression, rendering the mutants pan-aminoglycoside-susceptible. These data confirm the importance of an ArmZ-MexZ interaction for antimicrobial-inducible mexXY expression and intrinsic aminoglycoside resistance in P. aeruginosa.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Operón , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mutación , Unión Proteica , Conformación ProteicaRESUMEN
Screening of a transposon insertion mutant library of Pseudomonas aeruginosa for increased susceptibility to paromomycin identified a number of genes whose disruption enhanced susceptibility of this organism to multiple aminoglycosides, including tobramycin, amikacin, and gentamicin. These included genes associated with lipid biosynthesis or metabolism (lptA, faoA), phosphate uptake (pstB), and two-component regulators (amgRS, PA2797-PA2798) and a gene of unknown function (PA0392). Deletion mutants lacking these showed enhanced panaminoglycoside susceptibility that was reversed by the cloned genes, confirming their contribution to intrinsic panaminoglycoside resistance. None of these mutants showed increased aminoglycoside permeation of the cell envelope, indicating that increased susceptibility was not related to enhanced aminoglycoside uptake owing to a reduced envelope barrier function. Several mutants (pstB, faoA, PA0392, amgR) did, however, show increased cytoplasmic membrane depolarization relative to wild type following gentamicin exposure, consistent with the membranes of these mutants being more prone to perturbation, likely by gentamicin-generated mistranslated polypeptides. Mutants lacking any two of these resistance genes in various combinations invariably showed increased aminoglycoside susceptibility relative to single-deletion mutants, confirming their independent contribution to resistance and highlighting the complexity of the intrinsic aminoglycoside resistome in P. aeruginosa. Deletion of these genes also compromised the high-level panaminoglycoside resistance of clinical isolates, emphasizing their important contribution to acquired resistance.
Asunto(s)
Elementos Transponibles de ADN , Farmacorresistencia Bacteriana/genética , Eliminación de Gen , Genes Bacterianos , Mutagénesis Insercional , Pseudomonas aeruginosa/genética , Amicacina/farmacología , Antibacterianos/farmacología , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Biblioteca de Genes , Prueba de Complementación Genética , Gentamicinas/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Paromomicina/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Tobramicina/farmacologíaRESUMEN
Pan-aminoglycoside-resistant Pseudomonas aeruginosa mutants expressing the mexXY components of the aminoglycoside-accommodating MexXY-OprM multidrug efflux system but lacking mutations in the mexZ gene encoding a repressor of this efflux system and in the mexXY promoter have been reported (S. Fraud and K. Poole, Antimicrob. Agents Chemother. 55:1068-1074, 2011). Genome sequencing of one of these mutants, K2966, revealed the presence of a mutation within the predicted promoter region of the rplU-rpmA operon encoding ribosomal proteins L21 and L27, consistent with an observed 2-fold decrease in expression of this operon in the mutant relative to wild-type P. aeruginosa PAO1. Moreover, correction of the mutation restored rplU-rpmA expression and, significantly, reversed the elevated mexXY expression and pan-aminoglycoside resistance of the mutant. Reduced rplU-rpmA expression was also observed in a second mexXY-expressing pan-aminoglycoside-resistant mutant, K2968, which, however, lacked a mutation in the rplU-rpmA promoter region. Restoration of rplU-rpmA expression in the K2968 mutant following chromosomal integration of the rplU-rpmA operon derived from wild-type P. aeruginosa failed, however, to reverse the elevated mexXY expression and pan-aminoglycoside resistance of this mutant, although it did so for K2966, suggesting that the mutation impacting rplU-rpmA expression in K2968 also impacts other mexXY-related genes. Increased mexXY expression owing to reduced rplU-rpmA expression in K2966 and K2968 was dependent on PA5471, whose expression was also elevated in these mutants. Thus, mutational disruption of ribosome function, by limiting expression of ribosomal constituents, promotes recruitment of mexXY and does so via PA5471, reminiscent of mexXY induction by ribosome-disrupting antimicrobial agents. Interestingly, reduced rplU-rpmA expression was also observed in a mexXY-expressing pan-aminoglycoside-resistant clinical isolate, suggesting that ribosome-perturbing mutations have clinical relevance in the recruitment of the MexXY-OprM aminoglycoside resistance determinant.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas de la Membrana/genética , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Proteínas Ribosómicas/genética , Regulación Bacteriana de la Expresión Génica/genética , Operón/genética , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Bacteria encounter a myriad of stresses in their natural environments, including, for pathogens, their hosts. These stresses elicit a variety of specific and highly regulated adaptive responses that not only protect bacteria from the offending stress, but also manifest changes in the cell that impact innate antimicrobial susceptibility. Thus exposure to nutrient starvation/limitation (nutrient stress), reactive oxygen and nitrogen species (oxidative/nitrosative stress), membrane damage (envelope stress), elevated temperature (heat stress) and ribosome disruption (ribosomal stress) all impact bacterial susceptibility to a variety of antimicrobials through their initiation of stress responses that positively impact recruitment of resistance determinants or promote physiological changes that compromise antimicrobial activity. As de facto determinants of antimicrobial, even multidrug, resistance, stress responses may be worthy of consideration as therapeutic targets.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/efectos de la radiación , Fenómenos Fisiológicos Bacterianos , Farmacorresistencia Bacteriana , Estrés Fisiológico , Antibacterianos/uso terapéutico , Bacterias/aislamiento & purificación , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Humanos , Presión Osmótica , Estrés Oxidativo , TemperaturaRESUMEN
Pentachlorophenol (PCP) induced expression of the NalC repressor-regulated PA3720-armR operon and the MexR repressor-controlled mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa. PCP's induction of PA3720-armR resulted from its direct modulation of NalC, the repressor's binding to PA3720-armR promoter-containing DNA as seen in electromobility shift assays (EMSAs) being obviated in the presence of this agent. The NalC binding site was localized to an inverted repeat (IR) sequence upstream of PA3720-armR and overlapping a promoter region whose transcription start site was mapped. While modulation of MexR by the ArmR anti-repressor explains the upregulation of mexAB-oprM in nalC mutants hyperexpressing PA3720-armR, the induction of mexAB-oprM expression by PCP is not wholly explainable by PCP induction of PA3720-armR and subsequent ArmR modulation of MexR, inasmuch as armR deletion mutants still showed PCP-inducible mexAB-oprM expression. PCP failed, however, to induce mexAB-oprM in a mexR deletion strain, indicating that MexR was required for this, although PCP did not modulate MexR binding to mexAB-oprM promoter-containing DNA in vitro. One possibility is that MexR responds to PCP-generated in vivo effector molecules in controlling mexAB-oprM expression in response to PCP. PCP is an unlikely effector and substrate for NalC and MexAB-OprM--its impact on NalC binding to the PA3720-armR promoter DNA occurred only at high µM levels--suggesting that it mimics an intended phenolic effector/substrate(s). In this regard, plants are an abundant source of phenolic antimicrobial compounds and, so, MexAB-OprM may function to protect P. aeruginosa from plant antimicrobials that it encounters in nature.