RESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen of acute clinical importance. Combination treatment with an FtsZ inhibitor potentiates the activity of penicillin binding protein (PBP)-targeting ß-lactam antibiotics against MRSA. To explore the mechanism underlying this synergistic behavior, we examined the impact of treatment with the FtsZ inhibitor TXA707 on the spatial localization of the five PBP proteins expressed in MRSA. In the absence of drug treatment, PBP1, PBP2, PBP3, and PBP4 colocalize with FtsZ at the septum, contributing to new cell wall formation. In contrast, PBP2a localizes to distinct foci along the cell periphery. Upon treatment with TXA707, septum formation becomes disrupted, and FtsZ relocalizes away from midcell. PBP1 and PBP3 remain significantly colocalized with FtsZ, while PBP2, PBP4, and PBP2a localize away from FtsZ to specific sites along the periphery of the enlarged cells. We also examined the impact on PBP2a and PBP2 localization of treatment with ß-lactam antibiotic oxacillin alone and in synergistic combination with TXA707. Significantly, PBP2a localizes to the septum in approximately 15% of the oxacillin-treated cells, a behavior that likely contributes to the ß-lactam resistance of MRSA. Combination treatment with TXA707 causes both PBP2a and PBP2 to localize in malformed septum-like structures. Our collective results suggest that PBP2, PBP4, and PBP2a may function collaboratively in peripheral cell wall repair and maintenance in response to FtsZ inhibition by TXA707. Cotreatment with oxacillin appears to reduce the availability of PBP2a to assist in this repair, thereby rendering the MRSA cells more susceptible to the ß-lactam. IMPORTANCE MRSA is a multidrug-resistant bacterial pathogen of acute clinical importance, infecting many thousands of individuals globally each year. The essential cell division protein FtsZ has been identified as an appealing target for the development of new drugs to combat MRSA infections. Through synergistic actions, FtsZ-targeting agents can sensitize MRSA to antibiotics like the ß-lactams that would otherwise be ineffective. This study provides key insights into the mechanism underlying this synergistic behavior as well as MRSA resistance to ß-lactam drugs. The results of this work will help guide the identification and optimization of combination drug regimens that can effectively treat MRSA infections and reduce the potential for future resistance.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas do Citoesqueleto/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Sinergismo Farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/genética , Transporte Proteico/efeitos dos fármacos , beta-Lactamas/farmacologiaRESUMO
Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is in part a result of antimicrobial resistance, highlighting the need to uncover novel antibiotic targets and to discover new therapeutic agents. In the present study, we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf (sulfur mobilization)-dependent Fe-S cluster synthesis is essential in S. aureus We found that sufCDSUB were cotranscribed and that suf transcription was positively influenced by sigma factor B. We characterized an S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD (sufD*), resulting in decreased transcription of sufSUB Consistent with the transcriptional data, the sufD* strain had multiple phenotypes associated with impaired Fe-S protein maturation. They included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the tricarboxylic acid (TCA) cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD* strain also exhibited perturbed intracellular nonchelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Neutrófilos/microbiologia , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Proteínas de Bactérias/genética , Humanos , Proteínas Ferro-Enxofre/genética , Oxigênio/metabolismo , RNA Antissenso/análise , Espécies Reativas de Nitrogênio/metabolismo , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , VirulênciaRESUMO
BACKGROUND: Staphylococcus xylosus is a coagulase-negative, gram-positive coccus that is found in the environment and as a commensal organism on the skin and mucosal surfaces of animals. Despite the fact that S. xylosus is considered a nonpathogenic bacterium, several studies have linked S. xylosus to opportunistic infections in both animals and humans. During an investigation of mastitis-causing agents in the governorate of Basrah, Iraq, we identified an antibiotic-resistant strain of S. xylosus NM36 from a milk sample from a cow with chronic mastitis. In addition to robust biofilm formation, multiple antibiotic resistance phenotypes were found. To further understand the genetic background for these phenotypes, the full genome of S. xylosus NM36 was analyzed. RESULTS: The genome consisted of a single circular 2,668,086 base pairs chromosome containing 32.8% G + C. There were 2454 protein-coding sequences, 4 ribosomal RNA (rRNA) genes, and 50 transfer RNA (tRNA) genes in the genome. In addition, genetic variation was studied by searching sequence data against a representative reference genome. Consequently, single-nucleotide polymorphism analysis was conducted and showed that there were 46,610 single-nucleotide polymorphisms (SNPs), 523 insertions, and 551 deletions. In order to overcome antibiotics, S. xylosus NM36 had been armed with several antibiotic resistance genes from several groups and families. The genome annotation service in PathoSystems Resource Integration Center (PATRIC) and Rapid Annotation using Subsystem Technology (RAST) annotation servers showed that there are multiple antimicrobial resistance elements, including antibiotic inactivation enzymes (BlaZ family, FosB), antibiotic resistance gene clusters (TcaB, TcaB2, TcaR), proteins involved in methicillin resistance (LytH, FmtA, FemC, HmrB, HmrA), TetR family transcriptional regulators, and efflux pumps conferring antibiotic resistance (NorA). In addition, we investigated and categorized the biofilm and quorum-sensing elements of the NM36 strain and found that it has multiple subsets of biofilm regulators, confirming its pathogenic nature. CONCLUSIONS: These findings necessitate a reevaluation of microbial and clinical interventions when dealing with coagulase-negative staphylococci, particularly in the context of studies pertaining to public health. This is the first time, to our knowledge, that the entire genome of S. xylosus has been sequenced in Iraq.
RESUMO
Addressing the growing problem of antibiotic resistance requires the development of new drugs with novel antibacterial targets. FtsZ has been identified as an appealing new target for antibacterial agents. Here, we describe the structure-guided design of a new fluorescent probe (BOFP) in which a BODIPY fluorophore has been conjugated to an oxazole-benzamide FtsZ inhibitor. Crystallographic studies have enabled us to identify the optimal position for tethering the fluorophore that facilitates the high-affinity FtsZ binding of BOFP. Fluorescence anisotropy studies demonstrate that BOFP binds the FtsZ proteins from the Gram-positive pathogens Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae with Kd values of 0.6-4.6 µM. Significantly, BOFP binds the FtsZ proteins from the Gram-negative pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii with an even higher affinity (Kd = 0.2-0.8 µM). Fluorescence microscopy studies reveal that BOFP can effectively label FtsZ in all the above Gram-positive and Gram-negative pathogens. In addition, BOFP is effective at monitoring the impact of non-fluorescent inhibitors on FtsZ localization in these target pathogens. Viewed as a whole, our results highlight the utility of BOFP as a powerful tool for identifying new broad-spectrum FtsZ inhibitors and understanding their mechanisms of action.