PatA and PatB Form a Functional Heterodimeric ABC Multidrug Efflux Transporter Responsible for the Resistance of Streptococcus pneumoniae to Fluoroquinolones
Abstract:All bacterial multidrug ABC transporters have been shown to work as either homodimers or heterodimers. Two possibly linked genes, patA and patB from Streptococcus pneumococcus, that encode half-ABC transporters have been shown previously to be involved in fluoroquinolone resistance. We showed that the ΔpatA, ΔpatB, or ΔpatA/ΔpatB mutant strains were more sensitive to unstructurally related compounds, i.e., ethidium bromide or fluoroquinolones, than the wild-type R6 strain. Inside-out vesicles prepared from Esc… Show more
“…Turnover rates are stimulated if the substrate binds prior to hydrolysis but there is a considerable basal consumption of ATP (Figure 1—figure supplement 2). While there is evidence of ATP turnover by impaired NBSs, consideration of the reaction mechanism as well as previous biochemical studies of ABC heterodimers support an intrinsically slower rate at the degenerate NBS (Procko et al, 2006; Zutz et al, 2011; Boncoeur et al, 2012). Therefore, ADP and Pi are released from the consensus site prior to hydrolysis at the degenerate site enabling multiple rounds of asymmetric ATP hydrolysis without the complete disengagement of the NBDs to an apo conformation.10.7554/eLife.02740.025Figure 8.Distinct mechanisms of ABC heterodimers and homodimers.( A ) Asymmetric hydrolysis of ATP in an ABC heterodimer.…”
Section: Discussionmentioning
confidence: 94%
“…Modifications of the Walker B, signature and/or switch motifs sequences, which lead to selective catalytic impairment of one of the NBSs in a subclass of ABC exporters (Gao et al, 2000; Aleksandrov et al, 2002; Zhang et al, 2006; Procko et al, 2009; Boncoeur et al, 2012), are associated with fundamental differences in the conformational dynamics of the NBDs and in the power stroke of transport. In contrast to the ATP-induced association/dissociation cycle of NBDs in bacterial homodimers reported previously (Borbat et al, 2007; Zou et al, 2009) and further confirmed here, the NBDs of heterodimers form structurally asymmetric dimers as a consequence of ATP binding and its subsequent hydrolysis.…”
Multidrug ATP binding cassette (ABC) exporters are ubiquitous ABC transporters that extrude cytotoxic molecules across cell membranes. Despite recent progress in structure determination of these transporters, the conformational motion that transduces the energy of ATP hydrolysis to the work of substrate translocation remains undefined. Here, we have investigated the conformational cycle of BmrCD, a representative of the heterodimer family of ABC exporters that have an intrinsically impaired nucleotide binding site. We measured distances between pairs of spin labels monitoring the movement of the nucleotide binding (NBD) and transmembrane domains (TMD). The results expose previously unobserved structural intermediates of the NBDs arising from asymmetric configuration of catalytically inequivalent nucleotide binding sites. The two-state transition of the TMD, from an inward-to an outward-facing conformation, is driven exclusively by ATP hydrolysis. These findings provide direct evidence of divergence in the mechanism of ABC exporters.
“…Turnover rates are stimulated if the substrate binds prior to hydrolysis but there is a considerable basal consumption of ATP (Figure 1—figure supplement 2). While there is evidence of ATP turnover by impaired NBSs, consideration of the reaction mechanism as well as previous biochemical studies of ABC heterodimers support an intrinsically slower rate at the degenerate NBS (Procko et al, 2006; Zutz et al, 2011; Boncoeur et al, 2012). Therefore, ADP and Pi are released from the consensus site prior to hydrolysis at the degenerate site enabling multiple rounds of asymmetric ATP hydrolysis without the complete disengagement of the NBDs to an apo conformation.10.7554/eLife.02740.025Figure 8.Distinct mechanisms of ABC heterodimers and homodimers.( A ) Asymmetric hydrolysis of ATP in an ABC heterodimer.…”
Section: Discussionmentioning
confidence: 94%
“…Modifications of the Walker B, signature and/or switch motifs sequences, which lead to selective catalytic impairment of one of the NBSs in a subclass of ABC exporters (Gao et al, 2000; Aleksandrov et al, 2002; Zhang et al, 2006; Procko et al, 2009; Boncoeur et al, 2012), are associated with fundamental differences in the conformational dynamics of the NBDs and in the power stroke of transport. In contrast to the ATP-induced association/dissociation cycle of NBDs in bacterial homodimers reported previously (Borbat et al, 2007; Zou et al, 2009) and further confirmed here, the NBDs of heterodimers form structurally asymmetric dimers as a consequence of ATP binding and its subsequent hydrolysis.…”
Multidrug ATP binding cassette (ABC) exporters are ubiquitous ABC transporters that extrude cytotoxic molecules across cell membranes. Despite recent progress in structure determination of these transporters, the conformational motion that transduces the energy of ATP hydrolysis to the work of substrate translocation remains undefined. Here, we have investigated the conformational cycle of BmrCD, a representative of the heterodimer family of ABC exporters that have an intrinsically impaired nucleotide binding site. We measured distances between pairs of spin labels monitoring the movement of the nucleotide binding (NBD) and transmembrane domains (TMD). The results expose previously unobserved structural intermediates of the NBDs arising from asymmetric configuration of catalytically inequivalent nucleotide binding sites. The two-state transition of the TMD, from an inward-to an outward-facing conformation, is driven exclusively by ATP hydrolysis. These findings provide direct evidence of divergence in the mechanism of ABC exporters.
“…More recently, the overexpression of patA and patB genes was associated with FQ resistance in clinical isolates of S. pneumoniae and their inactivation restored susceptibility to FQ [55]. The patA (spr1887) and patB (spr1885) genes belong to the same operon and work together as heterodimers to make a functional ABC transporter [56]. Even though upregulation of patAB has been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are still unknown and may be due to mutations in rho-independent terminator or patAB gene duplication [57,58].…”
Purpose. Streptococcus pneumoniae is a commensal bacterium that normally colonizes the human nasopharyngeal cavity. Once disseminated, it can cause several diseases, ranging from non-invasive infections such as acute otitis media and sinusitis through to invasive infections with higher mortality, including meningitis and septicaemia. Since the identification of the first S. pneumoniae strain with decreased susceptibility to penicillin in the 1960s, antibiotic resistance among S. pneumoniae has increased disturbingly and the mechanisms of resistance have begun to unfold.Methodology. This work briefly reviewed the available data on the molecular mechanisms underlying antimicrobial resistance and its epidemiology among pneumococcal strains in Middle Eastern countries.Key findings. Both intrinsic and acquired mechanisms (mutations, acquisition of novel mobile genetic elements and sometimes gene duplication and overexpression) affect susceptibility to a large variety of antibiotics. In Middle Eastern countries, including Lebanon, Iran, Saudi Arabia and Turkey, surveillance showed a disturbing increase in the strength and prevalence of resistance to antibiotics over the years, especially in the last decade. However, no surveillance reports were found in other Middle Eastern countries, such as Syria and Iraq.Conclusion. In order to better survey, control and prevent the emergence of multidrug-and extremely drug-resistant S. pneumoniae strains, antimicrobial stewardship, national surveillance and public awareness programmes should be developed urgently in Middle Eastern countries.
“…The second efflux system was revealed while characterizing 13 putative efflux systems of the ATP-binding cassette (ABC) superfamily in S. pneumoniae TIGR4 (36). The ABC transporters SP2075 (PatA) and SP2073 (PatB) were shown to act as heterodimers (37) to enable resistance to several antimicrobial agents, including ethidium bromide (EtBr), dyes, and fluoroquinolones, and the overexpression of their genes is associated with fluoroquinolone resistance in S. pneumoniae clinical isolates (30).…”
The broad-spectrum fluoroquinolone ciprofloxacin is a bactericidal antibiotic targeting DNA topoisomerase IV and DNA gyrase encoded by the parC and gyrA genes. Resistance to ciprofloxacin in Streptococcus pneumoniae mainly occurs through the acquisition of mutations in the quinolone resistance-determining region (QRDR) of the ParC and GyrA targets. A role in low-level ciprofloxacin resistance has also been attributed to efflux systems. To look into ciprofloxacin resistance at a genome-wide scale and to discover additional mutations implicated in resistance, we performed whole-genome sequencing of an S. pneumoniae isolate selected for resistance to ciprofloxacin in vitro (128 g/ml) and of a clinical isolate displaying low-level ciprofloxacin resistance (2 g/ml). Gene disruption and DNA transformation experiments with PCR fragments harboring the mutations identified in the in vitro S. pneumoniae mutant revealed that resistance is mainly due to QRDR mutations in parC and gyrA and to the overexpression of the ABC transporters PatA and PatB. In contrast, no QRDR mutations were identified in the genome of the S. pneumoniae clinical isolate with low-level resistance to ciprofloxacin. Assays performed in the presence of the efflux pump inhibitor reserpine suggested that resistance is likely mediated by efflux. Interestingly, the genome sequence of this clinical isolate also revealed mutations in the coding region of patA and patB that we implicated in resistance. Finally, a mutation in the NAD(P)H-dependent glycerol-3-phosphate dehydrogenase identified in the S. pneumoniae clinical strain was shown to protect against ciprofloxacin-mediated reactive oxygen species.
Streptococcus pneumoniae is a major Gram-positive pathogen responsible for pneumonia, bacteremia, otitis media, and meningitis leading to considerable morbidity and mortality among children and elderly individuals (1). Penicillin, a -lactam antibiotic, has long been the mainstay against pneumococcal infections (2, 3), but the worldwide spread of antibiotic-resistant clones over the past decades has impaired its usefulness for dealing with S. pneumoniae infections (4-6). The rates of resistance against -lactams and macrolides among S. pneumoniae isolates have translated into an increased usage of fluoroquinolone antibiotics in the treatment of respiratory diseases (7-10).Fluoroquinolones are part of a class of synthetic broad-spectrum antibiotics that inhibit DNA synthesis in bacteria by targeting DNA gyrase (GyrA and -B subunits) and topoisomerase IV (ParC and -E subunits), two enzymes that are vital for DNA supercoiling and chromosome segregation, respectively (11,12). Although the worldwide prevalence of fluoroquinolone-resistant S. pneumoniae remains low in relation to -lactam resistance (Յ1%) (13-15), the dissemination of successful resistant clones has nonetheless increased the prevalence in some countries (16,17). Resistance to fluoroquinolones in S. pneumoniae arises in a stepwise fashion and results from alterations in the target binding site due to...
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