Identification of the
            <i>Haemophilus influenzae tolC</i>
            Gene by Susceptibility Profiles of Insertionally Inactivated Efflux Pump Mutants

Trepod, Catherine M.; Mott, John E.

doi:10.1128/aac.48.4.1416-1418.2004

Cited by 30 publications

(32 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Use of efflux-compromised E. coli strains proved essential for uncovering the mode of action of a new class of bacterial RNA polymerase inhibitor (CBR-703) (1), and this systematic efflux knockout approach has been applied more broadly in studies with Streptococcus pneumoniae (57) and Haemophilus influenzae (64). We know of no other published studies wherein P. aeruginosa efflux-compromised strains have been systematically employed toward the discovery of novel classes of antipseudomonal agents.…”

Section: Discussionmentioning

confidence: 99%

A Novel Indole Compound That Inhibits Pseudomonas aeruginosa Growth by Targeting MreB Is a Substrate for MexAB-OprM

Robertson¹,

Doyle²,

Du³

et al. 2007

J Bacteriol

View full text Add to dashboard Cite

Drug efflux systems contribute to the intrinsic resistance of Pseudomonas aeruginosa to many antibiotics and biocides and hamper research focused on the discovery and development of new antimicrobial agents targeted against this important opportunistic pathogen. Using a P. aeruginosa PAO1 derivative bearing deletions of opmH, encoding an outer membrane channel for efflux substrates, and four efflux pumps belonging to the resistance nodulation/cell division class including mexAB-oprM, we identified a small-molecule indole-class compound (CBR-4830) that is inhibitory to growth of this efflux-compromised strain. Genetic studies established MexAB-OprM as the principal pump for CBR-4830 and revealed MreB, a prokaryotic actin homolog, as the proximal cellular target of CBR-4830. Additional studies establish MreB as an essential protein in P. aeruginosa, and efflux-compromised strains treated with CBR-4830 transition to coccoid shape, consistent with MreB inhibition or depletion. Resistance genetics further suggest that CBR-4830 interacts with the putative ATP-binding pocket in MreB and demonstrate significant cross-resistance with A22, a structurally unrelated compound that has been shown to promote rapid dispersion of MreB filaments in vivo. Interestingly, however, ATP-dependent polymerization of purified recombinant P. aeruginosa MreB is blocked in vitro in a dosedependent manner by CBR-4830 but not by A22. Neither compound exhibits significant inhibitory activity against mutant forms of MreB protein that bear mutations identified in CBR-4830-resistant strains. Finally, employing the strains and reagents prepared and characterized during the course of these studies, we have begun to investigate the ability of analogues of CBR-4830 to inhibit the growth of both efflux-proficient and efflux-compromised P. aeruginosa through specific inhibition of MreB function.The activity of efflux-based drug extrusion mechanisms impact the overall effectiveness of current antimicrobial therapies and impair efforts to discover new chemotherapeutic treatments for many serious bacterial diseases (4,42,43,65). Few bacterial pathogens exemplify the importance of the interplay between xenobiotic efflux extrusion systems and the exclusion of antimicrobial agents at the level of the poorly permeable gram-negative outer membrane quite like Pseudomonas aeruginosa (46, 58). Indeed, this organism has the capacity to encode more than 50 potential multidrug transporters including 12 resistance nodulation/cell division (RND)-class transporters, which to date, represent the only clinically significant drug transporters that have been experimentally validated for P. aeruginosa (20,58).The MexAB-OprM tripartite pump confers basal resistance to P. aeruginosa to a number of chemically distinct antibiotics (58) and through gain-of-function mutations, including those in regulatory proteins (e.g., mexR or mexZ), may overproduce MexAB-OprM or other RND-class pumps which are significant contributors to multidrug resistance in clinical P. areuginosa isolates...

show abstract

Section: Discussionmentioning

confidence: 99%

A Novel Indole Compound That Inhibits Pseudomonas aeruginosa Growth by Targeting MreB Is a Substrate for MexAB-OprM

Robertson¹,

Doyle²,

Du³

et al. 2007

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…The substrate specificities of AcrAB EC -TolC EC and AcrAB HITolC HI are very similar and include both positively charged (dyes and erythromycin) and negatively charged (novobiocin) compounds (19,24). In contrast to E. coli, AcrAB HI -TolC HI does not protect H. influenzae from ␤-lactams, chloramphenicol, tetracycline, or fluoroquinolones.…”

mentioning

confidence: 90%

“…In all cases, the susceptibility profiles of AcrA HI , AcrB HI , or TolC HI null mutants were identical, suggesting that these proteins are components of a single pump. Mutations in other RND transporters identified by sequences searches of H. influenzae genome did not affect drug susceptibility (24). Therefore, AcrAB HI -TolC HI is a major, constitutively expressed multidrug efflux complex of H. influenzae and a potent target for the development of specific inhibitors of this transporter.…”

mentioning

confidence: 99%

“…Genetic and sequence studies showed that H. influenzae constitutively produces a homolog of AcrB EC (19). Similar to other RND transporters, H. influenzae AcrB (AcrB HI ) associates with AcrA HI , the periplasmic MFP, and the outer membrane channel TolC HI (24). Inactivation of any of the three proteins AcrA HI , AcrB HI , or TolC HI increased the susceptibility of H. influenzae to 16 antimicrobial compounds.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Drug-Induced Conformational Changes in Multidrug Efflux Transporter AcrB from Haemophilus influenzae

Dastidar

Mao²,

Lomovskaya

et al. 2007

J Bacteriol

View full text Add to dashboard Cite

In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance. Haemophilus influenzae, a gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces the multidrug efflux transporter AcrB (AcrB HI ). Similar to other RND transporters AcrB HI associates with AcrA HI , the periplasmic membrane fusion protein, and the outer membrane channel TolC HI . Here, we report that AcrAB HI confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolC EC ) protein. To investigate the intracellular dynamics of AcrAB HI , single cysteine mutations were constructed in AcrB HI in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrAB HI and in the presence or absence of AcrA HI and TolC EC . We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in AcrB HI .Multidrug efflux transporters belonging to the resistancenodulation-cell division (RND) superfamily of proteins are broadly represented in gram-negative bacteria. Previous studies established that these transporters play a major role in the intrinsic and induced resistance of gram-negative bacteria to multiple antimicrobial agents including clinically important antibiotics (reviewed in references 7, 11, 18, and 27). Their contribution to antibiotic resistance in clinical settings made RND-type transporters attractive targets of a search for specific and broad-spectrum inhibitors that could be used to increase the efficacy of antibiotics (8).RND-type multidrug transporters from various species share a high degree of sequence and, correspondingly, structure conservation. Structural and functional studies showed that RNDtype transporters exist as trimers, which are organized into two large domains: the transmembrane hydrophobic domain comprised by 36 transmembrane ␣-helices (TMS) with each protomer contributing 12 TMSs and the large periplasmic domain exposed to aqueous environment of the periplasm (17). This impressive structure, however, is not sufficient to provide multidrug resistance. RND transporters associate with two accessory proteins. The periplasmic membrane fusion proteins (MFPs) are believed to mediate the interaction between an RND transporter and an outer membrane channel belonging to the outer membrane factor family of proteins (18,27). Together, these interacting three components span the inner and outer membranes and the periplasm of gram-negative bacteria. Mutations in any of the three proteins completely abolish intrinsic multidrug resistance of gram-negative bacteria.In the three-component complexes, RND transpor...

show abstract

“…A later study suggested that AcrAB acted together with TolC and showed that the system also pumps out additional compounds such as ampicillin, fusidic acid, linezolid, puromycin, trimethoprim, cholate, Triton X-100, rhodamine 6G, and hexadecyltrimethylammonium bromide (371). A MATE family pump was cloned and produced resistance when expressed in an efflux-deficient E. coli host (16); however, its inactivation in H. influenzae produced little effect on antimicrobial susceptibility (371).…”

Section: Pasteurellaceaementioning

confidence: 99%

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

2015

View full text Add to dashboard Cite

SUMMARY The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.

show abstract

Identification of the Haemophilus influenzae tolC Gene by Susceptibility Profiles of Insertionally Inactivated Efflux Pump Mutants

Cited by 30 publications

References 12 publications

A Novel Indole Compound That Inhibits Pseudomonas aeruginosa Growth by Targeting MreB Is a Substrate for MexAB-OprM

A Novel Indole Compound That Inhibits Pseudomonas aeruginosa Growth by Targeting MreB Is a Substrate for MexAB-OprM

Drug-Induced Conformational Changes in Multidrug Efflux Transporter AcrB from Haemophilus influenzae

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

Contact Info

Product

Resources

About