Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy

Ayhan, Dilay Hazal; Tamer, Yusuf Talha; Akbar, Mohammed Ali; Bailey, Stacey; Wong, Michael; Daly, Seth M.; Greenberg, David E.; Toprak, Erdal

doi:10.1371/journal.pbio.1002552

Cited by 74 publications

(77 citation statements)

References 46 publications

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“…Our results also suggest potential therapeutic avenues for addressing the emergence of multidrug-resistant gonococcal strains. Selective knockdown of MtrCDE homologs in other bacteria via antisense RNA 59 and bacteriophages 60 has successfully re-sensitized resistant strains and enhanced antibiotic efficacy, and ectopic expression in N. gonorrhoeae of the mtrR repressor in a cephalosporin-resistant strain enhances gonococcal killing by β-lactam antibiotics in the mouse model 61 . Our population-wide estimated effect sizes for mtrC LOF mutations provide a prediction for the re-sensitization effect of MtrCDE knockdown across multiple genetic backgrounds and suggest particularly strong effects for the macrolide azithromycin (Supplementary Figure 4).…”

Section: Discussionmentioning

confidence: 99%

Increased antibiotic susceptibility inNeisseria gonorrhoeaethrough adaptation to the cervical environment

Mortimer

Hicks

et al. 2020

Preprint

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28Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and 29 antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that 30 have reverted to susceptibility regularly appear, prompting questions about which pressures 31 compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide 32 association on the largest collection of N. gonorrhoeae isolates to date (n=4882) to identify loss-33 of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased 34 antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical 35 isolates relative to urethral isolates (odds ratio (OR) = 3.74, 95% CI [1.98-6.70]). In support of a 36 model in which pump expression incurs a fitness cost in this niche, cervical isolates were also 37 enriched relative to urethral isolates in LOF mutations in the mtrCDE activator mtrA (OR = 8.60,) and in farA, a subunit of the FarAB efflux pump (OR = 6.25, 95% CI [3.90-39 9.83]). In total, approximately 1 in 3 cervical isolates (36.4%) contained a LOF mutation in either 40 the efflux pump components mtrC or farA or the activator mtrA. Our findings extend beyond N. 41 gonorrhoeae to other Neisseria: mtrC LOF mutations are rare (<1%) in the primarily 42 nasopharyngeal-colonizing N. meningitidis in a collection of 14,798 genomes but enriched in a 43 heterosexual urethritis-associated lineage (8.6%, p = 9.90×10 -5 ), indicating that efflux pump 44 downregulation contributes broadly to the adaptation of pathogenic Neisseria to the female 45 urogenital tract. Overall, our findings highlight the impact of integrating microbial population 46 genomics with host metadata and demonstrate how host environmental pressures can lead to 47 increased antibiotic susceptibility. 48 49 50 51Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea. 52Antibiotics have played a key role in shaping gonococcal evolution 1-3 , with N. gonorrhoeae 53 gaining resistance to each of the first line antibiotics used to treat it 4-6 . As N. gonorrhoeae is an 54 obligate human pathogen, the mucosal niches it infects-most commonly including the urethra, 55 cervix, pharynx, and rectum-must also influence its evolution 7 . The gonococcal phylogeny 56 suggests the interaction of these factors, with an ancestral split between a relatively drug-57 susceptible lineage circulating primarily in heterosexuals and a relatively drug-resistant lineage 58 circulating primarily in men who have sex with men 3 . 59 60 Despite the deeply concerning increase in antibiotic resistance reported in gonococcal 61 populations globally 8 , some clinical isolates of N. gonorrhoeae have become more susceptible 62 to antibiotics 9,10 . This unexpected phenomenon prompts questions about which environmental 63 pressures could be drivers of increased susceptibility and the mechanisms by which 64 suppression or reversion of resistance may occur. To address these questions, we analy...

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Section: Discussionmentioning

confidence: 99%

Increased antibiotic susceptibility inNeisseria gonorrhoeaethrough adaptation to the cervical environment

Mortimer

Hicks

et al. 2020

Preprint

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“…PPMOs (i) inhibited a large panel of clinical strains, (ii) are bactericidal, (iii) prevent and reduce biofilm, (iv) act synergistically with currently available antibiotics, and (v) reduce bacterial burden in vivo. Notably, similar 5=(RXR) 4 XB-conjugated PPMOs had no toxicity in cell culture (37) and demonstrated low toxicity at therapeutic doses in rodents (41,42,44,45). PPMOs are a promising choice for future clinical investigations since they have now demonstrated efficacy in many Gram-negative genera.…”

Section: Inhibition Of P Aeruginosa By Ppmosmentioning

confidence: 99%

Inhibition of Pseudomonas aeruginosa by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers

Howard

Sturge

Moustafa

et al. 2017

Antimicrob Agents Chemother

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Pseudomonas aeruginosa is a highly virulent, multidrug-resistant pathogen that causes significant morbidity and mortality in hospitalized patients and is particularly devastating in patients with cystic fibrosis. Increasing antibiotic resistance coupled with decreasing numbers of antibiotics in the developmental pipeline demands novel antibacterial approaches. Here, we tested peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which inhibit translation of complementary mRNA from specific, essential genes in P. aeruginosa. PPMOs targeted to acpP, lpxC, and rpsJ, inhibited P. aeruginosa growth in many clinical strains and activity of PPMOs could be enhanced 2-to 8-fold by the addition of polymyxin B nonapeptide at subinhibitory concentrations. The PPMO targeting acpP was also effective at preventing P. aeruginosa PAO1 biofilm formation and at reducing existing biofilms. Importantly, treatment with various combinations of a PPMO and a traditional antibiotic demonstrated synergistic growth inhibition, the most effective of which was the PPMO targeting rpsJ with tobramycin. Furthermore, treatment of P. aeruginosa PA103-infected mice with PPMOs targeting acpP, lpxC, or rpsJ significantly reduced the bacterial burden in the lungs at 24 h by almost 3 logs. Altogether, this study demonstrates that PPMOs targeting the essential genes acpP, lpxC, or rpsJ in P. aeruginosa are highly effective at inhibiting growth in vitro and in vivo. These data suggest that PPMOs alone or in combination with antibiotics represent a novel approach to addressing the problems associated with rapidly increasing antibiotic resistance in P. aeruginosa.

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“…These interactions however are crucial for assembly of active efflux complexes, and the potential to interchange the PAP used in an efflux system may provide the bacteria with adaptability and plasticity to circumvent antibacterial agents. In addition, the PAPs themselves have previously been highlighted as good targets against which to develop inhibitor molecules [18,21,22] making understanding these interactions increasingly important.…”

Section: Introductionmentioning

confidence: 99%

Identification of binding residues between periplasmic adapter protein (PAP) and RND efflux pumps explains PAP-pump promiscuity and roles in antimicrobial resistance

et al. 2019

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Active efflux due to tripartite RND efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. These pumps are also essential for Gram-negative pathogens to cause infection and form biofilms. They consist of an inner membrane RND transporter; a periplasmic adaptor protein (PAP), and an outer membrane channel. The role of PAPs in assembly, and the identities of specific residues involved in PAP-RND binding, remain poorly understood. Using recent high-resolution structures, four 3D sites involved in PAP-RND binding within each PAP protomer were defined that correspond to nine discrete linear binding sequences or "binding boxes" within the PAP sequence. In the important human pathogen Salmonella enterica, these binding boxes are conserved within phylogenetically-related PAPs, such as AcrA and AcrE, while differing considerably between divergent PAPs such as MdsA and MdtA, despite overall conservation of the PAP structure. By analysing these binding sequences we created a predictive model of PAP-RND interaction, which suggested the determinants that may allow promiscuity between certain PAPs, but discrimination of others. We corroborated these predictions using direct phenotypic data, confirming that only AcrA and AcrE, but not MdtA or MsdA, can function with the major RND pump AcrB. Furthermore, we provide functional validation of the involvement of the binding boxes by disruptive site-directed mutagenesis. These results directly link sequence conservation within identified PAP binding sites with functional data providing mechanistic explanation for assembly of clinically relevant RND-pumps and explain how Salmonella and other pathogens maintain a degree of redundancy in efflux mediated resistance. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a

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Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy

Cited by 74 publications

References 46 publications

Increased antibiotic susceptibility inNeisseria gonorrhoeaethrough adaptation to the cervical environment

Increased antibiotic susceptibility inNeisseria gonorrhoeaethrough adaptation to the cervical environment

Inhibition of Pseudomonas aeruginosa by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers

Identification of binding residues between periplasmic adapter protein (PAP) and RND efflux pumps explains PAP-pump promiscuity and roles in antimicrobial resistance

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