Diffusion of Antibiotics through the PilQ Secretin in Neisseria gonorrhoeae Occurs through the Immature, Sodium Dodecyl Sulfate-Labile Form

Abstract

In strains of Neisseria gonorrhoeae harboring the mtr and penB determinants that decrease permeation of antibiotics into the periplasm, mutation or deletion of the PilQ secretin of type IV pili increases resistance to penicillin by ϳ3-fold, indicating a role for PilQ in antibiotic permeation. In this study, we examined spontaneously arising mutants with decreased susceptibility to penicillin. One class of mutants had a phenotype indistinguishable from that of a previously characterized pilQ2 mutation that interfered with the formation of SDS-resistant PilQ multimers. A second class of mutants contained frameshift mutations in genes upstream of pilQ in the pilMNOPQ operon that increased resistance to levels similar to those of the pilQ2 mutation. Inframe deletions of these genes were constructed, but only the frameshift mutations increased antibiotic resistance, suggesting that the mutations had polar effects on PilQ. Consistent with this result, titration of wild-type PilQ levels revealed a direct correlation between resistance and expression levels of PilQ. To determine which form of PilQ, the monomer or the multimer, was responsible for antibiotic permeation, we manipulated and quantified these forms in different mutants. Deletion of PilW, which is responsible for the maturation of PilQ into SDS-resistant multimers, had no effect on resistance. Moreover, Western blot analysis revealed that while SDS-resistant multimer levels were decreased by 26% in frameshift mutants, the levels of PilQ monomers were decreased by 48%. These data suggest that immature, SDS-labile complexes, not mature, SDS-resistant PilQ complexes, serve as the route of entry of antibiotics into the periplasm. IMPORTANCEThe capacity of antibiotics to reach their target is crucial for their activity. In Neisseria gonorrhoeae, the PilQ secretin of type IV pili plays an important role in antibiotic influx when diffusion of antibiotics through porins is limited (e.g., in most resistant strains). On Western blots, PilQ exists both as a mature higher-order multimer and an immature, SDS-labile monomer. In this study, we examined spontaneously arising mutations in PilQ and in the genes upstream of PilQ in the pilMNOPQ operon that increase resistance to penicillin. We provide evidence that PilQ monomers associate by mass action to form immature multimers and that these complexes likely mediate the diffusion of antibiotics across the outer membrane. G onorrhea, the sexually transmitted infection (STI) caused byNeisseria gonorrhoeae, is the most prevalent STI worldwide, with an estimated 106 million infections per year (1). There is no vaccine against N. gonorrhoeae, and because patients do not develop immunological memory, there is a high rate of reinfection (2). Because of the absence of an adequate immune response, antibiotic therapy is the standard of care for curing gonorrheal infections, but antibiotic resistance has rendered many of the available therapeutics ineffective. Historically, gonorrhea has been treated with penicillin, tetracycli...

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake

“…Deletion of hofQ decreased the amount of eDNA while increasing the susceptibility to the tested β-lactams, supporting the hypothesis that eDNA can sequester and protect biofilm-associated cells from the activity of β-lactams. Although the A. actinomycetemcomitans Δ hofQ mutants were more susceptible to the tested β-lactams than the wild-type strain, the deletion of pilQ in N. gonorrhoeae made the strain more resistant to penicillin and decreased the transformation efficiency of the strain [36]. However, the N. gonorrhoeae strain used harbored determinant of penicillin resistance, mtrR and penB , which decrease antimicrobial permeation through outer membrane porins.…”

Heteroresistance to the model antimicrobial peptide polymyxin B in the emerging Neisseria meningitidis lineage 11.2 urethritis clade: mutations in the pilMNOPQ operon

“…Protein MW ladders were labeled on the left. levels of PilQ in both multimer and monomer forms (lanes 3 and 4), indicating a probable polar effect on PilQ protein expression, a phenomenon also observed in gonococci (Nandi et al, 2015).…”

“…As shown in Fig. , a high molecular weight multimer band, a monomer band and several smaller bands, presumably degraded products, were detected by the PilQ monoclonal antibodies, like the previously reported pattern (Nandi et al ., ). The pilQ frameshift mutants (3R4 and 33R1) resulting in premature truncation eliminated both of multimer and monomer bands (lanes 2 and 8).…”

“…Two mutants with pilQ T to P residue changes (14R2 and 17R1) yielded no multimer bands, while maintaining monomer bands at comparable intensities as the wild‐type strain (lanes 5 and 6). Finally, the pilM::IS1655 mutant (37R1) and the pilM frameshift mutant (45R2) showed reduced levels of PilQ in both multimer and monomer forms (lanes 3 and 4), indicating a probable polar effect on PilQ protein expression, a phenomenon also observed in gonococci (Nandi et al ., ).…”

“…PilQ facilitates the entry of a variety of antibiotics in the gonococci, including penicillin, ceftriaxone, vancomycin, tetracycline, rifampin and ciprofloxacin (Ropp et al ., ; Zhao et al ., ; Nandi et al ., ). An in vitro spontaneous mutation screen of two gonococcal isolates containing mosaic penA sequence with MICs to ceftriaxone ranging from 0.03 to 0.06 µg ml –1 identified mutants with increased MICs to ceftriaxone almost 10‐fold (0.5 µg ml –1 ).…”