Neisseria gonorrhoeae-derived outer membrane vesicles package β-lactamases to promote antibiotic resistance

Abstract

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea. The treatment of gonorrhoea is becoming increasingly challenging, as N. gonorrhoeae has developed resistance to antimicrobial agents routinely used in the clinic. Resistance to penicillin is wide-spread partly due to the acquisition of β-lactamase genes. How N. gonorrhoeae survives an initial exposure to β-lactams before acquiring resistance genes remains to be understood. Here, using a panel of clinical isolates of N. gonorrhoeae we show that the β-lactamase enzyme is packaged into outer membrane vesicles (OMVs) by strains expressing bla_TEM-1B or bla_TEM-106, which protects otherwise susceptible clinical isolates from the β-lactam drug amoxycillin. We characterized the phenotypes of these clinical isolates of N. gonorrhoeae and the time courses over which the cross-protection of the strains is effective. Imaging and biochemical assays suggest that OMVs promote the transfer of proteins and lipids between bacteria. Thus, N. gonorrhoeae strains secret antibiotic degrading enzymes via OMVs enabling survival of otherwise susceptible bacteria.

Keywords: OMV; TEM-1; antimicrobial resistance; gonorrhoea; lactamase; secretion system.

Figure 1.

(A)–(D) Confocal laser scanning microscopy of β-lactamase positive isolate N. gon-2 (A) and (B) or β-lactamase negative isolate N. gon-3 (C) and (D) labelled with anti-β-lactamase (A) and (C) or anti-PorB (B) and (D). DAPI (blue) was used to stain DNA. Scale bar 5 μm. Representative image of more than 500 bacterial cells in two biological samples. (E) Bacterial whole cell lysates (N. gon-1, N. gon-2, N. gon-3, and N. gon-4) and corresponding OMVs (NOMV-1, -2, -3, and -4) were analyzed by SDS-PAGE, and immunoblots for PorB and β-lactamase. Loading of the lysates was normalized based on optical density at 600 nm (OD₆₀₀) and OMV fractions to protein concentrations. Molecular weight markers are indicated on the left.

Figure 2.

Immunoblot analysis of β-lactamase in proteinase K and Triton X-100-treated OMVs. PorB, β-barrel outer membrane protein was used as the control. OMVs were treated with 0.5 μg/ml and 1.0 μg/ml of proteinase K. Some OMVs samples were treated with 0.1% Triton X-100 and some OMVs samples were further, in addition treated with proteinase K (1 μg/ml). Molecular weight markers are indicated on the left.

Figure 3.

β-lactamase containing OMVs enable replication antibiotics susceptible bacteria. (A) N. gon-3 growth was determined by optical density at 600 nm, OD₆₀₀, with or without 0.5 μg/ml AMX (0.5) in the presence or absence of NOMVs isolated from the clinical isolates (NOMV-1, -2, -3, and -4) in concentration of 50 μg/ml (50). (B) Growth of N. gon-3 with or without 0.006 μg/ml cefotaxime and NOMV-2 (50 μg/ml). The untreated N. gon-3 growth curve is the same as in (A), as experiments were performed simultaneously. Mean and standard deviation from three independent experiments are shown. (C) Growth (OD₆₀₀) of E. coli alone or in presence of AMX (2.0 μg/ml) or in combination of AMX (2.0 μg/ml) and OMVs isolated from E. coli lacking β-lactamase (EOMVs) or NOMV-2 (50.0 μg/ml). Mean and standard deviation from three independent experiments are shown.

Figure 4.

(A) Schematic of the experiments to determine CFUs and MIC of AMX. (B) Neisseria gonorrhoeae isolate N. gon-3 were incubated in control media, with AMX (0.5 μg/ml) or OMVs derived from resistant N. gon-2 (NOMV-2, 10 μg/ml). CFUs/ml were determent between 1 and 4 h. Mean and SEM from three independent experiments. Statistical analysis was determined by ANOVA (two way) and P < .01 are indicated with *.

Figure 5.

(A) The total membrane and periplasm (5 µg protein each) of N. gonorrhoeae strains N. gon-2 and -3 were probed for PorB, β-lactamase, and LOS. Ponceau staining was used a loading control. Molecular weight markers are indicated on the left. (B) Immunoblot analysis of whole bacterial lysate of N. gon-2, N. gon-3, and N. gon-2 treated with NOMV-2 adjusted to equal cell numbers (OD₆₀₀). Ponceau staining was used a loading control. (C) CFUs of N. gonorrhoeae MS11 exposed to NOMV-2 or control vehicle, washed and then treated with AMX (0.1 μg/ml) for 1 h. Mean and SEM from six independent experiments. *P < .05 (student’s t-test) (D). Escherichia coli treated with NOMV-2 for 1 h were imaged for LPS and PorB. Scale bar = 1 µm. Fluorescence intensity for LPS and PorB along the white line are shown on the right. (E) Flow cytometric analysis of unlabelled and FM4-64-labelled NOMV-2 (OMVs), and N. gon-3 treated with control or FM4-64-labelled NOMV-2 or labelled directly with FM4-64 showing count of events and fluorescence intensity. (F) NOMV-2 were labelled with R18 and then incubated with N. gon-3, Triton X-100 (TX100) or PBS. Unlabelled N. gono-2 and TX100 are included as control. Fluorescence intensity was measured over time (arbitrary units). Mean and SD from triplicate samples. *P < .01. Representative of two independent experiments.