Fig 1.
TEM images showing labile negative charged material on N. gonorrhoeae FA1090.
A. Cells from N. gonorrhoeae FA1090 strain treated with Alcian Blue–diamine L-lysine acetate method, show an easily visible layer of capsule-like material. B. The staining of N. gonorrhoeae FA1090 with Alcian Blue only, does not retain extracellular material on the bacterial cell surface. Scale bar = 1 μm.
Fig 2.
N. gonorrhoeae FA1090 exposes a polyP pseudo-capsule.
Immunofluorescence images show bacterial nucleoid in blue stained with DAPI and EcPPXc binding to polyP in green. Panel A shows a bacterial sub-population exposing the polyP pseudo-capsule, panels B and C exhibit localized polyP spots on the bacterial membrane, panel D shows polyP localization on the septum, whereas in the panels E and F, polyP is organized as a matrix. Panel G shows the results of the EcPPXc staining performed in the presence of exogenously added soluble polyP (25 mM). Scale bar = 2 μm.
Fig 3.
ppk and ppx deletion mutants differ in surface associated polyP.
The presence of polyP pseudo-capsule was assessed by flow cytometry analyses on intact N. gonorrhoeae FA1090 cells (A) and its isogenic mutants FA1090 Δppk1 (B), FA1090 Δppk2 (C), FA1090 Δppk1Δppk2 (D), FA1090 Δppx (E) after incubation with recombinant His-tagged EcPPXc coupled with anti-His monoclonal antibody combined with 488-conjugated secondary antibody. Blue filled profiles represent stained bacteria. Grey tinted areas indicate strains stained only with secondary antibodies used as negative control.
Fig 4.
The polyP pseudo-capsule is detected on BG27 clinical isolate and it is impaired by the ppk1 gene deletion.
To visualize the polyP pseudo-capsule, flow cytometry analyses (A) and confocal microscopy (B) were performed on the clinical isolate BG27 and BG27 Δppk1. A. The grey filled profile represents the negative control stained with the fluorescent secondary antibody only, whereas the green areas represent bacteria with polyP pseudo-capsule, indirectly stained taking advantage of the His-tag of the recombinant EcPPXc. B. Immunofluorescence images show bacterial nucleoid in blue stained with DAPI and EcPPXc binding to polyP in green. Scale bar = 5 μm.
Fig 5.
PolyP mutants display different survival rate in NHS, inversely correlating with complement fixation.
N. gonorrhoeae FA1090 (A) and BG27 (B) were incubated with 10% and 20% NHS. In the case of FA1090 the wild-type strain, the single Δppk1, Δppk2 and the double Δppk1Δppk2 mutants were tested, while in the case of BG27, the wild-type and the Δppk1 mutant were used. Results of serum resistance assays are specified as percentage of survival calculated as the ratio of colony forming unit (cfu) after incubation with NHS over cfu incubated with heat inactivated NHS. Data reported are representative of 3 biological replicates performed with 3 different lots of NHS. Standard deviations are represented by vertical bars (*p < 0.05, **p < 0.005, ***p < 0.001). The ability of the strains to trigger complement activation was measured by flow cytometry after incubation of bacteria with 10% NHS. The difference of the median fluorescence intensity (ΔMFI) is calculated by subtracting the MFI in presence of heat-inactivated NHS from the MFI relative to NHS and shows the bacterial population reacting with human anti-C9 antibody. Complement fixation results are representative of one biological replicates performed with one of the NHS lots tested.
Fig 6.
Soluble polyP restores survival in NHS and blocks complement deposition on serum sensitive F62 strain.
A. Serum resistance of F62 was measured in the presence of 10% NHS. Data are expressed as percentage of survival calculated as the ratio of cfu after incubation with NHS over cfu incubated with heat inactivated NHS. Histograms represent percentage of F62 survival in NHS (filled bar) and in NHS complemented with 50 μM polyP (stripped bar) or with 50 μM DNA (dotted bar). Error bars describe the standard deviation (****p = 0.0001). B. F62 polyP pseudo-capsule expression was assessed by flow cytometry through the binding of anti-His antibody targeting the recombinant EcPPXc. The shaded grey profile represents the negative bacterial population, while the red profile shows cells with polyP on their surface. C. The ability of soluble polyP to coat F62 surface was evaluated by flow cytometry. Red pic refers to the bacterial population coated by the incubation with 1 mM polyP and recognised by anti-His antibody binding the recombinant EcPPXc, whereas grey pic represents the negative control. D. C3 deposition was measured through flow cytometry in the presence of 10% NHS (yellow dotted profile), 10% heat inactivated NHS (grey filled profile), and 10% NHS complemented with soluble 50 μM polyP (red dotted profile). Fluorescence is given by the binding of human anti-C3 fluorescent-labelled antibody.
Fig 7.
Contribution of polyP in restoring serum resistance of sialylated F62 depends on NHS tested.
Serum resistance of F62, grown in the absence or presence of 0.5 μg/mL, 50 μg/mL and 500 μg/mL CMP-NANA (red, green, blue and purple bars, respectively), was measured in the presence of 10% NHS from different donors A and B. Data are expressed as percentage of survival calculated as the ratio of cfu after incubation with NHS over cfu incubated with heat inactivated NHS. Histograms represent percentage of F62 survival in NHS (filled bar) and in NHS complemented with 50 μM polyP (stripped bar). Error bars describe the standard deviation (**p = 0.005; ***p < 0.0005).
Fig 8.
PolyP pseudo-capsule protects N. gonorrhoeae from antibacterial activity of cathelicidin LL-37.
The antibacterial activity of LL-37 was assessed with a cfu counting assay, in which 105 cfu/mL of BG27 and BG27 Δppk1 (A), F62 (B) and SK92-679 (C) were incubated with increasing concentration of the peptide for 1 h at 37°C. The ability of the polyP to restore survival was evaluated by including in the bacterial suspension 20 μM polyP (broken line) or 20 μM DNA (black line). Surviving bacteria were established by plating serial dilution of incubated bacterial suspensions and compared to the number of bacteria present in the control without the peptide. Data are expressed as the mean ± standard deviation of three different experiments.
Fig 9.
The polyP pseudo-capsule potentiates the survival of engulfed N. gonorrhoeae by dHL60 and increases bacterial association.
A. Viability of phagocytized bacteria was assessed by cfu count assay in which dHL60 cells were previously incubated with N. gonorrhoeae BG27 and BG27 Δppk1 at multiplicity of infection (MOI) 50. Data are expressed as the percentage of viable bacteria in respect to total infecting bacteria, in the absence or presence of 50 μM polyP, after 1 h of infection. Vertical bars show the standard deviation (*p < 0.05, **p < 0.005). Cell-associated fluorescence volume (B) and percentage of internalized bacteria (C) were assessed through a fluorescent-based phagocytic assay in which dHL60 were infected at MOI 10 for 1 h with 488 –labelled BG27 and BG27 Δppk1, both in the absence or presence of 50 μM polyP. Cell-associated fluorescence volume was calculated using the 488 channel, that was used as value of adherence. The percentage of internalized bacteria (C) was calculated subtracting the extracellular fluorescence volume associated to the 546 channel (b) to the cell-associated fluorescence volume (a), according to the formula (a-b) *100/a (*p < 0.05).
Table 1.
List of strains used in this study.
Table 2.
Lists of plasmids used in this study.
Table 3.
List of oligonucleotides used in this study.