Figure 1.
NafA is a highly conserved protein among Neisseria species.
A. Schematic representation of NMC0982 (NafA). Regions corresponding to the three peptides used as immunogens are indicated. A transmembrane domain (TM) predicted by the TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html) is indicated as blue colour at the C-terminal. B. NafA was detected in whole cell lysate of FAM20 wild-type strain by western blot (w) using each peptide-generated antibody. The specific signal of NafA is indicated with an asterisk. Developed membranes (m) stained with amino black are also shown. C. BLAST alignment of NafA homologues from 19 different Neisseria strains and species, ordered by identity. The identity between NMC0982 (1. YP_975044) and NGO0887 (19. YP_207998) is 90.1%.
Figure 2.
NafA is induced and exposed at the bacterial surface upon host cells interaction.
FaDu cells were infected with the wild-type strain for 2 h (left column) or 6 h (right column). After washing away unbound bacteria, the cells were stained with anti-NafA peptide N antibody. Fluorescent (upper row) and Nomarski (lower row) images are shown. A 100 x objective lens was used. The inset shows higher magnification of the boxed area in the image. Arrow indicates a microcolony on the cell.
Figure 3.
The ΔNafA strain expresses more PilE protein than the wild-type strain.
Bacterial outer membrane proteins (A) and whole cell lysate samples (B) were prepared from strains indicated and separated by SDS-PAGE. A. Expression profiles of outer membrane protein were detected by Coomassie Brilliant Blue staining. PorB and Opa proteins were confirmed by MALDI-TOFMS. The lower panel shows the expression of NafA and Opa proteins in the outer membrane fraction, as detected by western blot analysis using anti-NafA peptide C antibody and anti-All Opa antibody, respectively. Arrows indicate non-specific bands (NS). B. Detection of NafA, Opa proteins and pilus components from whole bacterial lysate samples by western blot analysis. Equal amounts of whole cell lysates were loaded in each western blot analysis. C. The western blot analysis was performed as panel B. The signal intensities were analyzed using ImageJ software and relative value were presented when value of the wild-type strain was set as 1.0. D. Amounts of PilE produced by indicated strains were compared by whole cell ELISA using anti-pili (which mainly recognizes PilE, see Experimental procedures). The histogram shows the relative PilE expression levels of bacterial strains, OD450 value of the wild-type strain was set as 1.0. The error bars in C and D represent standard error of mean (SEM) from triplicate experiments. Statistically significant difference is indicated with single asterisk (P<0.05) or triple asterisks (P<0.001). E. Level of pilE mRNA were compared by quantitative RT-PCR. The histogram shows relative pilE mRNA levels normalized to the housekeeping genes, NMC0139 (50S ribosomal protein rplP; left three bars) or NMC0129 (30S ribosomal protein rpsJ; right three bars), and calculated in arbitrary units set to a value of 1 for the wild-type strain. The experiments were repeated using samples generated from two separate cDNA synthesises. Also, cDNA synthesized from a separate RNA preparation gave similar results (data not shown).
Figure 4.
NafA regulates bacterial piliation.
Piliation of the wild-type strain (A) and the ΔNafA strain (B, C) was analyzed by transmission electron microscope after negative staining. White arrows mark membrane blebs and black arrows mark pili. Arrowheads in panel B and C indicate thick bundled pilus structures. The inset in panel B shows a higher magnification of the boxed area, which displays the bundled pili. An overview of the ΔNafA strain piliation is shown in panel C. Black scale bars show 0.5 µm and white scale bars 50 nm.
Figure 5.
NafA negatively affects meningococcal attachment to host cells.
A. FaDu cells were infected with the wild-type strain, the ΔNafA strain, or the ΔNafA/NafA strain for 2 h (moi = 200). After washing and fixation, the cells were stained with an anti-N. meningitidis pili antibody, which also stains the whole bacterial surface. Cell nuclei were stained with DAPI. The images were acquired with 40 x (upper two rows) and 100 x (lower two rows) objectives. Signals from anti-pili channel (“bacteria”) and merged images with DAPI stained cells (“merge”) are shown. Arrows and arrowheads indicate the pilus network-like structures and pilus fibres, respectively. B. FaDu cells at 2.5×104 cells/well in 24-well plates were infected with 5×106 bacteria (moi = 200). After 2 h of infection, 4±0.07×104 bacteria were recovered from wells infected with the wild-type strain, i.e. about 1% of input bacteria were adhered to host cells. Adhesion of the bacterial strains in panel A was quantified and expressed as ratios relative to the wild-type strain, which was defined as 1. The error bars represent SEM of triplicate experiments. Statistically significant difference in adherence is indicated with two asterisks (P<0.001) or one asterisk (P<0.05).
Figure 6.
NafA regulates bacterial aggregation.
The wild-type strain, the ΔNafA strain, or the ΔNafA/NafA strain was suspended in GC-liquid (A), DMEM with 1% FCS (B), and DMEM without FCS (C). The formation of bacterial aggregates was analyzed at indicated time points using a living-cell microscope at 37°C in an atmosphere of 5% CO2. Arrows in panel A and B indicate small bacterial aggregates. The arrow and arrowhead in panel C show a loose and a spherical bacterial aggregate, respectively.
Figure 7.
Quantification of bacterial aggregation.
Aggregation was quantified by measuring the decrease in absorbance that occurs upon sedimentation of bacterial aggregates in GC liquid medium (A) or DMEM (B) under static conditions. Apparent differences in OD values (0–160 min) were due to aggregative properties, not due to growth rates.
Figure 8.
The ΔNafA strain displayed attenuated virulence during systemic infection.
Human CD46 transgenic mice were infected i.p. with the wild-type strain (closed circles) or the ΔNafA strain (open circles) at a dose of 1×108 CFU/mouse. A. Bacterial counts in blood (CFU/ml) were determined at 2 h, 6 h, and 24 h after challenge with each strain. Challenge with the ΔNafA strain resulted in lower levels of bacteremia than the wild-type strain at 6 h and 24 h post-infection. Statistically significant lower bacteremia compared to the wild type strain at each time point is indicated with an asterisk (P<0.05) or three asterisks (P<0.001). B. Survival rate of mice (n = 12 per group) after i.p. infection with the wild-type strain or the ΔNafA strain. The overall survival rates of the mice infected with the mutant were significantly lower (P<0.001) than those of the wild-type strain. C–E. Bacterial survival in whole blood collected from the human CD46 transgenic mice. Around 100 CFU bacteria were mixed with 10 µl whole blood at a 1∶1 volume ratio and incubated at 37°C in a 5% CO2 atmosphere for 1 h (C), 3 h (D), and 6 h (E). Bacteria were spread on GC agar plates and surviving bacteria were counted after overnight incubation. The data are presented as means ± S.E. of three independent experiments done in triplicate. Asterisk indicates significant difference (P<0.05). N.S. represents no significant difference.
Table 1.
Primers used for cloning.