Multilayer Regulation of Neisseria meningitidis NHBA at Physiologically Relevant Temperatures

Abstract

Neisseria meningitidis colonizes the nasopharynx of humans, and pathogenic strains can disseminate into the bloodstream, causing septicemia and meningitis. NHBA is a surface-exposed lipoprotein expressed by all N. meningitidis strains in different isoforms. Diverse roles have been reported for NHBA in heparin-mediated serum resistance, biofilm formation, and adherence to host tissues. We determined that temperature controls the expression of NHBA in all strains tested, with increased levels at 30−32 °C compared to 37 °C. Higher NHBA expression at lower temperatures was measurable both at mRNA and protein levels, resulting in higher surface exposure. Detailed molecular analysis indicated that multiple molecular mechanisms are responsible for the thermoregulated NHBA expression. The comparison of mRNA steady-state levels and half-lives at 30 °C and 37 °C demonstrated an increased mRNA stability/translatability at lower temperatures. Protein stability was also impacted, resulting in higher NHBA stability at lower temperatures. Ultimately, increased NHBA expression resulted in higher susceptibility to complement-mediated killing. We propose that NHBA regulation in response to temperature downshift might be physiologically relevant during transmission and the initial step(s) of interaction within the host nasopharynx. Together these data describe the importance of NHBA both as a virulence factor and as a vaccine antigen during neisserial colonization and invasion.

Keywords: 4CmenB; NHBA; Neisseria meningitidis; thermoregulation; vaccine.

Figure 1

NHBA expression and surface exposure are increased at lower temperatures. (a) Serogroup B N. meningitidis strains were grown overnight on GC agar plates at the indicated temperatures. Whole cell lysates were prepared and separated by SDS-PAGE prior to Western Blotting. NHBA, fHbp and Hfq proteins were detected using specific mouse-polyclonal antisera. Hfq served as loading control between different samples. In MC58 the full-length protein migrates with an apparent molecular weight of approximately 62 kDa (p3 long isoform), while the other bands right below 62 kDa and at approximately 49 kDa result from processing through NalP bacterial protease [39]. M11719 and 8047 express short isoforms (p20) migrating approximately 50 kDa and do not exhibit NalP processing. (b,c) The indicated strains were grown in GC broth at 30 °C or 37 °C until OD₆₀₀ 0.25. (b) Whole cell lysates were separated by SDS-PAGE and blotted using an anti-NHBA polyclonal antiserum. (c) NHBA surface exposure on MC58 and its Δnhba mutant strain at the indicated conditions. Mouse polyclonal antiserum against NHBA was used for flow cytometry analysis.

Figure 2

NHBA is highly expressed during the exponential phase and its expression at 30 °C is always higher than at 37 °C. (a) Growth profiles of MC58 strain in GC liquid medium at 30 °C (blue) and 37 °C (red). Samples were collected at the indicated consecutive time points (T1-T8) and whole cell lysates were analyzed for NHBA expression by Western blot. (b) φ indicates a non-specific band used as loading control and for relative protein quantification. (c) Relative protein quantification was calculated using ImageJ software, comparing early logarithmic phase (OD₆₀₀~0.25; T2_37°C; T3_30°C), late logarithmic phase (OD₆₀₀~0.85; T3_37°C; T5_30°C) and at stationary phase (OD₆₀₀~1.20; T6_37°C; T7_30°C) summing the signals of the full length and processed bands of NHBA for each timepoint and normalizing against the non-specific band (φ). (d) nhba mRNA steady state levels were quantified by qRT-PCR and relative expression levels were determined normalizing to the 16S RNA. Histograms represent the mean ± SEM from three independent biological replicates and were analyzed by Two-way ANOVA followed by uncorrected Fisher’s LSD multiple comparison test (*** p < 0.001; ** p < 0.01; * p < 0.05; ns: not significant).

Figure 3

NHBA thermoregulation acts at the post-transcriptional level. (a) Schematic representation of nhba mutants generated by ex-locus complementation. In the MC58 Δnhba strain background different mutants were generated by complementation in the NMB1428-NMB1429 locus. Kan^R: kanamycin resistance cassette; Clm^R: chloramphenicol resistance cassette; LacI: LacI repressor gene; Ptac: IPTG inducible promoter; mCherry: mCherry reporter gene. (b–d) MC58 wild-type and recombinant strains were grown in GC liquid medium at 30 °C or 37 °C until OD₆₀₀ 0.5 and supplemented with the indicated concentration of IPTG where needed. NHBA and mCherry protein expression were assessed by Western blotting using polyclonal mouse antiserum and monoclonal mouse antibody (ab167453, abcam), respectively. The φ and δ symbols indicate non-specific bands used as loading controls.

Figure 4

nhba transcript has longer half-life at 30 °C. Strain NGH38 was grown in GC broth until OD₆₀₀ 0.5 at the defined temperatures. RNA extracts were prepared at different timepoints after active transcription was stopped by adding rifampicin. NMB0838, fHbp, and nhba mRNA abundance were measured by qRT-PCR and normalized to 16S RNA. Relative RNA quantity was calculated as 2^−(Ct-Ct0). Data represent the mean of three independent biological replicates ± SD. One phase decay analysis for transcripts at 30 °C (blue line) and 37 °C (red line) are represented.

Figure 5

NHBA protein turnover is directly affected by temperature changes. The wild-type strain MC58 was grown in GC broth until OD₆₀₀ 0.5 at (a) 30 °C, and (b) 37 °C. Active translation was stopped by the addition of spectinomycin, the cultures split into equal volumes and incubated further at the indicated temperatures. Samples for whole cell extracts were collected and prepared at different timepoints thereafter. Protein samples were separated by SDS-PAGE prior to Western blotting. The NHBA band was detected using polyclonal mouse anti-NHBA serum. Relative protein quantification was calculated using ImageJ software, summing the signals of the full length and processed bands of NHBA for each timepoint and -normalizing against the non-specific band (φ) used as loading control, and plotted over time and the half-life quantified for each landing temperature after translation inhibition.

Figure 6

Correlation between NHBA expression, surface exposure and susceptibility to complement-mediated killing by anti-NHBA antibodies. MC58 Ptac_nhba strain was grown in MH broth + 0.25% glucose at 37 °C until OD₆₀₀ 0.25. IPTG was added at the indicated final concentrations. Bacteria were collected to perform (a) Western blotting, (b) FACS analysis and (c) serum bactericidal assay. (d–f) The relative quantification obtained by densitometry analysis of the NHBA-related bands in Western blot and FACS geometric mean calculation were estimated and, together with rSBA titers, were used to calculate the Pearson correlation coefficients. Data represent the median with the error range from three independent biological replicates. Line indicates the linear fit (d) or the semilog fit (e,f). Samples treated with 0.050 mM IPTG or with no IPTG were not taken into account to calculate the Pearson correlation coefficients, as these were found to be out of the linearity range of the assays. (g) MC58 wild-type strain was grown at 30 °C or 37 °C in MH broth + 0.25% glucose until OD₆₀₀ 0.25 was reached. Bacteria were collected and expression levels of NHBA and fHbp were determined by Western blotting, while surface exposure was confirmed by flow cytometry using polyclonal antisera. (h) Serum bactericidal titers were determined using baby rabbit complement as source of complement factors (rabbit SBA, rSBA). Data are representative of three independent biological replicates.