Available carbon source influences the resistance of Neisseria meningitidis against complement

Abstract

Neisseria meningitidis is an important cause of septicaemia and meningitis. To cause disease, the bacterium must acquire essential nutrients for replication in the systemic circulation, while avoiding exclusion by host innate immunity. Here we show that the utilization of carbon sources by N. meningitidis determines its ability to withstand complement-mediated lysis, through the intimate relationship between metabolism and virulence in the bacterium. The gene encoding the lactate permease, lctP, was identified and disrupted. The lctP mutant had a reduced growth rate in cerebrospinal fluid compared with the wild type, and was attenuated during bloodstream infection through loss of resistance against complement-mediated killing. The link between lactate and complement was demonstrated by the restoration of virulence of the lctP mutant in complement (C3(-/-))-deficient animals. The underlying mechanism for attenuation is mediated through the sialic acid biosynthesis pathway, which is directly connected to central carbon metabolism. The findings highlight the intimate relationship between bacterial physiology and resistance to innate immune killing in the meningococcus.

Figure 1.

NMB0543 is a homologue of the E. coli lactate permease. (A) Organization of predicted open reading frames around NMB0543 and location of the transposon insertion in the mutant, MC58Δ0543, and a putative transcription stop site is marked by . (B) Alignment of the predicted amino acid sequence of NMB0543 (accession no. Q9JRD7; EMBL) with LctP from E. coli (accession no. P33231; SwissProt). Identical residues are shown with an asterisk, conserved substitutions are shown with two dots, and semiconserved substitutions are shown with one dot.

Figure 2.

Identification of the N. meningitidis lactate permease. (A) Growth of wild-type N. meningitidis (MC58), and MC58Δ0543 in the presence of single carbon energy sources as indicated in the key. (B) Uptake of ¹⁴C L-lactate by MC58 and MC58Δ0543. (C) Growth of MC58 in defined media with single carbon sources (11 mM glucose or 2 mM lactate), and with both (10 mM glucose or 2 mM lactate).

Figure 3.

Growth of N. meningitidis in CSF. (A) Growth of wild-type N. meningitidis MC58 (open squares) and MC58ΔlctP (solid diamonds) in CSF. (B) The half hourly changes in optical density of cultures (A₆₀₀) are shown. The difference in the growth of the strains is statistically significant (Student's t test comparing changes in OD of the strains, P = 0.025).

Figure 4.

Utilization of carbon sources by N. meningitidis in CSF. (A) MC58 and (B) MC58ΔlctP were grown in CSF and the effect on carbon content was analyzed by ¹H-NMR. The peaks representing individual carbon sources are labeled. The time (in hours) when samples were taken during growth is indicated. (C) The percentage of lactate and glucose remaining in CSF during growth of MC58.

Figure 5.

The lactate permease mutant is attenuated during bloodstream infection. Competitive indices (C.I.) of the lactate permease mutant (MC58ΔlctP) and the complemented strain (MC58ΔlctP::lctP ^ect) in the infant rat model. Error bars show the standard deviation.

Figure 6.

LctP is required for resistance against complement- mediated lysis. (A) Relative resistance of MC58ΔlctP to complement- mediated lysis compared with MC58 in 50% normal human serum (NHS) or heat inactivated serum (HIS). The error bars show the standard error of the mean. (B) Complementation of MC58ΔlctP restores resistance against serum-mediated killing. (C) FACS analysis demonstrates that C3 deposition on MC58ΔlctP is higher than MC58. (D), The competitive index (C.I.) of wild type and lactate permease strain in wild-type and C3^−/− mice. The virulence of MC58ΔlctP is restored in complement deficient animals.

Figure 7.

LctP contributes to complement sensitivity in the absence of capsule. Relative resistance to complement of strains lacking capsule with (strain MC58ΔsiaD, shown by +LctP) or without (MC58ΔsiaDΔlctP, indicated with −LctP) the lactate permease. Assays were performed in the presence of human serum (3%, error bars indicate the standard error of the mean).

Figure 8.

LctP influences LPS sialylation. (A) Relative resistance to complement of strains lacking capsule and unable to sialylate their LPS with (MC58ΔsiaDΔlst) or without (MC58ΔsiaDΔlstΔlctP) the ability to utilize lactate. Error bars indicate the standard error of the mean. Western blot of cell lysates of strains (shown above each lane) probed with mAbs against (B) total LPS (L3,7,9), and a mAb against unsialylated LPS (3F11) before (C) or after (D) neuraminidase treatment. White lines indicate that intervening lanes have been spliced out.

Figure 9.

The metabolic fate of lactate in N. meningitidis. (A) Intermediary metabolism in MC58 with enzymes encoded on the horizontally acquired, capsule biosynthesis locus shown in dotted arrows. (B) Relative resistance to complement of MC58ΔsiaC and MC58ΔsiaCΔlctP. Assays were performed in 3% human serum, and the error bar shows the standard error of the mean.