Requirement of NMB0065 for connecting assembly and export of sialic acid capsular polysaccharides in Neisseria meningitidis

Abstract

Capsule expression in Neisseria meningitidis is encoded by the cps locus comprised of genes required for biosynthesis and surface translocation. Located adjacent to the gene encoding the polysialyltransferase in serogroups expressing sialic acid-containing capsule, NMB0065 is likely a member of the cps locus, but it is not found in serogroups A or X that express non-sialic acid capsules. To further understand its role in CPS expression, NMB0065 mutants were created in the serogroups B, C and Y strains. The mutants were as sensitive as unencapsulated strains to killing by normal human serum, despite producing near wild-type levels of CPS. Absence of surface expression of capsule was suggested by increased surface hydrophobicity and confirmed by immunogold electron microscopy, which revealed the presence of large vacuoles containing CPS within the cell. GC-MS and NMR analyses of purified capsule from the mutant revealed no apparent changes in polymer structures and lipid anchors. Mutants of NMB0065 homologues in other sialic acid CPS expressing meningococcal serogroups had similar phenotypes. Thus, NMB0065 (CtrG) is not involved in biosynthesis or lipidation of sialic acid-containing capsule but encodes a protein required for proper coupling of the assembly complex to the membrane transport complex allowing surface expression of CPS.

Fig. 1

(A) Genetic organization of the N. meningitidis capsule gene complex (cps) and the E. coli K1 capsule (kps) locus. The genes responsible for capsular biosynthesis (dotted arrows, syn (sia)ABCD), capsule transport (hatched arrows, ctrABCDEF; ctrEF were formerly known as lipA and lipB), and the E. coli K1 homologues of each are shown. The location of NMB0065 (black arrow) within the cps complex in the serogroup B strain NMB was similar to that of the published serogroup B MC58 genome sequence[45]. An IS1016-like transposase (indicated as a black bar) is present upstream of the NMB0065 homologue in serogroups Y and W135. OatC and OatWY are O-acetyltransferases specific for capsules of serogroup C and groups Y and W135, respectively. (B) The Clustal W protein sequence alignment of NMB0065 and NeuE [46]. The predicted polyprenyl-binding motif (PIRS) motif sequence in NeuE is highlighted in bold.

Fig. 2

(A) Schematic topology predictions of NMB0065 using four algorithms. The inner membranes are shown as thick black lines and the residue numbers of the predicted transmembrane segments are indicated. (B) PhoA activity assays. Two independent plasmids for each construct were examined together with the empty vector control. The error bars represent the standard deviation of triplicate measurements. This is a representative of two independent experiments. (C) Western blots with anti-PhoA monoclonal antibody. Expression of PhoA fusion proteins in each plasmid construct (configurations shown above the plasmid name) was assessed with and without induction by 0.2% arabinose. Equal numbers of cells were examined in each sample (Left panel). Whole cell lysate of the E. coli strain expressing the NMB0065-PhoA fusion was separated into soluble protein fraction (S) and total membrane fraction (M) and probed with anti-PhoA antibody. Equal amount of proteins (20 μg) were loaded in each lane (Right panel). (D) The expression of NMB0065 is not growth phase dependent. The NMB0065::lacZ reporter integrated at a permissive locus in strain NMB427 was grown in GC broth and samples collected at various growth phases. Data presented are the mean values and standard deviations of four independent cultures. The line graph shows the OD₆₀₀ values, while the corresponding β-galactosidase activities in Miller units were shown as gray bars. No significant difference in transcription was noted from the exponential to the early stationary phase.

Fig. 3

Bactericidal activity of normal human serum (NHS) against serogroup B and Y meningococci. Serum bactericidal assays were performed with serogroup B, and Y parent strains (NMB and GA0929), the corresponding NMB0065 mutant strains (RH2.2 and RH5.1) and the complemented serogroup B RH4.1 strain using 10% (black bars) and 25% (dotted bars) of NHS. The unencapsulated biosynthesis-deficient mutants were used as negative controls. Heat-inactivated serum (56°C for 30 min, 25%, hatched bars) was tested to confirm that serum bactericidal activity was due to complement-mediated lysis. Panel A, serogroup B (NMB), panel B, serogroup Y (GA0929). Data presented are the averages and standard deviations of two independent experiments.

Fig. 4

Electron photomicrographs of immunogold-labeled meningococcal cell sections. Panel (A) shows the wild-type parental strain NMB, (B) shows the unencapsulated mutant (synA) M7, and (C) shows the NMB0065 mutant. Labeling was performed with the serogroup B capsule-specific monoclonal antibody, 2-2-B, as the primary antibody, and the 10-nm gold-conjugated anti-immunoglobulin G/M antibody was used as the secondary antibody. The arrows indicate the capsule surrounding the wild-type strain but not the M7 or the NMB0065 mutant. The (v) indicates electron-translucent vacuoles containing internalized capsule of the NMB0065 mutant. Overviews of the wild type strain and the NMB0065 mutant are shown in Panels D and E, respectively.