Opc expression, LPS immunotype switch and pilin conversion contribute to serum resistance of unencapsulated meningococci

Abstract

Neisseria meningitidis employs polysaccharides and outer membrane proteins to cope with human serum complement attack. To screen for factors influencing serum resistance, an assay was developed based on a colorimetric serum bactericidal assay. The screening used a genetically modified sequence type (ST)-41/44 clonal complex (cc) strain lacking LPS sialylation, polysaccharide capsule, the factor H binding protein (fHbp) and MutS, a protein of the DNA repair mechanism. After killing of >99.9% of the bacterial cells by serum treatment, the colorimetric assay was used to screen 1000 colonies, of which 35 showed enhanced serum resistance. Three mutant classes were identified. In the first class of mutants, enhanced expression of Opc was identified. Opc expression was associated with vitronectin binding and reduced membrane attack complex deposition confirming recent observations. Lipopolysaccharide (LPS) immunotype switch from immunotype L3 to L8/L1 by lgtA and lgtC phase variation represented the second class. Isogenic mutant analysis demonstrated that in ST-41/44 cc strains the L8/L1 immunotype was more serum resistant than the L3 immunotype. Consecutive analysis revealed that the immunotypes L8 and L1 were frequently observed in ST-41/44 cc isolates from both carriage and disease. Immunotype switch to L8/L1 is therefore suggested to contribute to the adaptive capacity of this meningococcal lineage. The third mutant class displayed a pilE allelic exchange associated with enhanced autoaggregation. The mutation of the C terminal hypervariable region D of PilE included a residue previously associated with increased pilus bundle formation. We suggest that autoaggregation reduced the surface area accessible to serum complement and protected from killing. The study highlights the ability of meningococci to adapt to environmental stress by phase variation and intrachromosomal recombination affecting subcapsular antigens.

Figure 1. Serum killing assay of representative clones belonging to mutant classes I through III.

The data display means of two to three independent experiments. (A) Clones 1 and 14 represent mutant class I overexpressing the Opc protein (n = 31); (B) Clones 53 and 62 (mutant class II, n = 2) displayed altered LPS phenotype compared to the parental strain WUE4558; (C) Clones 2 and 77 showed pilin variation (mutant class III, n = 2).

Figure 2. Mutant class I: increased expression of Opc by clones with enhanced serum resistance.

(A) Coomassie blue stained SDS PAGE showing a prominent band at 26–28 kDa (arrow) in one of the resistant clones (clone 1). WUE4558 is the parental strain. (B) Detection of enhanced Opc-expression in clone 1 by Western blot developed with the Opc-specific monoclonal antibody B306. (C) The number of cytosine residues in the homopolymeric tract of the opc promoter is provided, which has been shown to dictate Opc expression .

Figure 3. Analysis of complement attack by flow cytometry of isogenic derivatives of DE9686 siaD- fhbp-.

(A) Enhanced vitronectin binding in the complemented mutant overexpressing Opc (DE9686 siaD- fhbp- opc- pAP1opc). (B) Membrane attack complex deposition on the Opc-positive strains DE9686 siaD- fhbp- and DE9686 siaD- fhbp- opc- pAP1opc in comparison to that on the opc knock out strain. (C) C3d load in DE9686 siaD-, fhbp- and the corresponding opc knock and opc complemented strain (D) Resistance towards 10% NHS of DE9686 siaD- fhbp-, DE9686 siaD- fhbp- opc-, and DE9686 siaD- fhbp- opc- pAP1opc. Data were compared by paired student's t-test.

Figure 4. Mutant class II: demonstration of LPS immunotype changes and their consequences with regards to serum resistance.

(A) Tricine gel electrophoresis revealed a double banding pattern for clone 53 and clone 62. As controls, strain MC58 (immunotype L3) and its pgm and lgtA (immunotype L8) mutants are shown, which have a truncation of four and two sugar residues, respectively. Clone 77 is shown as a further control, whose increased serum resistance is associated to pilin conversion, but not immunotype switches. (B) Flow cytometry analysis showing reduced deposition of membrane attack complex upon serum stress in strains DE9686 siaD- fhbp- lgtA- and DE9686 siaD- fhbp- lgtA- pAP1lgtC_#53 in comparison to strain DE9686 siaD-fhbp-. (C) Resistance towards 10% NHS was significantly enhanced in DE9686 siaD- fhbp- lgtA- and DE9686 siaD- fhbp- lgtA- pAP1lgtC_#53, when compared to DE9686 siaD- fhbp-. Data were compared by paired student's t-test. (D) Immuno dot blots demonstrating the immunotypes of clones 53 and 62 both expressing L1 and L8 LPS. The structure of the terminal sugar residues of meningococcal LPS is schematically depicted for the three type strains (WUE4558, L3; WUE679, L1 type strain; WUE2535, lgtA knock out strain, L8; for further information see Table 1).

Figure 5. Presence of LPS immunotypes L1, L3 and L8 in ST-41/44 cc and ST-32 cc meningococci.

Meningococcal LPS was analyzed by immuno dot blots. The isolates were obtained from invasive disease (German reference laboratory for meningococci) and healthy carriers , respectively.

Figure 6. Mutant class III: antigenic variation of pilin and its impact on autoaggregation.

(A) Immunoblot demonstrating class II pilin by antibody SM1(arrows). Both clones 2 and 77 revealed altered migration of PilE. (B) Autoaggregation assay: both clones 2 and 77 show accelerated rates of autoaggregation. Bacterial suspensions were kept without agitation for 150 min and the optical density was measured repeatedly to assess clearance of the suspension by autoaggregation and sedimentation.

Figure 7. Sequence analysis of pilin conversion events.

(A) Neighbor joining tree of Pilin amino acid sequences of individual colonies picked from WUE4558 and clones 2 and 77. The tree was computed with MEGA5 (http://www.megasoftware.net/; p-distance method; 168 positions of the aligned dataset). A bootstrap test was applied (2000 replicates). (B) Deduced amino acid sequences of the hypervariable region D flanked by two cysteine residues . A lysine residue previously associated to pilus bundle formation is highlighted by boxes.

Figure 8. Comparison of clones 2_2 and 2_6.

(A) Photograph showing autoaggregation of bacterial suspensions after 90 min. (B) Analysis of surface exposed pilin by ELISA. Clones 2_2 and 2_6 were compared with the parental strain WUE4558, the strain WUE3854 (pilE knock-out, negative control), and WUE3240 (MC58 siaD-, positive control). (C) Transmission electron micrographs of meningococci and pilus bundles obtained with a magnification of 16,000-fold. Pilus bundles were observed with clone 2_2, but not with clone 2_6. (D) Serum killing assay employing 10% normal human serum (NHS) on clones 2_2 and 2_6. Data were compared by a paired student's t-test.