A generic mechanism in Neisseria meningitidis for enhanced resistance against bactericidal antibodies

Neisseria meningitidis is a gram negative diplococcus and it is one of the leading causes of bacterial meningitis and septicemia. It has been shown that individuals with complement deficiency are sensitive to meningococcal infection, thus indicating the complement system is crucial for immunity against this pathogen. Furthermore, it has been shown that serum of individuals infected with N. meningitidis contains bactericidal antibodies. These antibodies initiate the activation of classical complement fixation pathway, which ultimately leads to insertion of membrane attack complex into the outer membrane of the organism and subsequently bacterial lysis.

Recently, a new vaccine has been introduced into countries across Europe and North America. This vaccine is N. meningitidis serogroup C conjugate (MCC) vaccine, as there have been rising incidences of N. meningitidis serogroup C (MenC) belonging to sequence type 11 (ST11). The MCC vaccine consists of alpha 2-9 linked polysialic capsular polysaccharide coupled to a carrier protein. The protection is offered by bactericidal antibodies directed against the capsule. This vaccine has been quite successful and dramatic decrease in the incidence of MenC disease has been observed. However, the continued use of this vaccine might lead to emergence of ST-11 strains expressing other capsular types or other strains of N. meningitidis might fill the ecological niche vacated by MenC. But, till now such concerns are largely speculative.

In the present study, the authors have described the identification and characterization of three MenC strains with enhanced resistance against bactericidal antibodies elicited by MCC vaccine. N. meningitidis contains an intergenic region (IGR) between the sia and ctr operons. These operons are involved in the capsule biosynthesis and export. The authors observed that an insertion sequence, IS1301 in this IGR was responsible for the resistance. This insertion results in increase transcription of these two operons, which leads to an increase in the amount of capsular polysaccharides and impairment of the alternative pathway of complement activation.

Due to this single genetic change the bacteria can evade killing by bactericidal antibodies. Given the widespread distribution of IS1301 in N. meningitidis genome and bacterium’s competence for DNA uptake, this genetic change is very important.

Here is the summary of major results:
1. Identification of MenC strains that are resistant to sertum bactericidal antibodies elicited by MCC vaccine: To identify the resistant strains, the sera from three patients who had been immunized with MCC vaccine and who had high level serum SBA titres against C11, a MenC strain were chosen. These were used to screen 109 MenC isolates from patients with meningococcal diseases. It was observed that three strains (patient isolates out of 109) were identified, against which the serum bactericidal antibodies (SBA) titres of all three immunized patients was <8. A SBA titre>8 is a proven correlate of protection against MenC disease and a titre less than that shows resistance. None of these three patients (from which R1, R2 and R3 were isolated) had received the meningococcal vaccine. These strains were sequence typed and they were observed to be C:2a:P1.5 and ST-11, subgroup ET-15 by MLST and fumC sequencing.
2. Strains are not resistant because of changes in the LPS sialylation or acetylation of capsular polysaccharides: To understand the basis of the increased resistance of R strains against immune sera, the authors first examined the degree of LPS sialylation of strains. This was done because this modification can contribute to the avoidance of complement mediated lysis. For these studies, five fully sensitive C:2a:P1.5 and ST-11/ET-15 strains were selected from the reference lab as controls. This analysis was done by using Western blot using mAb 3F11, which binds to unsialylated LPS. No consistent difference in the degree of LPS sialylation was observed between R strains and S strains. The authors next examined the role of alteration in acetylation status of the capsule in resistance. Acetylation of capsule is mediated by a transferase encoded by oatC, which is located in the capsule biosynthesis locus (cps). The oatC contains homopolymeric tracts that can mediate ON:OFF switching of acetylation after alteration in the tract length during DNA replication. The nucleotide sequence of oatC in the R and S strains indicated that the gene is predicted to be expressed in all the strains. Furthermore, FACS analysis revealed that capsules of all strains were acetylated.
3. Insertion of IS1301 in the capsule biosynthesis locus is responsible for increased resistance: To investigate the role of capsule itself in the resistance to SBA, the authors generated the capsule negative strains of R3 and S3 by inactivating siaD. SiaD encodes capsule specific polysialyl transferase. Now, the SBA titres of vaccinees sera were measured against these capsule deficient R3 and S3 strains. It was observed that the titres were unchanged against the S3 strain, while titres were increased (from <8 to >512) for the capsule deficient R3 strain. Thus, capsule deficient R3 strain became more susceptible to killing and thus, the expression of capsule is necessary for enhanced resistance of R3.
This observation suggests that R strain might harbor changes in the cps. To test this hypothesis the genomic DNA from strain R3:oatC was used to transform S3 and the transformants were analyzed for their sensitivity to bactericidal antibodies. The authors were able to identify transformants that had become resistant to killing (S3:T1). While others transformants remain fully sensitive (S3:T2 and S3:T3). To identify the particular regions of cps that might be responsible for the resistance, the overlapping fragments of the cps were PCR amplified from the transformants (S3:T1, S3:T2 abd S3:T3). One region of the cps yielded a significantly larger product from the transformant with higher resistance (S3:T1) compared with the sensitive transformants. To define the precise location of this polymorphic region, further PCRs were done. A nearly 1.1kb fragment was amplified from resistant strains, while a 300 bp product was obtained from sensitive strains. These results showed a direct correlation between the size of this region of cps and the ability of the strain to show resistance to SBAs. The authors further performed sequence analysis of this region and found out the presence of an insertion sequence IS1301 in the 134bp IGR between siaA and ctrA in all R strains and S3:T1 strains. IS1301 is an 844 bp mobile element that is present in multiple copies in the genomes of some meningococcal strains, including ST-11 isolates belonging to ET-15.

4. Insertion of IS1301 in the IGR increases capsule expression but does not affect its structure: To determine whether the insertion of IS1301 affected the antigenic properties of the capsular polysaccharide, immune sera were raised against S3, S3:R and R3 strains. Sera raised against S3 mediated killing of the homologous strain S3 but not S3:R or R3. Sera raised against S3:R and R3 were also able to elicit complement-mediated lysis of S3. This showed that the capsular polysaccharide from the R strains retain its immunogenecity. Even high concentration of same sear failed to induce killing of S3:R or R3. This suggested that the increase in resistance of the strain was not due to an alteration in the nature of antigens expressed. Furthermore, the authors studied the composition of the capsular polysaccharides of the strains by NMR spectroscopy. The spectrum of purified capsule from S3 was similar to that of S3:R and R3. To examine whether the insertion of IS1301 in the IGR affected the amount of capsule expressed, the authors performed quantitative real time PCR on siaA and ctrA, and compared the results with the transcripts levels of a house keeping gene, gdh. R3 and S3R strains showed a two to three fold increase in mRNA levels of both siaA and ctrA compared with S3. No significant difference was observed in the mRNA levels of gdh between the sensitive and resistant strains. The authors also determined levels of SiaA protein expressed by all the strains using Western blotting. It demonstrated that R3 and S3:R strains produced higher levels of SiaA protein than S3 strains. The authors also examined the strains by cyto-TEM after labeling live bacteria with the electron dense, cationic stain ferritin. This stain binds to negatively charged bacterial capsules. By using TEM, capsular polysaccharides were clearly visualized around strain S3. In contrast, there was much more capsule surrounding the R3 and S3:R strain.

5. Activation of the alternative complement pathway is reduced on the strains with enhanced resistance: To understand the underlying mechanisms for the resistance, the investigators examined the deposition of complements on the surface of bacteria. For this, the bacterial strains were incubated in immune serum, and the deposition of complements was examined. Both R3 and S3:R showed less deposition of C3 and MAC on their surfaces. Furthermore, the AP activity on the surface of strains was examined by blocking the CP and lectin pathways with magnesium and EGTA. This resulted in overall decrease in levels of complement factors deposited on the surface of bacteria. However, the levels of C3 and MAC present on the surface of R3 and S3:R were still less than S3 even in the absence of the CP and LP. This showed that strains with enhanced resistance had reduced activity of the AP on their surface.

The pubmed citation of this article is: Uria MJ, Zhang Q, Li Y, Chan A, Exley RM, Gollan B, Chan H, Feavers I, Yarwood A, Abad R, Borrow R, Fleck RA, Mulloy B, Vazquez JA, Tang CM. A generic mechanism in Neisseria meningitidis for enhanced resistance against bactericidal antibodies. J Exp Med. 2008 Jun 9;205(6):1423-34.