Antigen sequence typing of outer membrane protein (fetA) gene of Neisseria meningitidis serogroup A from Delhi & adjoining areas

Neisseria meningitidis is a human pathogen capable of causing meningitis, bacteraemia and some less common syndrome1. Meningococcal meningitis occurs as outbreaks in certain part of the world such as Asia and Africa. Meningococcaemia may occur with or without meningitis, and is highly lethal even when treated23.

Approximately 100,000 cases of acute bacterial meningitis occur worldwide each year. The incidence rate for meningococcal disease is < 1-3/100,000 and 10-25/100,000 in the developed and developing countries, respectively3. In India, the epidemic of cerebrospinal fever (earliest term for meningococcal meningitis) was reported in 1883-18844 and the earliest confirmed outbreak was in 1961-1962, followed by 1966-1967, 1985-1986, 2005-2006 in New Delhi and 2008-2009 in Meghalaya and Tripura5. Serogroup A has been reported repeatedly associated with all the outbreaks in India.

FetA is a member of the TonB-dependent class of outer membrane protein (OMP) of Gram-negative bacteria6. A protein model for FetA proposed 26 membrane spanning beta-sheet structures and 13 surface exposed loop structures7. Of these, loop 7, corresponded to a region of variable amino acid sequence which included the epitopes for several anti-FetA mouse monoclonal antibodies. Other shorter polymorphic regions were also located within the surface-exposed loops of the proposed structure8. This loop was designated as FetA variable region (VR).

FetA is a potential vaccine candidate against meningococci as it induces bactericidal activity. It has been observed in the serum of convalescent patients9, and experimental studies with monoclonal antibodies raised against FetA have demonstrated to have similar activity10. The antigenic change in outer membrane proteins can occur due to the host immune response or evolutionary pressure. Further, presence of highly variable FetA VR regions and the reports of occasional FetA deletions in invasive meningococcal11 and pharyngeal isolates appear to lessen its appeal as a potential vaccine candidate10. Due to highly divergent nature of FetA VR, 412 FetA VR peptides have been identified. These peptide sequences could be resolved into nine distinct families (F1, F2, F3, F4, F5, F6, F7, F8 and F9). The sequence changes were suggested to be a likely consequence of selection pressure imposed by the host immune responses12. Diversity of FetA allows it to be used as a marker of molecular characterization along with PorA VRs and multi locus sequence typing (MLST).

The precise characterization of N. meningitidis from cases of invasive meningococcal meningitis is essential for the management and control of meningococcal disease1314. In case of meningococci, the European Monitoring Group for Meningococci (EMGM) recommends the use of genogrouping, MLST and clonal complex15, porA VRs and fetA VR16 as methods of molecular characterization.

Antigen sequence typing of porA and fetA genes is now widely accepted and used for highly discriminating and precise typing of N. meningitidis17. This study was undertaken to characterize outer membrane protein FetA antigen of N. meningitidis isolates obtained from patients with invasive meningococcal meningitis from in Delhi and adjoining areas.

Material & Methods

A total of 15 isolates of N. meningitidis serogroup A obtained from the cases of invasive meningococcal meningitis in New Delhi, India, during 2005-2011, were included in this study. Eight of these were from occasional cases of meningococcal meningitis reported in 2008-2011. All these were isolated from routine diagnostic laboratories of Microbiology of the three hospitals of Delhi including All India Institute of Medical Sciences (AIIMS), Vardhman Mahavir Medical College (VMMC) and Safdarjung Hospital and National Center for Disease Control (NCDC). CSF (1-2 ml) was extracted from suspected cases of acute bacterial meningitis. Of these eight isolates, one was collected from AIIMS, four were collected from VMMC and Safdarjung Hospital and three were from NCDC. The remaining seven were taken from those stored at AIIMS from the outbreaks of meningococcal meningitis in Delhi and its adjoining areas during 2005-2006.

All isolates were stored at -70 °C in brain heart infusion broth with 10 per cent glycerol and recovered by plating on chocolate agar. For each isolate, the growth obtained from the surface of a single petri dish after overnight incubation in an atmosphere of 5 per cent CO₂ was used to prepare an opaque cell suspension in 500 μl deionized water and meningococcal DNA was extracted by using nucleic acid extraction kit (QIAGEN, Germany). For serogroup predictions, PCR was applied using primers designed for genes that are specific for each serogroup18. This was done by targeting orf-2 region of myn B gene for serogroup A.

All the PCRs were performed at the laboratory of Microbiology department in AIIMS, New Delhi. Primers mentioned in the Multi locus sequence typing website for FetA VR19 were used. The forward and reverse primers used were S1 5’-CGGCGCAAGCGTATTCGG-3’ and S8 5’-CGCGCCCAATTCGTAACCGTG-3’ (Fermentas, life Sciences, USA), respectively. Amplified product was of size 1200 bp. PCR products were prepared for sequencing using PCR product purification kit (Axyprep PCR cleanup kit, Axygen Biosciences USA). Sequencing reactions were performed using ABI PRISM Big Dye Terminator cycle sequencing ready reaction kit (Ver 3.1, Applied Biosystems, USA). Sequencing of DNA was carried out in sixteen capillaries ABI Prism 3130XL genetic analyzer (Applied Biosystems, USA). Consensus sequences were submitted to the PubMLST website19 and the fetA allele was obtained. DNA sequence of fetA gene was submitted to the genebank (Genebank Accession No. JN182195-J​N182196). General information of N. meningitidis isolate (AIIMS/08) was also submitted to the MLST database and id number 18485 obtained.

The study protocol was approved by the ethics committee of the AIIMS and patients’ consent was taken for the extraction of CSF.

Results & Discussions

Meningococcal meningitis is endemic in Delhi for the last 50 yr. The aim of the study was to document diversity in outer membrane protein fetA gene of N. meningitidis serogroup A isolated from invasive meningococcal meningitis cases in New Delhi, India during 2005-2011. All 15 isolates of N. meningitidis were serogroup A and carried the same fetA VR allele, F3-1 (Genebank accession No. JN182195-96). The nomenclature of all isolates as proposed by the EMGM was A: F3-1.

The findings add to our knowledge about the stability found in outer membrane protein of N. meningitidis serogroup A in Delhi, India, as no new allele was found. All of these were associated with the same type of fetA allele i.e. 3-1.

Reported studies showed that fetA 3-1 allele was found in both invasive and carrier state though mainly it is isolated from cases of invasive disease. Extensive MLST database19 search on reported studies showed that of the 49 isolates, 35 (71.4%) are from invasive disease including meningitis, six (12.2%) from carriers and the rest unspecified. f3-1 allele was most commonly associated with MLST clonal complex ST5 complex / subgroup III and porA type P1.20, 9 of serogroup A N. meningitides.

Earlier reported studies and search through global MLST database19 of serogroup A with sequence type ST5/clonal complex III in “meningitis belt” of Africa showed the allele type of fetA 3-1. However, it was noted that FetA characterization of N. meningitidis of serogroup A with sequence type ST5/clonal complex III was not done for all the entries in the database. Those few entries of Africa where FetA 3-1, were from Morocco in 1994, Burkinofaso in 2007, Slovenia in 2006 and Mali in 1997, 2009, 2010. Sequence type and genosubtyping were the same for all.

MLST database19 reveals that serogroup A sequence type 4789 of ST5/clonal complex III has earlier been reported from Bangladesh (2002), France (2006), Israel (2006), USA (2009), Italy (2009) and UK (2011). All the genosubtyping enteries for ST 4789 were found to be the same type P1.20, 9. The analysis of database reveals that USA, UK and Italy reported fetA allele as 3-1 similar to our findings. fetA allele 3-1 was found to be associated with other serogroups and sequence types in other geographical regions. Serogroup Y from Spain was also found to be associated with sequence type 4789 but further antigen sequence typing was not done19.

Extensive search of the database has led to the conclusion that serogroup A N. meningitidis appears to be a stable organism in relation with clonal complex, porA typing and fetA allele. The outer membrane protein (FetA) has potential to elicit immune response which has bactericidal activity. But variation in this region can result in the ineffectiveness of this as a vaccine component. According to a few studies available, stability is found in the genetic structure coding for fetA associated with serogroup A. Hence more studies on FetA typing are required from different parts of world in their respective serogroup, sequence type and porA type to know the degree of variation. Also the sequence diversity and length of the FetA VR make it suitable for use as a molecular marker.

In conclusion, serogroup A of N. meningitidis seems to be stable serogroup in its association with similar type of fetA allele. The limitation of this study was the small number of isolates characterized from a single outbreak and a few sporadic cases. Hence the degree of variation or stability of FetA antigen is likely to be underestimated.