Prevalence and phase variable expression status of two autotransporters, NalP and MspA, in carriage and disease isolates of Neisseria meningitidis

Abstract

Neisseria meningitidis is a human nasopharyngeal commensal capable of causing life-threatening septicemia and meningitis. Many meningococcal surface structures, including the autotransporter proteins NalP and MspA, are subject to phase variation (PV) due to the presence of homopolymeric tracts within their coding sequences. The functions of MspA are unknown. NalP proteolytically cleaves several surface-located virulence factors including the 4CMenB antigen NhbA. Therefore, NalP is a phase-variable regulator of the meningococcal outer membrane and secretome whose expression may reduce isolate susceptibility to 4CMenB-induced immune responses. To improve our understanding of the contributions of MspA and NalP to meningococcal-host interactions, their distribution and phase-variable expression status was studied in epidemiologically relevant samples, including 127 carriage and 514 invasive isolates representative of multiple clonal complexes and serogroups. Prevalence estimates of >98% and >88% were obtained for mspA and nalP, respectively, with no significant differences in their frequencies in disease versus carriage isolates. 16% of serogroup B (MenB) invasive isolates, predominately from clonal complexes ST-269 and ST-461, lacked nalP. Deletion of nalP often resulted from recombination events between flanking repetitive elements. PolyC tract lengths ranged from 6-15 bp in nalP and 6-14 bp in mspA. In an examination of PV status, 58.8% of carriage, and 40.1% of invasive nalP-positive MenB isolates were nalP phase ON. The frequency of this phenotype was not significantly different in serogroup Y (MenY) carriage strains, but was significantly higher in invasive MenY strains (86.3%; p<0.0001). Approximately 90% of MenB carriage and invasive isolates were mspA phase ON; significantly more than MenY carriage (32.7%) or invasive (13.7%) isolates. This differential expression resulted from different mode mspA tract lengths between the serogroups. Our data indicates a differential requirement for NalP and MspA expression in MenB and MenY strains and is a step towards understanding the contributions of phase-variable loci to meningococcal biology.

Figure 1. Variations in the size of the nalP deletion locus amongst N. meningitidis carriage isolates.

The nalP deletion loci were amplified with primers specific for flanking genes. Amplicons were grouped into four classes based on size; representative samples are shown. ΔnalP1 (V128; Dec), ΔnalP2 (V130; Nov), ΔnalP3 (V199; Nov) and ΔnalP4 (V206; Dec).

Figure 2. Genetic arrangement of the nalP locus in N. meningitidis isolates.

Unidirectional arrows represent ORFs. Repetitive elements are represented by different symbols. dRS3 consensus sequence: ATTCCCNNNNNNNNGGGAAT; REP4 consensus sequence: AAGACCGTCGGGCATCTGCAGCCGTC. Other repetitive sequences are not shown for clarity. Note that the figure is not drawn to scale but is a representation of the various repeat elements and genes present at the nalP locus in N. meningitidis MC58 and four nalP-negative carriage isolates representing ΔnalP1, ΔnalP2, ΔnalP3 and ΔnalP4 classes.

Figure 3. Immunoblot confirmation that nalP tract lengths of 7, 10 or 13Cs results in NalP expression.

Secreted protein preparations from representative strains were probed with rabbit anti-NalP antisera to confirm that only nalP homopolymeric tract lengths of 7, 10 or 13Cs are consistent with the gene being in-frame and phase ON. Secreted NalP fragments ranged in size between ca. 68–70kDa due to sequence polymorphisms between alleles , . Strains used (tract length in parentheses): V199; May (7), V1114; Nov (8), V78; Nov (9), V131; Nov (10), V182; Nov (11), V169; Dec (12) and V193; Nov (13).

Figure 4. Distribution of tract lengths for mspA (A) and nalP (B) in invasive and carriage isolates.

Invasive isolates shown are a sub-set of the 2010–11 UK isolates available in the Meningitis Research Foundation Meningococcus Genome Library database. Carriage isolates were from a study of meningococcal carriage in students at Nottingham University, UK during 2008–09. Black bars, carriage; grey bars, invasive. An ON PV state is produced by 6, 9 or 12Cs for mspA and by 7, 10 or 13Cs for nalP.