Uptake of Neisserial autotransporter lipoprotein (NalP) promotes an increase in human brain microvascular endothelial cell metabolic activity

Neisseria meningitidis is normally a human nasopharyngeal commensal but is also capable of causing lifethreatening sepsis and meningitis. N. meningitidis secretes several virulence-associated proteins including Neisserial autotransporter lipoprotein (NalP), an immunogenic, type Va autotransporter harboring an S8-family serine endopeptidase domain. NalP has been previously characterized as a cell-surface maturation protease which processes other virulence-associated meningococcal surface proteins, and as a factor contributing to the survival of meningococci in human serum due to its ability to cleave complement factor C3. Here, recombinant NalP (rNalP) fragments were purified and used to investigate the interaction of NalP with host cells. Flow cytometry and confocal microscopy demonstrated binding and uptake of rNalP into different human cell types. High-resolution microscopy confirmed that internalized rNalP predominantly localized to the perinuclear region of cells. Abolition of rNalP protease activity using site-directed mutagenesis did not influence uptake or subcellular localization, but inactive rNalP (rNalPS426A) was unable to induce an increase in human brain microvascular endothelial cell metabolic activity provoked by proteolytically-active rNalP. Our data suggests a more complex and multifaceted role for NalP in meningococcal pathogenesis than was previously understood which includes novel intra-host cell functions

Influence of the combination and phase variation status of the haemoglobin receptors HmbR and HpuAB on meningococcal virulence

Neisseria meningitidis can utilize haem, haemoglobin and haemoglobin–haptoglobin complexes as sources of iron via two TonB-dependent phase variable haemoglobin receptors, HmbR and HpuAB. HmbR is over-represented in disease isolates, suggesting a link between haemoglobin acquisition and meningococcal disease. This study compared the distribution of HpuAB and phase variation (PV) status of both receptors in disease and carriage isolates. Meningococcal disease (n = 214) and carriage (n = 305) isolates representative of multiple clonal complexes (CCs) were investigated for the distribution, polyG tract lengths and ON/OFF status of both haemoglobin receptors, and for the deletion mechanism for HpuAB. Strains with both receptors or only hmbR were present at similar frequencies among meningococcal disease isolates as compared with carriage isolates. However, >90 % of isolates from the three CCs CC5, CC8 and CC11 with the highest disease to carriage ratios contained both receptors. Strains with an hpuAB-only phenotype were under-represented among disease isolates, suggesting selection against this receptor during systemic disease, possibly due to the receptor having a high level of immunogenicity or being inefficient in acquisition of iron during systemic spread. Absence of hpuAB resulted from either complete deletion or replacement by an insertion element. In an examination of PV status, one or both receptors were found in an ON state in 91 % of disease and 71 % of carriage isolates. We suggest that expression of a haemoglobin receptor, either HmbR or HpuAB, is of major importance for systemic spread of meningococci, and that the presence of both receptors contributes to virulence in some strains

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

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