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

Abstract

Neisseria meningitidis is normally a human nasopharyngeal commensal but is also capable of causing life-threatening 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 sub-cellular localization, but inactive rNalP (rNalP) 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.

Crystal structure of a subtilisin-like autotransporter passenger domain reveals insights into its cytotoxic function

“…Indeed, the Ssp-S341A active site mutant had no subtilase activity and was unable to enter HEp-2 cells, while the native as well as other proteolytically active Ssp variants, including Ssp-ΔE2 and Ssp-ΔE2/ΔE3, which retained some protease activity, were internalised under the experimental conditions, albeit the latter protrusion mutants at possibly reduced rates. This dependence on subtilase activity for AT entry into eukaryotic cells contrasts with the subtilase AT NalP from N. meningitidis; the only other subtilase AT directly shown to enter cells and which does so independent of its subtilase activity 62 . The other AT group shown to enter eukaryotic cells are the (chymo)trypsin-like serine protease ATs.…”

“…As a number of ATs, such as the SPATE Pet and subtilase AT NalP from N. meningitidis , are known to enter mammalian cells to exert their effects 21 , 62 , we sought to determine if Ssp is also internalised by HEp-2 cells. Top10 supernatants containing Ssp were incubated with HEp-2 cells, washed, permeabilised, immunostained with an anti-Ssp polyclonal antibody and observed by confocal microscopy.…”

Phylogenetic Classification and Functional Review of Autotransporters

“…This likely results from degradation of host proteins as Fusolisin degrades IgA whereas NalP cleaves C3 of the complement system ( 247 , 248 ). Meanwhile, NalP can also enter a range of host cell types where it alters cellular metabolism ( 249 ). Notably, cytotoxicity, host cell internalization, and immunomodulation are also features of the Cluster A chymotrypsin-like proteases.…”