A characteristic of human pathogenic Neisseriae is the production and secretion of an immunoglobulin (Ig)A1-specific serine protease (IgA1 protease) that cleaves preferentially human IgA1 and other target proteins. Here we show a novel function for native IgA1 protease, i.e., the induction of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 from peripheral blood mononuclear cells. The capacity of IgA1 protease to elicit such cytokine responses in monocytes was enhanced in the presence of T lymphocytes. IgA1 protease did not induce the regulatory cytokine IL-10, which was, however, found in response to lipopolysaccharide and phytohemagglutinin. The immunomodulatory effects caused by IgA1 protease require a native form of the enzyme, and denaturation abolished cytokine induction. However, the proteolytic activity is not required for the cytokine induction by IgA1 protease. Our results indicate that IgA1 protease exhibits important immunostimulatory properties and may contribute substantially to the pathogenesis of neisserial infections by inducing large amounts of TNF-α and other proinflammatory cytokines. In particular, IgA1 protease may represent a key virulence determinant of bacterial meningitis.

Pathogenic Neisseria species, the causative agents of gonorrhoea and bacterial meningitis, encode a family of polymorphic exo-proteins which are autoproteolytically processed into several distinct extracellular components, including an IgA1 protease and an alpha-protein. IgA1 protease, a putative virulence determinant, is a sequence-specific endopeptidase known to cleave human IgA1, but additional target proteins have been postulated. The physical linkage of IgA1 protease and alpha-protein suggests a functional relationship of both precursor components. Previous work has shown that alpha-protein is essential neither for extracellular transport nor for the proteolytic activity of IgA1 protease. Intriguingly, alpha-proteins carry amino acid sequences reminiscent of nuclear location signals of viral and eukaryotic proteins. Here we demonstrate the functionality of these nuclear location signal sequences in transfected eukaryotic cells. Chimeric alpha-proteins show nuclear transport and selectively associate with nucleolar structures. More importantly, native purified alpha-proteins are capable of entering certain human primary cells from the exterior via an endocytotic route and accumulate in the nuclei. The neisserial alpha-proteins share several features with eukaryotic transcription factors, such as the formation of dimers via a heptad repeat sequence. We propose a role for alpha-proteins in the regulation of host-cell functions. As the alpha-proteins are covalently connected with IgA1 protease they may also serve as carries for the IgA1 protease into human cells where additional proteolytic targets may exist. Neisseria meningitidis, which locally colonizes the nasopharyngeal mucosa of many human individuals without apparently causing symptoms, secretes this nucleus-targeted factor in large quantities.

A unique feature of the immunoglobulin A1 (IgA1) protease from pathogenic Neisseriae, i.e. N. meningitidis and N. gonorrhoeae, is its co-secretion with an amphipathic a-protein. Polymerase chain reaction (PCR) analysis of the respective iga(alpha). gene region in 48 meningococcal strains revealed that this protein domain is conserved throughout all isolates in four different principal variants. Despite strain-dependent size and sequence variations, sequence analysis showed common structural characteristics. More than 80% of the amino acid sequence of all a-proteins is dependent on the five amino acids Q, E, A, K and R, resulting in a pI> 10. The sequences are highly conserved at the N-terminus and the C-terminus and contain long amphipathic alpha-helical stretches. These stretches have a strong probability of forming coiled coil conformations and comprise short repetitive sequence modules with pronounced similarities to T-cell epitopes. We therefore analyzed the T-cell response of 20 volunteer blood donors to four peptides, representing such predicted epitopes, and a recombinant meningococcal alpha-protein. Sixteen donors reacted against at least one peptide after culture of peripheral blood mononuclear cells in interleukin (IL)-2-rich medium, while two individuals showed a positive reaction only against an IgA1 protease-derived control peptide. From one donor, we established and maintained T-cell clones specific for purified alpha-protein. Characterization of the T-cell clones revealed a CD3- and a CD4-positive phenotype and the secretion of IL-2 and interferon-gamma (IFN-gamma),

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