The AmiC/NlpD Pathway Dominates Peptidoglycan Breakdown in Neisseria meningitidis and Affects Cell Separation, NOD1 Agonist Production, and Infection.

The human-restricted pathogen Neisseria meningitidis, which is best known for causing invasive meningococcal disease, has a nonpathogenic lifestyle as an asymptomatic colonizer of the human naso- and oropharyngeal space. N. meningitidis releases small peptidoglycan (PG) fragments during growth. It was demonstrated previously that N. meningitidis releases low levels of tripeptide PG monomer, which is an inflammatory molecule recognized by the human intracellular innate immune receptor NOD1. In the present study, we demonstrated that N. meningitidis released more PG-derived peptides than PG monomers. Using a reporter cell line overexpressing human NOD1, we showed that N. meningitidis activates NOD1 using PG-derived peptides. The generation of such peptides required the presence of the periplasmic N-acetylmuramyl-l-alanine amidase AmiC and the outer membrane lipoprotein NlpD. AmiC and NlpD were found to function in cell separation, and mutation of either amiC or nlpD resulted in large clumps of unseparated N. meningitidis cells instead of the characteristic diplococci. Using stochastic optical reconstruction microscopy, we demonstrated that FLAG epitope-tagged NlpD localized to the septum, while similarly tagged AmiC was found at the septum in some diplococci but was distributed around the cell in most cases. In a human whole-blood infection assay, an nlpD mutant was severely attenuated and showed particular sensitivity to complement. Thus, in N. meningitidis, the cell separation proteins AmiC and NlpD are necessary for NOD1 stimulation and survival during infection of human blood.

Functional analysis of the Gonococcal Genetic Island of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an obligate human pathogen that is responsible for the sexually-transmitted disease gonorrhea. N. gonorrhoeae encodes a T4SS within the Gonococcal Genetic Island (GGI), which secretes ssDNA directly into the external milieu. Type IV secretion systems (T4SSs) play a role in horizontal gene transfer and delivery of effector molecules into target cells. We demonstrate that GGI-like T4SSs are present in other ß-proteobacteria, as well as in α- and γ-proteobacteria. Sequence comparison of GGI-like T4SSs reveals that the GGI-like T4SSs form a highly conserved unit that can be found located both on chromosomes and on plasmids. To better understand the mechanism of DNA secretion by N. gonorrhoeae, we performed mutagenesis of all genes encoded within the GGI, and studied the effects of these mutations on DNA secretion. We show that genes required for DNA secretion are encoded within the yaa-atlA and parA-parB regions, while genes encoded in the yfeB-exp1 region could be deleted without any effect on DNA secretion. Genes essential for DNA secretion are encoded within at least four different operons.

Peptidoglycan fragment release from Neisseria meningitidis.

Neisseria meningitidis (meningococcus) is a symbiont of the human nasopharynx. On occasion, meningococci disseminate from the nasopharynx to cause invasive disease. Previous work showed that purified meningococcal peptidoglycan (PG) stimulates human Nod1, which leads to activation of NF-κB and production of inflammatory cytokines. No studies have determined if meningococci release PG or activate Nod1 during infection. The closely related pathogen Neisseria gonorrhoeae releases PG fragments during normal growth. These fragments induce inflammatory cytokine production and ciliated cell death in human fallopian tubes. We determined that meningococci also release PG fragments during growth, including fragments known to induce inflammation. We found that N. meningitidis recycles PG fragments via the selective permease AmpG and that meningococcal PG recycling is more efficient than gonococcal PG recycling. Comparison of PG fragment release from N. meningitidis and N. gonorrhoeae showed that meningococci release less of the proinflammatory PG monomers than gonococci and degrade PG to smaller fragments. The decreased release of PG monomers by N. meningitidis relative to N. gonorrhoeae is partly due to ampG, since replacement of gonococcal ampG with the meningococcal allele reduced PG monomer release. Released PG fragments in meningococcal supernatants induced significantly less Nod1-dependent NF-κB activity than released fragments in gonococcal supernatants and tended to induce less interleukin-8 (IL-8) secretion in primary human fallopian tube explants. These results support a model in which efficient PG recycling and extensive degradation of PG fragments lessen inflammatory responses and may be advantageous for maintaining meningococcal carriage in the nasopharynx.

Prevalence and detailed mapping of the gonococcal genetic island in Neisseria meningitidis.

The 57-kb gonococcal genetic island (GGI) encodes a type IV secretion system (T4SS) that is found in most strains of N. gonorrhoeae. This T4SS functions to secrete single-stranded DNA that is active in natural transformation. The GGI has also been found in some strains of N. meningitidis. We screened 126 isolates of N. meningitidis and found the GGI in 17.5% of strains, with the prevalence varying widely among serogroups. The GGI is found in a significant number of serogroup C, W-135, and X strains but was not found in strains of serogroup A, B, or Y. Through detailed PCR mapping and DNA sequencing, we identified five distinct GGI types in meningococci. DNA sequencing and a genetic assay revealed that the GGI was likely integrated into the meningococcal chromosome by the site-specific recombinase XerCD and that the GGI can be excised and lost from the genome. Functional studies showed that in contrast with the gonococcal T4SS, the meningococcal T4SS does not secrete DNA, nor does it confer Ton-independent intracellular survival. Deletion of T4SS genes did not affect association with or invasion of host cells. These results demonstrate that the GGI is found in a significant proportion of meningococcal strains and that while some strains carry multiple insertions and deletions in the GGI, other strains carry intact T4SS genes and may produce functional secretion systems.

The Gonococcal Genetic Island and Type IV Secretion in the Pathogenic Neisseria.

Eighty percent of Neisseria gonorrhoeae strains and some Neisseria meningitidis strains encode a 57-kb gonococcal genetic island (GGI). The GGI was horizontally acquired and is inserted in the chromosome at the replication terminus. The GGI is flanked by direct repeats, and site-specific recombination at these sites results in excision of the GGI and may be responsible for its original acquisition. Although the role of the GGI in N. meningitidis is unclear, the GGI in N. gonorrhoeae encodes a type IV secretion system (T4SS). T4SS are versatile multi-protein complexes and include both conjugation systems as well as effector systems that translocate either proteins or DNA-protein complexes. In N. gonorrhoeae, the T4SS secretes single-stranded chromosomal DNA into the extracellular milieu in a contact-independent manner. Importantly, the DNA secreted through the T4SS is effective in natural transformation and therefore contributes to the spread of genetic information through Neisseria populations. Mutagenesis experiments have identified genes for DNA secretion including those encoding putative structural components of the apparatus, peptidoglycanases which may act in assembly, and relaxosome components for processing the DNA and delivering it to the apparatus. The T4SS may also play a role in infection by N. gonorrhoeae. During intracellular infection, N. gonorrhoeae requires the Ton complex for iron acquisition and survival. However, N. gonorrhoeae strains that do not express the Ton complex can survive intracellularly if they express structural components of the T4SS. These data provide evidence that the T4SS is expressed during intracellular infection and suggest that the T4SS may provide an advantage for intracellular survival. Here we review our current understanding of how the GGI and type IV secretion affect natural transformation and pathogenesis in N. gonorrhoeae and N. meningitidis.