Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen Neisseria meningitidis and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 M LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain. N. meningitidis bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes, siaC and siaD, which further results in upregulation of capsule biosynthesis.Neisseria meningitidis (meningococci) is a gram-negative, aerobic diplococci that is an obligate human pathogen. Infections caused by N. meningitidis are an important cause of morbidity and mortality worldwide. Meningococci colonize the nasopharyngeal mucosa of approximately 10% of healthy individuals but can cross epithelial and endothelial cell barriers and enter the bloodstream, causing septicemia, with mortality rates of 20 to 50% (4). Meningitis occurs when bacteria transverse the blood cerebrospinal fluid, causing a fatal outcome in 15 to 20% of infected patients. Bacterial adherence is initially mediated by type IV pili with host cell receptors. PilT is the molecular motor responsible for pili retraction, which mediates a tight interaction. An important virulence factor of N. meningitidis is the endotoxin lipooligosaccharide (LOS), which is located in the bacterial outer cell membrane. Meningococcal LOS is composed of a conserved inner core of membraneassociated lipid A (16) to which variable ␣-and ␤-chains attach (13).As one of many first lines of defense against invading pathogens like Neisseria bacteria, epithelial cells produce antimicrobial peptides (AMPs). These peptides are effector molecules for the innate immune response, with both direct antimicrobial activity and a broad spectrum of immunomodulatory functions (18,22). LL-37 is the single known human cathelicidin and is expressed in various immune cells as well as in epithelial cells of inflamed skin, mouth, tongue, esophagus, and lungs. It has been shown that LL-37 interacts with bacterial membranes through both electrostatic and hydrophobic effects. It remains unknown whether LL-37 ultimately kills bacteria by formation of torroidal pores as described by Henzler Wildman et al. (11) or by detergent-like disintegration of the membrane via the carpet model as described by Shai (24), but increasing membrane permeability, osmotic swelling, and loss of the vital proton gradient are important char...

Summary N eisseria meningitidis, a major cause of bacterial meningitis and septicaemia, secretes multiple virulence factors, including the adhesion and penetration protein (App) and meningococcal serine protease A (MspA). Both are conserved, immunogenic, type Va autotransporters harbouring S6‐family serine endopeptidase domains. Previous work suggested that both could mediate adherence to human cells, but their precise contribution to meningococcal pathogenesis was unclear. Here, we confirm that App and MspA are in vivo virulence factors since human CD46‐expressing transgenic mice infected with meningococcal mutants lacking App, MspA or both had improved survival rates compared with mice infected with wild type. Confocal imaging showed that App and MspA were internalized by human cells and trafficked to the nucleus. Cross‐linking and enzyme‐linked immuno assay (ELISA) confirmed that mannose receptor (MR), transferrin receptor 1 (TfR1) and histones interact with MspA and App. Dendritic cell (DC) uptake could be blocked using mannan and transferrin, the specific physiological ligands for MR and TfR1, whereas in vitro clipping assays confirmed the ability of both proteins to proteolytically cleave the core histone H3. Finally, we show that App and MspA induce a dose‐dependent increase in DC death via caspase‐dependent apoptosis. Our data provide novel insights into the roles of App and MspA in meningococcal infection.

bMeningococcal disease is characterized by a fast progression and a high mortality rate. Cell-penetrating peptides (CPPs), developed as vectors for cargo delivery into eukaryotic cells, share structural features with antimicrobial peptides. A screen identified two CPPs, transportan-10 (TP10) and model amphipathic peptide (MAP), with bactericidal action against Neisseria meningitidis. Both peptides were active in human whole blood at micromolar concentrations, while hemolysis remained negligible. Additionally, TP10 exhibited significant antibacterial activity in vivo. Uptake of SYTOX green into live meningococci was observed within minutes after TP10 treatment, suggesting that TP10 may act by membrane permeabilization. Apart from its bactericidal activity, TP10 suppressed inflammatory cytokine release from macrophages infected with N. meningitidis as well as from macrophages stimulated with enterobacterial and meningococcal lipopolysaccharide (LPS). Finally, incubation with TP10 reduced the binding of LPS to macrophages. This novel endotoxin-inhibiting property of TP10, together with its antimicrobial activity in vivo, indicates the possibility to design peptide-based therapies for infectious diseases.

Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3=-5= exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis.