Endotoxin, Capsule, and Bacterial Attachment Contribute to Neisseria meningitidis Resistance to the Human Antimicrobial Peptide LL-37

Abstract

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...

Loss of Meningococcal PilU Delays Microcolony Formation and Attenuates Virulence In Vivo

“…The plasmid pJET1.2-⌬PilU_C was then introduced into the chromosome of the pilU mutant by transformation. To generate the pilT pilU double mutant strain, the FAM20 ⌬PilU strain was transformed with genomic DNA extracted from a previously constructed pilT-negative strain (13). Because pilT-negative Neisseria meningitidis cells are nontransformable, the pilU-complemented pilT pilU double mutant was generated by transforming the ⌬pilU_C strain in an identical manner.…”

Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence

“…Because the capsule is an important factor during adhesion to both biotic and abiotic surfaces (54), we sought to investigate the status of the capsule further. To determine whether these changes in expression affected the capsule in the ⌬pnp mutant, the amount of capsule was measured using whole-cell ELISA, as described previously (46). The ELISA revealed that there was no significant change in the amount of capsule present on the bacterial surface (Fig.…”

“…Capsular polysaccharides were quantified using whole-cell enzyme-linked immunosorbent assays (ELISA) with a monoclonal anti-serogroup C capsule antibody (code 95/678; National Institute for Biological Standards and Control), as described previously (46). Purified serogroup C polysaccharides (code 07/318; National Institute for Biological Standards and Control) were used as the positive control.…”

Serogroup-Specific Interaction of Neisseria meningitidis Capsular Polysaccharide with Host Cell Microtubules and Effects on Tubulin Polymerization

“…Moreover, there is evidence that the MenB capsule is important for meningococcal survival within human cells, and that capsular biosynthetic genes are upregulated in the intracellular environment (6). There is also evidence that the meningococcal capsule contributes to protect the bacteria against cationic antimicrobial peptides (CAMP), including the human cathelicidin LL-37 (6,7), while at the same time, by interacting with the capsule, LL-37 seems to inhibit the proinflammatory activity of the capsular polysaccharide (8). On the other hand, the expression of the capsular polysaccharide inhibits the colonization and invasion of the nasopharyngeal barrier by masking the meningococcal adhesins/invasins (9).…”