Neisseria meningitidis is a gram-negative bacterium that often asymptomatically colonizes the human nasopharyngeal tract. These bacteria cross the epithelial barrier can cause life-threatening sepsis and/or meningitis. Antimicrobial peptides are one of the first lines of defense against invading bacterial pathogens. Human beta-defensin 2 (hBD2) is an antimicrobial peptide with broad antibacterial activity, although its mechanism of action is poorly understood. Here, we investigated the effect of hBD2 on N. meningitidis. We showed that hBD2 binds to and kills actively growing meningococcal cells. The lethal effect was evident after 2 h incubation with the peptide, which suggests a slow killing mechanism. Further, the membrane integrity was not changed during hBD2 treatment. Incubation with lethal doses of hBD2 decreased the presence of diplococci; the number and size of bacterial microcolonies/aggregates remained constant, indicating that planktonic bacteria may be more susceptible to the peptide. Meningococcal DNA bound hBD2 in mobility shift assays and inhibited the lethal effect of hBD2 in a dose-dependent manner both in suspension and biofilms, supporting the interaction between hBD2 and DNA. Taken together, the ability of meningococcal DNA to bind hBD2 opens the possibility that extracellular DNA due to bacterial lysis may be a means of N. meningitidis to evade immune defenses.

Antimicrobial peptides (AMPs) play an important role in the defense against pathogens by targeting and killing invading microbes. Some pathogenic bacteria have been shown to negatively regulate AMP expression, while several commensals may induce AMP expression. The expression of certain AMPs, such as human betadefensin 2 (hBD2), can be induced via nuclear factor NF-kappa B, which, in turn, is negatively controlled by tumor necrosis factor alpha-induced protein 3 (TNFAIP3, or A20). In this work, we examined the expression of hBD1 and hBD2 during coincubation of pharyngeal epithelial cells with pathogenic Neisseria meningitidis and commensal lactobacilli. The Lactobacillus strains induced hBD2 expression in human pharyngeal cells, while the pathogen N. meningitidis did not. In coincubation experiments, meningococci were able to dampen the AMP expression induced by lactobacilli. We found that N. meningitidis induced the NF-kappa B inhibitor A20. Further, RNA silencing of A20 resulted in increased hBD2 expression after meningococcal infection. Since it is known that induction of A20 reduces NF-kappa B activity and thus hBD2 levels, meningococcal-mediated A20 induction could be a way for the pathogen to dampen AMP expression. Finally, treatment of N. meningitidis and lactobacilli with synthetic hBD2 reduced N. meningitidis viability more efficiently than Lactobacillus reuteri, explaining why maintaining low AMP levels is important for the survival of the pathogen.

The obligate human pathogen Neisseria meningitidis asymptomatically colonizes the upper respiratory tract, but crossing of the epithelial barrier can cause life-threatening meningitis and/or sepsis. N. meningitidis encounters numerous environmental challenges during colonization in the host, and has evolved different evasion strategies and virulence factors to ensure its survival. In contrast, Lactobacillus species are part of the human microbiota and their commensal colonization confers many benefits to the host, including the inhibition of pathogens.

The first cell type encountered by invading bacteria are epithelial cells and immune cells, which can effectively sense and respond to the presence of bacteria by alerting the immune system or by release of antimicrobial peptides. Antimicrobial peptides are small peptides that are able to directly kill bacteria, but also play a role in modulation of immune responses.  

This thesis focuses on the interaction between the human host and bacteria. Paper I shows that epithelial colonization by different bacterial species induces the transcription factor early growth response protein 1 (EGR1). Induction of EGR1 is mediated primarily by signaling through EGFR and ERK1/2 pathway. In paper II the ability of N. meningitidis and Lactobacillus to modulate expression of antimicrobial peptide human beta-defensin 2 (hBD2) in epithelial cells is compared. Expression of hBD2 is upregulated by lactobacilli. In contrast, N. meningitidis dampens this effect, likely mediated by induction of the host molecule A20, a negative regulator of NF-κB. Since N. meningitidis is susceptible to hBD2-mediated killing, exploitation of A20 may be an immune evasion mechanism. In paper III we demonstrate that hBD2 is able to kill N. meningitidis without causing membrane permeabilization. N. meningitidis DNA can bind hBD2 and thereby inhibit hBD2-mediated killing, presenting a possible evasion mechanism. Finally, paper IV shows that the absence of D-lactate dehydrogenase LdhA in N. meningitidis promotes aggregation and biofilm formation through increased autolysis-mediated release of extracellular DNA.

Neisseria meningitidis is a Gram-negative bacterium that asymptomatically colonizes the human nasopharyngeal mucosa. Pilus-mediated initial adherence of N. meningitidis to the epithelial mucosa is followed by the formation of three-dimensional aggregates, called microcolonies. Dispersal from microcolonies contributes to the transmission of N. meningitidis across the epithelial mucosa. We have recently discovered that environmental concentrations of host cell-derived lactate influences N. meningitidis microcolony dispersal. Here, we examined the ability of N. meningitidis mutants deficient in lactate metabolism to form biofilms. A lactate dehydrogenease A (idhA) mutant had an increased level of biofilm formation. Deletion of IdhA increased the N. meningitidis cell surface hydrophobicity and aggregation. In this study, we used FAM20, which belongs to clonal complex ST-11 that forms biofilms independently of extracellular DNA (eDNA). However, treatment with DNase I abolished the increased biofilm formation and aggregation of the ldhA-delicient mutant, suggesting a critical role for eDNA. Compared to wild-type, the IdhA-deficient mutant exhibited an increased autolytic rate, with significant increases in the eDNA concentrations in the culture supernatants and in biofilms. Within the IdhA mutant biofilm, the transcription levels of the capsule, pilus, and bacterial lysis genes were downregulated, while norB, which is associated with anaerobic respiration, was upregulated. These findings suggest that the absence of IdhA in N. meningitidis promotes biofilm formation and aggregation through autolysis-mediated DNA release.

The essential first step in bacterial colonization is adhesion to the host epithelial cells. The early host-responses post-bacterial adhesions are still poorly understood. Early growth response 1 (EGR1) is an early response transcriptional regulator that can be rapidly induced by various environmental stimuli. Several bacteria can induce EGR1 expression in host cells, but the involved bacterial characteristics and the underlying molecular mechanisms of this response are largely unknown. Here, we show that EGR1 can be induced in host epithelial cells by different species of bacteria independent of the adherence level, Gram-staining type and pathogenicity. However, bacterial viability and contact with host cells is necessary, indicating that an active interaction between bacteria and the host is important. Furthermore, the strongest response is observed in cells originating from the natural site of the infection, suggesting that the EGR1 induction is cell type specific. Finally, we show that EGFRERK1/2 and beta 1-integrin signaling are the main pathways used for bacteria-mediated EGR1 upregulation. In conclusion, the increase of EGR1 expression in epithelial cells is a common stress induced, cell type specific response upon host-bacteria interaction that is mediated by EGFRERK1/2 and beta 1-integrin signaling.