NafA Negatively Controls Neisseria meningitidis Piliation

Bacterial auto-aggregation is a critical step during adhesion of N. meningitidis to host cells. The precise mechanisms and functions of bacterial auto-aggregation still remain to be fully elucidated. In this work, we characterize the role of a meningococcal hypothetical protein, NMB0995/NMC0982, and show that this protein, here denoted NafA, acts as an anti-aggregation factor. NafA was confirmed to be surface exposed and was found to be induced at a late stage of bacterial adherence to epithelial cells. A NafA deficient mutant was hyperpiliated and formed bundles of pili. Further, the mutant displayed increased adherence to epithelial cells when compared to the wild-type strain. In the absence of host cells, the NafA deficient mutant was more aggregative than the wild-type strain. The in vivo role of NafA in sepsis was studied in a murine model of meningococcal disease. Challenge with the NafA deficient mutant resulted in lower bacteremia levels and mortality when compared to the wild-type strain. The present study reveals that meningococcal NafA is an anti-aggregation factor with strong impact on the disease outcome. These data also suggest that appropriate bacterial auto-aggregation is controlled by both aggregation and anti-aggregation factors during Neisseria infection in vivo

Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters

Neisseria 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

Imaging of Disease Dynamics during Meningococcal Sepsis

Neisseria meningitidis is a human pathogen that causes septicemia and meningitis with high mortality. The disease progression is rapid and much remains unknown about the disease process. The understanding of disease development is crucial for development of novel therapeutic strategies and vaccines against meningococcal disease. The use of bioluminescent imaging combined with a mouse disease model allowed us to investigate the progression of meningococcal sepsis over time. Injection of bacteria in blood demonstrated waves of bacterial clearance and growth, which selected for Opa-expressing bacteria, indicating the importance of this bacterial protein. Further, N. meningitidis accumulated in the thyroid gland, while thyroid hormone T4 levels decreased. Bacteria reached the mucosal surfaces of the upper respiratory tract, which required expression of the meningococcal PilC1 adhesin. Surprisingly, PilC1 was dispensable for meningococcal growth in blood and for crossing of the blood-brain barrier, indicating that the major role of PilC1 is to interact with mucosal surfaces. This in vivo study reveals disease dynamics and organ targeting during meningococcal disease and presents a potent tool for further investigations of meningococcal pathogenesis and vaccines in vivo. This might lead to development of new strategies to improve the outcome of meningococcal disease in human patients

Olfactory Nerve—A Novel Invasion Route of Neisseria meningitidis to Reach the Meninges

Neisseria meningitidis is a human-specific pathogen with capacity to cause septic shock and meningitis. It has been hypothesized that invasion of the central nervous system (CNS) is a complication of a bacteremic condition. In this study, we aimed to characterize the invasion route of N. meningitidis to the CNS. Using an intranasally challenged mouse disease model, we found that twenty percent of the mice developed lethal meningitis even though no bacteria could be detected in blood. Upon bacterial infection, epithelial lesions and redistribution of intracellular junction protein N-cadherin were observed at the nasal epithelial mucosa, especially at the olfactory epithelium, which is functionally and anatomically connected to the CNS. Bacteria were detected in the submucosa of the olfactory epithelium, along olfactory nerves in the cribriform plate, at the olfactory bulb and subsequently at the meninges and subarachnoid space. Furthermore, our data suggest that a threshold level of bacteremia is required for the development of meningococcal sepsis. Taken together, N. meningitidis is able to pass directly from nasopharynx to meninges through the olfactory nerve system. This study enhances our understanding how N. meningitidis invades the meninges. The nasal olfactory nerve system may be a novel target fo

Depletion of N-cadherin in olfactory epithelium upon <i>N. meningitidis</i> infection.

<p>CD46 transgenic mice were challenged i.n. with 10⁷ CFU of <i>N. meningitidis</i>. Mice challenged with PBS were set as uninfected control. At day 3 post-challenge, head tissue was collected and tissue sections were stained using N-cadherin and <i>N. meningitidis</i> antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014034#s4" target="_blank">Materials and Methods</a>. (A) In uninfected mice (left panels), strong and continuous N-cadherin staining (green) was identified in the apical layer (arrows) of the olfactory epithelium (OE) and bundles of olfactory nerve (arrowheads) in lamina propria (LP). In infected mice (right panels), the N-cadherin signal was significantly decreased in the corresponding regions. In merged image, bacteria (red) were detected at the olfactory mucosa surface of infected mice. Nuclei were stained with DAPI. (B) Quantification of N-cadherin staining intensity by software Image J. After infection, expression of N-cadherin was significantly decreased in both OE and the LP region. The data are presented as mean ± SD. Significant reductions of signal intensity compared with uninfected mice are indicated with asterisks (*, <i>p</i><0.05, nonparametric Mann-Whitney test). Abbreviations: OE, olfactory epithelium; LP, lamina propria; CP, cribriform plate.</p

Figure 5

<p>Translocation of <i>N. meningitidis</i> to the respiratory mucosa is dependent on PilC1. (A) Bioluminescent imaging of mice and nasal washes spread on GC-plates 24 h post infection. CD46 transgenic mice were infected i.v. with wild-type FAM20^LU or the PilC1-deficient mutant FAM20Δ<i>pilC1</i>^LU. Nontransgenic mice were challenged with FAM20^LU. Bacterial counts in the nasal washes (CFU/ml) are presented. Significantly fewer PilC1 mutants were recovered compared with wild-type bacteria (<i>P</i><0.05). (B) Immunohistochemical detection of <i>N. meningitidis</i> in nasal cavity sections of CD46 transgenic mice at 24 h post-infection. Bacteria (dark brown) were detected with rabbit anti-<i>N. meningitidis</i> antibody followed by HRP-conjugated goat anti-rabbit IgG secondary antibody, and stained with DAB. Control sections have been treated without the first antibody (w/o 1st Ab). Scale bar: 20 µm.</p

Primers used for generation of the pLKMp plasmid

*<p>Restriction enzyme cleavage sites are marked in bold. The DNA uptake sequence is underlined.</p

Meningococcal colonization triggers tissue damage of the olfactory epithelium.

<p>(A) 10⁷ CFU of <i>N. meningitidis</i> or PBS (uninfected control) was administrated i.n. to CD46 transgenic mice. At day 3 post-challenge, nasal tissue sections were collected and stained with Hematoxylin and Eosin. Hematoxylin stains negatively charged nuclei dark blue and eosin stains other tissue structures pink. The olfactory epithelium is thereby visualized as a region with a high density of heavily hematoxylin-stained cells. Images show the olfactory epithelial (OE) region of nasal mucosa and the luminal space of the nasal cavity (NC). Epithelial damage and atrophy was induced upon bacterial infection (right panels). Higher magnification (63×) of the box in upper panels is shown in lower panels. Arrows indicate infiltrated cells. Yellow bar: thickness of the OE. Abbreviations: LP, lamina propria; CP, cribriform plate; N, olfactory nerve. (B) Thickness of the olfactory epithelium was measured using a Carl Zeiss Axio Vision 2.05 image analysis system (Zeiss). Upon meningococcal infection, the thickness of the OE is significantly decreased compared to the uninfected mice (A, left panels). *, <i>p</i><0.05, nonparametric Mann-Whitney test.</p