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

Abstract

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.

Bacterial Factors Targeting the Nucleus: The Growing Family of Nucleomodulins

“…App and MspA are internalized by human cells and traffic to the nucleus. In vitro, MspA and App proteolytically cleave histone H3, and in vivo, they induce a dose-dependent increase in dendritic cell death via caspase-dependent apoptosis [100]. However, the functional link between these properties remains to be clarified.…”

Uptake of Neisserial autotransporter lipoprotein (NalP) promotes an increase in human brain microvascular endothelial cell metabolic activity

“…A HiTrap column pre-packed with five milliliters of Sephadex G-25 Superfine (GE Healthcare Lifesciences) equilibrated with 5 column volumes of phosphate buffered saline (PBS) was used for buffer exchange. His6-tagged recombinant E. coli trigger factor protein (rTF), encoded by the plasmid pCold TF (Takara Bio), was purified as previously described [18]. Protein concentration was measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific) following endotoxin removal using Pierce High Capacity Endotoxin removal spin columns (Thermo Fisher Scientific).…”

“…Alternatively proteins were transferred to nitrocellulose membranes and probed with rabbit polyclonal anti-NalP [6] or mouse anti-pentahistidine antibody (Qiagen) diluted 1:5,000 or 1:2,000, respectively, in blocking buffer (5% anti-mycotic solution. Dendritic cells were generated as previously [18], and subsequently grown in RPMI (Sigma) supplemented with 10% FBS and 1% antibiotic / anti-mycotic. Human brain (cerebral cortex) astrocytes (ScienCell) were cultured in astrocyte growth medium supplemented with 5% FBS, 1 × astrocyte growth supplement and 1% antibiotic / anti-mycotic.…”

“…NalP-mediated cleavage generates a functionally inactive C3a-like molecule, with the corresponding C3b-like fragment being rapidly inactivated by host serum factors resulting in the inhibition of C3b deposition on the bacterial surface and enhanced meningococcal serum resistance [17]. We hypothesized that, in addition to previously identified roles, NalP, like several other meningococcal autotransporter proteases [18][19][20], may also interact with host cells. Here we show uptake of NalP into various human cell types, and an increase in human brain microvascular endothelial cell metabolic activity which is dependent on NalP proteolytic activity.…”

A role for Fibroblast Growth Factor Receptor 1 in the pathogenesis ofNeisseria meningitidis

“…The process by which these steps occur is incompletely understood (Sokolova et al , 2004, Yazdankhah et al , 2004, Hill et al , 2010), but identified meningococcal adhesins include components of the type IV pilus (PilC1, PilC2 and PilQ), the outer membrane proteins Opa, Opc, Factor H-binding protein, PorA, PorB, HrpA, NadA, App and MspA, as well as lipooligosaccharide (LOS) (Merz et al , 2000, Hadi et al , 2001, Turner et al , 2006, Morand et al , 2009). Host cell receptors that have been identified include alpha actinin, integrins, CEACAMS, CD46, Complement receptor 3, GP96 scavenger receptor, laminin, platelet-activating factor, mannose receptor, Transferrin receptor 1, Laminin receptor and Galectin-3 (Merz et al , 2000, Morand et al , 2009, Orihuela et al , 2009, Quattroni et al , 2012, Alqahtani et al , 2014, Khairalla et al , 2015).…”