YgfZ contributes to secretion of cytotoxic necrotizing factor 1 into outer-membrane vesicles in Escherichia coli

Abstract

Cytotoxic necrotizing factor 1 (CNF1), a Rho GTPase-activating bacterial toxin, has been shown to contribute to invasion by meningitis-causing Escherichia coli K1 of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. However, CNF1 is a cytosolic protein and it remains unclear how its secretion occurs, contributing to E. coli invasion of HBMEC. To investigate the genetic requirement for CNF1 secretion in E. coli K1 strain RS218, we performed mini-Tn5 in vitro mutagenesis and constructed a transposon mutant library of strain NBC, in which b-lactamase was fused to the C-terminus of CNF1 in the chromosome of strain RS218. We identified a transposon mutant (NBC-1E6) that exhibited reduced b-lactamase activity in its culture supernatant and had the transposon inserted into the ygfZ gene. When ygfZ was deleted from the genome of strain RS218 (DygfZ), the translocation of CNF1 into HBMEC was impaired. Subcellular localization analysis of CNF1 demonstrated that YgfZ, a periplasmic protein, contributes to secretion of CNF1 into outer-membrane vesicles (OMVs). The DygfZ mutant was significantly defective in invasion of HBMEC compared to the parent E. coli K1 strain. The defects of the DygfZ mutant in CNF1 secretion into OMVs and translocation into HBMEC as well as invasion of HBMEC were abrogated by complementation with ygfZ. Taken together, our findings demonstrate that YgfZ contributes to CNF1 secretion into OMVs in meningitis-causing E. coli K1. INTRODUCTIONCNF1, the paradigm of the Rho GTPase-activating bacterial toxins (Boquet, 2001;Knust & Schmidt, 2010;Lemonnier et al., 2007), is frequently associated with uropathogenic and meningitis-causing Escherichia coli strains (Khan et al., 2002;Landraud, et al., 2000). CNF1 activates Rho GTPases by deamidation of glutamine 63, converting it into glutamic acid, thus inhibiting GTPhydrolysing activity, resulting in constitutive activation of Rho GTPase. CNF1 has been shown to contribute to polymerization of F-actin, increased formation of stress fibres and phagocytosis (Caprioli et al., 1983;Falzano et al., 1993;Flatau et al., 1997;Schmidt et al., 1997; VouretCraviari et al., 1999). CNF1-like toxins have also been found in other bacteria, including the dermonecrotic toxin of Bordetella spp. and the CNFc from Yersinia pseudotuberculosis (Knust & Schmidt, 2010;Kume et al., 1986;Lockman et al., 2002).We have shown that CNF1 contributes to invasion by E. coli K1 of human brain microvascular endothelial cells (HBMEC) and penetration into the brain via the interaction with its receptor, 37 laminin receptor precursor (37LRP)/67 laminin receptor (67LR) (Chung et al., 2003;Khan et al., 2002;Kim et al., 2005). CNF1 is a cytoplasmic protein and its secretion is, therefore, a strategy utilized by meningitis-causing E. coli K1 to invade the blood-brain barrier (Khan et al., 2002;Kim, 2003Kim, , 2008. However, it remains incompletely understood how CNF1 secretion occurs. No typical signal peptide is found in the CNF1 sequence. Recent studies have ...

Human Meningitis-Associated Escherichia coli

“…Increased expression of 67-kDa LR has been shown to be associated with invasive and metastatic properties of a variety of tumors (82), and it remains speculative whether CNF1-expressing E. coli has any role in malignant transformation of certain cancers. Although CNF1 is shown to interact with 37-kDa LRP/67-kDa LR on the cell surface of HBMEC, resulting in RhoA activation and increased internalization of CNF1-expressing E. coli , CNF1 is a bacterial cytoplasmic protein (83, 84), and it remains unclear how it is secreted into the outer membrane and interacts with 37-kDa LRP/67-kDa LR on the blood-brain barrier. Taken together, these findings indicate that meningitis-causing E. coli invades HBMECs through ligand-receptor interactions.…”

“…It, however, remains unclear how CNF1 secretion occurs across the bacterial inner membrane and outer membranes. Elucidation of the mechanisms involved in CNF1 secretion is, therefore, likely to enhance our knowledge on the pathogenesis of E. coli meningitis and also help in developing a novel strategy targeting CNF1 secretion in prevention and therapy of E. coli meningitis (83, 84). Taken together, these findings suggest that modulation of bacterial secretion systems (CNF1 secretion, type II secretion, type III secretion, type VI secretion) represents a novel target for prevention and therapy of E. coli meningitis.…”

The effects of cytotoxic necrotizing factor 1 expression in the uptake ofEscherichia coliK1 by macrophages and the onset of meningitis in newborn mice

“…20 E44-infected cells also showed a greater number of microspikes (protrusions) compared to uninfected cells. Macrophages infected with Dcnf1 displayed a lower amount of microspikes, which may be due to minimal Cdc42 activation.…”

“…Microspike formation mediated by Rho GTPases modulates bacterial invasion in RAW 264.7 macrophages CNF1 constitutively activates Rho GTPases, such as RhoA, Rac1, and Cdc42, by the deamidation of glutamine residues and inhibition of the GTP-hydrolyzing activity, promoting actin polymerization. [20][21][22] To examine the specific role of Rho GTPases in macrophages infected with E. coli K1 strains, we pre-treated the cells with Y-27632 (inhibitor of Rho-associated protein kinase p160ROCK), NSC 23766 (Rac1 inhibitor), ¡/¡ mice were infected with bacteria and stained for actin. The number of microspikes were counted in a blinded fashion by analyzing Z-stacks and expressed as a percentage of cells with microspikes.…”

“…[20][21][22] Previous studies have demonstrated that human brain microvascular endothelial cells (HBMEC), an in vitro blood-brain barrier model, treated with CNF1 are highly susceptible to E. coli K1 invasion in a RhoAdependent mechanism. Lack of CNF1 in E. coli K1 significantly inhibits its invasion in HBMEC and the onset of meningitis in a newborn rat model.…”