Yersinia pestisAil recruitment of C4b‐binding protein leads to factor I‐mediated inactivation of covalently and noncovalently bound C4b

Abstract

The outer membrane protein Ail of Yersinia pestis mediates several virulence functions, including serum resistance. Here, we demonstrate that Ail binds C4b-binding protein (C4BP), the primary fluid-phase regulator of the classical and lectin pathways. Noncovalent binding of C4 and C4b to Ail was also observed. C4BP bound to Ail can act as a cofactor to the serine protease factor I (fI) in the cleavage of fluid-phase C4b. Employing a panel of C4BP alpha-chain mutants, we observed that the absence of complement control protein domain 6 and 8 reduced binding to Ail. Immunoblot analysis of normal human serum (NHS)-treated bacteria revealed minimal C4b alpha'-chain complexes with bacterial outer membrane targets. Addition of the anti-C4BP monoclonal antibody MK104 to NHS restored C4b-alpha' chain target complexes, suggesting that C4b binds covalently to targets on the Y. pestis surface. C4b bound to Ail noncovalently was also cleaved in a C4BP and fI-dependent manner, leaving the C4c fragment bound to Ail. MK104 also prevented the cleavage of noncovalently bound C4b. Collectively, these data suggest that when C4BP is bound to Ail, fI can cleave and inactivate C4b that has bound covalently to bacterial surface structures as well as C4b bound noncovalently to Ail. Keywords: Ail r C4b-binding protein r Complement r YersiniaAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionYersinia pestis is a rod-shaped, Gram negative, nonmotile, facultative anaerobic bacterium, and one of the most virulent pathogens known to man. Over the course of three pandemics, this pathogen has claimed millions of human lives throughout history. Y. pestis can be transferred from a rodent carrier to a human host via Correspondence: Dr. Derek K. Ho e-mail: derek.ho@helsink.fi the bite of an infected flea, or by direct human-to-human transmission in the case of pneumonic plague. Y. pestis remains endemic in certain parts of the world and is a reemerging disease in Africa [1]. The high mortality if left untreated, the absence of an effective vaccine, the emergence of antibiotic resistant strains [2], and the possibility of bioterrorism justify continued research into this pathogen [3].Y. pestis possesses a battery of mechanisms that disarm the host immune response. For example, Y. pestis produces a tetra-acylated LPS that is a poor inducer of TLR 4 [4]. The pYV (pCD1) virulence plasmid encodes a type III secretion system and associated C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 742-751 Immunity to infection 743 effector proteins (Yops), which confer the ability to disrupt host cell signaling and the actin cytoskeleton [5]. Plasmid pPCP1 encodes the plasminogen activator Pla, which degrades fibrin clots and extracellular matrix components, facilitating bacterial dissemination. Plasmid pMT1 encodes the antiphagocytic capsular antigen, F1 [6]. Y. pestis is highly resistant to the bactericidal action of serum due to the presen...

Yersinia pestis uses the Ail outer membrane protein to recruit vitronectin

“…The finding that Y. pestis Ail binds C4BP, the primary fluid-phase regulator of the classical and lectin pathways, in addition to vitronectin is consistent with previous findings (Ho et al, 2014;Ngampasutadol et al, 2005). Importantly, many other pathogens bind both C4BP and vitronectin , suggesting that there may be a distinct advantage to recruiting both inhibitors.…”

“…However, the mechanisms by which Ail facilitates adhesion to host cells and mediates complement resistance are not well understood. Ail has been shown to bind the ECM proteins fibronectin and laminin, which contribute to bacterial adhesion, and to the complement inhibitor C4BP, which contributes to complement resistance (Ho et al, 2014;Tsang et al, 2010;Yamashita et al, 2011). Here, we demonstrate that Y. pestis Ail also actively recruits the multifunctional glycoprotein vitronectin to the bacterial surface.…”

“…The extracellular loops mediate interactions with components of the extracellular matrix (ECM) (fibronectin, laminin and heparan sulfate proteoglycans) and complement system [C4b-binding protein (C4BP)] that contribute to bacterial attachment/ invasion and serum resistance, respectively Ho et al, 2014;Tsang et al, 2010;Yamashita et al, 2011). Co-crystallization studies with a heparin analogue in conjunction with site-directed mutagenesis identified two heparin-binding sites (HBS) on Ail formed by residues from extracellular loops 2 and 3 (HBS-1) and extracellular loop 1 (HBS-2) (Yamashita et al, 2011).…”

Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

“…4A), confirming data from a previous report by Kolodziejek et al (33). Mice infected with the ⌬lpp ⌬ail and ⌬lpp ⌬msbB double mutants and the ⌬lpp ⌬msbB ⌬ail triple mutant had increased survival rates (20,40, and 100%, respectively). Clinically, animals infected with WT CO92 or mutants that provided minimal attenuation in mice had ruffled fur, hunched back, and lethargy, and they were unable to groom and tended to huddle together.…”

“…Ail (attachment-invasion locus), also referred to as OmpX, is a major contributor to serum resistance and complement evasion in Y. pestis (31)(32)(33)(34) and accounts for 20 to 30% of the total outer membrane proteins in yersiniae at 37°C (35)(36)(37). Ail proteins of Y. enterocolitica and Y. pestis are ϳ69% homologous (34) and bind, as well as regulate, several mediators of the complement system, e.g., complement protein 4-binding protein (38)(39)(40) and complement factor H (FH) (41)(42)(43). In addition to serum resistance, Ail of Y. pestis facilitates the adhesion/invasion of bacteria in host cells (33,34,(44)(45)(46), inhibits inflammatory responses (32,44), and assists in the translocation of damaging Yersinia outer membrane proteins (Yops) to host cells (44,45,47,48).…”