Identification of OprF as a Complement Component C3 Binding Acceptor Molecule on the Surface of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a versatile opportunistic pathogen that can cause devastating persistent infections. Complement is a highly conserved pathway of the innate immune system, and its role in the first line of defense against pathogens is widely appreciated. One of the earliest events in the complement cascade is the conversion of C3 to C3a and C3b, the latter typically binds to one or more acceptor molecules on the pathogen surface. We previously demonstrated that complement C3b binding acceptors exist on the P. aeruginosa surface. In the current study, we utilized either C3 polyclonal or C3b monoclonal antibodies in a farWestern technique followed by mass spectroscopy to identify the C3b acceptor molecule(s) on the P. aeruginosa surface. Our data provide evidence that OprF (an outer membrane porin, highly conserved in the Pseudomonadaceae) binds C3b. An oprFdeficient P. aeruginosa strain exhibits reduced C3 deposition compared to the wild type. We observed reduced internalization of oprF-deficient bacteria by neutrophils after opsonization compared with wild-type P. aeruginosa. Heterologous expression of OprF significantly enhanced C3b binding and increased serum-mediated bactericidal effects in complement-susceptible Escherichia coli. Furthermore, the predicted secondary structure of the C-terminal, surface-exposed region of OprF has high structural identity to the OmpA domain of several other Gram-negative bacteria, one of which is known to bind C3b. Therefore, these findings provide new insights into the biology of complement interactions with P. aeruginosa and other Gram-negative bacteria. Pseudomonas aeruginosa is an opportunistic nosocomial human pathogen that causes a wide range of clinical symptoms and infections in immunocompromised patients. This bacterium is often the causative agent of acute and chronic infections of the airway, sepsis, burn wounds, and skin infections. Excessive infiltration of neutrophils at the site of infection and the presence of complement have been observed in inflamed tissue and blood (1, 2). Serum sensitivity is directly dependent upon recognition of the bacterial surface by antibodies and complement (3). Prior studies also suggested that complement binds to the P. aeruginosa surface and enhances phagocytosis by acting as an opsonin (4). In this study, we sought to understand the consequences of complement interaction with P. aeruginosa. C3 is the central component of the complement system, playing a crucial role in the three major complement component activation processes. C3b, a product of C3 activation, binds covalently to bacterial surfaces through either a reactive intramolecular ester or an amide bond (5). C3b also participates in the formation of C5 convertase that cleaves C5 and initiates the sequential deposition of the remaining complement components to form the C5b-9 membrane attack complex (MAC), which may result in direct killing of microorganisms (6).The human complement system plays an important role in the clearance of early pulmonary P. aeruginosa inf...

Inflammation: A Double-Edged Sword in the Response to <b><i>Pseudomonas aeruginosa</i></b> Infection

“…The antimicrobial activity of these serum proteins is enhanced in the acidic and hypocalcemic conditions characteristic of infected and inflamed tissue [64]. The P. aeruginosa outer membrane porin OprF serves as a binding acceptor molecule for C3b to initiate formation of the membrane attack complex (MAC) [65], but another bacterial protein, dihydrolipoamide dehydrogenase (Lpd), can inhibit MAC deposition and limit complement damage [66]. Of interest, P. aeruginosa can stimulate neutrophils to undergo neutrophil extracellular trap (NET) formation with activation and deposition of the C5b-9 MAC on NETs, potentially contributing to local host cell damage and death [67].…”

Otopathogenic Pseudomonas aeruginosa Enters and Survives Inside Macrophages

“…Since bacterial outer membrane proteins (OMPs) play a crucial role in interaction of pathogens with immune cells (Confer and Ayalew, 2013), we determined the role of P. aeruginosa OprF in bacterial survival inside macrophages. OprF is the major OMP of P. aeruginosa and has been demonstrated to play an important role in interaction of this pathogen with host cells (Nestorovich et al, 2006; Bouffartigues et al, 2012; Reusch, 2012; Mishra et al, 2015). We observed that survival of otopathogenic P. aeruginosa inside macrophages requires bacterial oprF expression.…”

Sensing Mg2+ contributes to the resistance of Pseudomonas aeruginosa to complement‐mediated opsonophagocytosis

“…The ligand blot experiments described here failed to detect binding of C3 to OprF, as described Mishra et al . (). It is possible that the human serum used by these researchers contained specific antibodies against OprF.…”

“…Mishra et al . used 20% normal human serum (Mishra et al ., ), while we used 2 μg/ml of C3 or 0.2% normal human serum. Indeed, in preliminary experiments using 20 μg/ml C3 or 20% normal human serum, we were able to detect OprF as a C3 binding molecule.…”

“…To date, two C3 binding molecules have been identified on the P. aeruginosa surface, LPS (Jensen et al, 1993) and OprF (Mishra et al, 2015). To investigate whether the differences in the binding of C3 between PAO1 grown in low or high Mg 21 concentration were associated with changes in the LPS, the binding of C3 to LPS purified from PAO1 grown in LB or LB supplemented with 3 mM Mg 21 was analysed by enzyme-linked immunosorbent assay (ELISA).…”