Recombinant Haemophilus influenzae OmpP2

In Gram-negative bacteria, porins not only contribute to bacterial homeostasis, but also are involved in adherence to and invasion of host cells. Haemophilus parasuis outer membrane protein P2 (OmpP2), a member of the porin family, is an important surface protein involved in serum resistance. To further determine the features of OmpP2, the ability of ompP2 deficient mutant (Delta ompP2) of a H. parasuis SC096 strain to interact with porcine umbilicus veins endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15) was evaluated in this study. The Delta ompP2 mutant exhibited dramatically increased ability to adhere to and invade PUVEC and PK-15 cells. Conversely, pretreatment of cell lines with purified native OmpP2 porins significantly inhibited adhesion and invasion of the SC096 strain to the both host cells. To explain the unexpected phenomenon, a 2-dimensional gel electrophoresis-based proteomics comparison was performed between the wild-type SC096 and Delta ompP2 mutant strains. There were 55 differentially expressed proteins identified from mutant. Among them, three overexpressed proteins of the ADP-L-glycero-D-mannoheptose-6-epimerase RfaD, thymidylate synthase ThyA and putative macrophage infectivity potentiator-related protein Mip were confirmed as molecular targets that modulated adherence and invasion capacities of the Delta ompP2 mutant. Collectively, the OmpP2 of the H. parasuis SC096 strain mediated the adherence to and invasion of cells and loss of OmpP2 expression resulted in promoted cell-adherence and invasion properties which were due to the overexpression of RfaD, ThyA and Mip. (C) 2012 Elsevier B.V. All rights reserved.

Haemophilus influenzae has an absolute requirement for NAD ( factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with K-S values of similar to8 and 4mM, respectively, in 0.1 M KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.