Characterization of the porin proteins OmpP2A and OmpP2B of Haemophilus ducreyi
Davie, Jeremiah J.
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Haemophilus ducreyi is an obligate human pathogen and the causative agent of the sexually transmitted, genital ulcerative disease chancroid. 35000HP, the only H. ducreyi strain to a have completely sequenced genome, contains only two known porin proteins, OmpP2A and OmpP2B. While these proteins have been described to function as porins in black lipid bilayer assays, the specificities and properties of these proteins have not been characterized. Sequence conservation and protein expression studies were performed with clinical isolates from both known clonal lineages of H. ducreyi. Sequence analysis identified a series of conserved nucleotide deletions resulting in frame-shift mutations present in the non-expressed porin genes of a selected subset of strains that resulted in pseudogene formation. In 35000HP, loss of OmpP2A and OmpP2B expression in the mutant 35000HP::P2AB resulted in no obvious growth defect or phenotype. A proteomics-based comparison resulted in the identification of 231 proteins present in membrane-associated protein samples, of which a subset of 56 proteins were differentially expressed at a level of 1.5-fold or greater in the porin-deficient strain 35000HP::P2AB relative to 35000HP. Proteins identified in these studies suggested a decreased membrane stability phenotype, which was verified by disk diffusion assay. Additionally, growth comparisons of the single and double-porin deficient mutants to that of 35000HP in media of altered osmotic conditions indicate that the differential expression of OmpP2A and OmpP2B is required for optimal growth. Also, Phenotype Microarray screens of 190 carbon sources indicate that OmpP2A and OmpP2B are required for the optimal acquisition of hexose sugars, nucleoside sugars and organic acids. Furthermore, when grown in nutrient limiting conditions, the expression of either porin is sufficient for optimal growth but the loss of both porins results in a pronounced growth defect that can be ablated by supplementation with exogenous nutrients. OmpP2A and OmpP2B appear to be classical, non-specific porins whose expression is required for optimal adaptation to environmental stress. Overall, these studies have enhanced our knowledge regarding the basic biology, stress response systems, metabolic activity range and membrane permeability properties of H. ducreyi while simultaneously ascribing a specific function for OmpP2A and OmpP2B and characterizing a global compensatory response to their disruption.