Role of OmpA1 and OmpA2 in Aggregatibacter actinomycetemcomitans and Aggregatibacter aphrophilus serum resistance

Abstract

Aggregatibacter actinomycetemcomitans and Aggregatibacter aphrophilus belong to the HACEK group of fastidious Gram-negative organisms, a recognized cause of infective endocarditis. A. actinomycetemcomitans is also implicated in aggressive forms of periodontitis. We demonstrated that A. aphrophilus strains, as A. actinomycetemcomitans are ubiquitously serum resistant. Both species encode two Outer membrane protein A paralogues, here denoted OmpA1 and OmpA2. As their respective pangenomes contain several OmpA1 and OmpA2 alleles, they represent potential genotypic markers. A naturally competent strain of A. actinomycetemcomitans and A. aphrophilus, respectively were used to elucidate if OmpA1 and OmpA2 contribute to serum resistance. Whereas OmpA1 was critical for survival of A. actinomycetemcomitans D7SS in 50% normal human serum (NHS), serum resistant ompA1 mutants were fortuitously obtained, expressing enhanced levels of OmpA2. Similarly, OmpA1 rather than OmpA2 was a major contributor to serum resistance of A. aphrophilus HK83. Far-Western blot revealed that OmpA1AA, OmpA2AA, and OmpA1AP can bind to C4-binding protein, an inhibitor of classical and mannose-binding lectin (MBL) complement activation. Indeed, ompA1 mutants were susceptible to these pathways, but also to alternative complement activation. This may at least partly reflect a compromised outer membrane integrity but is also consistent with alternative mechanisms involved in OmpA-mediated serum resistance.

Outer membrane vesicle-mediated serum protection inAggregatibacter actinomycetemcomitans

“…Whether A. actinomycetemcomitans OMVs may also be involved in serum resistance is not known. A functional role of A. actinomycetemcomitans OMVs in complement interaction would be consistent with in vitro observations that the vesicles, in an OmpA1-and OmpA2-dependent manner, respectively, could bind to C4-binding protein [21], a major inhibitor of classical and MBL complement activation [34]. Lipopolysaccharide (LPS) is one of the most abundant components of OMVs, including those released by A. actinomycetemcomitans, and is displayed on the outer surface of the vesicles [23][24][25].…”

“…An A. actinomycetemcomitans ompA1 ompA2 double mutant strain, i.e. D7SS ompA1::spe, ompA2::kan [Spe r , Km r ] [21] was used as a serum-sensitive test strain. SA3138 is a serotype a A. actinomycetemcomitans wildtype strain [40].…”

“…The outer membrane protein, Omp100 (also referred to as ApiA) was previously shown to be required for full serum resistance of some serotype b, and d A. actinomycetemcomitans strains, and to physically interact with the alternative complement pathway negative regulator, Factor H in vitro [17,20]. We recently demonstrated that the major outer membrane protein, OmpA1 was critical for serum survival of the A. actinomycetemcomitans serotype a model strain, D7SS [21]. Interestingly, serum resistant ompA1 mutants were fortuitously obtained, which expressed increased levels of the paralogue, OmpA2.…”

“…Interestingly, serum resistant ompA1 mutants were fortuitously obtained, which expressed increased levels of the paralogue, OmpA2. Thus, OmpA2, which normally seems to be expressed at low levels in vitro, could apparently operate as a functional homologue to OmpA1 in A. actinomycetemcomitans serum resistance [21]. As several complement activation pathways, i.e., classical, alternative, and mannose-binding lectin (MBL) complement activation were needed to completely eliminate serum-sensitive ompA1 ompA2 double mutant A. actinomycetemcomitans derivatives [21], it is plausible that serum resistance in this species, similar to in Acinetobacter baumanii [22], is highly complex, and depend on multiple gene products.…”

“…A. actinomycetemcomitans OMVs have been shown to deliver virulence factors, such as leukotoxin, and cytolethal distending toxin (CDT) to human cells [25,26], and to internalize into the host cells to act as a trigger of innate immunity [27]. Proteomics, and Western blot analysis of A. actinomycetemcomitans OMVs have identified several additional vesicle-associated proteins that can contribute to evasion of the immune defense, including the IL1β-binding lipoprotein, BilRI, Omp100, OmpA1, and OmpA2, and a Factor H-binding protein homologue [21,28,29]. A role of OMVs in contributing to bacterial serum resistance has been demonstrated in a number of bacterial species, including Moraxella catarrhalis, Neisseria gonorrhoeae, Porphyromonas gingivalis, and Vibrio cholerae [30][31][32][33].…”