Identification of a Moraxella catarrhalis outer membrane protein exhibiting both adhesin and lipolytic activities

Abstract

The UspA1 and Hag proteins have previously been shown to be involved in the ability of the Moraxella catarrhalis wild-type strain O35E to bind to human Chang and A549 cells, respectively. In an effort to identify novel adhesins, we generated a plasmid library of M. catarrhalis DNA fragments, which was introduced into a nonadherent Escherichia coli strain. Recombinant E. coli bacteria were subsequently enriched for clones that gained the ability to bind to Chang and A549 cells, yielding the plasmid pELFOS190. Transposon mutagenesis of this plasmid identified the potential adhesin gene mcaP (M. catarrhalis adherence protein). Sequence analysis revealed that McaP is related to autotransporter proteins and has substantial similarity with the GDSL family of lipolytic enzymes, particularly the Moraxella bovis phospholipase B. Expression of the mcaP gene product by E. coli increased adherence to Chang, A549, and 16HBE14o(-) polarized human bronchial cells 50- to 100-fold. Spectrophotometric assays with p-nitrophenol derivatives also demonstrated that McaP is an esterase. Furthermore, thin-layer chromatography revealed that McaP cleaves both phosphatidylcholine and lysophosphatidylcholine. McaP releases fatty acids and glycerophosphorylcholine upon cleavage of phosphatidylcholine, thus exhibiting phospholipase B activity. The construction and characterization of isogenic M. catarrhalis O35E mutants demonstrated that the lack of McaP expression abolishes esterase activity and considerably decreases adherence to several human cell lines.

FIG. 1.

Binding of E. coli recombinant cells to Chang monolayers in a visual adherence assay.

FIG. 2.

SDS-PAGE analysis of M. catarrhalis strains and E. coli recombinant cells. (A) Whole-cell lysates prepared from E. coli TOP10 recombinant cells harboring the plasmid pBR322 (lane 1) or pJTmcaP (lane 2), resolved by SDS-PAGE, and stained with Coomassie blue. (B) Ten microliters of heat extract prepared from E. coli cells harboring pBR322 (lane 1) or pJTmcaP (lane 2). (C) Proteins present in outer membrane vesicles that were prepared from strain O35E (lane 1) and its isogenic mcaP mutant O35E.M (lane 2). The arrowheads indicate the position at which the mcaP gene product migrates. Positions of molecular mass markers are shown to the left in kilodaltons.

FIG. 3.

Lipolytic activities of E. coli recombinant cells (A) and M. catarrhalis strains (B). Lipid substrates composed of 4 (C₄)- to 16 (C₁₆)-carbon chains are described in Materials and Methods. Units = [(OD₄₁₀ at 15 min − OD₄₁₀ at 0 min)/OD₅₈₀]/15 min, where OD₄₁₀ is the optical density at 410 nm.

FIG. 4.

Thin-layer chromatography analysis of McaP phospholipase activity. (A) PC substrate. Lanes 1, unprocessed fatty acids (FA) and DAG. Lane 2, unprocessed PC. Lanes 3 and 4, digestion products of PC incubated with pBR322 or pJTmcaP extracts, respectively. Lanes 5 to 8, unprocessed LPC, GPC, DAG, and phosphatidic acid (PA), respectively. Lanes 5 to 8 also represent the digestion products of PLA, PLB, PLC, and PLD, respectively (as indicated by the brackets). (B) LPC substrate. Lane 1, unprocessed LPC. Lanes 2 and 3, digestion products of LPC incubated with pBR322 or pJTmcaP extracts, respectively. Lanes 4 and 5, products of LPC digestion with commercial PLA₂ or PLB. Arrowheads indicate the locations of GPC bands (A) and LPC bands (B). Arrows denote the chloroform-methanol-water solvent front.

FIG. 5.

Adherence of M. catarrhalis strains to human cell lines in vitro. The adherence of the M. catarrhalis mutants O35E.M, O35E.ZCS, and O35E.ZCSM is expressed as the percentage (± standard error) of that of the parent strain O35E. The asterisk indicates that the decrease in O35E.ZCSM adherence, compared to the adherence of O35E.ZCS, is statistically significant. Expression (+) or lack thereof (−) of selected proteins is indicated.