Eric R. Lafontaine

The Moraxella catarrhalis Porin-Like Outer Membrane Protein CD Is an Adhesin for Human Lung Cells

ABSTRACT The outer membrane protein CD (OMPCD) of Moraxella catarrhalis is an outer membrane protein with several attributes of a potential vaccine antigen. We isolated four transposon mutants of strain O35E on the basis of their reduced binding to A549 human lung cells in microcolony formation assays, and we determined that they contain a transposon in ompCD. We also found that these transposon insertions had pleiotropic effects: mutants grew slower, became serum sensitive, bound ∼10-fold less to A549 cells, and appeared transparent when grown on solid medium. We confirmed that these various phenotypes could be attributed solely to disruption of ompCD by constructing the isogenic strain O35E.CD1. O35E-ompCD was cloned, and recombinant Escherichia coli bacteria expressing the gene product exhibited a 10-fold increase in adherence to A549 cells. This is the first report of M. catarrhalis ompCD mutants, and our findings demonstrate that this gene product is an adhesin for human lung cells.

The Hag Protein of Moraxella catarrhalis Strain O35E Is Associated with Adherence to Human Lung and Middle Ear Cells

ABSTRACT Previous studies have demonstrated that the Moraxella catarrhalis surface antigen UspA1 is an adhesin for Chang human conjunctival cells. The present report demonstrates that lack of UspA1 expression does not affect the adherence of strain O35E to A549 human lung cells or primary cultures of human middle ear epithelial (HMEE) cells. These results imply that another molecule mediates the adherence of M. catarrhalis to these two cell lines. To identify this adhesin, strain O35E was mutagenized with a transposon and 1,000 mutants were screened in a microcolony formation assay using A549 cells. Nine independent isolates exhibited an 8- to 19-fold reduction in adherence and contained a transposon in the same locus. Nucleotide sequence data and PCR analysis indicated that the transposons were inserted in different locations in the gene encoding the surface protein Hag. Quantitative assays using one representative transposon mutant, O35E.TN2, showed considerably decreased binding to A549 as well as HMEE cells. However, this mutant adhered at wild-type levels to Chang conjunctival cells. These findings suggest that the M. catarrhalis Hag protein is an adhesin for cell lines derived from human lung and middle ear tissues.

Moraxella catarrhalis strain O35E expresses two filamentous hemagglutinin-like proteins that mediate adherence to human epithelial cells.

ABSTRACT Two-partner secretion (TPS) systems are a family of proteins being rapidly identified and characterized in a growing number of gram-negative bacteria. TPS systems mediate the secretion of proteins, many involved in virulence traits such as hemolysis, adherence to epithelial cells, inhibition of bacterial growth, and immunomodulation of the host. A TPS system typically consists of a transporter located in the bacterial outer membrane (OM) which is responsible for the recognition and secretion of at least one large exoprotein. Two of the better-characterized TPS systems specify the Bordetella pertussis FHA and Haemophilus influenzae HMW1/HMW2 proteins. We identified three gene products of Moraxella catarrhalis strain O35E that resemble TPS proteins and designated them MhaC (transporter), MhaB1 (exoprotein), and MhaB2 (exoprotein). Western blot analysis using anti-MhaC, or antibodies reacting to both MhaB1 and MhaB2 (MhaB-reactive), revealed that these antigens are expressed in the OM of 63% of isolates tested. Mutations in the mhaC gene specifying the putative transporter of the M. catarrhalis wild-type strains O35E, O12E, and McGHS1 resulted in the absence of MhaB1/MhaB2 in the OM of mutants. These results are therefore consistent with the Mha proteins functioning as a TPS system. Furthermore, we discovered that these mhaC mutants exhibit markedly decreased binding to human epithelial cells relevant to pathogenesis by M. catarrhalis (Chang, HEp2, A549, and/or 16HBE14o−). Expression of O12E MhaC and MhaB1 in a nonadherent strain of Escherichia coli was found to increase the adherence of recombinant bacteria to HEp2 monolayers by sevenfold, thereby demonstrating that this M. catarrhalis TPS system directly mediates binding to human epithelial cells. The construction of isogenic mutants in the mhaB1 and mhaB2 genes of strain O35E also suggests that the MhaB proteins play distinct roles in M. catarrhalis adherence.

The Moraxella catarrhalis Autotransporter McaP Is a Conserved Surface Protein That Mediates Adherence to Human Epithelial Cells through Its N-Terminal Passenger Domain

ABSTRACT The protein McaP was previously shown to be an adhesin expressed by the Moraxella catarrhalis strain O35E, which also displays esterase and phospholipase B activities (J. M. Timpe et al., Infect. Immun. 71:4341-4350, 2003). In the present study, sequence analysis suggests that McaP is a conventional autotransporter protein that contains a 12-stranded β-barrel transporter module (amino acids [aa] 383 to 650) linked to a surface-exposed passenger domain exhibiting lipolytic activity (aa 62 to 330). An in-frame deletion removing most of this predicted N-terminal passenger domain was engineered, and Escherichia coli expressing the truncated McaP protein exhibited greatly reduced adherence to A549 human lung epithelial cells compared to E. coli expressing wild-type McaP. Site-directed mutagenesis of a serine residue at position 62 of McaP, predicted to be important for the lipolytic activity of the protein, resulted in loss of hydrolysis of p-nitrophenyl ester of caproate. E. coli expressing this mutated McaP, however, adhered to A549 monolayers at levels greater than recombinant bacteria expressing the wild-type adhesin. These results indicate that the predicted passenger domain of McaP is involved in both the binding and the lipolytic activity of the molecule and demonstrate that the adhesive properties of McaP do not require its lipolytic activity. Sequence analysis of mcaP from eight Moraxella catarrhalis strains revealed that the gene product is highly conserved at the amino acid level (98 to 100% identity), and Western blot analysis demonstrated that a panel of 16 isolates all express McaP. Flow cytometry experiments using antibodies raised against various portions of McaP indicated that its predicted passenger domain as well as transporter module contain surface-exposed epitopes. In addition to binding to the surface of intact bacteria, these antibodies were found to decrease adherence of M. catarrhalis to A549 human lung cells by up to 47% and to reduce binding of recombinant E. coli expressing McaP by 98%. These results suggest that McaP should be considered as a potential vaccine antigen.

Hag directly mediates the adherence of Moraxella catarrhalis to human middle ear cells.

ABSTRACT Moraxella catarrhalis is a human pathogen that causes otitis media in young children and lung infections in patients with chronic obstructive pulmonary disease. In this study, the role of the surface protein Hag in the adherence of multiple M. catarrhalis strains was examined. The hag genes of four clinical isolates were disrupted with a spectinomycin resistance cassette, and the binding of isogenic mutants to primary cultures of human middle ear epithelial cells (HMEE), as well as A549 pneumocytes, was measured. These experiments revealed that the attachment of most mutants to both cell types was 10-fold less than that of their wild-type progenitors. To determine whether Hag directly mediates adherence to human cells, the hag genes from three M. catarrhalis isolates were cloned and expressed in a nonadherent Escherichia coli cloning strain. At least 17-fold more E. coli bacteria expressing Hag attached to HMEE cells than an adherence-negative control. Surprisingly, Hag expression did not increase the binding of recombinant E. coli to A549 monolayers. Our data demonstrate that the involvement of Hag in M. catarrhalis adherence to A549 and HMEE cells is conserved among isolates and that Hag directly mediates binding to HMEE cells.

Regions important for the adhesin activity of Moraxella catarrhalis Hag

BackgroundThe Moraxella catarrhalis Hag protein, an Oca autotransporter adhesin, has previously been shown to be important for adherence of this respiratory tract pathogen to human middle ear and A549 lung cells.ResultsThe present study demonstrates that adherence of M. catarrhalis isogenic hag mutant strains to the human epithelial cell lines Chang (conjunctival) and NCIH292 (lung) is reduced by 50–93%. Furthermore, expressing Hag in a heterologous Escherichia coli background substantially increased the adherence of recombinant bacteria to NCIH292 cells and murine type IV collagen. Hag did not, however, increase the attachment of E. coli to Chang cells. These results indicate that Hag directly mediates adherence to NCIH292 lung cells and collagen, but is not sufficient to confer binding to conjunctival monolayers. Several in-frame deletions were engineered within the hag gene of M. catarrhalis strain O35E and the resulting proteins were tested for their ability to mediate binding to NCIH292 monolayers, middle ear cells, and type IV collagen. These experiments revealed that epithelial cell and collagen binding properties are separable, and that residues 385–705 of this ~2,000 amino acid protein are important for adherence to middle ear and NCIH292 cells. The region of O35E-Hag encompassing aa 706 to 1194 was also found to be required for adherence to collagen. In contrast, β-roll repeats present in Hag, which are structural features conserved in several Oca adhesins and responsible for the adhesive properties of Yersinia enterocolitica YadA, are not important for Hag-mediated adherence.ConclusionHag is a major adherence factor for human cells derived from various anatomical sites relevant to pathogenesis by M. catarrhalis and its structure-function relationships differ from those of other, closely-related autotransporter proteins.The Moraxella catarrhalis Hag protein, an Oca autotransporter adhesin, has previously been shown to be important for adherence of this respiratory tract pathogen to human middle ear and A549 lung cells. The present study demonstrates that adherence of M. catarrhalis isogenic hag mutant strains to the human epithelial cell lines Chang (conjunctival) and NCIH292 (lung) is reduced by 50–93%. Furthermore, expressing Hag in a heterologous Escherichia coli background substantially increased the adherence of recombinant bacteria to NCIH292 cells and murine type IV collagen. Hag did not, however, increase the attachment of E. coli to Chang cells. These results indicate that Hag directly mediates adherence to NCIH292 lung cells and collagen, but is not sufficient to confer binding to conjunctival monolayers. Several in-frame deletions were engineered within the hag gene of M. catarrhalis strain O35E and the resulting proteins were tested for their ability to mediate binding to NCIH292 monolayers, middle ear cells, and type IV collagen. These experiments revealed that epithelial cell and collagen binding properties are separable, and that residues 385–705 of this ~2,000 amino acid protein are important for adherence to middle ear and NCIH292 cells. The region of O35E-Hag encompassing aa 706 to 1194 was also found to be required for adherence to collagen. In contrast, β-roll repeats present in Hag, which are structural features conserved in several Oca adhesins and responsible for the adhesive properties of Yersinia enterocolitica YadA, are not important for Hag-mediated adherence. Hag is a major adherence factor for human cells derived from various anatomical sites relevant to pathogenesis by M. catarrhalis and its structure-function relationships differ from those of other, closely-related autotransporter proteins.

Moraxella catarrhalis Coaggregates with Streptococcus pyogenes and Modulates Interactions of S. pyogenes with Human Epithelial Cells

ABSTRACT The pathogens Streptococcus pyogenes and Moraxella catarrhalis colonize overlapping regions of the human nasopharynx. We have found that M. catarrhalis can dramatically increase S. pyogenes adherence to human epithelial cells and that species-specific coaggregation of these bacteria correlates with this enhanced adherence.

Hag mediates adherence of Moraxella catarrhalis to ciliated human airway cells.

ABSTRACT Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.

Identification of Burkholderia mallei and Burkholderia pseudomallei adhesins for human respiratory epithelial cells

BackgroundBurkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms.ResultsComparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649) that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells) and A549 (type II pneumocytes), as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures.A second YadA-like gene product highly similar to BoaA (65% identity) was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705). The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to thrive inside J774A.1 murine macrophages, suggesting a possible role for these proteins in survival within professional phagocytic cells.ConclusionsThe boaA and boaB genes specify adhesins that mediate adherence to epithelial cells of the human respiratory tract. The boaA gene product is shared by B. pseudomallei and B. mallei whereas BoaB appears to be a B. pseudomallei-specific adherence factor.

Identification of domains of the Hag/MID surface protein recognized by systemic and mucosal antibodies in adults with chronic obstructive pulmonary disease following clearance of Moraxella catarrhalis.

ABSTRACT Moraxella catarrhalis is a common cause of respiratory tract infection in the setting of chronic obstructive pulmonary disease (COPD). Adults with COPD acquire and clear strains of M. catarrhalis from the respiratory tract continuously and develop strain-specific protection following clearance of a strain. In previous work, we identified Hag/MID (Moraxella immunoglobulin D-binding protein), a large multifunctional surface protein that acts as an adhesin and hemagglutinin, as a target of antibody responses in adults with COPD after clearance of M. catarrhalis. The goal of the present study was to characterize the domains of Hag/MID to which humans make antibodies, including both systemic and mucosal antibody responses. Analysis of recombinant peptide constructs, which spanned the M. catarrhalis strain O35E Hag/MID protein, with well-characterized serum and sputum samples revealed that most adults with COPD made antibodies directed toward a region of the molecule bounded by amino acids 706 to 863. Serum immunoglobulin G (IgG) and IgA purified from sputum both recognized the same domain. Some flanking sequence of this fragment was necessary for the epitope(s) in this region to maintain its conformation to bind human antibodies. These results reveal that humans consistently generate both systemic and mucosal antibody responses to an immunodominant region of the Hag/MID molecule, which was previously shown to overlap with several biologically relevant domains, including epithelial cell adherence, IgD binding, collagen binding, and hemagglutination.