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

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia pseudomallei </it>and <it>Burkholderia mallei </it>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 <it>B. pseudomallei </it>and <it>B. mallei </it>adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms.</p> <p>Results</p> <p>Comparative sequence analyses identified a gene product in the published genome of <it>B. mallei </it>strain ATCC23344 (locus # BMAA0649) that resembles the well-characterized <it>Yersinia enterocolitica </it>autotransporter adhesin YadA. The gene encoding this <it>B. mallei </it>protein, designated <it>boaA</it>, was expressed in <it>Escherichia coli </it>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 <it>boaA </it>gene in <it>B. mallei </it>ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the <it>B. pseudomallei </it>strains K96243 and DD503 were also found to contain <it>boaA </it>and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures.</p> <p>A second YadA-like gene product highly similar to BoaA (65% identity) was identified in the published genomic sequence of <it>B. pseudomallei </it>strain K96243 (locus # BPSL1705). The gene specifying this protein, termed <it>boaB</it>, appears to be <it>B. pseudomallei</it>-specific. Quantitative attachment assays demonstrated that recombinant <it>E. coli </it>expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a <it>boaB </it>mutant of <it>B. pseudomallei </it>DD503 showed decreased adherence to these respiratory cells. Additionally, a <it>B. pseudomallei </it>strain lacking expression of both <it>boaA </it>and <it>boaB </it>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.</p> <p>Conclusions</p> <p>The <it>boaA </it>and <it>boaB </it>genes specify adhesins that mediate adherence to epithelial cells of the human respiratory tract. The <it>boaA </it>gene product is shared by <it>B. pseudomallei </it>and <it>B. mallei </it>whereas BoaB appears to be a <it>B. pseudomallei</it>-specific adherence factor.</p

Moraxella catarrhalis Strain O35E Expresses Two Filamentous Hemagglutinin-Like Proteins That Mediate Adherence to Human Epithelial Cells▿

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

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells▿ §

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

Timeline of vaccination experiments.

<p>Timeline of vaccination experiments.</p

Recovery of WT <i>M. catarrhalis</i> O35E from the nasopharynx of immunized chinchillas three days post infection.

<p>Results are expressed as the mean (± std error) CFU/gr of nasopharyngeal tissues (note the log scale). The asterisk indicates that the reduction in the number of bacteria is statistically significant (Wilcoxon signed rank test, <i>P</i> value is shown in parentheses). Control and His-tagged MhaB groups were tested in parallel on three separate occasions. Each column represents 12 animals (groups of n = 4 animals/experiment).</p

Recovery of <i>M. catarrhalis</i> from the nasopharynx of chinchillas three days post-infection.

<p>Animals were inoculated with ∼1×10⁹ CFU. Results are expressed as the mean (± standard error) CFU/gr of nasopharyngeal tissues. Strains were tested in parallel on two separate occasions. Each column represents 12 animals. The asterisk indicates that the reduction in the number of bacteria is statistically significant (Wilcoxon signed rank test).</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Western blot analysis of serum from chinchillas inoculated with the WT <i>M. catarrhalis</i> strain O35E.

<p>Equivalent amounts of whole cell lysates (WT <i>M. catarrhalis</i> O35E, <i>uspA2</i> KO strain O35E.2, <i>hag</i> transposon mutant strain O35E.TN2, and <i>ompCD</i> KO strain O35E.CD1) were resolved by SDS-PAGE, transferred to PVDF and probed with the indicated primary Abs. <u>Panels A and B:</u> Pre- and post-infection serum samples were pooled and used as primary Abs at a dilution of 1∶250. Goat α-rat IgG conjugated to HRP were used as secondary Abs. <u>Controls:</u> The murine monoclonal Abs 10F3 (Panel C, α-CopB), 5D2 (Panel D, α-Hag), 17H4 (Panel E, α-UspA2) and 1D3 (Panel F, α-OMPCD) were used as primary Abs in combination with goat α-mouse HRP-(IgG+IgA+IgM) secondary Abs. These controls were included to verify the identity of proteins recognized by post-infection chinchilla serum in panel B. MW markers are shown to the left of in kDa.</p

Inhibition of adherence with samples from chinchillas immunized with His-tagged MhaB protein.

<p>The WT <i>M. catarrhalis</i> strains O35E, O12E, and McGHS1 were preincubated for 30 min at 37°C with pooled samples from chinchillas sham-vaccinated with PBS (black bars) or with pooled samples from chinchillas immunized with His-tagged MhaB at dilutions of 1∶50, 1∶100, 1∶200 and/or 1∶2000. These bacteria were then used to perform adherence assays. The adherence of <i>M. catarrhalis</i> preincubated with samples from chinchillas vaccinated with PBS was set at 100%. The adherence of <i>M. catarrhalis</i> preincubated with samples from chinchillas immunized with His-tagged MhaB is expressed as the percentage (±standard error) of that of <i>M. catarrhalis</i> preincubated with samples from chinchillas vaccinated with PBS. Assays were performed in triplicate on three separate occasions. The asterisks indicate that the reduction in adherence is statistically significant (<i>P</i> values <0.05, paired <i>t</i> test). Post-boost samples taken on Day 44 of vaccination experiments (see Fig. 3) were pooled and used in these assays.</p