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

Abstract

Background: Burkholderia 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.

Results: Comparative 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.

Conclusions: The 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.

Figure 1

Structural features of the boaA and boaB gene products. Different regions of the predicted B. mallei ATCC23344 BoaA (A), B. pseudomallei K96243 BoaA (B) and B. pseudomallei K96243 BoaB (C) proteins are depicted with the positions of residues defining selected domains. The horizontal brackets outline selected regions of the BoaA and BoaB proteins and the percent identity between these regions is shown below the brackets. Transporter modules (OM anchors) and helical linkers were identified using the PSIPRED secondary structure prediction algorithm. The colored boxes show the relative position and number of repeated SLST motifs.

Figure 2

Sequence comparison of boaA and boaB gene products. The last 93 residues of selected boaA and boaB gene products are shown with the positions of the aa defining these regions in parentheses. Perfectly conserved aa are shown in black text over white background. Residues unique to BoaA proteins are shown in blue text over a yellow background. Residues unique to BoaB proteins are shown in white text over a blue background. Bm = B. mallei, Bp = B. pseudomallei.

Figure 3

Analysis of recombinant E. coli strains. Panel A: Total RNA was isolated from E. coli strains, reverse-transcribed to cDNA, and the relative levels of boaA or boaB transcript were determined by qRT-PCR. Each bar represents 4 different samples collected on 2 separate occasions. The Y-axis corresponds to the levels of boaA or boaB transcript normalized to recA and the error bars correspond to the standard error. Negative controls in which the reverse transcriptase enzyme was not added to reaction mixtures were included in all experiments (data not shown). Panel B: Proteins present in Sarkosyl-insoluble OM protein preparations were resolved by SDS-PAGE, transferred to PVDF membranes and analyzed by western blot with antibodies against BoaA (lanes 1-3) or BoaB (lanes 4-6). Lanes 1 & 4, E. coli (pCC1.3); lanes 2 & 5, E. coli (pSLboaB); lanes 3 & 6, E. coli (pSLboaA). MW markers are shown to the left in kilodaltons. Panel C: Non-permeabilized E. coli strains were fixed onto glass slides and fluorescently-labeled with DAPI (blue) and with α-BoaA or α-BoaB antibodies (red). Bacteria were visualized by microscopy using a Zeiss LSM 510 Meta confocal system. Representative microscopic fields are shown. Panel D: E. coli strains were incubated with A549 and HEp2 cells for 3-hr and with NHBE cultures for 6-hr. Epithelial cells were washed to remove unbound bacteria, lysed, diluted, and spread onto agar plates to enumerate bound bacteria. The results are expressed as the mean percentage (± standard error) of inoculated bacteria adhering to epithelial cells. Asterisks indicate that the increased adherence of the indicated strains, compared to E. coli carrying the control plasmid pCC1.3, is statistically significant (P < 0.05). These attachment assays were performed in duplicate on at least 3 separate occasions.

Figure 4

Quantitative reverse-transcriptase PCR analysis of B. mallei and B. pseudomallei strains. Total RNA was isolated from B. pseudomallei (Bp) DD503 and B. mallei (Bm) ATCC23344, reverse-transcribed to cDNA, and the relative levels of boaA or boaB transcript was determined by qRT-PCR. Each bar represents 4 different samples collected on 2 separate occasions. The Y-axis corresponds to levels of boaA or boaB transcript normalized to recA and the error bars correspond to the standard error. A primer set for Borrelia burgdorferi recA was used as a control to further demonstrate primer specificity (see bars labeled as control). Of note, negative controls in which the reverse transcriptase enzyme was not added to reaction mixtures were included in all experiments and the results were equivalent to the Borrelia burgdorferi controls (data not shown).

Figure 5

Adherence of B. mallei and B. pseudomallei strains to human respiratory epithelial cells. The effects of boaA and boaB mutations on the adherence of B. pseudomallei (Bp) DD503 and B. mallei (Bm) ATCC23344 to monolayers of A549 (panels A and D) and HEp2 (panels B and E) cells and cultures of NHBE (panels C and F) was measured in duplicate on at least 3 separate occasions. The results are expressed as the mean percentage (± standard error) of inoculated bacteria adhering to epithelial cells. Asterisks indicate that the difference between the adherence of the mutant and that of the parental strain is statistically significant (P < 0.05).

Figure 6

Uptake and growth of B. pseudomallei strains in J774A.1 murine macrophages. J774A.1 cells (duplicate wells in each of two 24-well tissue culture plates) were infected with B. pseudomallei strains at an MOI of 10 and incubated for 1-hr to allow phagocytosis of the organisms. Following incubation, the monolayers were incubated for 2-hr in medium containing gentamicin to kill extracellular bacteria. After gentamicin treatment (i.e. 3-hr post infection), the wells of one plate were washed, lysed, serially diluted, and spread onto agar plates to determine the number of bacteria phagocytosed by macrophages. The results of this first part of the experiments (i.e. bacterial uptake) are shown in panel A and are expressed as the percentage of bacteria (± standard error) used to infect macrophages that were phagocytosed. The wells of the other tissue culture plate inoculated with B. pseudomallei strains were washed once, fresh medium without antibiotics was added to wells, and the plate was incubated for an additional 5-hr. Following this incubation (i.e. 8-hr post-infection), the wells were processed as described above in order to enumerate bacterial numbers. The results of this second part of the experiments (i.e. intracellular growth of phagocytosed bacteria) are shown in panel B and are expressed as a growth/uptake ratio (± standard error) obtained by dividing the number of bacteria/well at 8-hr post infection by the number of bacteria/well at the 3-hr post infection time point. These experiments were repeated on at least 3 separate occasions. The asterisk indicates that the difference between the intracellular growth of the double mutant strain DD503.boaA.boaB and that of its parent isolate DD503 is statistically significant (P < 0.05). Panel C shows the total number of bacteria in the inoculum (grey bars), the number of phagocytosed bacteria (open bars, 3-hr post infection) and the total number of bacteria/well at the end point of the experiment (black bars, 8-hr post infection).