To test this hypothesis, the B. mallei ATCC23344 boaA and B. selleck pseudomallei DD503 boaB genes were cloned into the E. coli strain EPI300. This organism does not normally adhere well to human epithelial cells [61, 62, 66] and
therefore provides an appropriate heterologous genetic background RG7112 in vivo for examining the adhesive properties of BoaA and BoaB. To verify gene expression, RNA was prepared from E. coli harboring the plasmids pCC1.3 (control), pSLboaA (specifies B. mallei ATCC23344 boaA) and pSLboaB (specifies B. pseudomallei DD503 boaB), and analyzed by quantitative Reverse-Transcriptase PCR (qRT-PCR). Fig 3A demonstrates that the boaA and boaB genes are expressed by recombinant bacteria and that the primers used in these experiments are specific for their corresponding genes. Sarkosyl-insoluble OM proteins were also extracted from E. coli cells and analyzed by western blot to ensure production of the Burkholderia proteins. Fig 3B shows that α-BoaA antibodies (Abs) react with a band of 130-kDa in the OM of E. coli expressing boaA (lane 3) whereas SCH727965 chemical structure Abs against BoaB bind to a 140-kDa antigen in E. coli expressing boaB (lane 5). These molecular weights (MWs) are
consistent with the predicted masses of the gene products (Table 1). 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 Sitaxentan (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.