Characterization of BcaA, a putative classical autotransporter protein in Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3' to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

Fig 1

bcaA and bcaB appear to form an operon. (A) Genetic organization of the bcaA and bcaB locus and description of the strains used in this study. A visual representation of the primer pairs used for RT-PCR is shown. (B) The putative domains of BcaA and BcaB proteins drawn to scale. (C) RT-PCR analysis of the operon structure of bcaA and bcaB. gDNA, genomic DNA. Primer pairs 1 and 2, 3 and 4, and 5 and 6 were used as indicated in panel A.

Fig 2

Immunoblot of whole-cell lysates of strain Bp340 with no tag and the Bp340::pCCS12HA1 strain. Strains were separated by SDS-PAGE and stained with anti-HA antibody.

Fig 3

Average numbers of plaques formed in an A549 monolayer by Bp340, the Bp340ΔbcaA strain (A), the Bp340ΔbcaB strain (B), and the respective complementation strains. Assays were performed three times in duplicate, and the results were combined. Data are means ± standard errors of the means (SEM). **, P < 0.0080; ***, P < 0.0001 (by one-way ANOVA with Tukey's posttest at a 95% confidence interval).

Fig 4

CFU recovered after 2-h adherence and invasion assays with Bp340, the Bp340ΔbcaA strain (A), the Bp340ΔbcaB strain (B), and the respective complementation strains. Assays were performed three times in duplicate, and the results were combined. Data are means ± SEM. ***, P < 0.0001 (by one-way ANOVA with Tukey's posttest at a 95% confidence interval).

Fig 5

Six- to 8-week-old female BALB/c mice were inoculated with 500 CFU of B. pseudomallei Bp340 (white circles), the Bp340ΔbcaA strain (A), the Bp340ΔbcaB strain (B) (light gray circles), and the respective complementation strains (dark gray circles). Each circle represents the number of CFU recovered from a mouse. The horizontal lines represent the average numbers of CFU. The dashed line represents the lower limit of detection. ***, P < 0.0001. Animal experiments were performed twice for each strain, and the results were combined.

Fig 6

Histological analysis of B. pseudomallei-infected tissues. Representative tissue samples from BALB/c mice infected i.n. with Bp340 or the Bp340ΔbcaA and Bp340ΔbcaA::attTn7bcaA strains (∼500 CFU) or from mock (PBS)-infected control animals are shown.