The sensor kinase BfmS controls production of outer membrane vesicles in Acinetobacter baumannii

Abstract

Background: Acinetobacter baumannii is an important opportunistic pathogen responsible for various nosocomial infections. The BfmRS two-component system plays a role in pathogenesis and antimicrobial resistance of A. baumannii via regulation of bacterial envelope structures. This study investigated the role of the sensor kinase, BfmS, in localization of outer membrane protein A (OmpA) in the outer membrane and production of outer membrane vesicles (OMVs) using wild-type A. baumannii ATCC 17978, ΔbfmS mutant, and bfmS-complemented strains.

Results: The ΔbfmS mutant showed hypermucoid phenotype in the culture plates, growth retardation under static culture conditions, and reduced susceptibility to aztreonam and colistin compared to the wild-type strain. The ΔbfmS mutant produced less OmpA in the outer membrane but released more OmpA via OMVs than the wild-type strain, even though expression of ompA and its protein production were not different between the two strains. The ΔbfmS mutant produced 2.35 times more OMV particles and 4.46 times more OMV proteins than the wild-type stain. The ΔbfmS mutant OMVs were more cytotoxic towards A549 cells than wild-type strain OMVs.

Conclusions: The present study demonstrates that BfmS controls production of OMVs in A. baumannii. Moreover, BfmS negatively regulates antimicrobial resistance of A. baumannii and OMV-mediated host cell cytotoxicity. Our results indicate that BfmS negatively controls the pathogenic traits of A. baumannii via cell envelope structures and OMV production.

Keywords: Acinetobacter baumannii; BfmS; Cytotoxicity; OmpA; Outer membrane vesicle.

Fig. 1

Production of OmpA in the outer membrane fraction of transposon-inserted A. baumannii mutant strains. Bacteria were cultured in LB broth for 24 h and proteins (10 μg) in the outer membrane fractions were separated on a 12% SDS-PAGE gel. MW, molecular weight marker; WT, A. baumannii ATCC 17978; #691 and #692 mutant strains, Transposon was inserted in the open reading frame of the A1S_0749 (bfmS) gene. Western blot analysis was performed to identify 38 kDa-OmpA. Protein samples resolved on 12% SDS-PAGE gel were transferred to nitrocellulose membranes and immunoblotted with a polyclonal anti-rabbit OmpA immune sera

Fig. 2

Production of OmpA protein and expression of ompA gene in A. baumannii strains. a SDS-PAGE analysis of bacterial proteins. The bacterial lysates and outer membrane fractions corresponding to 10 μg of protein were separated on a 12% SDS-PAGE gel. Proteins precipitated from the culture supernatants (200 ml) were resuspended in 200 μl of PBS and then 15 μl of the samples were separated on 12% SDS-PAGE gel. Lane MW, molecular weight marker; 1, A. baumannii ATCC 17978; 2, ΔbfmS mutant OH0790; 3, ΔbfmS-complemented OH0883. b Transcription levels of ompA in the three A. baumannii strains were determined using qPCR. The data are mean ± SD expression levels of the target gene in each strain relative to expression of this gene in A. baumannii ATCC 17978. Data were obtained from three independent experiments

Fig. 3

Characteristics of the ΔbfmS mutant strain. a A. baumannii strains were grown in LB broth under shaking or static conditions and then OD₆₀₀ was determined at the indicated times. The data are representative of three experiments with similar results. b A. baumannii strains were cultured overnight on blood agar plates. c Transcription levels of bfmS and bfmR in A. baumannii strains were determined using qPCR. The data are mean ± SD expression levels of the target genes in each strain relative to expression of these genes in A. baumannii ATCC 17978. Data were obtained from three independent experiments

Fig. 4

Biofilm formation, expression of the csuCD genes, and interactions with host cells in A. baumannii strains. a Biofilms formed on 5 ml polystyrene tubes were stained with crystal violet. The amount of crystal violet eluted from the biofilms with ethanol was quantified as the OD₅₇₀ normalized to total bacterial growth (OD₆₀₀). The data are presented as mean ± SD of three independent experiments. ** p < 0.01 compared to wild-type ATCC 17978. b Transcription levels of csuC and csuD in the three A. baumannii strains were determined using qPCR. Data are mean ± SD expression levels of the target genes in each strain relative to expression of these genes in A. baumannii ATCC 17978. The data were obtained from three independent experiments. c Adherence to and invasion of A549 cells by A. baumannii strains. A549 cells were infected with the A. baumannii strains at MOI 100 for 3 h, and then the cell monolayers were lysed with Triton X-100. Dilutions of the bacterial lysates were plated on LB agar, and CFUs were counted. The data are presented as mean ± SD of three independent experiments

Fig. 5

OMV production and its protein profile in A. baumannii strains. (a and b) Production of OMVs from A. baumannii strains. OMVs were isolated from A. baumannii cultured in LB broth. a The size and number of OMV particles isolated from three A. baumannii strains were determined using nanoparticle tracking analysis. The data are representative of three independent experiments with similar results. b The protein concentration of OMVs isolated from 1 L of bacterial culture was measured using a modified BCA assay. The data are presented as mean ± SD of two independent experiments. ** p < 0.01 compared to wild-type ATCC 17978. c SDS-PAGE and western blot analyses of OMV proteins. Protein samples were resolved by SDS-PAGE in 12% gels, transferred to nitrocellulose membranes, and immunoblotted with a polyclonal anti-rabbit OmpA immune sera. Lane MW, molecular weight marker; 1, A. baumannii ATCC 17978; 2, ΔbfmS mutant OH0790; 3, ΔbfmS-complemented OH0883

Fig. 6

Pathogenic effect of OMVs derived from three A. baumannii strains. a Biofilm formation of A. baumannii strains cultured with OMVs from different A. baumannii strains. A. baumannii was inoculated in polystyrene tubes and then OMVs (5 μg/ml) obtained from different A. baumannii strains were added to bacterial culture media. Biofilms formed on polystyrene tubes were stained with crystal violet. The amount of crystal violet eluted from the biofilms with ethanol was quantified as the OD₅₇₀ normalized to total bacterial growth (OD₆₀₀). The data are presented as mean ± SD of three independent experiments. b Cytotoxicity of A549 cells treated with OMVs from A. baumannii strains. Cells were treated with various concentrations of A. baumannii OMVs for 24 h. Cell viability was determined using the MTT assay. Data are presented as mean ± SD of three independent experiments. + p < 0.05 compared to untreated control cells. * p < 0.05 comparing the same concentration of OMVs from A. baumannii ATCC 17978