Comparative proteomic analysis of outer membrane protein 43 (omp 43)-deficient Bartonella henselae

Abstract

Outer membrane proteins (OMPs) of Gram-negative bacteria constitute the first line of defense protecting cells against environmental stresses including chemical, biophysical, and biological attacks. Although the 43-kDa OMP (OMP43) is major porin protein among Bartonella henselae-derived OMPs, its function remains unreported. In this study, OMP43-deficient mutant B. henselae (Δomp43) was generated to investigate OMP43 function. Interestingly, Δomp43 exhibited weaker proliferative ability than that of wild-type (WT) B. henselae. To study the differences in proteomic expression between WT and Δomp43, two-dimensional gel electrophoresis-based proteomic analysis was performed. Based on Clusters of Orthologus Groups functional assignments, 12 proteins were associated with metabolism, 7 proteins associated with information storage and processing, and 3 proteins associated with cellular processing and signaling. By semi-quantitative reverse transcriptase polymerase chain reaction, increases in tldD, efp, ntrX, pdhA, purB, and ATPA mRNA expression and decreases in Rho and yfeA mRNA expression were confirmed in Δomp43. In conclusion, this is the first report showing that a loss of OMP43 expression in B. henselae leads to retarded proliferation. Furthermore, our proteomic data provide useful information for the further investigation of mechanisms related to the growth of B. henselae.

Keywords: Bartonella; omp43; outer membrane protein; proliferation; proteomics.

Fig. 1. (A) Coomassie blue stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Bartonella henselae wild-type strain, omp43 deficient mutant strain (Δomp43), and purified outer membrane protein 43 protein (OMP43). M, protein marker; WT, wild-type strain cell lysate. (B) Western blotting analysis of WT and Δomp43.

Fig. 2. Growth curve of Bartonella henselae. Viable cell counts (reported as OD₆₀₀) on blood agar plates were determined at 24 h intervals after plating of individual cultures. OD, optical density; WT, wild-type strain; Δomp43, mutant strain. Data significance was evaluated with a Student's t-test; ^*p < 0.01, ^**p = 0.0562.

Fig. 3. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of Bartonella henselae wild-type strain (WT, left) and mutant strain (Δomp43, right). The pH gradient is indicated at the bottom, and molecular mass standard is indicated to the left. The analyzed target proteins by reverse transcriptase polymerase chain reaction are labeled on the panels. Three independent sets of cultures were analyzed, but only one representative set grown on BAP is shown here. MW, molecular weight.

Fig. 4. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of target gene mRNAs (tldD, efp, ntrX, pdhA, purB, ATPA, Rho, and yfeA) between the wild-type (WT) and mutant (Δomp43) strains. (A) Agarose gel electrophoresis showing representative RT-PCR results. (B) Relative intensities of the PCR bands were quantified by scanning densitometry. Each datum represents the mean ± SD (error bars) obtained from triplicate cultures of a representative experiment out of three performed. Data are expressed as ratios of target mRNA normalized to 16S mRNA. Data significance was evaluated with a Student's t-test; ^*p < 0.05, ^**p < 0.01.