Overexpression of outer membrane protein A (OmpA) increases aminoglycoside sensitivity in mycobacteria

Abstract

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex infection, is a leading cause of death worldwide from a single infectious agent. The emergence of drug resistance Mtb clinical strains makes the situation more serious. The role of Mtb outer membrane protein A (OmpA) in antimicrobial resistance remains unclear. This study aimed to evaluate the effect of OmpA expression on mycobacterial drug resistance. In this study, a Mycobacterium smegmatis (Ms) strain overexpressing OmpA (Ms-OmpA) and a Mycobacterium bovis (Mb) strain overexpressing OmpA (Mb-OmpA) were constructed, and their susceptibility to anti-TB drugs was determined by performing the minimal inhibitory concentrations (MICs), the plate assay and the macrophage infection assays.

Results: The streptomycin MIC of the overexpressing strain was 2-fold lower than those of the wide-type (Ms) and empty plasmid strains (pMV-261) as well as amikacin and gentamicin. Moreover, both the plate and the macrophage infection assays indicate that overexpression of OmpA increases streptomycin sensitivity in Mycobacteria. The other aminoglycosides like amikacin and gentamicin have the same phenotypes as streptomycin on the plates for the virulent strain Mb-OmpA. The porin inhibitor spermidine can increase streptomycin tolerance in the overexpressing strain, and overexpressing OmpA can increase the intracellular accumulation of hydrophilic ethidium bromide, which indicates that porin protein OmpA contributes to aminoglycosides sensitivity in Mycobacteria.

Conclusions: In this study, we have characterized the contribution of OmpA in the antimicrobial resistance phenotype of Mycobacteria, which may provide valuable insights for understanding antibiotic resistance and designing new strategies for TB treatment.

Keywords: Mycobacterium bovis; Mycobacterium smegmatis; Mycobacterium tuberculosis complex; Aminoglycosides; Drug resistance; OmpA; Streptomycin.

Fig. 1

Over-expression of OmpA in Mycobacteria. (A) Identification of OmpA gene in Ms by PCR. (1) DL2000DNA Marker. (2) PCR products of Ms-pMV261. (3) PCR products of Ms-OmpA. (B) Identification of OmpA overexpression in Ms. (1) Ms (Wild type). (2) The Ms-OmpA whole bacteria. (3) Ms-OmpA lysates. (4) The pellets of Ms-OmpA. The OmpA immunized mouse serum was used as the primary antibody for detection. (C) OmpA alignments to other reference strains, Acinetobacter baumannii(Abi), Aggregatibacter actinomycetemcomitans(Aas), Burkholderia ambifaria(Baa), Cronobacter sakazakii(Csi), Haemophilus influenza(Hie), Histophilus somni 2336(Hsi), Klebsiella pneumonia(Kpe), Mannheimia haemolytica(Mha), Pasteurella multocida(Pma), Riemerella anatipestifer(Rar), Pseudomonas aeruginosa PA96(Paa), Salmonella enterica serovar Typhimurium(Sest), Yersinia pestis biovar Microtus str. 91,001(Ypbm), Escherichia coli str. K-12 substr. MG1655(E.coli), Chlamydia pneumoniae TW-183 (Cpe), Mycobacterium tuberculosis(Mtb), Mycobacterium bovis(Mb), and a phylogenetic tree of 10 species. (D) The growth kinetics of Ms, Ms-OmpA, Ms-pMV261

Fig. 2

Over-expression of OmpA increase aminoglycoside sensitivity in Mycobacteria. (A ∼ B) Overexpression of OmpA can increase mycobacteria to 0.5, 0.75 µg/ml streptomycin and 0.5 µg/ml amikacin. (C) Measurement of growth curve for wild type, vector pMV261, and over-expression strain Ms-OmpA with 0.5 µg/ml streptomycin. (D) Macrophages were infected by the strains and treated with 0.5 µg/ml streptomycin for 6 h and 12 h, respectively. The survival rate was calculated by CFU counts on the plates. (E) Over-expression of OmpA enhanced the Mb-OmpA susceptibility to 0.2 µg/ml streptomycin, 0.2 µg/ml amikacin, 0.8 µg/ml gentamicin

Fig. 3

OmpA works as a porin protein for aminoglycosides’ uptake. (A) Spermidine affects the growth inhibition of streptomycin on Ms. Spermidine, as a porin inhibitor, can bind to porin and reduce the permeability of protein channels, spm: spermidine. The growth of Ms under streptomycin pressure in the presence and absence of spermidine was detected by measuring absorbance values at 600 nm. (B) Cell wall permeability assay. Mycobacterium smegmatis was incubated with 2 µg/ml of hydrophilic substrate ethidium bromide and the fluorescence intensity was measured with excitation light at 540 nm and emission light at 630 nm. (C) The model of OmpA in uptakes of aminoglycosides. The OmpA allows the transition of hydrophilic small-molecule compounds. Overexpressing OmpA can transport more hydrophilic antibiotics such as the aminoglycosides into the bacteria, which leads to the increase of Mycobacteria’s sensitivity to the antibiotics