Molecular analysis of resistance to streptogramin A compounds conferred by the Vga proteins of staphylococci

Abstract

The Vga and Msr resistance determinants, encoded by mobile genetic elements in various staphylococcal strains, belong to a family of ATP-binding cassette (ABC) proteins whose functions and structures are ill defined. Their amino acid sequences are similar to those of proteins involved in the immunity of streptomycetes to the macrolide-lincosamide-streptogramin antibiotics that they produce. Sequence analysis of the genomes of the gram-positive bacteria with low G+C contents revealed that Lmo0919 from Listeria monocytogenes is more closely related to Vga variants than to Msr variants. In the present study we compared the antibiotic resistance profiles conferred by the Vga-like proteins in two staphylococcal hosts. It was shown that Vga(A), the Vga(A) variant [Vga(A)v], and Lmo0919 can confer resistance to lincosamides and streptogramin A compounds, while only Vga(B) is able to increase the level of resistance to pristinamycin, a mixture of streptogramin A and streptogramin B compounds. By using polyclonal antibodies, we found that the Vga(A) protein colocalized with the beta subunit of the F(1)-F(0) ATPase in the membrane fractions of staphylococcal cells. In order to identify functional units in these atypical ABC proteins, such as regions that might be involved in substrate specificity and/or membrane targeting, we analyzed the resistance phenotypes conferred by various plasmids carrying parts or modified versions of the vga(A) gene and we determined the subcellular localization of the gene products. Only polypeptides composed of two ABC domains were detected in the cell membranes. No region of drug specificity was identified. Resistance properties were dependent on the integrities of both Walker B motifs.

FIG. 1.

Progeny of the plasmids used. Abbreviations: PvegII, constitutive promoter; RBS, ribosome-binding site of the vga(A) gene; B, BamHI; E, EcoRI; X, XbaI; H, HindIII, S, SmaI; K, KpnI.

FIG. 2.

Alignment of the proteins studied. Identical residues are boxed. Highly conserved functional motifs of ABC proteins (WALKER A, LID, SIGNATURE, WALKER B, and SWITCH) are indicated. Vga(A), Vga(A)v, and Lmo0919 are characterized by shorter helical domains encompassing residues between the LID and the SIGNATURE motifs of the N-terminal ABC domain.

FIG. 3.

Analysis of lysates extracted from S. epidermidis BM3302 transformants and probed with antibodies raised against MalE-Vga(A). Lanes: 1, 10 ng of purified MalE protein; 2, strain BM3302 transformed by pRB474; 3, strain BM3302 transformed by pIP1845 expressing Vga(A); 4, strain BM3302 transformed by pIP1861 expressing Vga(B); 5, strain BM3302 transformed by pIP1863 expressing Vga(A)v; 6, strain BM3302 transformed by pIP1867 expressing Lmo0919.

FIG. 4.

Analysis of membranes (M) and ribosomes (R) extracted from two S. epidermidis clinical isolates and probed with various immune sera. The first part (lanes 1) of each panel corresponds to strain BM10385 carrying vga(A), and the second part (lanes 2) corresponds to strain IPF69 carrying msr(A). (A) Immunoblot probed with antibodies raised against MalE-Vga(A); (B) immunoblot probed with antibodies raised against the β subunit of the F₁-F₀ ATPase; (C) immunoblot probed with antibodies raised against the L24 protein.

FIG. 5.

Analysis of the cytoplasmic (C) and membrane (M) fractions from S. epidermidis BM3302 transformants probed with antibodies raised against MalE-Vga(A). Lanes: 1 and 5, strain BM3302 transformed by pIP1835 expressing the first half of Vga(A); 2 and 6, strain BM3302 transformed by pIP1837 expressing the second half of Vga(A); 3 and 7, strain BM3302 transformed by pIP1845 expressing Vga(A); 4 and 8, strain BM3302 transformed by pRB474, used as a control.

FIG. 6.

Analysis of the membrane fractions from S. epidermidis BM3302 transformants probed with antibodies raised against MalE-Vga(A). Lanes: 1, 10 ng of MalE protein; 2, strain BM3302 transformed by pRB474; 3, strain BM3302 transformed by pIP1838 expressing Vga(A) mutated in the second Walker B motif; 4, strain BM3302 transformed by pIP1845 expressing a wild-type Vga(A) protein; 5, strain BM3302 transformed by pIP1877 expressing the chimera Msr(A)-Vga(A); 6, strain BM3302 transformed by pIP1879 expressing the chimera Vga(A)-Msr(A); 7, strain BM3302 transformed by pIP1887 expressing Vga(A) mutated in the first Walker B motif.