Development of a High-Throughput Cloning Strategy for Characterization of Acinetobacter baumannii Drug Transporter Proteins

Abstract

Heterologous expression of membrane proteins in Escherichia coli often requires optimization to overcome problems with toxicity of the recombinant protein to the host cell. A number of Gateway-based destination vectors were constructed to investigate expression of membrane proteins using a high-throughput approach. These vectors were tested using putative drug transporter proteins from the multidrug and toxic compound extrusion (MATE) family and the resistance-nodulation-cell division superfamily encoded by the human pathogen Acinetobacter baumannii. Active transport of antibiotics and antiseptics mediated by efflux proteins contributes to the high level of multidrug resistance observed in A. baumannii. Substrates for 4 of the 5 putative efflux proteins investigated were identified using the expression vectors designed in this study. Additionally, a Gateway-based suicide vector was designed for construction of specific A. baumannii insertion disruption mutants. This knockout cloning strategy was tested and shown to be successful in inactivating AbeM4, a putative MATE family protein. Therefore, we have shown that the Gateway-based vectors constructed in this study are versatile tools that can be used for manipulation and characterization of membrane proteins.

Roles of Efflux Pumps from Different Superfamilies in the Surface-Associated Motility and Virulence of Acinetobacter baumannii ATCC 17978

“…This gene encodes a permease homologous (99% identity) to the AdeJ component corresponding to the AdeIJK RND efflux pump described in A. baumannii strain BM4454 (14). The AbeM3 transporter, encoded by the gene A1S_3371, and the transporter encoded by the gene A1S_0710, both identified in A. baumannii ATCC 17978, were chosen as members of the MATE and SMR efflux pump superfamilies, respectively (15,16). From the ABC efflux pump superfamily, the genes A1S_0536 and A1S_1535 were selected.…”

“…Moreover, this mutant was highly susceptible to previously untested compounds, including deoxycholate (DC). The A1S_3371 MATE efflux pump mutant only showed a slight difference in tetracycline even though no antimicrobial resistances were previously associated with this transporter (15). The A1S_0710 SMR efflux pump mutant exhibited greater susceptibility than the wild type to compounds such as SDS and DC.…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…AbeM hyperexpression (with moderately increased expression levels of AdeABC and AdeIJK) was observed in imipenemresistant isolates (559). Three additional AbeM homologs, AbeM2, -3, and -4, were also identified, with no demonstrated role of AbeM4 in MDR (560). Although the clinical importance of AbeM pumps remains unknown, the abeM gene was used with other resistance genes (adeB, adeR, ampC, and ompA) to assess the genetic linkage of multidrug-resistant endemic isolates (529).…”

Filamentous hemagglutinin adhesin FhaB limits i A baumannii i biofilm formation

“…Bacterial pathogens secreting virulence proteins serve many divergent functions in order to adhere, survive, multiply and disseminate within mammalian hosts(5). High multidrug resistance level observed in A. baumannii is attributed to active antibiotics and antiseptics transport mediated by efflux proteins (15).Virulence factors assigned for the translocation mechanism are known as the type V secretion system. In this system, proteins are secreted out of cells without the mediation of other proteins (16).…”