Expression of a novel class of bacterial Ig-like proteins is required for IncHI plasmid conjugation

Antimicrobial resistance (AMR) is currently one of the most important challenges to the treatment of bacterial infections. A critical issue to combat AMR is to restrict its spread. In several instances, bacterial plasmids are involved in the global spread of AMR. Plasmids belonging to the incompatibility group (Inc)HI are widespread in Enterobacteriaceae and most of them express multiple antibiotic resistance determinants. They play a relevant role in the recent spread of colistin resistance. We present in this report novel findings regarding IncHI plasmid conjugation. Conjugative transfer in liquid medium of an IncHI plasmid requires expression of a plasmid-encoded, large-molecular-mass protein that contains an Ig-like domain. The protein, termed RSP, is encoded by a gene (ORF R0009) that maps in the Tra2 region of the IncHI1 R27 plasmid. The RSP protein is exported outside the cell by using the plasmid-encoded type IV secretion system that is also used for its transmission to new cells. Expression of the protein reduces cell motility and enables plasmid conjugation. Flagella are one of the cellular targets of the RSP protein. The RSP protein is required for a high rate of plasmid transfer in both flagellated and nonflagellated Salmonella cells. This effect suggests that RSP interacts with other cellular structures as well as with flagella. These unidentified interactions must facilitate mating pair formation and, hence, facilitate IncHI plasmid conjugation. Due to its location on the outer surfaces of the bacterial cell, targeting the RSP protein could be a means of controlling IncHI plasmid conjugation in natural environments or of combatting infections caused by AMR enterobacteria that harbor IncHI plasmids. Author summary Dissemination of antimicrobial resistance (AMR) among different bacterial populations occurs due to mainly the presence of plasmids that encode AMR determinants. IncHI plasmids are one of the groups of bacterial plasmids that confer AMR to several enterobacteria. Recently, resistance to one of the last-resort antibiotics (colistin) for some multidrug-resistant infections has spread very rapidly. IncHI plasmids represent 20% of all plasmids transmitting colistin resistance worldwide and 40% in Europe. When analyzing the interactions of the IncHI1 plasmid R27 with Salmonella, we identified a large-molecular-mass protein that is encoded by this plasmid and is exported to the external medium. The R27 plasmid gene coding for that protein (R0009) is widespread among IncHI plasmids. In this report, we characterize the protein, termed RSP. The presented data show that RSP plays a relevant role in IncHI plasmid conjugation and suggest that the protein is retained on the outer surface of the bacterial cells and facilitates cell-to-cell contact before plasmid DNA transfer. Considering that IncHI plasmids significantly contribute to AMR dissemination within enterobacteria, the findings reported in this paper suggest that the identified protein can be a target to control both IncHI-mediated AMR dissemination and infections caused by AMR enterobacteria that harbor these plasmids.