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

Abstract

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.

Fig 1

Diagram representing the RSP protein (A) and the alignment of the C-terminal region from the RSP protein that shows similarity to the SiiE protein from Salmonella Thyphimurium (B). A) The putative bacterial Ig-like domain (Big 3) that maps between the 331 and 470 amino acid residues was identified using the Conserved Domain algorithm (https://www.ncbi.nlm.nih.gov/cdd/) and the C-terminal region identified using Phyre2 program that shows similarity to SiiE are shown. B) Alignment of the C-terminal region encompassing amino acids 1146 to 1391 from the RSP protein that shows similarity to the SiiE protein from Salmonella Thyphimurium. The alignment was generated using the Phyre2 program. The query sequence is the RSP protein, and the template sequence is the SiiE protein, as indicated in the figure.

Fig 2. Subcellular localization of the RSP protein.

A) Detection of the RSP protein in the secretome of the strains SL1344 and SL1344 (R27). The arrow points to the RSP protein. B) Immunodetection of the Flag-tagged RSP protein with anti-Flag antibodies in the different cellular compartments. RSP-Flag is indicated. Experiments were repeated three times. A representative experiment is shown.

Fig 3. The RSP protein is required for conjugation of the R27 plasmid.

Transfer frequencies of the R27 and R27 Δrsp plasmids. The recipient strain was the SL1344 ibplac strain. The donor strains were SL1344 (R27), SL1344 (R27 Δrsp) and SL1344 (R27 Δrsp pLG338-rsp). The data shown are the means and standard deviations of three independent experiments. Statistical analysis showed a significant difference (*P-value < 0.001; **P-value < 0.005). n.d., not detectable.

Fig 4. Temperature- and growth medium-dependent expression of the rsp gene.

Effect of the growth medium and growth temperature on transcription of the rsp gene determined by using a rsp::lacZ transcriptional fusion. Samples were collected from cultures grown in LB (A) or M9 media (B) at 25 and 37°C., either at the exponential (O.D._{600 nm} 0.4) or early stationary (O.D._{600 nm} 2.0) growth phases. β-galactosidase activity is expressed as Miller units. The data shown are the means and standard deviations of three independent experiments. Statistical analysis showed a significant difference (*P-value < 0.001, **P-value < 0.005). n.s., not significant.

Fig 5. Expression of the RSP protein reduces cell motility.

Relative motility of the strains SL1344, SL1344 (R27), SL1344 (R27 Δrsp) and SL1344 (R27 Δrsp pLG338-rsp). Motility of the strain SL1344 (measured as the diameter of the growth zone in the agar plate) was considered as 100%. The results are the means of three independent experiments. Standard deviations are shown.

Fig 6. The R27 T4SS trhC ATPase is required for RSP export.

A) SDS-PAGE analysis of the secretomes of different constructs of the strain SL1344 containing different R27 derivatives. Coomassie blue staining (A) and immunostaining with Flag-specific antibodies (B). C) Immunodetection of the RSP protein in the cytoplasm and periplasmic fractions of the different constructs. Inactivation of the R27 thrC gene interferes with RSP export in the strain SL1344 (R27). RSP export is restored in the strain SL1344 (R27 ΔtrhC) by providing in trans the plasmid pBR322-thrC. The arrow points to the RSP protein. Experiments were performed three times. A representative experiment is shown.

Fig 7. Detection of the RSP protein by immunogold electron microscopy.

The plasmid-free SL1344 strain is shown in panel (A). The strain SL1344 (R27) is in panels (B, D-F). The strain SL1344 (R27Δrsp) is shown in panel (C). Samples in panels (A-D and F) were labeled with rabbit anti-RSP polyclonal antibodies and goat anti-rabbit IgG conjugated to 12 nm gold particles. The control experiment is shown in panel (E) and was labeled with goat anti-rabbit IgG conjugated to 12 nm gold particles in the absence of a specific antibody. Arrows point to the RSP protein associated with the flagella. Bars represent 0,5 μm.

Fig 8. Conjugation frequencies of R27 and R27 Δrsp plasmids using SL1344 and SL1344 ΔflgE as donor strains.

Conjugations were performed either in liquid (A) or on nitrocellulose filters (B, C). The data shown are the means and standard deviations of three independent experiments. Statistical analysis showed significant differences (*P-value < 0.005; **P-value < 0.001), n.s., not significant.

Fig 9. Opsonization of RSP-Flag enhances bacterial phagocytosis by macrophages.

Strain SL1344 cells expressing either RSP (wild-type R27) or RSP-Flag (RSP-Flag R27) were opsonized with anti-Flag antibodies or left nonopsonized. Macrophages were infected for 30 min with Salmonella cells (either opsonized or nonopsonized) at an MOI of 15. Negative control cells were not infected. The results are the mean of 2 independent experiments each of which was performed using biological triplicates. One-way ANOVA, Tuckey´s post hoc (***P < 0.0001; **P < 0.01).