Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance

Reviewer #1 (Public Review):

The study by Prieto et al. faces the increasingly serious problem of bacterial resistance to antimicrobial agents. This work has an important element of novelty proposing a new approach to control antibiotic resistance spread by plasmids. Instead of targeting the resistance determinant, plasmid-borne proteins are used as antigens to be bound by specific nanobodies (Nbs). Once bound plasmid transfer was inhibited and Salmonella infection blocked. This in-depth study is quite detailed and complex, with many experiments (9 figures with multiple panels), rigorously carried out. Results fully support the authors' conclusions. Specifically, the authors investigated the role of two large molecular weight proteins (RSP and RSP2) encoded by the IncHI1 derivative-plasmid R27 of Salmonella. These proteins have bacterial Ig-like (Big) domains and are expressed on the cell surface, creating the opportunity for them to serve as immunostimulatory antigens. Using a mouse infection model, the authors showed that RSP proteins can properly function as antigens, in Salmonella strains harboring the IncHI1 plasmid. The authors clearly showed increased levels of specific IgG and IgA antibodies against these RSP proteins proteins in different tissues of immunized animals. In addition, non-immunized mice exhibited Salmonella colonization in the spleen and much more severe disease than immunized ones.

However, the strength of this work is the selection and production of nanobodies (Nbs) that specifically interact with the extracellular domain of RSP proteins. The procedure to obtain Nbs is lengthy and complicated and includes the immunization of dromedaries with purified RPS and the construction of a VHH (H-chain antibody variable region) library in E. coli. As RSP is expressed on the surface of E. coli, specific Nbs were able to agglutinate Salmonella strains harboring the p27 plasmid encoding the RSP proteins.
The authors demonstrated that Nbs-RSP reduced the conjugation frequency of p27 thus limiting the diffusion of the amp resistance harbored by the plasmid. This represents an innovative and promising strategy to fight antibiotic resistance, as it is not blocked by the mechanism that determines, in the specific case, the amp resistance of p27 but it targets an antigen associated with HincHI- derivative plasmids. Thus, RPS vaccination could be effective not only against Salmonella but also against other enteric bacteria. A possible criticism could be that Nbs against RSP proteins reduce the severity of the disease but do not completely prevent the infection by Salmonella.

Reviewer #2 (Public Review):

Summary:

This manuscript aims to tackle the antimicrobial resistance through the development of vaccines. Specifically, the authors test the potential of the RSP protein as a vaccine candidate. The RSP protein contains bacterial Ig-like domains that are typically carried in IncHl1 plasmids like R27. The extracellular location of the RSP protein and its role in the conjugation process makes it a good candidate for a vaccine. The authors then use Salmonella carrying an IncHl plasmid to test the efficacy of the RSP protein as a vaccine antigen in providing protection against infection of antibiotic-resistant bacteria carrying the IncHl plasmid. The authors found no differences in total IgG or IgA levels, nor in pro-inflammatory cytokines between immunized and non-immunized mice. They however found differences in specific IgG and IgA, attenuated disease symptoms, and restricted systemic infection.

The manuscript also evaluates the potential use of nanobodies specifically targeting the RSP protein by expressing it in E. coli and evaluating their interference in the conjugation of IncHl plasmids. The authors found that E. coli strains expressing RSP-specific nanobodies bind to Salmonella cells carrying the R27 plasmid thereby reducing the conjugation efficacy of Salmonella.

Strengths:

- The main strength of this manuscript is that it targets the mechanism of transmission of resistance genes carried by any bacterial species, thus making it broad.

- The experimental setup is sound and with proper replication.

Weaknesses:

- The two main experiments, evaluating the potential of the RSP protein and the effects of nanobodies on conjugation, seem as parts of two different and unrelated strategies.

- The survival rates shown in Figure 1A and Figure 3A for Salmonella pHCM1 and non-immunized mice challenged with Salmonella, respectively, are substantially different. In the same figures, the challenge of immunized mice and Salmonella pHCM1 and mice challenged with Salmonella pHCM1 with and without ampicillin are virtually the same. While this is not the only measure of the effect of immunization, the inconsistencies in the resulting survival curves should be addressed by the authors more thoroughly as they can confound the effects found in other parameters, including total and specific IgG and IgA, and pro-inflammatory cytokines.

- Overall the results are inconsistent and provide only partial evidence of the effectiveness of the RSP protein as a vaccine target.

- The conjugative experiments use very long conjugation times, making it harder to asses if the resulting transconjugants are the direct result of conjugation or just the growth of transconjugants obtained at earlier points in time. While this could be assessed from the obtained results, it is not a direct or precise measure.

- While the potential outcomes of these experiments could be applied to any bacterial species carrying this type of plasmids, it is unclear why the authors use Salmonella strains to evaluate it. The introduction does a great job of explaining the importance of these plasmids but falls short in introducing their relevance in Salmonella.