Targeting Bacterial Immunoglobulin-like Domains on Plasmids to Combat Antimicrobial Resistance

Core Concepts

Plasmid-encoded proteins containing bacterial immunoglobulin-like domains are promising targets for vaccines and nanobody-based therapeutics to combat the spread of antimicrobial resistance.

Abstract

The content discusses the potential of targeting plasmid-encoded proteins containing bacterial immunoglobulin-like (Big) domains as a strategy to combat antimicrobial resistance (AMR). Key highlights:

IncHI plasmids, which are commonly found in AMR bacteria, encode large proteins containing Big domains that are located on the bacterial surface, such as in flagella or conjugative pili.

Immunization of mice with the RSP protein, one of the Big domain-containing proteins encoded by IncHI plasmids, provided partial protection against infection by an AMR Salmonella strain harboring an IncHI plasmid. Immunized mice showed reduced clinical symptoms, Salmonella counts in the spleen, and inflammatory cytokine expression.

The authors generated nanobodies (Nbs) targeting the RSP protein and demonstrated that the Nb-expressing E. coli cells could significantly interfere with the conjugative transfer of the IncHI R27 plasmid from Salmonella to recipient cells.

Since Big domain-containing proteins are also encoded by other plasmid groups like IncA/C and IncP2, targeting these proteins could be a valuable strategy to combat infections caused by bacteria harboring diverse AMR plasmids.

The use of antigens directly linked to AMR determinants, such as the Big domain-containing proteins, can provide protection beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

Stats

Infection with the antibiotic-resistant Salmonella strain SL1344 (pHCM1) reduced mouse survival by about 40%, while ampicillin treatment only improved survival in mice infected with the antibiotic-sensitive SL1344 (WT) strain.
Immunization with the RSP protein increased the levels of RSP-specific IgG in serum and RSP-specific IgA in the intestinal tract of mice.
Immunization with the RSP protein significantly improved the survival rate of mice and reduced the incidence of clinical signs after challenge with the SL1344 (pHCM1) strain.
The E. coli strain expressing the RSP-specific nanobody Nb3 reduced the conjugation frequency of the R27 plasmid from the Salmonella SL1344 (R27) strain by up to 1000-fold.

Quotes

"Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids."
"Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids."

Key Insights Distilled From

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

by Prieto,A., M... at www.biorxiv.org 12-12-2023

https://www.biorxiv.org/content/10.1101/2023.12.12.571265v1

Deeper Inquiries

How could the RSP-specific nanobodies be further developed and optimized for therapeutic applications against AMR infections

To further develop and optimize the RSP-specific nanobodies for therapeutic applications against AMR infections, several key steps can be taken:

Affinity Maturation: Through techniques like phage display or yeast display, the nanobodies can be subjected to rounds of selection to enhance their binding affinity to the RSP protein. This process, known as affinity maturation, can lead to the generation of nanobodies with higher specificity and potency.

Engineering for Stability: Nanobodies can be engineered for improved stability, solubility, and resistance to proteases. By introducing specific mutations or modifications, the nanobodies can withstand harsh conditions in the body and maintain their functionality for longer periods.

Multivalency: Creating multivalent nanobody constructs by linking multiple nanobodies together can enhance their binding avidity and efficacy. Multivalent nanobodies can improve target engagement and neutralization of the RSP protein, leading to better therapeutic outcomes.

In vivo Studies: Conducting in vivo studies to evaluate the pharmacokinetics, biodistribution, and efficacy of the RSP-specific nanobodies in animal models of AMR infections. These studies can provide valuable insights into the potential therapeutic benefits and dosing regimens for clinical translation.

Humanization: If the nanobodies are derived from camelids, humanization may be necessary to reduce immunogenicity and enhance their compatibility for human use. Humanized nanobodies have a lower risk of eliciting immune responses and can be better tolerated in patients.

Combination Therapy: Exploring the use of RSP-specific nanobodies in combination with other antimicrobial agents or immunomodulators to enhance their therapeutic effects. Synergistic interactions with existing treatments can improve the overall efficacy of the nanobodies in combating AMR infections.

By implementing these strategies, the RSP-specific nanobodies can be optimized for clinical applications, offering a promising avenue for the development of novel therapies against AMR infections.

What other plasmid-encoded proteins with immunoglobulin-like domains could be explored as potential vaccine or therapeutic targets against AMR

In addition to the RSP protein, several other plasmid-encoded proteins with immunoglobulin-like domains could be explored as potential vaccine or therapeutic targets against AMR. Some examples include:

Proteins from IncA/C Plasmids: Proteins encoded by IncA/C plasmids, such as AcrA, AcrB, and AcrD, which are involved in multidrug efflux mechanisms, could be targeted to disrupt antibiotic resistance in Gram-negative bacteria.

Proteins from IncP2 Plasmids: Immunoglobulin-like proteins encoded by IncP2 plasmids, like TrbI and TrbJ, which are involved in plasmid transfer and stability, could be potential targets for interfering with the spread of AMR genes.

Proteins from IncF Plasmids: IncF plasmids encode proteins like TraF and TraH, which play crucial roles in conjugative transfer. Targeting these proteins could disrupt the horizontal transfer of resistance genes among bacterial populations.

Proteins from IncN Plasmids: IncN plasmids carry genes encoding proteins like KorB and KorA, which are involved in plasmid replication and partitioning. Inhibiting these proteins could hinder the maintenance and dissemination of AMR plasmids.

Exploring a diverse range of plasmid-encoded proteins with immunoglobulin-like domains for their potential as vaccine or therapeutic targets can provide a comprehensive approach to combating antimicrobial resistance across different bacterial species and resistance mechanisms.

What are the broader implications of targeting plasmid-encoded proteins rather than pathogen-specific virulence factors for developing broadly effective antimicrobial resistance interventions

Targeting plasmid-encoded proteins rather than pathogen-specific virulence factors for developing antimicrobial resistance interventions has several broader implications:

Broad-Spectrum Activity: Plasmid-encoded proteins are often conserved among different bacterial strains and species, making them attractive targets for developing broad-spectrum antimicrobial interventions. By targeting these proteins, it may be possible to combat a wide range of AMR pathogens simultaneously.

Interference with Horizontal Gene Transfer: Plasmid-encoded proteins are frequently involved in horizontal gene transfer mechanisms, facilitating the spread of AMR genes among bacterial populations. By targeting these proteins, interventions can disrupt the transfer of resistance determinants and limit the dissemination of AMR.

Reduced Selective Pressure: Inhibiting plasmid-encoded proteins can reduce the selective pressure for the maintenance of AMR plasmids in bacterial populations. This approach may help in slowing down the emergence and spread of resistant strains, contributing to the overall containment of AMR.

Combinatorial Strategies: Targeting plasmid-encoded proteins can be combined with other antimicrobial approaches, such as antibiotics or vaccines targeting specific pathogens. This combinatorial strategy can offer synergistic effects and enhance the overall efficacy of AMR interventions.

One Health Approach: By focusing on plasmid-encoded proteins, interventions can address AMR not only in human health but also in veterinary and environmental settings. This holistic "One Health" approach can help in tackling AMR from a multidisciplinary perspective.

Overall, targeting plasmid-encoded proteins provides a promising avenue for developing effective and versatile interventions against antimicrobial resistance, with the potential to impact a wide range of bacterial pathogens and resistance mechanisms.