Absence of PmrAB-mediated phosphoethanolamine modifications of Citrobacter rodentium lipopolysaccharide affects outer
Charles Viau1, Valerie Le Sage, Daniel K Ting
1Department of Microbiology and Immunology, McGill University, 3775 University St., Montreal, QC, Canada H3A 2B4.
Abstract:
The PmrAB two-component system of enterobacteria regulates a number of genes whose protein products modify lipopolysaccharide (LPS). The LPS is modified during transport to the bacterial outer membrane (OM). A subset of PmrAB-mediated LPS modifications consists of the addition of phosphoethanolamine (pEtN) to lipid A by PmrC and to the core by CptA. In Salmonella enterica, pEtN modifications have been associated with resistance to polymyxin B and to excess iron. To investigate putative functions of pEtN modifications in Citrobacter rodentium, ΔpmrAB, ΔpmrC, ΔcptA, and ΔpmrC ΔcptA deletion mutants were constructed. Compared to the wild type, most mutant strains were found to be more susceptible to antibiotics that must diffuse across the LPS layer of the OM. All mutant strains also showed increased influx rates of ethidium dye across their OM, suggesting that PmrAB-regulated pEtN modifications affect OM permeability. This was confirmed by increased partitioning of the fluorescent dye 1-N-phenylnaphthylamine (NPN) into the OM phospholipid layer of the mutant strains. In addition, substantial release of periplasmic β-lactamase was observed for the ΔpmrAB and ΔpmrC ΔcptA strains, indicating a loss of OM integrity. This study attributes a new role for PmrAB-mediated pEtN LPS modifications in the maintenance of C. rodentium OM integrity.
Insights
The PmrAB system modifies lipopolysaccharide (LPS) in Citrobacter rodentium, impacting outer membrane (OM) integrity. These phosphoethanolamine (pEtN) modifications are crucial for maintaining OM permeability and bacterial resistance.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- The PmrAB two-component system regulates lipopolysaccharide (LPS) modifications in enterobacteria.
- Phosphoethanolamine (pEtN) addition to LPS by PmrC and CptA is known to confer resistance to antibiotics and iron stress in Salmonella enterica.
Purpose of the Study:
- To investigate the role of PmrAB-mediated pEtN LPS modifications in the outer membrane (OM) integrity and function of Citrobacter rodentium.
- To determine if pEtN modifications influence OM permeability and antibiotic susceptibility in C. rodentium.
Main Methods:
- Construction and characterization of C. rodentium deletion mutants for pmrAB, pmrC, cptA, and pmrC cptA.
- Assessment of antibiotic susceptibility, ethidium dye influx, and 1-N-phenylnaphthylamine (NPN) partitioning to evaluate OM permeability.
- Measurement of periplasmic β-lactamase release to assess OM integrity.
Main Results:
- Mutant strains lacking PmrAB-mediated pEtN modifications exhibited increased susceptibility to antibiotics that cross the OM.
- All mutant strains showed enhanced ethidium dye influx and NPN partitioning, indicating increased OM permeability.
- Significant release of periplasmic β-lactamase was observed in ΔpmrAB and ΔpmrC ΔcptA mutants, signifying compromised OM integrity.
Conclusions:
- PmrAB-regulated pEtN LPS modifications are essential for maintaining the integrity of the C. rodentium outer membrane.
- These modifications play a critical role in regulating OM permeability and protecting against antibiotic entry.
