Summary
Francisella tularensis is a Gram-negative, intracellular bacterium that causes the zoonotic disease tularemia. Intracellular pathogens, including F. tularensis, have evolved mechanisms to survive in the harsh environment of macrophages and neutrophils, where they are exposed to cell membrane-damaging molecules. The bacterial cell wall, primarily composed of peptidoglycan (PG), maintains cell morphology, structure, and membrane integrity. Intracellular Gram-negative bacteria protect themselves from macrophage and neutrophil killing by recycling and repairing damaged PG – a process that involves over 50 different PG synthesis and recycling enzymes. Here, we identified a PG recycling enzyme, L,D-carboxypeptidase A (LdcA), of F. tularensis that is responsible for converting PG tetrapeptide stems to tripeptide stems. Unlike E. coli LdcA and most other orthologs, F. tularensis LdcA does not localize to the cytoplasm and also exhibits L,D-endopeptidase activity, converting PG pentapeptide stems to tripeptide stems. Loss of F. tularensis LdcA led to altered cell morphology and membrane integrity, as well as attenuation in a mouse pulmonary infection model and in primary and immortalized macrophages. Finally, an F. tularensis ldcA mutant protected mice against virulent Type A F. tularensis SchuS4 pulmonary challenge.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
This revised manuscript clarifies some speculative points in the previous manuscript, while providing new data to further characterize F. tularensis L,D-carboxypeptidase (LdcA) function and better define the putative catalytic triad. Major changes include: 1. Prior manuscript noted periplasmic localization of LdcA but revised manuscript notes that LdcA may be outer membrane-associated or periplasmic; 2.Better descriptions of peptidoglycan (PG) recycling and metabolism pathways and products, including new enzymatic studies using double point mutants in the putative catalytic triad; 3. New TEM images and revised descriptions of bacterial phenotypes; 4. Comparisons of OD600 and CFU at multiple time points for wild-type and mutant bacterial strains; 5. New studies on antibiotic sensitivity of Type A strain SchuS4 LdcA mutant; 6. New descriptions of antimicrobial, detergent, and stressor assay findings.