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A periplasmic L,D-carboxypeptidase is important for cell morphology, membrane integrity, and virulence in Francisella tularensis

Abstract F. tularensis is a Gram-negative, intracellular bacterium that causes the zoonotic disease tularemia. Intracellular pathogens, including F. tularensis, have evolved mechanisms to allow survival in the harsh environment of macrophages and neutrophils, where they are exposed to cell membrane-damaging molecules. One mechanism that protects intracellular Gram-negative bacteria from macrophage or neutrophil killing is the ability to recycle and repair damaged peptidoglycan (PG), – a process that requires over 50 different PG synthesis and recycling enzymes. In addition to PG repair, PG recycling occurs during cell division and plays critical roles in maintaining cell morphology, structure, and membrane integrity. 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 prototypic LdcA homologs, F. tularensis LdcA is outer membrane-associated 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, a F. tularensis LdcA mutant protected mice against virulent Type A F. tularensis SchuS4 pulmonary challenge. Importance PG is well known to play important roles in protecting bacteria from external insults and osmotic stress. However, pathogenic bacteria modify their PG architecture to avoid antibiotic activity and/or encode PG recycling and repair pathways to promote bacterial virulence. Prototypic Gram-negative bacteria, including E. coli and Pseudomonas aeruginosa, recycle PG in a multi-enzyme pathway, including the cytoplasmic L,D-carboxypeptidase, LdcA. Here, we demonstrated that a previously unstudied protein from the intracellular bacterium F. tularensis contains an LdcA conserved domain, this F. tularensis LdcA is outer membrane-associated, and deletion of this LdcA ortholog increases bacterial outer membrane thickness, resulting in bacteria that are more resistant to various stressors (e.g., H2O2, NaCl, low pH), yet are completely attenuated in a mouse pulmonary infection model. Finally, we demonstrated that the F. tularensis LdcA mutant can be used as a live, attenuated vaccine to protect against pulmonary challenge with fully virulent F. tularensis.