A Francisella tularensis L,D‐carboxypeptidase plays important roles in cell morphology, envelope integrity, and virulence

Abstract

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 envelope‐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.

Horizontal-Acquisition of a Promiscuous Peptidoglycan-Recycling Enzyme Enables Aphids To Influence Symbiont Cell Wall Metabolism

“…Interestingly, we observed a decrease in pentapeptide abundance for Ap LdcA and an increase for Ec LdcA. The former suggests that Ap LdcA acts as an l , d -endopeptidase that converts pentapeptides directly to tripeptides, an activity previously reported for LdcA homologs from Novosphingobium aromaticivorans and Francisella tularensis ( 29 , 31 ). An explanation for the latter observation is described in the following section.…”

“…1 ). Since the Buchnera genome encodes no recognizable l , d -carboxypeptidases or endopeptidases and LdcA homologs from intracellular pathogens collectively exhibit both of these functions ( 29 , 30 ), it is possible that Ap LdcA could be responsible for producing these stem peptides in Buchnera .…”

“…Despite its role in PGN recycling, a cytoplasmic process involving soluble muropeptides, there is growing evidence that LdcA is an efficient starting point for the evolution of novel enzyme activities that modify the cell wall ( 29 , 30 ). In aphids, the use of LdcA in their symbiotic association with Buchnera implies a derived ability of Ap LdcA to target symbiont cell walls.…”

“…Enzymes shown in green indicate those for which aphid-encoded homologs exist. Organism abbreviations, shown in parentheses, denote the species for which the reaction has been demonstrated for the enzyme homolog: Eco , E. coli ( 27 , 67 ); Nar , Novosphingobium aromaticivorans ( 31 ); Ftu , Francisella tularensis ( 29 ); Ngo , Neisseria gonorrhoeae ( 30 ); and Pae , Pseudomonas aeruginosa ( 68 ). Organism abbreviations shown in orange designate enzymes capable of utilizing both muropeptides and cell wall as substrates.…”

“…While E. coli LdcA ( Ec LdcA) is known to utilize only solubilized PGN fragments (muropeptides), LdcA homologs from some intracellular pathogens are exported to the periplasm and display a shift in substrate tolerance, modifying the polymeric cell wall in addition to soluble muropeptides ( 29 , 30 ). Furthermore, LdcA homologs exhibit multifunctionality, demonstrating endopeptidase activities in addition to their carboxypeptidase function ( 29 – 31 ). We hypothesized that Ec LdcA might display a similar shift in activity that could enable aphids to control or support its symbionts.…”