LD-transpeptidase-mediated cell envelope remodeling enables developmental transitions and survival in <i>Coxiella burnetii</i> and <i>Legionella pneumophila</i>

<i>Coxiella burnetii</i> and <i>Legionella pneumophila</i> are two phylogenetically related bacterial pathogens that exhibit extreme intrinsic resistance when they enter into a dormancy-like state. This enables both pathogens to survive extended periods in growth-limited environments. Survival is dependent upon their ability to undergo developmental transitions into two phenotypically distinct variants, one specialized for intracellular replication and another for prolonged survival in the environment and host. We currently lack an understanding of the mechanisms that mediate these developmental transitions. Here, we performed peptidoglycan (PG) glycoproteome analysis and showed significant enrichment of PG structures catalyzed by LD-transpeptidases (LDTs) in the survival variants of <i>C. burnetii</i> and <i>L. pneumophila</i>. This is supported by the upregulation of LDTs, resulting in susceptibility to carbapenem antibiotics. Furthermore, deletion of the most upregulated LDT, <i>lpg</i>1386, in <i>L. pneumophila</i> significantly changes PG architecture, survival, and susceptibility to antibiotics. Significantly regulated by RpoS, a stationary-phase sigma factor, LDT-dependent PG remodeling is differentially activated by the host intracellular growth environment compared to axenic culture. In addition, β-barrel tethering, a newly discovered mechanism of LDT-mediated cell envelope stabilization, seems not to be specific to the survival variants. Interestingly, an outer membrane (OM) long-chain fatty acid transporter (Lpg1810) is tethered to PG in <i>L. pneumophila</i>. Collectively, these findings show that LDT-mediated PG remodeling is a major determinant of developmental transitions and survival in <i>C. burnetii</i> and <i>L. pneumophila</i>. Understanding this mechanism might inform new therapeutic approaches for treating chronic infections caused by these pathogens, as well as suggest new methods to decontaminate environmental reservoirs during outbreaks.IMPORTANCE<i>Coxiella burnetii</i> and <i>L. pneumophila</i> cause Q Fever and Legionnaire's disease in humans, respectively. There is a lack of effective treatments for fatal chronic infections caused by these pathogens, particularly chronic Q Fever. These bacteria survive long term in nutrient-limited environments by differentiating into phenotypically distinct survival variants. Our study revealed that LDTs, a group of PG remodeling enzymes, play a prominent role in the phenotypic differentiations of these bacteria. We show that LDT-targeting carbapenems are effective against the survival variants, thus demanding the exploration of carbapenems for treating chronic infections caused by these pathogens. We report the tethering of a unique OM fatty acid transporter to PG in <i>L. pneumophila</i> that could indicate a novel function of tethering, that is, the uptake of nutrient substrates. Homologs of this transporter are widely present in the Methylobacteriaceae family of bacteria known to survive in water systems like <i>Legionella</i>, thus suggesting a potentially conserved mechanism of bacterial survival in nutrient-limited environments.