<i>Chlamydia trachomatis</i> restricts signaling through NOD2 until late in the pathogen’s developmental cycle

Pathogenic chlamydial species restrict their peptidoglycan (PG) to the division septum of their replicative forms. PG is a microbe-associated molecular pattern, and two of its major pattern recognition receptors in human cells are nucleotide-binding oligomerization domain-containing proteins 1 and 2 (NOD1 and NOD2, respectively). It has been proposed that this unique morphological feature is evidence of pathoadaptation by the microbe, permitting PG-dependent cell division while also reducing the bacterium's recognition by innate immune receptors. <i>Chlamydia trachomatis</i>-infected cells activate NOD1 signaling within 8-12 hours of exposure to the bacterium, roughly coinciding with the microbe's transition from its infectious to replicative forms. Here, we report that, unlike NOD1 signaling, <i>Chlamydia</i>-induced NOD2 signaling does not occur until later in the pathogen's developmental cycle. Both <i>C. trachomatis</i> and the related murine pathogen <i>Chlamydia muridarum</i> signal late in infection in HEK293 reporter cell lines expressing either human or murine-derived NOD2 receptors. NOD2 signaling can be modulated by disruption of the chlamydial amidase enzyme, AmiA_CT, interrupting the microbe's developmental cycle, and treatment with inhibitors of lipooligosaccharide or PG biosynthesis/assembly. These results mirror prior observations with Chlamydia-induced TLR9 signaling, leading us to hypothesize that Chlamydia-induced NOD2 signaling results from lytic events that occur sporadically during the transition between the pathogen's developmental forms. Given our finding that pre-treating cells with NOD2-stimulatory ligands reduces chlamydial inclusion size and delays the developmental cycle, we hypothesize that the microbe preferentially degrades its PG during development to reduce the generation of NOD2 ligands.