LipL21 lipoprotein binding to peptidoglycan enables Leptospira interrogans to escape NOD1 and NOD2 recognition
(A) PG 0.5 h and PG 4 h from L. interrogans Manilae L495 and Fiocruz L1-130 strains, were loaded on coomassie-stained gels (upper panels; protein gels), revealing a 21-kDa protein corresponding to the LipL21. The protein specificity was checked by immunodetection using LipL21 antiserum (lower panels; western blot, (WB)). (B) LipL21 expression checked by immunodetection on bacterial extracts from L. interrogans Manilae L495 (lipl21+), the (lipl21-) M58 mutant and the complemented C5M58 strain (lipl21-/+). (C) HPLC separation profiles of muropeptides after digestion by mutanolysin of L. interrogans Manilae lipl21+, lipl21- and lipl21-/+ peptidoglycans, extracted with the 0.5 h protocol. As positive control for the mutanolysin digestion, E. coli PG was extracted with the leptospiral 0.5 h protocol. D) Growth curves of L. interrogans Manilae lipl21+, lipl21- and lipl21-/+ strains in EMJH medium at 28°C, with agitation. (E) Muropeptides or 6 μg of PGs extracted from the parental Manilae strain (lipl21+), the LipL21 mutant (lipl21-) and the complemented strain (lipl21-/+) were co-transfected with the reporters and NOD1 plasmids in HEK293T cells. 24 h after transfection, luciferase activity was measured. MurTriDAP (MTP) (100 nM), the NOD1 agonist was used as positive control and water as negative control (water). Data are expressed as the mean ± SEM of relative light units representing luciferase activity normalized with respect to β-galactosidase activity. This graph is representative of 3 independent experiments. The unpaired t test was used to compare the recognition of each PG by hNOD1 transfected cells to water as a negative control (none). A p value < 0.05 was considered significant. p values: *** p < 0.001. Non significant differences for the + and -/+ PG are not indicated.