Host cell adhesion and translocation of lig-transformed L. biflexa Interactions of Patoc wt, Patoc ligA, and Patoc ligB strains with mammalian host cells were assayed by examining adherence of leptospires to MDCK cells and translocation of leptospires across polarized MDCK cell monolayers. Adherence of L. interrogans strain Fiocruz L1-130 and Patoc ligA, but not Patoc wt and Patoc ligB, to MDCK cells was found to significantly increase in a time-dependent manner in

two experiments (PF-6463922 clinical trial Figure 3). After a 240 min incubation period, approximately four times more Patoc ligA adhered to MDCK cells than Patoc wt and Patoc ligB. The number of adherent Patoc ligA leptospires per cell at 240 min incubation point was comparable (0.23 and 1.02 in experiments 1 and 2, respectively) to that observed for the pathogenic L. interrogans strain Fiocruz L1-130 (0.16 and Fludarabine 0.73 in experiments 1 and 2, respectively). Figure 3 Association of L. biflexa transformants with MDCK monolayers. Adhesion of MDCK epithelial cells GDC-0994 with L. interrogans (L1-130), L. biflexa wild-type strain (wt), and ligA- (ligA), and ligB- (ligB) L. biflexa transformants. Results were determined after 30, 60, and 240 minutes exposure, followed by extensive washing of non-adherent bacteria. The bars show the mean number of bacteria associated per host cell ± standard deviation carried out in 10 random fields in two independent

experiments. The numbers of adherent leptospires/cell between the L. biflexa wild-type strain and the ligA- and ligB- L. biflexa transformants were Rucaparib clinical trial statiscally different at 240 minutes (P < 0.05).

Patoc ligA and ligB strains did not demonstrate enhanced ability to translocate across MDCK monolayers in comparison with Patoc wt in three experiments (representative experiment in Figure 4). As reported previously [30], we found that a small proportion (< 1%) of Patoc wt was able to translocate across MDCK monolayers after a 240 min incubation period. Proportions of translocating leptospires recovered from the lower transwell chamber were not significantly different between Patoc wt and Patoc ligA and ligB during the assay's time course (Figure 4). In contrast, > 6% of the inoculum of pathogenic L. interrogans strain Fiocruz L1-130 was recovered in the lower chamber after 240 min of incubation (Figure 4). As previously reported [30], recovery of L. interrogans strain Fiocruz L1-130 was not associated with significant alterations in the TER (Figure 4), indicating that disruption of tight junctions of the monolayers did not occur during the translocation process. Together these findings indicate that whereas expression of LigA in the saprophyte Patoc was associated with an enhanced host cell adherence phenotype similar to that observed with pathogenic leptospires, it did not impart the ability to efficiently invade and translocate across polarized host cell monolayers. Figure 4 Translocation assays.