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. 2019 Apr 10;10:756. doi: 10.3389/fmicb.2019.00756

FIGURE 2.

FIGURE 2

Insertional inactivation of ptr leads to impaired recovery of C. trachomatis from IFNγ-induced stress. (A) LGV-L2 ptr knock out (L2 ptr::GII), LGV-L2 transformed with the plasmid vector pBOMB-ptrF expressing FLAG-tagged Ptr (L2 PTR-F) and LGV-L2 ptr knock-out transformed with pBOMB-ptrF (L2 ptr::GII PTR-F), display similar levels of infectious progeny generation compared to C. trachomatis LGV-L2 wild type (L2 wt) strain in the untreated (UT) condition. The number of infectious bacteria was quantified at 30 hpi and normalized to the input (mean ± SEM, n = 3, Student’s t-test). (B) The ptr knock-out strain L2 ptr::GII exhibits decreased generation of infectious progeny upon recovery from IFNγ-induced stress (IFN-Rec), while this phenotype is rescued by expression of Ptr from a plasmid vector in the L2 ptr::GII PTR-F strain. HeLa cells were pre-treated with IFNγ (15 ng/mL) for 24 h and infected with the indicated strains. At 24 hpi, IFNγ was removed, cells were supplemented with tryptophan (100 mg/L) and incubated for various times post-infection to assess recovery. Infectious progeny post-recovery was quantified at 48, 56, and 72 hpi and normalized to the input (mean ± SEM). Statistical analysis was performed by Student’s t-test (n = 3; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). (C) L2 ptr::GII produces infectious progeny at similar levels than L2 wt upon recovery from penicillin-induced stress (PEN-Rec). HeLa cells were infected with the indicated strains and supplemented with penicillin (1 IU/mL) at the time of infection. At 24 hpi penicillin was removed, cells were replenished with complete medium and incubated up to 48, 56, or 72 hpi for recovery. Since pBOMB-ptrF carries ampicillin resistance, L2 PTR-F and L2 ptr::GII PTR-F are not affected by penicillin. In all cases, infectious progeny was normalized by input (mean ± SEM, n = 3). Statistical analysis was performed by Student’s t-test [non-significant (ns), P > 0.05]. (D) Representative TEM images of the untreated (UT, 30 hpi) and of IFN-Rec (48 hpi) conditions for HeLa cells infected the L2 wt, L2 ptr::GII and L2 ptr::GII PTR-F strains. Insets highlight C. trachomatis particles displaying typical ultrastructural features of EBs and RBs. Scale bars represent 2 μm. (E) Quantification of EBs/inclusion and EBs/RBs visualized by TEM during IFN-Rec. Values represent the mean ± SEM of at least three replicates. Statistical significance was calculated by Student’s t-test (∗∗P < 0.01 and ∗∗∗P < 0.001). (F) HeLa cells were treated with IFNγ for 24 h and infected with either L2 wt, L2 ptr::GII or L2 ptr::GII PTR-F strains. For the recovery condition (Rec), IFNγ was removed at 24 hpi and cells were replenished with full media supplemented with tryptophan. For the non-recovery condition (NoRec) IFNγ was continuously present during infection. Genome copy number was assessed by real-time PCR during IFNγ treatment and upon recovery at the indicated times post-infection using primers specific for ompA. Data points represent the mean of three replicates.