Fig 1.
(A) Diagram illustrating infection and exposure beginning at T0, immediately after infection. Host cells were infected with Chlamydia (C.) pecorum or C. trachomatis serovar E, exposed to DAMPs in incubation medium, and incubated for 35 or 39 hours, at which time samples were collected for analysis. (B) DAMP exposure beginning at T14, 14 hours after infection. DAMPs were added directly to incubation medium. (C) DAMP exposure beginning at T0, followed by 35/39-hour incubation, media change for continued DAMP exposure or recovery in the absence of DAMPs, and 24 hours of additional incubation.
Fig 2.
Determination of optimal DAMP concentrations for inhibition of C. pecorum inclusion development.
HeLa cells were infected with C. pecorum and exposed to the DAMPs (A) cAMP, (B) ATP, or (C) EHNA/ADO (ADO concentration varies, EHNA = 25 μM) in incubation medium immediately after infection. Cells were incubated for 35 hours, fixed and labeled with anti-LPS and DAPI, and inclusion size was determined (mean ± SD; ** p ≤ 0.0001, *p ≤ 0.01, t test; n = 50 inclusions per coverslip from a single experiment). Arrows indicate concentrations selected for use in the study.
Fig 3.
DAMPs reduce chlamydial inclusion size.
HeLa cells were infected with C. pecorum or C. trachomatis serovar E and exposed to cAMP (1 mM), 8BrcAMP (1 mM), ATP (1 mM), apyrase (2.5 U), apyrase (2.5 U) followed by ATP (1 mM), ADO (50 μM), EHNA (25 μM), or ADO (50 μM) plus EHNA (25 μM) in incubation medium immediately after infection. Cells were incubated for 35 hours (C. pecorum) or 39 hours (C. trachomatis), fixed and labeled with anti-LPS and DAPI. Number of inclusions per nucleus was determined and percent infection was calculated for C. pecorum (A) and C. trachomatis (B) (mean ± SD; *p ≤ 0.05, t test; n = 3). Mean inclusion size was determined for C. pecorum (C and E) and C. trachomatis (D and F) (mean ± SD; *p ≤ 0.05, t test; n = 3).
Fig 4.
cAMP, ATP, and EHNA/ADO inhibit C. pecorum inclusion development without inducing aberrant body formation.
HeLa cells were infected with C. pecorum and exposed to the DAMPs cAMP (1 mM), ATP (1 mM), or ADO (50 μM, plus 25 μM EHNA) in incubation medium immediately after infection. Cells were incubated for 35 hours and fixed for further processing. Cells were labeled with anti-LPS (green) and DAPI (blue) for immunofluorescence microscopic analysis (left). Cells were processed by standard methods for transmission electron microscopic analysis (right). Representative images are shown for C. pecorum-infected mock- (A and B), cAMP- (C and D), ATP- (E and F), and EHNA/ADO-exposed (G and H) cells. Immunofluorescence microscopy images represent one experiment of three independently repeated experiments with similar results. Transmission electron microscopy images represent a single experiment.
Fig 5.
cAMP, ATP, and EHNA/ADO inhibit C. trachomatis inclusion development without inducing aberrant body formation.
HeLa cells were infected with C. trachomatis serovar E and exposed to the DAMPs cAMP (1 mM), ATP (1 mM), or ADO (50 μM, plus 25 μM EHNA) in incubation medium immediately after infection. Cells were incubated for 39 hours and fixed for further processing. Cells were labeled with anti-LPS (green) and DAPI (blue) for immunofluorescence microscopic analysis (left). Cells were processed by standard methods for transmission electron microscopic analysis (right). Representative images are shown for C. trachomatis-infected mock- (A and B), cAMP- (C and D), ATP- (E and F), and EHNA/ADO-exposed (G and H) cells. Immunofluorescence microscopy images represent one experiment of three independently repeated experiments with similar results. Transmission electron microscopy images represent a single experiment.
Fig 6.
cAMP, ATP and EHNA/ADO reduce the number of bacteria per inclusion.
HeLa cells were infected with C. pecorum or C. trachomatis serovar E and exposed to the DAMPs cAMP (1 mM), ATP (1 mM), or ADO (50 μM, plus 25 μM EHNA) in incubation medium immediately after infection. Cells were incubated for 35 hours (C. pecorum) or 39 hours (C. trachomatis), fixed, and processed by standard methods for TEM analysis. Total number of bacteria (EB, IB, RB, and AB) in ten inclusions per condition was determined, and mean number of bacteria per inclusion (A and B) was calculated (mean ± SD; *p ≤ 0.0001, t test; n = 10 inclusions from a single experiment). A representative image of an inclusion in mock-exposed, C. trachomatis-infected cells (C) shows EB (dark, 0.25–0.5 μm), IB (dark center and pale periphery), RB (pale, 0.5–1 μm) and AB (pale, <2 μm). Analyses of the mean relative proportion of EB, RB, IB and AB per each experimental group are shown for C. pecorum (D) and C. trachomatis (E).
Fig 7.
DAMPs reversibly reduce chlamydial infectivity in a species-specific manner.
HeLa cells were infected with C. pecorum or C. trachomatis serovar E and exposed to cAMP (1 mM), ATP (1 mM), or ADO (50 μM, plus 25 μM EHNA) in incubation medium immediately after infection and incubated for 35 hours (C. pecorum) or 39 hours (C. trachomatis). Infected monolayers were then collected and processed for titration by sub-passage. Number of inclusions was determined and inclusion forming units (IFU) per ml (IFU/ml) was calculated for C. pecorum (A) and C. trachomatis (B) (mean ± SD; *p ≤ 0.05, t test; n = 3). In some wells, incubation medium on cells was replaced with fresh incubation medium with DAMPs for continued exposure or without DAMPs for recovery. These cells were incubated a further 24 hours before processing for titration by sub-passage. Number of inclusions was determined and IFU/ml was calculated for C. pecorum (C) and C. trachomatis (D) (mean ± SD, *p ≤ 0.05, t test; values are derived from duplicate determinations within a representative experiment, and represent results obtained from two independent experiments).
Fig 8.
cAMP, ATP and EHNA/ADO reversibly reduce C. pecorum inclusion size.
HeLa cells were infected with C. pecorum (A-L) and exposed to cAMP (1 mM; D-F), ATP (1 mM; G-I), or ADO (50 μM, plus 25 μM EHNA; J-L) in incubation medium immediately after infection and incubated for 35 hours (A-C = mock exposed). At 35 hours post infection cells were fixed and labeled with anti-LPS (green) and DAPI (blue) (left). In replicate wells, incubation medium on cells was replaced with fresh incubation medium with DAMPs for continued exposure (middle) or without DAMPs for recovery (right). These cells were incubated a further 24 hours before fixation and labeling with anti-LPS and DAPI. Images represent one experiment of two independently repeated experiments with similar results.
Fig 9.
cAMP, ATP and EHNA/ADO reversibly reduce C. trachomatis inclusion size.
HeLa cells were infected with C. trachomatis serovar E (A-L) and exposed to cAMP (1 mM; D-F), ATP (1 mM; G-I), or ADO (50 μM, plus 25 μM EHNA; J-L) in incubation medium immediately after infection and incubated for 39 hours (A-C = mock exposed). At 39 hours post infection cells were fixed and labeled with anti-LPS (green) and DAPI (blue) (left). In replicate wells, incubation medium on cells was replaced with fresh incubation medium with DAMPs for continued exposure (middle) or without DAMPs for recovery (right). These cells were incubated a further 24 hours before fixation and labeling with anti-LPS and DAPI. Images represent one experiment of two independently repeated experiments with similar results.
Fig 10.
cAMP, ATP and EHNA/ADO inhibit inclusion development less dramatically when added at 14 hpi.
HeLa cells were infected with C. pecorum or C. trachomatis serovar E and exposed to 1 mM cAMP, 1 mM ATP, or 50 μM ADO plus 25 μM EHNA in incubation medium 14 hours after infection. Cells were fixed and labeled with anti-LPS (green) and DAPI (blue) at 35 hours post infection (C. pecorum) or 39 hours post infection (C. trachomatis). Number of inclusions per nucleus was determined and percent infection was calculated for C. pecorum (A) and C. trachomatis (G) (mean ± SD; p > 0.05 in all cases, t test; n = 3). Mean inclusion size was determined for C. pecorum (B) and C. trachomatis (H) (mean ± SD; *p ≤ 0.05, t test; n = 3). Representative images are shown for C. pecorum-infected mock- (C), cAMP- (D), ATP- (E), and EHNA/ADO-exposed (F) cells and for C. trachomatis-infected mock- (I), cAMP- (J), ATP- (K), and EHNA/ADO-exposed (L) cells. Immunofluorescence microscopy images represent one experiment of three independently repeated experiments with similar results.