Chlamydia Serine Protease Inhibitor, targeting HtrA, as a New Treatment for Koala Chlamydia infection

Abstract

The koala, an iconic marsupial native to Australia, is a threatened species in many parts of the country. One major factor in the decline is disease caused by infection with Chlamydia. Current therapeutic strategies to treat chlamydiosis in the koala are limited. This study examines the effectiveness of an inhibitor, JO146, which targets the HtrA serine protease for treatment of C. pecorum and C. pneumoniae in vitro and ex vivo with the aim of developing a novel therapeutic for koala Chlamydia infections. Clinical isolates from koalas were examined for their susceptibility to JO146. In vitro studies demonstrated that treatment with JO146 during the mid-replicative phase of C. pecorum or C. pneumoniae infections resulted in a significant loss of infectious progeny. Ex vivo primary koala tissue cultures were used to demonstrate the efficacy of JO146 and the non-toxic nature of this compound on peripheral blood mononuclear cells and primary cell lines established from koala tissues collected at necropsy. Our results suggest that inhibition of the serine protease HtrA could be a novel treatment strategy for chlamydiosis in koalas.

Figure 1. C. pecorum IPTaLE and C. pecorum MarsBar one step growth curve.

C. pecorum IPTaLE and C. pecorum MarsBar were harvested at 20, 28, 36, 44, 52 and 60 h PI (as indicated on the x-axis) to measure inclusion forming units. Error bars indicate the standard error of the mean obtained from triplicate infected wells (MOI 0.3).

Figure 2. C. pecorum inclusion vacuole size shows similar morphology among isolates from different anatomical sites.

Cultures were infected at an MOI 0.3. Cultures were fixed and labelled at times indicated to the left of the figure. Chlamydia are shown in green and host cells red. Isolates examined were C. pecorum IPTaLE, C. pecorum MarsBar and C. pecorum DBDeUG.

Figure 3. JO146 inhibition during mid-replicative phase of the Chlamydia developmental cycle.

Infectious yield at 44 h PI (C. pecorum) and 72 h PI (C. pneumoniae) of isolates treated with JO146 at mid-replicative phase (MOI 0.3). Error bars indicate the standard error of the mean obtained from experimental replicates (minimum of 3). The different koala Chlamydia isolates tested were (A) C. pecorum ocular isolates; C. pecorum IPTaLE, C. pecorum AWH1, and C. pecorum AWH7, (B) C. pecorum UGT isolates; C. pecorum DBDeUG, C. pecorum MarsBar, C. pecorum AWH2, C. pecorum AWH4, C. pecorum PM11, and C. pecorum PM15, and (C) C. pneumoniae The concentration of JO146 used is indicated by grey scale shading (legend to the right). The presence of # indicates when the treatment was lethal and no inclusion forming units were detected.

Figure 4. Infectious progeny yield at completion of the chlamydial developmental cycle following 8 h treatment with JO146.

Media, JO146 (50, 100, 150 μM) and DMSO was added to C. pecorum IPTaLE and to C. pneumoniae LPCoLN cultured at an MOI of 0.3 in McCoy B and HEp-2 cells respectively, at 16 h PI and left for 8 h. All wells were then washed with pre-warmed media and further incubated. The cultures were harvested for subsequent measurement of infectious progeny at the completion of the developmental cycle (C. pecorum IPTaLE 44 h PI, C. pneumoniae LPCoLN 72 h PI). Error bars indicate the standard error of the mean obtained from triplicate infected wells.

Figure 5. Infectious yield after treatment with JO146 for extended time.

Media, JO146 (50, 100, 150 μM) and DMSO was added to (A) C. pecorum IPTaLE and to (B) C. pneumoniae LPCoLN cultured at an MOI of 0.3 in McCoy B and HEp-2 cells respectively, at 16 h PI. The cultures were harvested for subsequent measurement of infectious progeny at time points extending further than completion of their developmental cycle. Shown are mean IFU/ml with error bars indicating the standard error of the mean obtained from triplicate infected wells.

Figure 6. Chlamydial inclusion vacuole size fails to progress over time in cell culture after treatment with 150 μM JO146.

(A) Cultures treated with 150 μM JO146 or DMSO control at 16 h PI (MOI 0.3). Cultures were fixed and labelled at times indicated to the left and treatment conditions indicated are at the top. C. pecorum IPTaLE were cultured in McCoy B cells, C. pneumoniae LPCoLN in HEp-2 cells. Chlamydia are shown in green and host cells red. (B) Quantitative representation of the inclusion size of JO146 and DMSO control treatments at each time point. IFU/FOV = Average Inclusion Forming Units per Field of View ± SE (n = 18). (C) Average inclusion size ± SE where C. pecorum IPTaLE (n = 5), C. pneumoniae LPCoLN (n = 4).

Figure 7. JO146 efficacy on Chlamydia infected koala endometrial primary epithelial cells.

Uterine primary epithelial cells were extracted from the uterus of a euthanized koala and infected with C. pecorum IPTaLE, C. pecorum DBDeUG, or C. pecorum MarsBar at an MOI of 0.5 or 1.0 and treated at 16 h PI with 100 μM JO146 or DMSO control. Shown is the mean percent infectivity (at 30 h PI, or 14 h after JO146 addition) with error bars indicating the standard error of the mean obtained from triplicate infected wells. The x axis indicates the different koala Chlamydia isolates and MOI.