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. 2023 May 1;95(5):e01826-20.
doi: 10.1128/AAC.01826-20. Epub 2021 Feb 16.

A 2-pyridone amide inhibitor of transcriptional activity in Chlamydia trachomatis

Carlos Núñez-Otero  1 , Wael Bahnan  2 , Katarina Vielfort  2 , Jim Silver  2 , Pardeep Singh  3 , Haitham Elbir  2 , Fredrik Almqvist  4 , Sven Bergström  5 , Åsa Gylfe  6
Affiliations

A 2-pyridone amide inhibitor of transcriptional activity in Chlamydia trachomatis

Carlos Núñez-Otero et al. Antimicrob Agents Chemother. .

Abstract

Chlamydia trachomatis is a strict intracellular bacterium that causes sexually transmitted infections and eye infections that can lead to life-long sequelae. Treatment options are limited to broad-spectrum antibiotics that disturb the commensal flora and contribute to selection of antibiotic-resistant bacteria. Hence, development of novel drugs that specifically target C. trachomatis would be beneficial. 2-pyridone amides are potent and specific inhibitors of Chlamydia infectivity. The first generation compound KSK120, inhibits the developmental cycle of Chlamydia resulting in reduced infectivity of progeny bacteria. Here, we show that the improved, highly potent second-generation 2-pyridone amide KSK213 allowed normal growth and development of C. trachomatis and the effect was only observable upon re-infection of new cells. Progeny elementary bodies (EBs) produced in the presence of KSK213 were unable to activate transcription of essential genes in early development and did not differentiate into the replicative form, the reticulate body (RB). The effect was specific to C. trachomatis since KSK213 was inactive in the closely related animal pathogen C. muridarum and in C. caviae The molecular target of KSK213 may thus be different in C. trachomatis or non-essential in C. muridarum and C. caviae Resistance to KSK213 was mediated by a combination of amino acid substitutions in both DEAD/DEAH RNA helicase and RNAse III, which may indicate inhibition of the transcriptional machinery as the mode of action. 2-pyridone amides provide a novel antibacterial strategy and starting points for development of highly specific drugs for C. trachomatis infections.

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Figures

FIG 1
FIG 1
Chemical structures of KSK120 and KSK213.
FIG 2
FIG 2
C. trachomatis inclusions grow with normal morphology and numbers in the presence of KSK213. (A) HeLa cells infected with C. trachomatis L2 were treated with 10 μM, 5 μM, or 2.5 μM KSK213 or just 0.1% DMSO for 45 hpi prior to fixation. The number, area, and immunostaining intensity of the Chlamydia inclusions were quantified by automated microscopy and compared to DMSO-treated controls. Bars show means, and error bars show standard deviation from three experiments. Statistical significance (*) was assessed by Kruskal-Wallis (P < 0.05) and Dunn’s posttest (P < 0.05). (B) Representative transmission electron micrographs of Chlamydia inclusions in HeLa cells treated with 10 μM KSK213 (right) or 0.5% DMSO (left) (scale bar 5 μm, top) and a closeup of the bacteria inside the inclusion (scale bar 1 μm, bottom).
FIG 3
FIG 3
C. trachomatis L2 EBs produced in the presence of KSK213 enter new HeLa cells but fail to differentiate into RBs in the first 5 h after reinfection. (A) Differential staining of attached and internalized EBs upon reinfection of cells after 0.1% DMSO or 2.5 μM KSK213 treatment. The bars show, in comparison to the DMSO control, the total number of attached and internalized EBs per cell and the fraction of internalized EBs out of the total number of EBs. Statistical significance was assessed by Mann-Whitney U test; ns, no statistical significance. (B) Representative TEM pictures of individual bacteria reinfecting cells after treatment with 0.5% DMSO (left) or 10 μM KSK213 (right) at 3 hpi (top) and 5 hpi (bottom). Gray arrows point to intermediate bodies (IBs), black arrows point to reticulate bodies (RBs), and white arrows point to elementary bodies (EBs). *, cell nuclei.
FIG 4
FIG 4
KSK213 treatment reduces early gene expression in C. trachomatis L2 upon reinfection of HeLa cells. (A) Expression of seven genes at 1 hpi normalized to 16S rRNA levels. Bars show the fold change after 10 μM KSK213 treatment compared to the 0.5% DMSO control. (B) 16S rRNA levels at 3 and 5 hpi normalized to the levels at 1 hpi. Bars show the fold change after 10 μM KSK213 treatment compared to the 0.5% DMSO control. Statistical analysis comparing KSK213 to DMSO treatment was assessed by Mann-Whitney U test. *, P < 0.05; **, P < 0.01.
FIG 5
FIG 5
Amino acid substitutions in DEAD/DEAH box helicase (HepAG571S) and RNase III (RncP177T) mediate resistance to KSK213 and cross-resistance to KSK120. (A) Dose-dependent inhibition by KSK213 in the reinfection assay with the C. trachomatis L2 strain HepAG571SRncP177T and the wild type (WT). (B) Representative transmission electron micrographs of HepAG571SRncP177T at 48 hpi. An inclusion on the left and a closeup of bacteria within the inclusion on the right. (C) Growth inhibition by doxycycline (Dox), azithromycin (Azi), and ofloxacin (Ofl) in HepAG571SRncP177T and WT. Bars show average number of inclusions as percent of the 0.5% DMSO control. (D) Inhibition of Chlamydia infectivity by 2.5 μM KSK120 in the reinfection assay with the C. trachomatis L2 strains HepAG571SRncP177T and WT. (E) Inhibition of Chlamydia infectivity by 0.1 μM KSK213 in the reinfection assay with the C. trachomatis L2 strains WT, UhpCA394T, and HepAG571SRncP177T. Bars in panels D and E show average number of inclusions upon reinfection after KSK213 treatment in percent of the 0.5% DMSO control. Error bars in all panels show standard deviation. Statistical significance was assessed by Kruskal-Wallis and Dunn’s posttest or Mann-Whitney U test in panels C, D, and E. *, P < 0.05; **, P < 0.01; ns, no statistical significance.
FIG 6
FIG 6
KSK213 reduces the infectivity of different C. trachomatis serovars but is inactive in C. muridarum and C. caviae. HeLa cells infected with C. trachomatis serovar A or D (A) or Vero cells infected with C. trachomatis L2, C. muridarum, or C. caviae (B) were incubated for 46 to 48 h (36 h for C. muridarum) prior to cell lysis and reinfection of fresh cells. Inclusion numbers upon reinfection were quantified 44 hpi and presented as a percent of a DMSO-treated control. Bars show mean, and error bars show standard deviation of three experiments.
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