Pkn1 kinase is a critical regulator of cell division in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular, developmentally regulated pathogen with a reduced genome as a consequence of adapting to the host cell environment. Despite possessing only eight transcription factors, its developmental cycle is tightly regulated. Hence, we hypothesize that factors like post-transcriptional and post-translational modifications play significant roles in controlling developmental progression. Chlamydia encodes serine/threonine kinases, including an ortholog of Pkn1. In this study, we characterized the function of Pkn1 in chlamydial biology and revealed its role as a cell division regulator. We designed an anhydrotetracycline (aTc)-inducible construct encoding Pkn1 tagged with six histidines at the C-terminus (Pkn1_6xH). Wild-type Pkn1 localized primarily at the division septum whereas a kinase-null Pkn1 isoform localized in the cytosol. To further explore the association of Pkn1 with the divisome, we designed a plasmid encoding an aTc-inducible CRISPRi system targeting pkn1 ( pkn1 KD). Intriguingly, decreased pkn1 transcript levels resulted in enlarged chlamydial cells with irregular coccoid shapes akin to penicillin-treated cells, indicating a block in cell division when pkn1 transcripts are reduced. These effects were complemented by expressing an additional wild-type copy, but not a kinase-null copy, of pkn1 during knockdown. Using a bacterial two hybrid system, we identified an interaction between Pkn1 and MreB, a cytoskeletal protein forming a ring at the septum in Chlamydia . We showed the co-localization of these proteins in Chlamydia and that inhibition of MreB function disrupts Pkn1 localization. Overall, our data indicate an essential function for Pkn1 activity in regulating cell division in Chlamydia .

The Serine/Threonine protein kinase superfamily is present in prokaryotes, and Pkn1 is a member of this superfamily in Chlamydia trachomatis . Chlamydial Pkn1 was previously characterized as a S/T protein kinase in vitro , but no in vivo studies had been performed to characterize its function in Chlamydia . In this study, we identified the in vivo function of Pkn1 as a regulator of chlamydial cell division. Pkn1 localized to the chlamydial division septum, and disrupting its expression blocked cell division. Our findings are important in understanding the molecular mechanism of chlamydial cell division and contribute to identifying new targeted therapeutic strategies for chlamydial infectious diseases.