A novel C. trachomatis protease is required for efficient recovery from IFNg-induced persistence

PANZETTA, MARÍA E.; LUJÁN, AGUSTÍN L.; BASTIDAS, ROBERT J.; DAMIANI, MARÍA T.; VALDIVIA, RAPHAEL H.; SAKA HÉCTOR A

Spetses

Otro; EMBO/FEBS Joint Lecture Course IUBMB Advanced School The New Microbiology; 2019

EMBO/FEBS IUBMB Advanced School The New Microbiology

Chlamydia trachomatis (CT) is the most common sexually transmitted bacterial pathogen in humans and a frequent cause of asymptomatic, long-lasting, persistent infections leading to serious complications, particularly in young women. Chlamydia displays a unique obligate intracellular lifestyle involving the infectious elementary body (EB) and the replicative reticulate body (RB). Once inside the cells, CT resides in a vacuole, termed an “inclusion”, which successfully avoids interaction with the lysosomal pathway. In the presence of stressors such as gamma-interferon (IFNg) or beta-lactam antibiotics, CT undergoes an interruption in its replication cycle and enters a viable but non-cultivable, “persistent” state, usually associated with the presence of enlarged, aberrant reticulate bodies. Upon removal of the stressors, the bacteria resume cell division and developmental transitions. CT has been considered a genetically intractable pathogen until very recently, thus the bacterial factors regulating Chlamydia persistence remain poorly elucidated. Aiming to identify the genetic determinants involved in chlamydial persistence, we carried out a high throughput screening using a collection of ~800 fully sequenced CT mutants obtained by chemical mutagenesis and identified 8 mutants with defects in recovery upon IFNg and/or penicillin-induced stress. We have focused our study on a mutant strain (M275) carrying 4 point mutations, one of them leading to a nonsense mutation in ptr, encoding a poorly characterized putative protease. After IFNγ-induced stress, M275 shows a significant decrease in progeny generation, a blockade in RB/EB transition and a slowdown in replication compared to the Wt strain. We used lateral gene transfer to map the mutation/s responsible for the persistence defect. The recombinant analysis linked the defect to either ptr or bioF (encoding a 7-keto-8-aminopelargonate synthase) mutations. Finally, through targeted insertional gene inactivation we identified ptr as a gene required for resistance to IFNg-induced stress. A ptr-null (ptr::GII) strain reproduced the persistence defect observed in M275 and this defect was restored by complementation of Ptr expression. The ptr::GII mutant displayed reduced infectious progeny generation and genome replication during recovery post IFNg-induced stress, indicating that Ptr is required for a rapid exit from persistence. By immunofluorescence with anti-Ptr antibodies developed in our lab, we observed that this rotease is secreted into the inclusion lumen. Finally, we used a murine genital tract model of infection and found impaired clearance at 14 days post-infection for ptr::GII suggesting that a reduced replication rate upon IFN removal may enhance the ability of this bacteria to establish a long-lasting infection in the uterus. Overall, our findings indicate that ptr is required for rapid recovery upon IFNg-induced stress in vitro and that lack of Ptr results in impaired clearance of C. trachomatis in mice.