The Chlamydia trachomatis type III secretion substrates CT142, CT143, and CT144 are secreted into the lumen of the inclusion

Abstract

Chlamydia trachomatis is a human bacterial pathogen causing ocular and genital infections. It multiplies exclusively within an intracellular membrane-bound vacuole, the inclusion, and uses a type III secretion system to manipulate host cells by injecting them with bacterially-encoded effector proteins. In this work, we characterized the expression and subcellular localization in infected host cells of the C. trachomatis CT142, CT143, and CT144 proteins, which we previously showed to be type III secretion substrates. Transcriptional analyses in C. trachomatis confirmed the prediction that ct142, ct143 and ct144 are organized in an operon and revealed that their expression is likely driven by the main σ factor, σ66. In host cells infected by C. trachomatis, production of CT142 and CT143 could be detected by immunoblotting from 20–26 h post-infection. Immunofluorescence microscopy of infected cells revealed that from 20 h post-infection CT143 appeared mostly as globular structures outside of the bacterial cells but within the lumen of the inclusion. Furthermore, immunofluorescence microscopy of cells infected by C. trachomatis strains carrying plasmids producing CT142, CT143, or CT144 under the control of the ct142 promoter and with a C-terminal double hemagglutinin (2HA) epitope tag revealed that CT142-2HA, CT143-2HA or CT144-2HA showed an identical localization to chromosomally-encoded CT143. Moreover, CT142-2HA or CT144-2HA and CT143 produced by the same bacteria co-localized in the lumen of the inclusion. Overall, these data suggest that the CT142, CT143, and CT144 type III secretion substrates are secreted into the lumen of the inclusion where they might form a protein complex.

Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to InvasiveChlamydia trachomatisBiovars

“…Staining patterns varied, ranging from small punctate particles to larger particles with outer membrane-like morphologies. Similar staining patterns have been reported by others for CT143 (28) and for plasmid-regulated chromosomal loci CT049 and CT50 (26). It is not understood why plasmid-regulated proteins exhibit atypical inclusion staining properties.…”

“…(27,28), CT702 (hypothetical) (19), and CT412 (PmpA) (19). Importantly, the genes corresponding to these proteins were also found to be the most significantly downregulated genes transcriptionally in C. trachomatis L2 PϪ compared to Pϩ (5,18).…”

“…CT142 to -144 are encoded in an operon (28). It has been suggested that CT142, -143, and -144 might form a protein complex (28).…”

“…It has been suggested that CT142, -143, and -144 might form a protein complex (28). In contrast to CT049 to -051, CT142 and CT143 are highly conserved among C. trachomatis isolates.…”

The Human Centrosomal Protein CCDC146 Binds Chlamydia trachomatis Inclusion Membrane Protein CT288 and Is Recruited to the Periphery of the Chlamydia-Containing Vacuole

“…Once established, the transformed strains were cultured in the presence of 10 U/ml of penicillin, or 500 μg/ml of spectinomycin, and plaque purified using Vero cells, as described (Nguyen and Valdivia, 2013 ). Infection of mammalian cells by C. trachomatis and quantification of infection progeny was done as previously described (da Cunha et al, 2017 ).…”