Chlamydia trachomatis secretion of hypothetical protein CT622 into host cell cytoplasm via a secretion pathway that can be inhibited by the type III secretion system inhibitor compound 1

Abstract

Using antibodies raised with C. trachomatis fusion proteins, we localized a hypothetical protein encoded by the ORF ct622 in the cytoplasm of C. trachomatis-infected mammalian cells. The detection was specific since the antibody labelling of CT622 protein was removed by preabsorption with CT622 but not other fusion proteins. We similarly confirmed that CT621, a known secretion protein encoded by a hypothetical ORF downstream of ct622, was secreted into host cell cytosol. Proteins CT622 and CT621 displayed a similar secretion pattern, with both intra-inclusion and host cell cytosol localization, that was distinct from that of CPAF (chlamydial protease/proteasome-like activity factor). However, the expression and secretion kinetics differed significantly between CT622 and CT621: CT622 mRNA was detected at 2 h, protein at 6 h and secretion of protein into host cell cytoplasm at 36 h post-infection, while CT621 mRNA was detected at 8 h, protein at 16 h and secretion at 24 h. The secretion of both CT622 and CT621 was blocked by N9-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide (compound 1), an inhibitor known to target the type III secretion system of bacteria. These results suggest that CT621 and CT622 may fulfil different functions during chlamydial intracellular growth. Further characterization of these proteins may generate important information for understanding chlamydial pathogenesis.

Mutations in hemG Mediate Resistance to Salicylidene Acylhydrazides, Demonstrating a Novel Link between Protoporphyrinogen Oxidase (HemG) and Chlamydia trachomatis Infectivity

“…Subsequent studies indicate that SAHs block Chlamydia growth but not entry into cells (17)(18)(19)(20)(21), which supports the prevalent notion that the T3S is essential during the middle and late stages of the Chlamydia developmental cycle (22). Subsequent studies indicate that secretion or localization of predicted T3S effectors is altered by SAHs (19,(23)(24)(25)(26). Because the growth inhibition of Chlamydia by SAHs is reversed by exogenous iron, it has been postulated that iron chelation by SAHs may be responsible for their antichlamydial properties (27,28).…”

Identification of Antigen-Specific Antibody Responses Associated with Upper Genital Tract Pathology in Mice Infected with Chlamydia muridarum

“…Due to the difficulty in expressing the upstream fragment, the TC0912 fusion protein used in the current study was encoded by the downstream fragment alone and thus represents a homolog of CT622 or CTA0675. Interestingly, CT622 was recently found to secrete into the host cell cytosol (22). In addition, the gene coding for CTA0675 is highly polymorphic among various ocular isolates (9,25).…”

“…The internalized EB differentiates into a noninfectious but metabolically active reticulate body (RB). The RB makes new proteins, not only for organism multiplication, but also for secretion into the inclusion lumen, inclusion membrane (33,45), and host cell cytosol (5,22,24,56,62,63). After replication, the progeny RBs differentiate back into EBs to spread to nearby cells.…”

Characterization of Chlamydia trachomatis Plasmid-Encoded Open Reading Frames

“…Hoechst 33258 (blue; Sigma) was used to visualize DNA (blue). A rabbit antichlamydial organism antibody (R1L2 [25]) plus a goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 488 (green; Jackson ImmunoResearch, West Grove, PA) were used to visualize chlamydial organisms. The mouse antiGlgA (glycogen synthase A) antibody (raised with a glutathione S-transferase [GST]-GlgA fusion protein [our unpublished data]) plus a goat anti-mouse IgG conjugated with Cy3 (red; Jackson ImmunoResearch) were used to visualize GlgA.…”