Chlamydia trachomatis recruits protein kinase C during infection

Abstract

Chlamydia trachomatis is a significant pathogen with global and economic impact. As an obligate intracellular pathogen, C. trachomatis resides inside the inclusion, a parasitophorous vacuole, and depends on the host cell for survival and transition through a biphasic development cycle. During infection, C. trachomatis is known to manipulate multiple signaling pathways and recruit an assortment of host proteins to the inclusion membrane, including host kinases. Here, we show recruitment of multiple isoforms of protein kinase C (PKC) including active phosphorylated PKC isoforms to the chlamydial inclusion colocalizing with active Src family kinases. Pharmacological inhibition of PKC led to a modest reduction of infectious progeny production. PKC phosphorylated substrates were seen recruited to the entire periphery of the inclusion membrane. Infected whole cell lysates showed altered PKC phosphorylation of substrates during the course of infection. Assessment of different chlamydial species showed recruitment of PKC and PKC phosphorylated substrates were limited to C. trachomatis. Taken together, PKC and PKC substrate recruitment may provide significant insights into how C. trachomatis manipulates multiple host signaling cascades during infection.

Keywords: Chlamydia; microdomains; phosphorylation; protein kinase C.

Figure 1.

PKC is recruited to the chlamydial inclusion and PKC inhibitors reduce C. trachomatis IFUs. (A) HeLa cells were infected with C. trachomatis for 36 hours and prepared for immunofluorescence microscopy. Phospho-PKC-pan antibody was used to detect endogenous levels of phosphorylated PKCs recruited to the chlamydial inclusion. (B) Phospho-PKC-pan recruitment was monitored over a time course of infection. Shown are 12, 24, 36 and 48 hours post-infection. (C) HeLa cells were infected with C. trachomatis L2 and treated with staurosporine (0.5 µM) or Go6893 (0.5 µM) with the inhibitors being present throughout infection. At 48 hours post-infection, the cells were lyzed to release Chlamydia and cell lysates were serially diluted and used to infect HeLa cell monolayers. Infection was allowed to proceed for 18 hours, cells were fixed with cold methanol, processed for microscopy and 30 fields of view were counted for each condition in triplicate. Error bars indicated standard deviation. Scale bar, 10 µm. *P < 0.001.

Figure 2.

Multiple isoforms of PKC are recruited to the inclusion microdomains by C. trachomatis. HeLa cells were infected with C. trachomatis L2 for 18 hours, fixed and prepared for immunofluorescence microscopy. (A) Endogenous levels of PKCα showing colocalization with active Src kinases (pY419-Src). Multiple infected and uninfected cells are shown. (B) Endogenous levels (irrespective of phosphorylation state) of PKCα, PKDδ and PKD/PKCµ were assessed for colocalization with active Src kinases (pY419-Src). (C) Phosphospecific antibodies were used to detect the different phosphorylated isoforms of PKC also colocalizing with active Src Kinases (pY419-Src). Scale bar, 10 µm.

Figure 3.

PKC phosphorylated substrates are recruited to the entire periphery of the C. trachomatis inclusion. HeLa cells were infected with C. trachomatis L2 at an MOI of 0.5 for 18 hours, fixed in cold fixative and processed for immunofluorescence microscopy. (A) Phospho (Ser)-PKC substrates are shown surrounding the chlamydial inclusion (Chlamydia detected with anti-Chlamydia LPS). Multiple infected and uninfected cells can be seen for comparison. (B) Phospho (Ser)-PKC substrates and Akt substrates were detected with phosphospecific antibodies for recruitment to the chlamydial inclusion (Chlamydia detected with anti-Chlamydia LPS). (C) Active Src kinases (pY49-Src) are shown colocalizing in discrete microdomain overlapping the phospho-(Ser)-PKC. Scale bar, 10 µm.

Figure 4.

PKC and PKC substrate recruitment are limited to C. trachomatis serovars. HeLa cell monolayers were infected with C. trachomatis serovar D (42 hours), C. trachomatis serovar B, (42 hours), C. muridarum (18 hours), C. caviae (18 hours) and C. pneumoniae (42 hours) at an MOI of ∼0.5, fixed in cold methanol and processed for immunofluorescence microscopy. (A) Total phospho-PKC recruitment as detected by a phospho-PKC-pan antibody (arrows indicate discrete regions of phospho-PKC recruitment) and (B) recruitment of phosphorylated PKC substrates are shown. All Chlamydia species were detected with anti-Chlamydia LPS antibody. Scale bar, 10 µm.

Figure 5.

PKC substrates are differentially phosphorylated during C. trachomatis infection. Chlamydia trachomatis L2-infected HeLa cell lysates with and without chloramphenicol treatment were collected at 4, 12, 24, 36 and 48 hours post-infection and probed for serine-phosphorylated PKC substrates. Mock infected HeLa lysates, C, serve as a control and total protein reference. GAPDH was used as a loading control and Hsp60 was used to detect Chlamydia. Molecular mass is shown in kilodaltons (kD).