Cleavage of p65/RelA of the NF-κB pathway by Chlamydia

Abstract

Chlamydia trachomatis is a bacterial pathogen that infects the eyes and urogenital tract. Ocular infection by this organism is the leading cause of preventable blindness worldwide. The infection is also a leading cause of sexually transmitted disease in the United States. As obligate intracellular pathogens, chlamydiae have evolved sophisticated, yet undefined, mechanisms to maintain a favorable habitat for intracellular growth while avoiding harm to the host. We show here that chlamydiae have the ability to interfere with the NF-B pathway of host inflammatory response. We found that Chlamydia infection did not promote I B␣ degradation, a prerequisite for NF-B nuclear translocation/activation, nor induce p65/RelA nuclear redistribution. Instead, it caused p65 cleavage into an N terminus-derived p40 fragment and a p22 of the C terminus. The activity was specific because no protein cleavage or degradation of NF-B pathway components was detected. Moreover, murine p65 protein was resistant to cleavage by both human and mouse biovars. The chlamydial protein that selectively cleaved p65 was identified as a tail-specific protease (CT441). Importantly, expression of either this protease or the p40 cleavage product could block NF-B activation. A hallmark of chlamydial STD is its asymptomatic nature, although inflammatory cellular response and chronic inflammation are among the underlying mechanisms. The data presented here demonstrate that chlamydiae have the ability to convert a regulatory molecule of host inflammatory response to a dominant negative inhibitor of the same pathway potentially to minimize inflammation.CT441 ͉ inflammation ͉ protease

Chlamydial CT441 Is a PDZ Domain-Containing Tail-Specific Protease That Interferes with the NF-κB Pathway of Immune Response

“…Therefore, both CT823 and CT858 proteins were expected to exhibit a different substrate specificity from that of CT441 even though the substrate for CT823 is unknown. Indeed, neither protein displayed p65 cleavage activity (14). Instead, CT858 was reported to degrade proteins, including the RFX5 and USF-1 transcription factors.…”

“…Constructs for the expression of HAtagged CT441, CT823, and CT858 as mature peptides were described previously (14). A standard PCR protocol was used to generate CT441 S455A and K481T.…”

“…The cells were lysed 24 h posttransfection, and protein expression and cleavage of the endogenous p65 protein were detected by Western blotting analyses. Alternatively, the cell lysates were used as substrates in in vitro protein cleavage assays as described previously (14).…”

Structural Basis of the Proteolytic and Chaperone Activity of Chlamydia trachomatis CT441

“…For SRAP1 cleavage by recombinant CT441, purified SRAP1 (6.25 g) was incubated with CT441 (5.2 g) at 37°C or 4°C. Samples incubated at 37°C were collected after 5 min, 10 min, 30 min, 1 h, 2 h, and 4 h; samples incubated at 4°C were collected after 6 h and 24 h. As a control, CT441°w as incubated with SRAP1 for 4 h or 24 h. For SRAP1 production in eukaryotic host cells, HEK293 cells were transiently transfected with a SRAP1-encoding plasmid (4 g/well; Origene) 24 h after seeding, using TurboFect (Thermo Scientific) in transfection medium (Opti-MEM), followed by medium exchange to RPMI 1640 (5% FCS) 5 . Intracellular localization of proteins was visualized using the primary antibodies mouse anti-CT441 (1:1,000; provided by G. Zhong) and rabbit anti-SRAP1 (1:250; Santa Cruz), as well as the corresponding Cy5-donkey anti-mouse (1:250, Cell Signaling) and FITC-goat anti-rabbit (1:100; Cell Signaling) secondary antibodies.…”

“…A second chlamydial protease, designated CT441 for C. trachomatis, shares significant amino acid sequence similarity with tailspecific proteases (Tsps) from other species (e.g., 25% identity with Tsp from Escherichia coli) and was first proposed by Lad et al to interfere with host antimicrobial and inflammatory responses (5,6); however, in later reports, a role of CT441 and CPAF in the cleavage of NF-B during the chlamydial infection has been put into question (4,7). Unique regions that show no similarities to any characterized domain are present at the N terminus of Tsp proteins.…”