Identification of MAVS splicing variants that interfere with RIGI/MAVS pathway signaling.

The mitochondrial anti-viral signaling protein (MAVS), also known as CARDIF, IPS-1, KIAA1271 and VISA, is a mitochondria associated protein that regulates type I interferon production through coordinated activation of NF-kappaB and IRF3. The N-terminal CARD domain of MAVS interacts with RIGI helicase of upcapped RNA detection and the putative TRAF2 and TRAF6 binding motifs modulate protein interaction for NF-kappaB activation. MAVS is encoded by a single gene composed of 6 exons but is generally detected as multiple protein bands after separation by SDS-PAGE. In an effort to identify MAVS variants with diverse biological functions, we isolated three splicing variants and named them MAVS 1a (exon 2 deletion), 1b (exon 3 deletion) and 1c (exon 6 deletion), respectively. MAVS 1a and 1b, due to a frame shift by exon deletion, encode 131 and 124 aa residues, respectively. Except the first 39 aa residues encoded by exon 1, MAVS 1a does not share sequence homology with known proteins, it instead contains a putative TRAF2-binding motif and interacts with TRAF2 and RIP1. MAVS 1b shares the first 97 residues with wt MAVS and 27 aa residues of unknown protein. Unlike MAVS that activates both NF-kappaB and IRF3 pathways, expression of MAVS 1b selectively activates an IFNbeta but not an IL8 promoter. MAVS 1b interacts with RIP1 and FADD and exhibits anti-viral activity against VSV infection. This study uncovers MAVS splicing variants of diverse biological function.

Chlamydial CT441 is a PDZ domain-containing tail-specific protease that interferes with the NF-kappaB pathway of immune response.

Chlamydia species are bacterial pathogens that affect over 140 million individuals worldwide. Ocular infection by Chlamydia trachomatis is the leading cause of preventable blindness, and urogenital tract infection by Chlamydia causes sexually transmitted disease. As obligate intracellular organisms, Chlamydia species have evolved mechanisms to evade the host immune system, including the degradation of the transcription factors regulatory factor X5 and upstream stimulation factor 1, which are required for the expression of major histocompatibility complex molecules I and II by CPAF and cleavage of p65 of the NF-kappaB pathway by the encoded CT441 protein. Here, we report the characterization of CT441 as a tail-specific protease. CT441 contains a PDZ domain of protein-protein interactions and a Ser/Lys dyad catalytic unit. Mutation at either Ser455 or Lys481 in the active site ablated CT441 activity of p65 cleavage. In addition, we found that the production of CT441 Tsp, which was detected at the middle and late stages of an infection, correlated with p65 cleavage activity. In addition to high homology, human and mouse p65 proteins also contain an identical C-terminal tail of 22 amino acid (aa) residues. However, only human p65 was susceptible to cleavage. Using molecular biology approaches, we mapped the p65 cleavage site(s) to a region that differs from that of mouse p65 by 6 aa residues. Additionally, the substitution of T352 with a proline inhibited p65 cleavage. Together, the study demonstrates that CT441 is a tail-specific protease that is capable of interfering with the NF-kappaB pathway of host antimicrobial and inflammatory responses.

Cleavage of p65/RelA of the NF-kappaB pathway by Chlamydia.

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-kappaB pathway of host inflammatory response. We found that Chlamydia infection did not promote IkappaBalpha degradation, a prerequisite for NF-kappaB 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-kappaB 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-kappaB 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.

Activation of lipid metabolism contributes to interleukin-8 production during Chlamydia trachomatis infection of cervical epithelial cells.

Chlamydia trachomatis infection is the most common cause of bacterial sexually transmitted diseases. Infection of the urogenital tract by C. trachomatis causes chronic inflammation and related clinical complications. Unlike other invasive bacteria that induce a rapid cytokine/chemokine production, chlamydial infection induces delayed inflammatory response and proinflammatory chemokine production that is dependent on bacterial growth. We present data here to show that the lipid metabolism required for chlamydial growth contributes to Chlamydia-induced proinflammatory chemokine production. By gene microarray profiling, validated with biochemical studies, we found that C. trachomatis LGV2 selectively upregulated PTGS2 (COX2) and PTGER4 (EP4) in cervical epithelial HeLa 229 cells. COX2 is an enzyme that catalyzes the rate-limiting step of arachidonic acid conversion to prostaglandins, including prostaglandin E2 (PGE2) and other eicosanoids, whereas EP4 is a subtype of cell surface receptors for PGE2. We show that Chlamydia infection induced COX2 protein expression in both epithelial cells and peripheral blood mononuclear cells and promoted PGE2 release. Exogenous PGE2 was able to induce interleukin-8 release in HeLa 229 epithelial cells. Finally, we demonstrated that interleukin-8 induction by Chlamydia infection or PGE2 treatment was dependent on extracellular signal-regulated kinase/mitogen-activated protein activity. Together, these data demonstrate that the host lipid remodeling process required for chlamydial growth contributes to proinflammatory chemokine production. This study also highlights the importance of maintaining a balanced habitat for parasitic pathogens as obligate intracellular organisms.

Up-regulation of the JAK/STAT1 signal pathway during Chlamydia trachomatis infection.

Chlamydia trachomatis infection is the most common cause of sexually transmitted disease, leading to female pelvic inflammatory disease and infertility. The disease process has been linked to cellular response to this bacterial pathogen. This obligate intracellular pathogen infects macrophages, fibroblast cells, and epithelial and endothelial cells. We show in this study that infection of cervical epithelial cells, the primary target of Chlamydia trachomatis, leads to up-regulation and activation of the JAK/STAT signal pathway. Specifically, Chlamydia trachomatis infection of HeLa 229 cells selectively induces STAT1, STAT2, and IFN-stimulated transcription factor 3gamma expression and promotes STAT1 activation. The up-regulation of STAT1 is dependent on bacterial replication, because treatment of infected cells with antibiotics prevents STAT1 up-regulation. By analysis of the gene transcriptional and cytokine expression profiles of host cells combined with the use of neutralizing Abs, we show that IFN-beta production is critical for STAT1 induction in epithelial cells. Finally, we demonstrate that the host up-regulates STAT1 to restrict bacterial infection, because Chlamydia propagates more efficiently in STAT1-null or STAT1 knockdown cells, whereas Chlamydia growth is inhibited in cells with up-regulated STAT1 expression. This study demonstrates that the infected cells up-regulate the host innate antimicrobial response to chlamydial infection. It also highlights the importance of cellular response by nonimmune cells in host clearance of chlamydial infection.