Despite aggressive antimicrobial treatment regimens, over 100 million persons are infected annually worldwide with the sexually-transmitted obligate intracellular bacterial pathogen, Chlamydia trachomatis. During infection, C. trachomatis secretes effector proteins into the host cytosol that interfere with host signaling networks to dampen host immune defenses. Unfortunately infections are asymptomatic in the majority of men and women, development of long-term protective immunity against reinfection is inhibited, and therefore many infections become chronic and spur secondary pathologies like infectious blindness (trachoma), pelvic inflammatory disorder, ectopic pregnancy, and infertility. The serine protease chlamydial protease-like activity factor (CPAF) has emerged as a central chlamydial virulence factor due to its importance in lipid and membrane transport, actin cytoskeleton structure and dynamics, microtubule-based motors, lysosomal recognition of the inclusion, ERK/MEK signaling pathways, and the onset of programmed cell death and inflammation signaling. Using an inhibitor-based chemical biology approach, we recently developed two classes of CPAF inhibitors, and observed that inhibition of CPAF activity inhibited bacterial replication, destabilized the integrity of the parasitophorous inclusion vacuole housing replicating chlamydia, and resulted in bacterial cell death. We also determined that CPAF inhibition reprogrammed infected cells to re-initiate host immune defense responses to infection by secreting pro-inflammatory cytokines and activating an inflammasome-dependent programmed host cell death pathway. Based on these collective data, we hypothesize that CPAF inhibitors may form the basis for a new class of small molecule 'antimicrobial vaccines'that not only kill bacteria, but also may enhance adaptive and acquired immunity responses to chlamydial infections. Our goals for this proposal are to understand the role CPAF in C. trachomatis pathogenesis, to gain insight into the molecular level details of CPAF protease function, inhibition and identification of targets, and to determine the extent to which CPAF inhibitors can clear infections, rescue host immune defense activity, and protect against reinfection in a clinically relevant murine animal model of chlamydial genital tract infection.

Public Health Relevance

Infections from the intracellular bacterial pathogen Chlamydial trachomatis affect over 100 million worldwide annually, causing serious human health problems such as pelvic inflammatory disease, ectopic pregnancy, blindness and infertility. Chlamydia live inside human cells and use secretion of effector molecules such as the protease CPAF to degrade proteins that trigger host immune defenses and to assist in bacterial growth and division, making this enzyme an important new antimicrobial and antivirulence target. In this proposal we wish to understand the role of CPAF in chlamydial infection biology, to gain insight into the molecular level details of CPAF function and identify its targets within the host cell, an examining the ability of CPAF inhibitors to slow or reverse infection progression in a mouse genital tract model of chlamydial infection.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Synthetic and Biological Chemistry B Study Section (SBCB)
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Hiltke, Thomas J
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Duke University
Schools of Arts and Sciences
United States
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Maksimchuk, Kenneth R; Alser, Katherine A; Mou, Rui et al. (2016) The Chlamydia trachomatis Protease CPAF Contains a Cryptic PDZ-Like Domain with Similarity to Human Cell Polarity and Tight Junction PDZ-Containing Proteins. PLoS One 11:e0147233
Biniossek, Martin L; Niemer, Melanie; Maksimchuk, Ken et al. (2016) Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics. Mol Cell Proteomics 15:2515-24
Kokes, Marcela; Dunn, Joe Dan; Granek, Joshua A et al. (2015) Integrating chemical mutagenesis and whole-genome sequencing as a platform for forward and reverse genetic analysis of Chlamydia. Cell Host Microbe 17:716-25
Snavely, Emily A; Kokes, Marcela; Dunn, Joe Dan et al. (2014) Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches. Pathog Dis 71:336-51