The major objectives of this Sexually Transmitted Infections Co-operative Research Center (STI-CRC) entitled """"""""Innate and Adaptive Immunity in Experimental and Human Gonococcal Infection in Women"""""""" are directed to providing a diverse and comprehensive understanding of the innate and adaptive immune mechanisms associated with gonococcal infection. We will emphasize basic host responses and innate immune mechanisms in infection with Neisseria gonorrhoeae with the immediate aim of improving understanding of gonococcal immunology and pathogenesis and a longer term goal to gain insights that will direct efforts to facilitate the prevention of gonococcal infections in humans, particularly women. We will use a promising animal model to examine these mechanisms and separately employ a generalized translational approach that will emphasize the links between immunobiology and clinical epidemiology to assess the potential of vaccine candidates in humans, which will then be investigated in the context of the humanized mouse model of infection. Five research projects and four service cores are proposed. In the first project (Douglas T Golenbock, MD, PL), we will address a basic and fundamental question in lipopolysaccharide (LPS, called lipooligosaccharide [LOS] in the case of N. gonorrhoeae) biology to understand the binding of LOS derived lipid A with its direct ligand that serves as an intermediary structure to activate and permit signaling of toll-like receptor (TLR)4. In Project 2 (Robin Ingalls, MD, PL), we will determine if naturally occurring mutations in gonococcal LOS or polymorphisms in the TLR4 adaptor Mai, account for differences in the host inflammatory response to infection. In Project 3 (Caroline A Genco, PhD, PL), we will examine the role that TLRs and cytosolic nucleotide oligomerization domain (NOD) protein receptors (NOD-like receptors [NLRs]) play as receptors for Neisseria ligands. In Project 4 (Sanjay Ram, MD, PL), we will examine novel roles for the alternative complement pathway (ACP) enhancing molecule. Properdin, in blocking TLR4 mediated signaling by LOS and separately, in amplifying potentially protective vaccine induced antibody function. In Project 5 (Peter A. Rice, MD), we will examine peptide mimics of gonococcal LOS as potential vaccine candidates in a humanized mouse model of gonococcal infection while also determining if natural antibodies against the candidates protect exposed women from gonococcal infection. The Center will have five Cores (Clinical, Laboratory, Animal Statistical, and administrative) providing support to 4 or 5 of the 5 Projects. PROJECT 1: THE TLR4/MD-2 SIGNALING PATHWAY IN GONOCCOCAL DISEASE (Golenbock, D) PROJECT 1 DESCRIPTION (provided by applicant): Neisseria gonorrhoeae causes a variety of disease syndromes including pelvic inflammatory disease [PID]. PID can lead to chronic pelvic pain, ectopic pregnancy and infertility. Those syndromes caused by N. gonorrhoeae have, in common, intense inflammation mediated by inflammatory cells. This inflammation is primarily the result of the interaction of Neisserial LPS (LOS) with the LPS receptor complex: TLR4 and MD- 2. The components of the LPS receptor were identified a decade ago, yet it is still poorly understood, 1) how the binding of lipid A to MD-2 results in the formation of an active receptor complex, and, 2) how a signal is subsequently transmitted resulting in the production of proinflammatory mediators such as TNFa and IL-1?. In this proposal, we describe plans to determine how MD-2, once bound to lipid A, acquires the ability to activate TLR4. The approach builds upon our success in purifying MD-2, a small molecule with 7 cysteine residues that has a notorious tendency to form inactive multimers. We plan to resolve the structure of MD-2 in the absence and presence of activating ligand, and in the presence of TLR4 to determine what conformational changes in TLR4/MD-2 induce signaling. We shall then focus our energies on TLR4-related adapter molecules involved in cell signaling. We have previously analyzed 5 single nucleotide polymorphisms (SNPs) in the adapter protein known as Mai (used by both TLR2 and TLR4). Two SNPs are of great interest: S180L and D96N. As part of another NIH funded project, we have begun to generate mice carrying these lesions and are screening patient samples for the presence of D96N. Mai knock out mice and knock-in mice carrying the mouse equivalent of D96N or S180L will be tested in the mouse model of GC infection by Dr. Ingalls (PL, project 2). We will perform similar molecular genetic studies of the 6 known SNPs in MyD88, the downstream adapter that interacts with Mai and an important adapter for at least 8 of the TLRs. Should any of the SNPs display a phenotype, we will generate knock-in mice and screen patient samples to determine relevancy. Finally, we will attempt to define the interaction of Mai and MyD88 by biochemical means, culminating in an attempt to co crystallize the Mal/MyD88 dimer.
Gonorrhea is a common sexually transmitted infection worldwide. Women usually have few or no symptoms associated with infection, which often leads to delays in treatment and the development of complications such as pelvic inflammatory disease and infertility and increased likelihood of acquiring HIV infection. A better understanding of natural immunity to this pathogen will lead to treatments that can lessen the infectious complications and support the development of protective vaccine strategies.
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