Andre J. Ouellette - US grants

The goal of the proposed studies is to understand mechanisms of mucosal defense by characterizing antibacterial peptide components of mouse Paneth cells and their effects on pathogenic microorganisms. Cryptdin is one of several Paneth cell defensins, and cryptdin and other enteric defensins are hypothesized to constitute a family of antimicrobial peptides produced by these epithelial granulocytes to protect against invasion and colonization of the small bowel by bacteria. Experiments are proposed to isolate cryptdin and intestinal defensins from small bowel and determine their antibacterial activities in vitro, to characterize enteric defensin genes in Paneth cells, and to define genetic loci in Salmonella typhimurium that confer resistance to cryptdin and enteric defensins. Specifically, one series of objectives is to purify cellular and secreted forms of intestinal defensins from mouse small bowel and determine their peptide sequences, to characterize natural and synthetic intestinal defensins with regard to antimicrobial activity against virulent and avirulent strains of enteric pathogens, and to determine their cellular, sub cellular, and extracellular distribution using light and electron microscopic immunohistochemical techniques. Secondly, cDNAs corresponding to enteric defensins will be isolated and characterized, the intestinal cells that express such sequence will be identified by in situ hybridization, and the structure and organization of the murine cryptdin gene family will be defined. Thirdly, the relationship between resistance to enteric defensins and Salmonella virulence will be investigated by analyzing genetic loci of S. typhimurium that confer resistance to cryptdins, by identifying S. typhimurium phoP mutants with suppressor mutations of the cryptdin sensitive phenotype and determining the genetic basis of the reversion, by characterizing bacterial genes and proteins that confer resistance to cryptdins, and by constructing S. typhimurium strains with mutations in genes essential to cryptdin resistance. By delineating the role of enteric defensins in the mucosal barrier, the factors that regulate their expression, and specific mechanisms of resistance to cryptdins, a component of mucosal host defense will have been characterized at the level of the peptide effectors, the genes encoding the peptides, and the molecular targets of the peptides in the microorganism. Completion of these aims should provide insight into molecular mechanisms of intestinal defense against enteric bacterial pathogens.

The objective of these studies is to define the molecular basis of mucosal host defense by investigating the synthesis of endogenous antimicrobial peptides by Paneth cells. Paneth cell defensins, or cryptdins, have been implicated as components of the mucosal barrier, because they are abundant granule components with diverse primary structures, release into the lumen, and potent microbicidal proximal region of chromosome 8 that have highly-conserved, two-exons structures. Despite diverse primary structures of mature cryptdin peptides, their prepro-coding regions and propieces are almost identical, suggesting that certain residues within Paneth cell defensin precursors have roles in the packaging of cryptdin genes along the proximal-distal axis in adult and developing small intestine, to investigate precursors of potential importance in posttranslational processing. The first aim is to determine whether individual crypts differ in the expression of specific cryptdin genes; amplified cDNAs from isolated crypts along the promixmal- distal axis will be assayed for cryptdin isoform sequences by hybridization to peptide-specific oligonucleotides. Similarly, the kinetics of cryptdin isoform mRNA appearance will be determined during intestinal development in neonatal mice to test whether cryptdin genes are induced selectively or in concert. The second aim is to test whether amino acid changes in cryptdins influence or modulate biological activity; the biochemical and antimicrobial properties of recombinant peptides corresponding to naturally-occurring and mutagenized cryptdins will be characterized. The third aim will test whether cryptdin precursors contain determinants in targeting enteric defensins to Paneth cell granules; the effect of natural and mutated cryptdin-1 propieces on the distribution of chimeric reporter gene targeting will be measured in Paneth cells from isolated crypts and compared with packaging in mouse macrophage cell lines to establish whether defensin trafficking to granules occurs by conserved or cell-specific mechanisms. These studies should characterize the molecular distribution of enteric defensins, define functional determinants for biological activity, and provide knowledge of events leading to their release into the lumen.

DESCRPTION (provided by applicant):The fifth semi-annual Gordon Research Conference on Antimicrobial Peptides (AMPs) will meet in Ventura, California in March 6-11, 2005. The emergence of antibiotic-resistant microorganisms underscores the need to study of endogenous substances. AMPs are endogenous, gene-encoded antibiotics found in every species investigated, including invertebrates, plants, vertebrates, and mammals, including humans. Evidence shows that AMPs mediate innate host defense in a variety of biological settings. In addition to AMP microbicidal action by membrane disruptive mechanisms, studies show that certain mammalian AMPs also have receptor-mediated inflammation-associated activities, supporting the hypothesis that AMPs are multifunctional host defense effector molecules. Knowledge of the roles of AMPs in innate immune responses will contribute to improved understanding of the pathophysiology of human diseases, including cystic fibrosis, sexually transmitted and opportunistic infections, periodontitis, inflammatory bowel disease, and others. The conference will focus on this relatively young, rapidly expanding multidisciplinary field of study. The program will be divided into 9 sessions of oral presentations and two poster sessions. All participants will be urged to present in one of these formats. The oral presentations will include keynote addresses by Professors Michael Zasloff and Robert E. W. Hancock, 7 oral sessions (20 min lecture + 10 min discussion), and 10 short talks selected from key posters, with attention to include junior and student participants. A short oral presentation will be presented by the recipient of a newly-established Career Achievement Award. The themes of the oral sessions are: AMP Diversity and Phylogenetics, Regulation of AMP Synthesis, Mechanisms of AMP Action, Pathogenesis and Microbial Resistance Mechanisms, AMP Model Systems and Innate Immunity, Biochemical/Biophysical Parameters of AMP Action, and Microbial Responses to Antimicrobials. Ample time for organized discussion and informal interactions between participants has been included. In addition, one session, "Hot Corner and Late Breaking News" will be devoted to discussing topics at the cutting edge of current investigation. The organizational philosophy is to encourage diverse participation, and maintain an eye towards gender issues, junior scientists, minorities, and individuals with physical limitations.

DESCRIPTION (provided by applicant): The purpose of this research is to investigate the role of alpha-defensins in innate immunity of the rhesus macaque (Macaca mulatta). The goals of the proposed studies are to define the determinants of primate alpha-defensin bactericidal activity, their mechanisms of action, and to characterize mechanisms that regulate myeloid (RMADs) and Paneth cell (REDs) alpha-defensin biosynthesis at the transcriptional and posttranslational levels. We propose to quantitate the expression of RED and RMAD mRNAs and peptides in small bowel mucosa, neutrophils and NK cells, characterize and compare their mechanisms of action and the structural basis of that activity, investigate mechanisms of proRED and proRMAD activation in Paneth cells and myeloid cells, and identify cis-acting structural components of RMAD and RED genes that regulate the specificity of their transcription. In Aim #1, the microbicidal activities of REDs 1-6 will be tested against varied bacterial species, and membrane disruptive activities of natural and mutant REDs and RMADs will be characterized and quantitated. The hypothesis that mutagenesis alters peptide structure will be tested by determining NMR structures of native and mutant RED4 and RMAD4 peptides in solution and in the presence of rapidly-tumbling phospholipid bicelles to test the hypothesis that specific residue positions interact with phospholipid bilayers. In Aim #2, we will test the hypothesis that alpha-defensin precursor processing is mediated by lineage-specific proteolytic events by measuring the extent of RED and RMAD precursor processing in intact monkey small intestine and in rhesus neutrophils, NK cells, and monocytes. We will identify the respective processing enzymes, determine whether precursor proteolysis is coincident with activation of bactericidal activity, and determine the effect of mutations at conserved alpha-defensin positions on peptide folding, disulfide bond formation, activation, and membrane binding and disruption. Thus, in Aim #3, the hypotheses that primate alpha-defensin gene expression is regulated by lineage-specific, cis-acting elements will be tested by quantitating the expression and distribution of individual RED and RMAD mRNAs in enteric and myeloid tissues and by identifying determinants of lineage-specific alpha-defensin gene transcription. Potential lineage-specific gene regulatory regions will be assayed by functional analyses of RED and RMAD promoter constructs and chimeric minigenes in cultured monocytes.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To test the hypothesis that N and C terminal regions in mouse pro-alpha defensin are in proximity and the distance between these regions increases on mutagenizing the acidic residues.

DESCRIPTION (provided by applicant): The proposed studies will test the hypothesis that induced imbalance in Wnt/¿-catenin signaling results in Paneth cell depletion, dysbiosis of the resident microbiota, inflammation, enhanced susceptibility to infection by pathogenic bacteria, and that these effects can be reversed or ameliorated using a specific modulator of the Wnt/¿-catenin signaling pathway. Anatomically restricted to the small bowel, Paneth cells release ¿-defensins and host defense molecules, and maintenance of Paneth cell homeostasis is essential to small intestinal health and integrity. The Wnt signaling cascade has a critical role i gut ontogeny, and Wnt signaling mediated by ¿- catenin p300 coactivator complexes is critical for Paneth cell differentiation. ¿-catenin/CBP-mediated transcription maintains stem cell potency; in contrast, ¿-catenin/p300 transcription complexes initiate differentiation programs of more limited proliferative capacity. The primary ¿-catenin co-activators CBP and p300 share extensive similarity but interact with ¿-catenin via their distinctive N termini, which has enabled development of small molecule antagonists that specifically inhibit their association with ¿-catenin and participation in ¿-catenin-mediated transcription. For example, ICG-001 promotes Wnt-dependent differentiation by binding to CBP specifically, but not to p300, blocking CBP/¿-catenin transcription without 1) interfering with p300, 2) altering levels of ¿-catenin, but still ) allowing CBP to participate in other transcription complexes. Conversely, IQ-1 antagonizes the ¿-catenin-p300 interaction but not CBP associations, providing a means for inducing selective Paneth cell deficiency. Thus, we will test the hypothesis that antagonism of ¿- catenin/p300 signaling in mice with IQ-1 induces Paneth cell depletion and dysfunction. The impact of disrupting Paneth cell homeostasis will be determined by monitoring the time course of IQ-1 induced Paneth cell deficiency and coincident changes in expression of Paneth cell specific markers and induced inflammation evident as infiltration and translocation of myeloid and lymphoid cells. Effects of Paneth cell deficiency on the composition of the prokaryotic and eukaryotic microbiota and on barrier integrity inferred from translocation of gut bacteria in vivo will be characterized. After establishing Paneth cell deficiency, the effects of drug withdrawal and of ICG-001 administration to correct co-activator imbalance and restore Paneth cell differentiation and barrier integrity will be determined, and Paneth cell autonomous responses to IQ-1 and ICG- 001 will be determined on crypt organoids ex vivo. The effect of reestablishing Paneth cell lineage allocation and crypt homeostasis on reversing prokaryotic and eukaryotic dysbiosis also will be determined. We anticipate that IQ-1-induced Paneth cell depletion will evoke proinflammatory responses, induce dysbiosis, and enhance susceptibility to bacterial translocation and that ICG-001 will selectively enable p300/¿-catenin driven transcription to promote Wnt-dependent Paneth cell differentiation and accelerate recovery of small bowel homeostasis.

The emergence of carbapenem-resistant Enterobacteriaceae (CRE) has created a major and urgent public health threat, because carbapenems are drugs of last resort for treatment of infections caused by a growing number of multidrug resistant (MDR) bacterial pathogens. The objective of this project is to develop a novel therapeutic for treatment of CRE infections by capitalizing on the unique host defense-modulating properties of macrocyclic theta (?)-defensins in animal models of infection. Preliminary studies provide evidence that ?- defensins and engineered analogs can be developed as agents to treat carbapenemase-producing Klebsiella pneumoniae (KPC-Kp). Pharmacokinetic (PK) studies and analyses of cytokine responses in ?-defensin- treated septic mice demonstrate that these novel cyclic peptides augment host responses to bacteria by modu- lating the release of proinflammatory mediators and attenuating NF?B activation, enhancing host defense in a manner independent of the pathogen?s antibiotic resistance profile. In Aim 1, we will identify a lead therapeutic peptide (LTP) from a series of macrocyclic peptides bioinspired by ?-defensins. Criteria for selection of the LTP will include a) efficacy in the mouse KPC-Kp sepsis model, b) peptide solubility and stability, c) lack of acute toxicity, d) ease of synthetic production, e) extent of bacterial burden reduction in vivo, f) pharmacody- namic (PD) response of additional correlative biomarkers, g) dose-dependent efficacy in a second infection model, and h) single-dose PK. Since half of the lead series of peptides is already in hand, down selection to the LTP can be completed rapidly and certainly within the mandated two-year period. In Aim 2, we will perform single and multiple-dose PK of injectable LTP in mice and NZW rabbits, and use 14C-labeled LTP to define peptide absorption, distribution, metabolism, and excretion (ADME) in mice. Preliminary toxicity studies will be performed to determine maximum tolerated dose in mice. In Aim 3, we propose to express a precyclic-LTP in E. coli using the pET-28a system; the precyclic product is efficiently cyclized in vitro with the recently discov- ered cycloconvertase that produces ?-defensins in vivo. We will also produce cyclic LTP using a robust re- combinant expression system in planta, in collaboration with one of the project?s commercial partners. These studies will capitalize on recent success in producing fully cyclized ?-defensin in tobacco leaf. Collectively, the results of Aim 3 studies will provide a system for efficient, low cost production of the LTP. The significance of the project lies in its promise for development of a unique peptide-based therapeutic for treatment of CRE in- fections. We hypothesize that peptide-based immunomodulatory therapeutics will offer advantages for CRE treatment over agents that seek to target a particular resistance component or function solely as microbicides, representing an innovative approach. Based on published work, existing preliminary data, resources already in place, and established corporate partnerships, we believe the proposed research plan has great potential to introduce a paradigm changing approach for treating CRE and other MDR pathogens.