Discovery and Characterization of Novel Virulence Factors from Pathogenic Bacteria

1.  OVERVIEW AND GLOBAL OBJECTIVES.

Recent evidence exposes an impending catastrophe involving bacterial resistance to the present regime of antibiotics and an industry pipeline that is ill-equipped to provide new compounds, especially since the emergence of multi-drug resistant bacteria (1).  In recent years, the growing bacterial drug resistance problem has dampened the enthusiasm of pharmaceutical companies in the pursuit of alternative antibacterials (1,2). The pipeline for developing novel anti-infective agents consists largely of new derivatives of a small core of antibiotic classes including β-lactams, quinolones, macrolides and glycopeptides (3). Methicillin-resistant S. aureus (MRSA) infections have reached epidemic proportions in the U.S. (4), and are spreading through sports centres, schools, and gymnasiums, affecting athletes and school children, causing death in a matter of weeks. In Canada, the high incidence of flesh-eating diseases, fatalities due to drinking water contamination (Walkerton, May 2000; 7 deaths), Listeria monocytogenes contamination at Maple Leaf Foods (Toronto, Aug 2008; 20 deaths), E. coli O157 contamination of food (500 deaths annually in the U.S.), Salmonella-induced food poisoning such as contamination of vegetables (4 million U.S. cases/y), and the emergence of multi-drug resistance bacteria (5), clearly warrants the need to search for novel antimicrobials.

Many bacterial pathogens use toxins as tools to cause infection by modifying, maiming or killing host cells.  The bacterial mono-ADP-ribosyltransferases (mARTs) are a family of protein toxins that covalently transfer ADP-ribose from NAD+ to host proteins, DNA, RNA or even antibiotics (6).  Each mART toxin modifies one or more specific host protein(s) that usually yields a unique pathology. We recently developed a tactic based on fold-recognition methods that allows us to identify prospective, new mART toxin family members from bacterial genomes (7,8) (Table 1). These new toxins can serve as targets in the development of novel therapeutics for treatment of bacterial infections and can provide important insights into the toxin-enzyme active-site architecture and host cell invasion and cytotoxic activity.

To facilitate the discovery and characterization of mART toxins, we established S. cerevisiae for toxin gene expression under the control of the Cu2+-inducible CUP1 promoter, where an active toxin causes a growth-defect phenotype in yeast (9). We use this novel yeast-based assay to validate the cytotoxicity of newly data-mined, putative mART toxins and we compare our yeast results with toxin activity against mammalian cells. We have also shown that our best lead compounds are potent inhibitors of the diphtheria toxin (DT) group (10), and also show inhibitor potency against the cholera toxin (CT) group. Previously, we discovered and characterized a novel mART from V. cholerae, which we named cholix toxin (9,11,12).  Remarkably, an immunotoxin constructed from cholix domains II and III has recently been prepared for cancer therapeutics (13). More recently, we characterized P. luminescens Photox toxin (14,15), A. hydrophila VahC toxin, and B. cereus Certhrax toxin (man. in prep).  We recently determined the crystal structures of cholix (11), VahC, and Certhrax in complex with tightly-binding inhibitors, which revealed important insights into the architecture of the mART toxin active site, the chemical requirements for an inhibitor platform, and provided critical insights into the mechanisms of host cell entry and cytotoxicity.  Thus, we are now uniquely poised to develop inhibitors of mART toxins for use as therapeutics in the treatment of bacterial pathogens and to characterize toxins from a wide-range of medically relevant bacteria.

Aim#1. Identification and characterization of new mARTs.

In order to provide novel targets for antivirulence therapy, and to reveal the phylogeny of the mART toxin family, R. Fieldhouse, Ph.D. student, developed an in silico strategy for identifying new mARTs from the sequenced genomes of pathogenic bacteria (7).  Our search strategy combines fold recognition analyses with a pattern-based primary sequence search, which reduces reliance on sequence similarity and advances us toward true structure-based mART toxin family expansion as evidenced in our recent publication (8). Our goal is to expand the mART toxin family using this innovative approach.  What makes our strategy unique and cutting edge is that we use fold-recognition searches extensively rather than relying on PSI-BLAST or secondary structure prediction.  Our genomic data mining combines pattern- and structure-based searches.  A bioiniformatic tool set allows us to discover new toxins, classify, rank them on a relative toxicity scale in yeast, and assess their structure and function. Thus, we routinely use in silico methods to probe structure, secretion, cell entry, activation, NAD+ substrate binding, target identification, intracellular target binding and reaction mechanism (7,8). We recently detailed our success in expanding the mART toxin family and its corresponding phylogenetic tree with a seminal publication (8) and our contribution to expansion of the CT-group within the mART family.

We believe a computational approach is especially justified for several reasons.  (i) Challenges in cloning, expression, purification and crystallization hampers experimental results validation, (ii) mARTs sequences become available faster than biochemical studies can be conducted, (iii) toxin family members may be key players in current bacterial outbreaks and are often excellent drug targets against antibiotic (antivirulence) resistance, and (iv) it is safer to conduct initial investigations in silico due to potential containment issues.

Our research strategy for the identification and characterization of mART toxins has finally come to fruition and is shown in Fig. 1 below.  The approach is unique in the world in that it ranges from bioinformatics (toxin discovery by data mining) to three-dimensional structure determination leading into antivirulence (drug) discovery.  It  involves several steps, including establishing a collaboration with a laboratory with expertise in the microbiology of the bacterial pathogen for characterizing the role of the associated mART in that organism as we demonstrated for cholix toxin (11,23).  We have successfully applied our bioinformatics approach (7) to identify new mART toxins from a number of pathogenic bacteria (8) and with the characterization of V. cholerae cholix toxin (11), P. luminescens Photox (13,14) and A. hydrophila VahC.

One of our long-term goals is to build the phylogenetic tree of the mART toxin family since we are only now just beginning to harvest the fruit of our labours. After several years of toiling to develop the tools to discover and characterize this toxin family, we have finally prevailed and the field is ripe for the harvest. We propose that the characterization of new, members of the mART family will greatly facilitate our understanding of the reaction mechanism, cellular activation, substrate binding, physiological targets, inhibitor versatility/relevance, and cellular intoxication mechanism(s).  Furthermore, these new mART toxins provide novel targets for antivirulence strategies against the offending bacterial pathogens (15).

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  6. Holbourn, K. P., C. C. Shone, and K. R. Acharya. 2006. A family of killer toxins. Exploring the mechanism of ADP-ribosylating toxins. FEBS J. 273:4579-4593.
  7. Fieldhouse, R. J. and A. R. Merrill. 2008. Needle in the haystack: structure-based toxin discovery. Trends Biochem. Sci. 33:546-556.
  8. Fieldhouse, R. J., Z. Turgeon, D. White, and A. R. Merrill. 2010. Cholera- and anthrax-like toxins are among several new ADP-ribosyltransferases. PLoS Comput. Biol. 6:e1001029.
  9. Turgeon, Z., D. White, R. Jorgensen, D. Visschedyk, R. J. Fieldhouse, D. Mangroo, and A. R. Merrill. 2009. Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins. FEMS Microbiol. Lett. 300:97-106.
  10. Turgeon, Z., R. Jorgensen, D. Visschedyk, P. R. Edwards, S. Legree, C. McGregor, R. J. Fieldhouse, D. Mangroo, M. Schapira, and A. R. Merrill. 2011. Newly discovered and characterized antivirulence compounds inhibit bacterial mono-ADP-ribosyltransferase toxins. Antimicrob. Agents Chemother. 55:983-991.
  11. Jorgensen, R., A. E. Purdy, R. J. Fieldhouse, M. S. Kimber, D. H. Bartlett, and A. R. Merrill. 2008. Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae. J. Biol. Chem. 283:10671-10678.
  12. Turgeon, Z., R. Jorgensen, D. Visschedyk, P. R. Edwards, S. Legree, C. McGregor, R. J. Fieldhouse, D. Mangroo, M. Schapira, and A. R. Merrill. 2010. Newly Discovered and Characterized Antivirulence Compounds inhibit Bacterial Mono-ADP-ribosyltransferase Toxins. Antimicrob. Agents Chemother.
  13. Sarnovsky, R., T. Tendler, M. Makowski, M. Kiley, A. Antignani, R. Traini, J. Zhang, R. Hassan, and D. J. FitzGerald. 2010. Initial characterization of an immunotoxin constructed from domains II and III of cholera exotoxin. Cancer Immunol. Immunother. 59:737-746.
  14. Visschedyk, D. D., A. A. Perieteanu, Z. J. Turgeon, R. J. Fieldhouse, J. F. Dawson, and A. R. Merrill. 2010. Photox, a novel actin-targeting mono-ADP-ribosyltransferase from Photorhabdus luminescens. J. Biol. Chem. 285:13525-13534.
  15. Perieteanu, A. A., D. D. Visschedyk, A. R. Merrill, and J. F. Dawson. 2010. ADP-ribosylation of cross-linked actin generates barbed-end polymerization-deficient F-actin oligomers. Biochemistry 49:8944-8954.