Project Leader:

Dr. Jo-Anne Dillon

View a U of S video of Dr. Dillon

Overview
Bacterial cell division and growth are interrelated and have implications for the organisms’ ability to cause infections. Our laboratory investigates the mechanisms of round (coccal) bacterial division, the regulation of cell division genes and the association of cell division with pathogenicity, using Gram-negative Neisseria gonorrhoeae and Gram-positive Enterococcus faecalis as models. Neisseria gonorrhoeae is an exclusively human pathogen which causes over 62 million new infections (gonorrhoea) each year and for which there are no vaccines to control its spread. E. faecalis is an opportunistic human pathogen causing nosocomial (i.e., in-hospital) infections and is an important veterinary pathogen with significant economic impact. Both N. gonorrhoeae and E. faecalis can develop resistance to all antibiotics used for treatment. Thus, the investigation of the cell biology of each organism with an emphasis on cell division may lead to new insight for the exploitation of non-traditional antimicrobial targets. As part of a national and international research collaboration The International Collaboration on Gonococci (ICG), our laboratory is also working to reveal the molecular mechanisms of antimicrobial resistance in N. gonorrhoeae and its molecular epidemiology.

Background:

Bacterial cell division occurs through a number of sequential processes with the involvement of multiple proteins. These processes include the selection of mid-cell sites, the assembly of a protein ring (the FtsZ-ring) at the cell centre and subsequent recruitment of a protein cascade (divisome) leading to complete separation to produce two equal daughter cells. Our laboratory has pioneered the study of cell division in Neisseria gonorrhoeae, a round Gram-negative microorganism that divides in alternating perpendicular planes, unlike rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, which divide in parallel planes. Using an array of methods including genomics, bioinformatics, biochemistry, structural modeling, molecular microbiology and fluorescence microscopy, we identified and characterized the gene organization of both the division cell wall (dcw) cluster as well as the min cluster which includes the min genes- minC, minD and minE, responsible for mid-cell site selection. How round cells identify the mid-cell and initiate the division pathway cascade is complex. Many key cell division proteins move within the cell and contribute to bacterial subcellular architecture. Using targeted mutagenesis to alter structure, we have been able to ascertain important functional domains on the Min proteins. We will continue to study the structure/function relationships of proteins involved in cell division in N. gonorrhoeae.

Gram-positive bacteria possess different division machinery components. For example, B. subtilis lacks the MinE homologue although it contains MinC and MinD. Instead, a protein (DivIVA) replaces MinE and acts with MinC and MinD to specify the division site in B. subtilis. E. faecalis possesses DivIVA but lacks any Min proteins. DivIVA from E. faecalis is essential and plays important roles in chromosomal segregation. A novel DivIVA binding protein in E. faecalis has also been identified in our laboratory. Current studies are focusing on the role of the DivIVA-related protein complex in the cell division mechanisms in E. faecalis and B. subtilis.

Studies on the association of cell division with pathogenicity are in their conceptual infancy. Cell division and growth are interrelated and have implications for bacterial invasiveness, as one of the main requirements for pathogenicity is the ability to grow in a host. It has been determined that an increase in gonococcal growth rate enhances pathogenicity. Studies with Salmonella typhimurium and the uropathogenic Escherichia coli indicated that the host cell response induced filamentous growth, an indicator of cell division arrest. Thus, we are investigating various cell division mutants of N. gonorrhoeae for properties of pathogenicity and their responses to the host defense system.

Gonorrhoea infections cause a heavy public health burden worldwide due to the high prevalence of the pathogen causing this disease, N. gonorrhoeae, and its association with HIV transmission and resistance to commonly used antibiotics. Gonorrhoea control methods comprise correct diagnosis and effective treatment coupled with epidemiological strategies to block transmission. However, the inexorable development of resistance in N. gonorrhoeae has become a major and increasing problem in effective treatment and control. The bacteria’s global distribution and the high prevalence of resistance to penicillins, erythromycin, tetracyclines and quinolones has rendered these drugs useless for the treatment of gonorrhoea; third-generation cephalosporins are the only antimicrobials effective today. Noticeably, gonococcal isolates with reduced susceptibility to third-generation cephalosporins have recently been reported. The antimicrobial resistance of N. gonorrhoeae to various antibiotics is associated with mutations in various alleles.

Transmission patterns of gonorrhoea can be determined using methods of epidemiology and molecular epidemiology. Various molecular tools have been developed to investigate gonococcal strain transmission, such as porB-based sequence analysis and the Neisseria gonorrhoeae-multi-antigen sequence typing (NG-MAST). Such molecular epidemiology analyses can disclose sexual networks unrevealed in traditional epidemiology and partner tracing. The understanding of the epidemiological and microbiological determinants for gonorrhoea spread can influence policy development regarding more effective interventions

Objectives:

1. To investigate molecular mechanisms of cell division in N. gonorrhoeae and E. faecalis.
2. To characterize the role of cell division mutations on the regulation of gene expression and bacterial pathogenicity.
3. To determine molecular mechanisms of antimicrobial resistance in N. gonorrhoeae and transmission patterns of sexually transmitted infections by molecular epidemiology.
   

Progress:

An overview of our recent findings:

1. N. gonorrhoeae (Ng) determines its mid-cell sites through the Min system and nucleoid occlusion. Acting as cell division inhibitors, MinC interacts with MinD. The N-terminus of MinCNg mediates interaction with FtsZNg but does not affect interaction with MinDNg or homodimerization of MinCNg (Greco-Stewart et al 2007). The C-terminus of MinENg acts as a topological specificity factor by modulating MinD activity in bacterial cell division and by determining the dynamics of the MinCD complex (Eng et al 2006, Ramirez-Arcos et al 2002). A conserved polar region in the cell division site determinant MinD is required for responding to MinE-induced oscillation but not for localization within coiled arrays (Szeto et al 2005). The N-terminus of MinD contains determinants which affect its dynamic localization and enzymatic activity (Szeto et al 2004).
   
   
2. DivIVA is an essential protein in E. faecalis (Ef) and plays a pivotal role in proper cell division and chromosome segregation (Ramirez-Arcos et al 2005). DivIVAEf self-interacts forming a complex in vitro comprising 10-12 monomers. The coiled-coil structures in this protein are important for its oligomerization and biological function (Rigden et al 2008).
   
   
3. An international research collaboration on the antimicrobial susceptibility of N. gonorrhoeae has been carried out in South America and the Caribbean (Dillon et al 2006) and China (Yang et al 2006). Mutations in quinolone-resistant determinants of gyrA and parC in N. gonorrhoeae isolates from Shanghai have been identified (Yang et al 2006). Circulating clusters of N. gonorrhoeae isolates have been characterized by porB sequence analysis. porB DNA sequence typing has been sufficiently discriminatory to differentiate N. gonorrhoeae isolates and is congruent with epidemiological linkages. Novel porB sequences of N. gonorrhoeae and novel mutations of PorB proteins have been identified (Liao et al 2008).
   

Selected publications : ________________________________________________ VIDO publications

1. M. Liao, K. Bell1, W-M Gu, Y. Yang, N.F. Eng, W. Fu, L. Wu, C-G Zhang, Y. Chen, A.M. Jolly, and J.R. Dillon. 2008. Clusters of circulating Neisseria gonorrhoeae strains and association with antimicrobial resistance in Shanghai. J. Antimicrob. Chem. In press January 2008, paper ID: DKM544.
2. M.D. Rigden, C. Baier, S. Ramirez-Arcos, M. Liao, M. Wang and J.R. Dillon. 2008. Identification of the coiled-coil domains of Enterococcus faecalis DivIVA that mediate oligomerization and their importance for biological function. J. Biochem. Manuscript ID: JB-07-10-0380. Under final revision.
3. Greco-Stewart, V. S. Ramirez-Arcos, M. Liao and J.R. Dillon. 2007. N-terminus determinants of MinC from Neisseria gonorrhoeae mediate interaction with FtsZ but do not affect interaction with MinD or homodimerization. Arch. Microbiol. 187: 451-458.
4. Yang Yang, Mingmin Liao, Wei-Ming Gu, Kelli Bell, Lei Wu, Nelson F. Eng, Chu-Guang Zhang, Yue Chen, Ann M. Jolly, and Jo-Anne R. Dillon. 2006. Antimicrobial susceptibility and molecular determinants of quinolone resistance in Neisseria gonorrhoeae isolates from Shanghai. J. Antimicrob. Chemother. 58: 868-872. 
5. Dennis Ramos, Thierry Ducat, Jenny Cheng, Nelson F. Eng, Jo-Anne R. Dillon and Natalie K Goto. 2006. Conformation of the cell division regulator MinE: evidence for interactions between the topological specificity and anti-MinCD domains. Biochem. 45(14):4593-4601.
6. Eng, N.F., Szeto, J., Acharya, S., Tessier, D., and J.R. Dillon. 2006. The C-terminus of MinE from Neisseria gonorrhoeae acts as a topological specificity factor by modulating MinD activity in bacterial cell division. Res. Microbiol. 157(4):333-344.
7. J.R. Dillon, Ruben, M., H. Li, Borthagaray, G., Marquez, C., Fiorito, S., Galarza, P., Portilla, J.L., Leon, L., Agudelo CI, Sanabria OM, Maldonado A, and Prabhakar P. 2006. Challenges in the control of gonorrhea in South America and the Caribbean: monitoring the development of resistance to anitibiotics. Sex. Trans. Dis. 33(2):87-95.
8. Liao M., P.S. Ruddock, A.S. Rizvi, S.H. Hall, F.S. French, and J.R. Dillon. 2005. A cationic peptide of the male reproductive tract, HE2α, displays potent antimicrobial activity against Neisseria gonorrhoeae, Staphylococcus aureus, and Enterococcus faecalis. J. Antimicrob. Chemother. 56: 957-961.
9. Ruddock, P.S., M. Liao, B. Foster, L. Lawson, J.T. Arnason and J.R. Dillon. 2005. Garlic natural health products exhibit variable constituent levels and antimicrobial activity against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis. Phytother. Res. 19:327-334.
10. Szeto, J., N.F. Eng, S. Acharya, M. Rigden, and J.R. Dillon. 2005. A conserved polar region in the cell division site determinant MinD is required for responding to MinE-induced oscillation but not for localization within coiled arrays. Res. Microbiol. 156: 17-29.
11. Ramirez-Arcos, S., M. Liao, S. Marthaler, M. Rigden, and J.R. Dillon. 2005. Enterococcus faecalis divIVA: an essential gene involved in cell division, cell growth, and chromosome segregation. Microbiol. 151: 1381-1393.
12. Szeto, J., Acharya, S., Eng, N.F., and J.R. Dillon. 2004. The N-terminus of MinD contains determinants which affect its dynamic localization and enzymatic activity. J. Bacteriol. 186: 7175-7185.
13. Ramirez-Arcos, S., V. Greco, H. Douglas, D. Tessier, D. Fan, J. Wang, J. Szeto and J.R. Dillon. 2004. Conserved glycines in the C-terminus of MinC mediate protein are implicated in their functionality as cell division inhibitors. J. Bacteriol. 186: 2841-2855.
14. Greco, V., L.-K. Ng, R Catana, H. Li, and J.R. Dillon. 2003. Molecular epidemiology of tetracycline resistant Neisseria gonorrhoeae (TRNG) in Canada: temporal and geographical trends and prevalence from 1986 to 1997. Microb. Drug Resist. 9:353-360
15. Sosa, J., S. Ramirez-Arcos, M. Ruben, H. Li, R. Llanes, A. Llop, and J.R. Dillon. 2003. High percentages of resistance to tetracycline and penicillin and reduced susceptibility to azithromycin characterize the majority of strain types of Neisseria gonorrhoeae isolates in Cuba, 1995-1998. Sex. Trans. Dis. 30:443-448.
16. Ramirez-Arcos, S., J. Szeto, J.R. Dillon and W. Margolin. 2002. Conservation of dynamic localization among MinD and MinE orthologs: Oscillation of Neisseria gonorrhoeae proteins in Escherichia coli. Mol. Microbiol. 46: 493-504.
17. Márquez, C.,J.R. Dillon, V. Rodriguez and G. Borthagaray. 2002. Detection of a novel tetM determinant in tetracycline-resistant Neisseria gonorrhoeae from Uruguay, 1996-1999. Sex. Trans. Dis. 29:792-797.
18. Hill, S.A., D. S. Samuels, C. Nielsen, S.W. Knight, F. Pagotto and J.R. Dillon. 2002. Integration host factor interactions with Neisseria gene sequences: Correlation between predicted binding sites and in vitro binding of Neisseria-derived IHF Protein. Mol. Cell Probes. 16:153-158.
19. Ramirez-Arcos, S., H. Salimnia, I. Bergevin, M. Paradis,J.R. Dillon. 2001. Expression of Neisseria gonorrhoeae cell division genes ftsZ, ftsE and minD is influenced by environmental conditions. Res. Microbiol. 152:781-791
20. Szeto, J., S. Ramirez-Arcos, C. Raymond, L.D. Hicks, C.M. Kay, and J.R. Dillon. 2001. Gonoccoccal MinD affects cell division in Neisseria gonorrhoeae and Escherichia coli and exhibits a novel self interaction. J. Bacteriol. 183:6253-6264.
21. Pagotto, F., and J.R. Dillon. 2001. Multiple origins and replication proteins influence the biological properties of β-lactamase-producing plasmids from Neisseria gonorrhoeae. J. Bacteriol. 183:5472-5481.
22. Dillon, J.R., H. Li, J. Sealy, M. Ruben, The Caribbean Gonococcal Antimicrobial Susceptibility Program (GASP) Network and P. Prabhakar. 2001. Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from three Caribbean countries - Trinidad, Guyana and St. Vincent. Sex. Trans. Dis. 28:508-514.
23. Dillon, J.R., J-P.A. Rubabaza, A. Schwartz Benzaken, J.C.G. Sardinha, H. Li, M.G. Campos Bandeira, and E. Dos Santos Fernando Filho. 2001. Reduced susceptibility to azithromycin and high percentages of penicillin and tetracycline resistance in Neisseria gonorrhoeae isolates from Manaus, Brazil. Sex. Trans. Dis. 28:521-526.
24. Ramirez-Arcos, S., T.J. Beveridge, J. Szeto, C. Victor, F. Francis and J.R. Dillon. 2001. Deletion of the cell division inhibitor MinC results in lysis of Neisseria gonorrhoeae. Microbiol. 147:225-237.
25. Bernatchez, S., F. Francis, H. Salimnia, T.J. Beveridge, H. Li and J.R. Dillon. 2000. Genomic, transcriptional and phenotypic analysis of ftsE and ftsX of Neisseria gonorrhoeae. DNA Res. 7:75-81.
26. Francis, F., S. Ramirez-Arcos, H. Salimnia, C. Victor and J.R. Dillon. 2000. Organization and transcription of the division cell wall (dcw) cluster in Neisseria gonorrhoeae. Gene 251:141-151.
27. Pagotto, F., H. Salimnia, P.A. Totten and J.R. Dillon. 2000. Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication. Gene 244:13-19.
28. Pagotto, F., T. Aman,L.-K. Ng, K.-H. Yeung, M. Brett and J.R. Dillon. 2000. Sequence analysis of the family of penicillinase-producing plasmids of Neisseria gonorrhoeae based on DNA sequencing. Plasmid 43:24-34.
29. Salimnia, H., A. Radia, S. Bernatchez, T.J. Beveridge and J.R. Dillon. 2000. Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae. Arch. Microbiol. 173:10-20.

Former graduate students and research associates:

PDFs/Research associates:

Ph.D. students:

Nelson F. Eng (Ph.D.), A MinE study: Functional characterization of the cell division protein MinE from the Gram-negative coccus Neisseria gonorrhoeae. Completed in 2007.

Jason Szeto (Ph.D.). The role of MinD in Neisseria gonorrhoeae cell division. Completed in 2004.

Franco Pagotto (Ph.D.). Genetic and molecular characterization of origins of replication from b-lactamase-producing plasmid of Neisseria gonorrhoeae. Completed in 2001.  

Stephane Bernatchez (Ph.D.). Molecular cloning and characterization of the ftsEX genes of Neisseria gonorrhoeae CH811 encoding a putative ABC transporter and identification of their flanking genes. Completed in 1998.

James Ng (Ph.D.), Characterization of Peptostreptococcus species by Aminopeptidase profiles and genotyping methods. Completed in 1996.

Fiona S. Lawson, (Ph.D.). Carbamoylphosphate synthetase (CPS) genes of Neisseria gonorrhoeae and other Neisseria species: Novel gene organization, variable intergenic sequences, characterization of naturally occurring mutants, and evolution of CPS genes. Completed in 1996.

François J. Picard, (Ph.D.). Genetics of arginine and proline biosynthesis in Neisseria gonorrhoeae. Completed in 1991.

Kwok-Him Yeung (Ph.D.). Genetic analysis and molecular characterization of the naturally-occuring penicillinase-producing plasmids in Neisseria gonorrhoeae. Completed in 1989.  

M.Sc. students:

Sudeep Acharya (M.Sc.). Biochemical structural and functional analysis of the cell division site determinant MinDNG: To divide or not to divide. Completed in 2006.

Patrick Ruddock (M.Sc.). Plant derived compounds with antimicrobial activity towards Neisseria gonorrhoeae. Completed in 2005.  

Susan Marthaler (M.Sc.). Uncoiling the mysteries of DivIVA Ef: The biological functioning of the Enterococcus faecalis cell development protein. Completed in 2005.

Marc D. Rigden (M.Sc.). Structural and biochemical analysis of DivIVA from Enterococcus faecalis. Completed in 2005.

Valerie Greco (M.Sc.). Functional analysis of the role of conserved Glycine residues and N-terminus motifs in the cell division inhibitor MinC from Neisseria gonorrhoeae. Completed in 2004.

Finola Francis (M.Sc.). Transcriptional analysis of cell division genes in Neisseria gonorrhoeae. Completed in 1998.

Avni Radia (M.Sc.). Molecular characterization of the cell division gene ftsZ in Neisseria gonorrhoeae. Completed in 1997.

Abu Tholib Aman (M.Sc.). Structural studies of the β-Lactamase-producing plasmids of Neisseria gonorrhoeae. Completed in 1994.

Hui Li (M.Sc.). Evaluation of molecular typing methods to discriminate between isolates of Neisseria gonorrhoeae: Restriction endonuclease analysis, ribotyping and pulsed field gel electrophoresis. Completed in 1993.

Benoit R. Gauthier (M.Sc.). Development of a novel shuttle system for the transfer of Gonococcal genes between Escherichia coli and Neisseria gonorrhoeae. Completed in 1990.

Kimberly Hannah (M.Sc.). Physical and genetic analysis of a conjugative plasmid of Neisseria gonorrhoeae and its role in the evolution of gonococcal strains. Completed in 1988.

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