EID Faculty

Profile

B.V.Sc., University of Bristol School of Veterinary Science
Ph.D., Cornell University College of Veterinary Medicine 

Research

Molecular biology, biochemistry and developmental biology of helminth parasites and the immunobiology of helminth infections

Figure 1: Scanning electron micrograph of a pair of Schistosoma mansoni

Helminths, or parasitic worms, including nematodes, flukes and tapeworms, collectively infect approximately 2 billion people worldwide, or about a third of the world population. The majority of infected people reside in developing countries in tropical and temperate climate zones, where helminths constitute a significant public health concern. Helminth infections are also of concern to U.S. service personnel, Peace Corps workers and civilians that visit these endemic areas. Many helminthiases are ancient diseases, recognized as scourges of humankind since antiquity, while others have risen to prominence recently and constitute emerging or re-emerging infections. Helminth infections can range from mild and unapparent to severe, with considerable morbidity and mortality, depending on the species of parasite involved and various other host and environmental factors.

Helminths are metazoans, with significantly larger genomes than other pathogens such as viruses, bacteria and protists. Consequently, helminths exhibit diverse cellular and molecular processes and complex developmental programs. Together, these features make helminths a challenging and exciting area of research and there is much to be learned about these important pathogens.

Schistosomiasis, caused by blood flukes of the genus Schistosoma, is a potentially serious case of liver, intestine and urinary system pathology that affects approximately 200 million people worldwide. Because efficacious vaccines for schistosomiasis have proved difficult to develop, the long-term objective of our studies is to develop new immunotherapies and chemotherapies aimed at inhibiting schistosome development in the definitive human host, thus simultaneously preventing the pathology associated with schistosome infection and blocking parasite transmission. Our previous studies, using a murine model of Schistosoma infection, have demonstrated that schistosomes require signals from host CD4+ T cells to complete their development normally, suggesting that blocking interactions between schistosomes and host T cells might provide a novel approach to interfering with parasite development. Currently we are focused on further understanding the role CD4+ T cells play in facilitating the development and reproduction of Schistosoma blood flukes and on elucidating how schistosomes respond to signals from the host immune system, from signal transduction to gene transcription.

Figure 2: H&E-stained section of liver showing a juvenile Schistosoma mansoni developing in the portal venule of a wild type C57BL/6 mouse. Note the perivascular accumulations of lymphocytes, macrophages and eosinophils

Selected Publications

• Fraga, L.A.O., Lamb, E.W., Moreno, E.C., Chatterjee, M., Dvorak, J., Delcroix, M., Sajid, M., Caffrey, C.R. and Davies, S.J. (2010) Rapid Induction of IgE Responses to a Worm Cysteine Protease During Pre-patent Schistosome Infection. BMC Immunology 11(1):56
• Florin, D.A., Lawyer, P., Rowton, E., Schultz, E., Wilkerson, R., Davies, S.J., Lipnik, R., ad Keep, L. (2010) Population dynamics of Lutzomyia shannoni (Diptera: Psychodidae) at the Patuxent National Wildlife Research Refuge, Marlyand. J. Am. Mosq. Control Assoc. 26(3):337?339

• Florin, D.A., Lawyer, P., Rowton, E., Schultz, E., Wilkerson, R., Davies, S.J., Lipnik, R., and Keep, L. (2010) Morphological Anomalies in Two Lutzomyia (Psathyromyia) shannoni (Diptera: Psychodidae: Phlebotominae) Specimens Collected From Fort Rucker, Alabama, and Fort Campbell, Kentucky. J. Med. Entomol. 47(5): 952-956
• Lamb, E.W., Walls, C.D., Pesce, J.T., Riner, D.K., Maynard, S.K., Crow, E.T., Wynn, T.A., Schaefer, B.C., and Davies, S.J. (2010) Blood fluke exploitation of non-cognate CD4+ T cell help to facilitate parasite development. PLoS Pathogens 6(4):e1000892  * Selected for "Faculty of 1000 Biology"*

• Swierczewski, B.E. and Davies, S.J. (2010) Conservation of protein kinase A catalytic subunit sequences in the schistosome pathogens of humans. Exp. Parasitol. 125(2):156-60

• Swierczewski, B.E. and Davies, S.J. (2010) Developmental regulation of protein kinase A expression and activity in Schistosoma mansoni. Int. J. Parasitol. 40(8):929-35 

• Fraga, L.A.O., Torrero, M.N., Tocheva, A.S., Mitre, E., and Davies, S.J. (2010) Induction of Type 2 Responses by Schistosome Worms During Pre-Patent Infection. J. Infect. Dis. 201, 464-472 

• Swierczewski, B.E. and Davies, S.J. (2009) A Schistosome cAMP-Dependent Protein Kinase Catalytic Subunit is Essential for Parasite Viability. PLoS Negl. Trop. Dis. 3(8), e505 

• Lamb, E.W., Crow, E.T., Lim, K.C., Liang, Y.-s., Lewis, F.A., and Davies, S.J. (2007) Conservation of CD4+ T cell-dependent developmental mechanisms in the blood fluke pathogens of humans. Int. J. Parasitol.37, 405-415 

• Blank, R.B., Lamb, E.W., Tocheva, A.S., Crow, E.T., Lim, K.C., McKerrow, J.H. and Davies, S.J. (2006) The Common Gamma Chain Cytokines IL-2 and IL-7 Indirectly Modulate Blood Fluke Development Via Effects on CD4+ T cells. J. Infect. Dis. 194, 1609-1616

Research:

Many infections are caused by the formation of microbial biofilms on surfaces in the human body.  Biofilms are organized communities of microbes attached to a surface, or to each other, and are encased in a self-produced extracellular matrix.  Biofilm growth provides protection from adverse environmental conditions, enables evasion of the host immune defenses, and confers resistance to remarkably high concentrations of antimicrobial agents.  Pathogenic bacteria can form biofilms on surfaces throughout a host and on virtually any implantable medical device.  Biofilm infections pose a significant challenge to human health because of their chronic nature and the difficulty associated with treating them.

 

The Frank lab studies relationships between bacterial pathogens and their hosts in biofilm-associated infections.  In particular, our efforts focus on Enterococcus faecalis, a Gram-positive bacterium that is both a human commensal and an opportunistic pathogen.  The enterococci are exceptionally robust, recalcitrant to many classes of antimicrobial agents, and highly proficient at acquiring virulence factors via horizontal gene transfer.  These characteristics have enabled E. faecalis and other enterococci to emerge as leading causes of healthcare-associated infections.  In immunosuppressed patients, E. faecalis can cause a myriad of infections with biofilm etiology, including endocarditis, surgical site infections, and catheter-associated urinary tract infections.

 

The questions we are currently investigating grew from initial experiments evaluating E. faecalis gene expression during infection in animal models of endocarditis and subdermal abscess formation.  By pairing these models of biofilm-associated infection with genetic and transcriptomic techniques, we established that (1) an E. faecalis intramembrane metalloprotease called Eep is a biofilm infection-associated virulence factor, and (2) when E. faecalis is introduced into a mammalian host as a pathogen, there is marked genome-wide upregulation of antisense RNA transcripts that occurs concomitantly with activation of a conserved bacterial stress response known as the stringent response.  We are now following up these observations with a combination of in vitro- and in vivo-based approaches.  High throughput sequencing and genetic techniques will be employed to define the sensing pathways and regulatory circuits involved in triggering the bacterial transcriptional and stress responses observed in the host environment.  We are also interested in identifying additional biofilm-associated virulence factors in E. faecalis and how they affect interactions between the bacterium and its host.  Finally, we are pursuing the mechanisms by which E. faecalis colonizes endovascular tissues as part of a broader interest in understanding how biofilm-forming bacteria may affect human cardiovascular health.

Research Areas and Interests

The research in my lab revolves around the molecular biology and pathogenesis of human viruses. We have a long standing interest in human T-lymphotropic virus type 1 (HTLV-1) and human immunodeficiency virus (HIV), and more recently in Kaposi sarcoma-associated herpesvirus/human herpesvirus type 8 (KSHV/HHV-8). We are particularly interested in the mechanisms of action of viral regulatory proteins and how they impact cellular mRNA transcription, cell cycle control, and signal transduction pathways to effect viral replication and pathogenesis, especially, oncogenesis. Through the study of the HTLV-1 trans-activator/oncoprotein, Tax, we have recently found that persistent and potentially oncogenic activation of I-κB kinases (IKKs)/NF-κB by Tax triggers a cellular senescence checkpoint response. This checkpoint response is induced by hyperactivated p65/RelA and is mediated by two cyclin-dependent kinase inhibitors, p21 and p27, in a p53- and pRb-independent manner. It is often impaired in cancer cells with constitutively activated NF- B. Our recent data indicate that de-regulation of G1 cyclin-dependent kinases can dampen the senescence checkpoint response to facilitate chronic NF-κB hyperactivation. We believe this represents a critical step in leukemia development. Interestingly, we have found the anti-sense protein of HTLV-1, HBZ, which down-regulates NF-κB and HTLV-1 trans-activation by Tax, can mitigate or prevent Tax-induced senescence. Most recently, we have found that similar to -herpesviruses such as EBV and KSHV, HTLV-1 infection can lead to two alternative outcomes - productive infection accompanied by senescence or latent infection followed by clonal expansion - based on the relative expression of regulatory proteins: Tax, Rex, and HBZ. When Tax/Rex expression is robust and dominant over HBZ, productive infection ensues with expression of structural proteins and NF-κB hyper-activation, which induces senescence. When Tax/Rex expression is muted and HBZ is dominant, latent infection is established with expression of regulatory (Tax/Rex/HBZ) but not structural proteins. HBZ maintains viral latency by down-regulating Tax-induced NF-κB activation and senescence, and by inhibiting Rex-mediated expression of viral structural proteins. Current efforts in the lab concentrate on understanding how chronic NF-κB activation induces cellular senescence and how the senescence response becomes impaired in cancer cells whose NF-κB signaling pathway is chronically activated. Cell-free systems are also being established to elucidate the mechanism by which Tax activates IKKs. Finally, we are actively investigating the mechanisms underlying HTLV-1 latency and reactivation with the objective of facilitating virus control in infected persons to prevent progression to disease.

A model for HTLV-1 Leukemogenesis. HTLV-1 is transmitted by cell-to-cell contact. The expression levels of Tax and HBZ modulate the outcomes of infection. Robust viral replication stimulated by Tax is accompanied by cellular senescence. HBZ moderates trans-activation by Tax, thereby down-regulates viral replication and Tax expression to allow oligoclonal expansion of infected T cells. Cytotoxic T lymphocyte (CTL) killing can control virus replication in asymptomatic carriers and select for cells that carry latent proviral DNA. HTLV-1-infected cells develop chromosomal instability. Loss of p16INK4a, p15INK4b, and other tumor suppressors and constitutive Jak/Stat activation may contribute to the inactivation of the senescence checkpoint to allow persistent Tax expression and NF-?B activation. Loss of Tax expression is favored because Tax is a primary CTL target and has a propensity to induce genomic instability and cellular senescence. Inactivation of the senescence checkpoint can facilitate potent NF-?B activation by Tax at the early stage of leukemogenesis and aid the development of Tax-independent NF-?B activation later. The mitogenic activity of HBZ mRNA may help sustain the ATL tumor phenotype.

Immunoblots of HeLa cells transduced with LV-Tax or LV-GFP. Cell lysates were prepared and immunoblotted using cyclin B1, human securin (Securin), p21CIP1/WAF1, p27KIP1, p16INK4a, Tax, and actin antibodies as described. (RIght) Expression of the senescence-associated ?-galactosidase (SA-?-Gal) in HeLa cells transduced with LV-Tax. Asynchronously growing HeLa cells (2.5 x 104 cells/well in 6-well plates) were transduced with LV-Tax or LV-GFP at an m.o.i. of 5, grown for 3 days, and stained with X-Gal overnight at 37ºC.

HTLV-1 Tax-expressing HeLa-FUCCI (fluorescent ubiquitin cell cycle indicator) cells bypass mitosis and become senescent. Ad-Tax-transduced cells were released from G1/S arrest and photographed every hour for 140 hours. The image of cells at the 140th hour is shown. Image processing software was used to move the Tax-expressing senescent cell (top) in close proximity to the normal growing colony (bottom). Cells with red, yellow, and green nuclei are in G1, G1/S, and S/G2 phases of the cell cycle respectively. Time-lapses movies showing the progression of Tax-expressing cells through cell cycle can be found here.

• Movie 1
• Movie 2

Selected Publications:

Philip S., M. A. Zahoor, H. Zhi, Y. K. Ho, and C. -Z. Giam. 2014. Regulation of Human T-Lymphotropic Virus Type I Latency and Reactivation by HBZ and Rex. PLoS.Pathog. 10:e1004040.

Zahoor M. A., S. Philip, H. Zhi, and C. Z. Giam. 2014. NF- B Inhibition Facilitates the Establishment of Cell Lines that Chronically Produce HTLV-1 Viral Particles. J. Virol. 88:3496-504

Zhi H., M. A. Zahoor, A. Shudofsky, and C. -Z. Giam. 2014. KSHV vCyclin Counters the Senescence/G1 Arrest Response Triggered by NF- B Hyper-activation. Oncogene doi: 10.1038/onc.2013.567. [Epub ahead of print]

Ho, Y. K., H. Zhi, D. DeBiaso, S. Philip, H. -M. Shih, and C. -Z. Giam. 2012. HTLV-1 Tax-Induced Rapid Senescence Is Driven by Activated IKK and p65/RelA. J Virol. 86:9474-83.

Zhi, H., L. Yang, Y. L. Kuo, Y. K. Ho, H. M. Shih, and C. -Z. Giam. 2011. NF-kappaB hyper-activation by HTLV-1 tax induces cellular senescence, but can be alleviated by the viral anti-sense protein HBZ. PLoS.Pathog. 7:e1002025.

Yang, L., N. Kotomura, Y. K. Ho, H. Zhi, S. Bixler, M. J. Schell, and C. -Z. Giam. 2011. Complex cell cycle abnormalities caused by human T-lymphotropic virus type 1 Tax. J.Virol. 85:3001-3009. (cover of March 20, 2011 issue)

Zhang, L., H. Zhi, M. Liu, Y. L. Kuo, and C. -Z. Giam. 2009. Induction of p21(CIP1/WAF1) expression by human T-lymphotropic virus type 1 Tax requires transcriptional activation and mRNA stabilization. Retrovirology. 6:35.

Liu, M., L. Yang, L. Zhang, B. Liu, R. Merling, Z. Xia, and C. -Z. Giam. 2008. Human T-cell leukemia virus type 1 infection leads to arrest in the G1 phase of the cell cycle. J.Virol. 82:8442-8455.

Merling, R., C. Chen, S. Hong, L. Zhang, M. Liu, Y. L. Kuo, and C. -Z. Giam. 2007. HTLV-1 Tax mutants that do not induce G1 arrest are disabled in activating the anaphase promoting complex. Retrovirology. 4:35.

Giam, C. Z. and K. T. Jeang. 2007. HTLV-1 Tax and adult T-cell leukemia. Front Biosci. 12:1496-1507.

Soung, N. K., Y. H. Kang, K. Kim, K. Kamijo, H. Yoon, Y. S. Seong, Y. L. Kuo, T. Miki, S. R. Kim, R. Kuriyama, C. -Z. Giam, C. H. Ahn, and K. S. Lee. 2006. Requirement of hCenexin for proper mitotic functions of polo-like kinase 1 at the centrosomes. Mol.Cell Biol. 26:8316-8335.

Zhang, L., M. Liu, R. Merling, and C. -Z. Giam. 2006. Versatile reporter systems show that transactivation by human T-cell leukemia virus type 1 Tax occurs independently of chromatin remodeling factor BRG1. J.Virol. 80:7459-7468.

Kuo, Y. L. and C. -Z. Giam. 2006. Activation of the anaphase promoting complex by HTLV-1 tax leads to senescence. EMBO J. 25:1741-1752.

Liu, B., S. Hong, Z. Tang, H. Yu, and C. -Z. Giam. 2005. HTLV-I Tax directly binds the Cdc20-associated anaphase-promoting complex and activates it ahead of schedule. Proc.Natl.Acad.Sci.U.S.A 102:63-68.

Current Lab Members

Afrin Begum	Michelle Pilligua-Lucas
Kristie Connolly, Ph.D.	Nazia Rahman
Claire Costenoble-Caherty	Erica Raterman, Ph.D.
Carolina Gomez	Rachel Rowland
Isabelle Leduc, Ph.D.	Riley Sennett
Adriana Le Van	Leah Vincent

 

Past Lab Members

 

Undergraduates & McNeese State Summer Interns

Marie-Eve Pelletier	Michael Authement
William Ferguson	Joseph Tod Guidry
Beau Hanks	Brandon Haynes
Jana Jones	Katheryn Leonards
Raphael Herd	Jason Pelligua
Ben Ferguson	Allison Fusilier
James Robinson	Cathryn Frey

 

 

Research Overview

Gonorrhea is the second in incidence among the reportable diseases in the United States and a significant source of morbidity and mortality in women due to the serious nature of ascended infection and its resultant complications. Gonorrhea also impacts neonatal health and is a co-factor in the spread of the human immunodeficiency virus. The rapid spread of antibiotic resistance seriously threatens current control measures and new treatments and a gonorrhea vaccine are needed.

As a pathogen, Gc is fascinating due to its many sophisticated adaptation mechanisms. The primary research interests of the Jerse laboratory are i.) animal modeling of Gc genital tract infections ii.) understanding the mechanisms utilized by Gc to evade host innate defenses in the female genital tract; iii.) studying the in vivo fitness costs and benefits of antibiotic resistance mutations. A second focus is the development of gonorrhea vaccines against surface factors known to play a role in infection and the pre-clinical testing of vaginal microbicides and antibiotics against gonorrhea. We also developed of a female mouse model of Gc and chlamydial coinfection for pathogenesis studies and to facilitate the development of dual therapies against these two common STI pathogens.

Animal Modeling

Female Mouse Model of Gc Genital Tract Infection

• Jerse, A. E. (1999). "Experimental gonococcal genital tract infection and opacity protein expression in estradiol-treated mice." Infect Immun 67(11): 5699-708.
• Dalal, S. J., J. S. Estep, I. E. Valentin-Bon and A. E. Jerse (2001). "Standardization of the Whitten Effect to induce susceptibility to Neisseria gonorrhoeae in female mice." Contemp Top Lab Anim Sci 40(2): 13-7.
• Packiam, M., S. J. Veit, D. J. Anderson, R. R. Ingalls and A. E. Jerse (2010). "Mouse strain-dependent differences in susceptibility to Neisseria gonorrhoeae infection and induction of innate immune responses." Infect Immun 78(1): 433-40.
• Jerse A.E., H. Wu, M. Packiam, R.A. Vonck, A.A. Begum , and L.E. Garvin. 2011. Estradiol-treated female mice as surrogate hosts for Neisseria gonorrhoeae genital tract infections. Front. Microbiol. 2:107.

 

Gc/Chlamydia Co-infection Model

• Vonck, R. A., T. Darville, C. M. O'Connell and A. E. Jerse (2011). "Chlamydial infection increases gonococcal colonization in a novel murine coinfection model." Infect Immun 79(4): 1566-77.

 

 

Gc/Lactobacillus Co-colonization Model

• St Amant, D. C., I. E. Valentin-Bon and A. E. Jerse (2002). "Inhibition of Neisseria gonorrhoeae by Lactobacillus species that are commonly isolated from the female genital tract." Infect Immun 70(12): 7169-71.
• Muench, D. F., D. J. Kuch, H. Wu, A. A. Begum, S. J. Veit, M. E. Pelletier, A. A. Soler-Garcia and A. E. Jerse (2009). "Hydrogen peroxide-producing lactobacilli inhibit gonococci in vitro but not during experimental genital tract infection." J Infect Dis 199(9): 1369-78.
• Jerse, A. E., E. T. Crow, A. N. Bordner, I. Rahman, C. N. Cornelissen, T. R. Moench and K. Mehrazar (2002). "Growth of Neisseria gonorrhoeae in the female mouse genital tract does not require the gonococcal transferrin or hemoglobin receptors and may be enhanced by commensal lactobacilli." Infect Immun 70(5): 2549-58.

 

Host Responses to Infection

• Song, W., S. Condron, B. T. Mocca, S. J. Veit, D. Hill, A. Abbas and A. E. Jerse (2008). "Local and humoral immune responses against primary and repeat Neisseria gonorrhoeae genital tract infections of 17beta-estradiol-treated mice." Vaccine 26(45): 5741-51.
• Packiam M., Veit S.J., Wu H., Mavrogiorgos N., A.E. Jerse, and R.R. Ingalls (2012). Protective and immunoregulatory role of toll-like receptor 4 in experimental gonococcal infection of female mice. Mucosal Immun. 5:19-29.
• Hobbs, M. M., J. E. Anderson, J. T. Balthazar, J. L. Kandler, R. W. Carlson, J. Ganguly, A. A. Begum, J. A. Duncan, J. T. Lin, P. F. Sparling, A. E. Jerse, and W. M. Shafer (2013). Lipid A's structure mediates Neisseria gonorrhoeae fitness during experimental infection of mice and men. MBio. 4(6):e00892-13.
• Packiam, M., R. D. Yedery, A. A. Begum, R. W. Carlson, J. Ganguly, G. D. Sempowski, M. S. Ventevogel, W. M. Shafer, and A. E. Jerse (2014). Phosphoethanolamine decoration of Neisseria gonorrhoeae lipid A plays a dual immunostimulatory and protective role during experimental genital tract infection. Infect Immun. 82(6):2170-9.

 

Adaptation to the Female Genital Tract

• Jerse, A. E., N. D. Sharma, A. N. Simms, E. T. Crow, L. A. Snyder and W. M. Shafer (2003). "A gonococcal efflux pump system enhances bacterial survival in a female mouse model of genital tract infection." Infect Immun 71(10): 5576-82.
• Wu, H. and A. E. Jerse (2006). "Alpha-2,3-sialyltransferase enhances Neisseria gonorrhoeae survival during experimental murine genital tract infection." Infect Immun 74(7): 4094-103.
• Soler-Garcia, A. A. and A. E. Jerse (2007). "Neisseria gonorrhoeae catalase is not required for experimental genital tract infection despite the induction of a localized neutrophil response." Infect Immun 75(5): 2225-33.
• Wu, H., A. A. Soler-Garcia and A. E. Jerse (2009). "A strain-specific catalase mutation and mutation of the metal-binding transporter gene mntC attenuate Neisseria gonorrhoeae in vivo but not by increasing susceptibility to oxidative killing by phagocytes." Infect Immun 77(3): 1091-102.

• Exley, R. M., H. Wu, J. Shaw, M. C. Schneider, H. Smith, A. E. Jerse and C. M. Tang (2007). "Lactate acquisition promotes successful colonization of the murine genital tract by Neisseria gonorrhoeae." Infect Immun 75(3): 1318-24.
• Hobbs, M. M., J. E. Anderson, J. T. Balthazar, J. L. Kandler, R. W. Carlson, J. Ganguly, A. A. Begum, J. A. Duncan, J. T. Lin, P. F. Sparling, A. E. Jerse, and W. M. Shafer (2013). Lipid A's structure mediates Neisseria gonorrhoeae fitness during experimental infection of mice and men. MBio. 4(6):e00892-13.
• Packiam, M., R. D. Yedery, A. A. Begum, R. W. Carlson, J. Ganguly, G. D. Sempowski, M. S. Ventevogel, W. M. Shafer, and A. E. Jerse (2014). Phosphoethanolamine decoration of Neisseria gonorrhoeae lipid A plays a dual immunostimulatory and protective role during experimental genital tract infection. Infect Immun. 82(6):2170-9.

 

Opacity Protein Selection in vivo

• Simms, A. N. and A. E. Jerse (2006). "In vivo selection for Neisseria gonorrhoeae opacity protein expression in the absence of human carcinoembryonic antigen cell adhesion molecules." Infect Immun 74(5): 2965-74.
• Cole, J. G., N. B. Fulcher and A. E. Jerse (2010). "Opacity proteins increase Neisseria gonorrhoeae fitness in the female genital tract due to a factor under ovarian control." Infect Immun 78(4): 1629-41.
• Hobbs M.M., P.F. Sparling, M.S. Cohen, W.M. Shafer, C.D. Deal, and A.E. Jerse. 2011. Experimental gonococcal infection in male volunteers: cumulative experience with Neisseria gonorrhoeae strains FA1090 and MS11mkC. Front. Microbiol. 2:123.

 

Antibiotic Resistance and Fitness

• Warner, D. M., J. P. Folster, W. M. Shafer and A. E. Jerse (2007). "Regulation of the MtrC-MtrD-MtrE efflux-pump system modulates the in vivo fitness of Neisseria gonorrhoeae." J Infect Dis 196(12): 1804-12.
• Warner, D. M., W. M. Shafer and A. E. Jerse (2008). "Clinically relevant mutations that cause derepression of the Neisseria gonorrhoeae MtrC-MtrD-MtrE Efflux pump system confer different levels of antimicrobial resistance and in vivo fitness." Mol Microbiol 70(2): 462-78.
• Kunz A.N., A.A. Begum, H. Wu, J.A. D'Ambrozio, J.M. Robinson, W.M. Shafer, M.C. Bash, and A.E. Jerse. 2012. Impact of Fluoroquinolone resistance mutations on gonococcal fitness and in vivo selection for compensatory mutations. J. Infect. Dis. 205:1821-1829.

 

 

Product Development

Vaccines

• Plante, M., Jerse A.E., Hamel J., Coutre F., Rioux C.R., Brodeur B.R., and D. Martin. 2000. Intranasal immunization with gonococcal outer membrane preparations reduces the duration of vaginal colonization of mice by Neisseria gonorrhoeae. J. Infect. Dis. 182:848-855.
• Cole, J. G. and A. E. Jerse (2009). "Functional characterization of antibodies against Neisseria gonorrhoeae opacity protein loops." PLoS One 4(12): e8108.
• Garvin, L. E., M. C. Bash, C. Keys, D. M. Warner, S. Ram, W. M. Shafer and A. E. Jerse (2008). "Phenotypic and genotypic analyses of Neisseria gonorrhoeae isolates that express frequently recovered PorB PIA variable region types suggest that certain P1a porin sequences confer a selective advantage for urogenital tract infection." Infect Immun 76(8): 3700-9.
• Zhu W., C.J. Chen, C.E. Thomas, J.E. Anderson, A.E. Jerse, and P.F. Sparling. 2011. Vaccines for gonorrhea: can we rise to the challenge? Front. Microbiol. 2:124.

 

Vaginal microbicides

• Spencer, S. E., I. E. Valentin-Bon, K. Whaley and A. E. Jerse (2004). "Inhibition of Neisseria gonorrhoeae genital tract infection by leading-candidate topical microbicides in a mouse model." J Infect Dis 189(3): 410-9.
• Zeitlin L., Hoen T.E., Achilles S.L., Hegarty T.A., Jerse A.E., Kreider J.W., Olmsted S.S., Whaley K.J., Cone R.A., and T.R. Moench. 2001. "Tests of BufferGel for contraception and prevention of sexually transmitted diseases in animal models." Sex. Transm. Dis. 28:417-423.

 

Research: Cellular and Molecular Mechanisms of HIV Pathogenesis and HIV Vaccine Development

Memory CD4 T cells is the primary target of HIV infection. Mucosal tissues are enriched for these memory CD4 T cells thereby making these tissues a central player in the immunopathogenesis and persistence of HIV infection. Mucosal CD4 T cells are the first to get infected as HIV breaches the mucosal barrier and serve to actively amplify infection. As the infection explodes out of the mucosa it ravages all of the memory CD4 T cells that are present in the entire immune system very early during the acute phase. This early phase of infection is accompanied by a massive loss of memory CD4 T cells. The cells that are lost represent the entire memory CD4 T cell repertoire that an individual has accumulated over his lifetime. Given the central importance of these cells in the development of immune responses onset of immunodeficiency occurs very early during infection. Protecting the mucosal CD4 T cells from infection thereby is a central goal for the development of HIV vaccines and therapy. Using state of the art technologies such as multi-color flow cytometry, real time qPCR and microarray based approaches we hope to gain a better understanding of the correlate of protection in the mucosa, and develop strategies that can better protect the mucosal CD4 T cell compartment. Research in my laboratory is focused on:

1. Understanding and delineating the correlates of immunity in mucosal tissues.
2. Developing vaccine based strategies to protect the mucosal CD4 T cell compartment.
3. Understanding why CD8 T cells fail to control viral infection.
4. Developing strategies aimed at reducing the viral reservoir in mucosal and other lymphoid tissues.
5. Understanding the mechanisms underlying the reactivation of secondary viral infections such as EBV and CMV in mucosal tissues during immunodeficiency.

Understanding and delineating the correlates of immunity in mucosal tissues

Protecting the mucosal CD4 T cell from infection is critical to stop the dissemination of infection. However the exact nature of protective immunity required in the mucosal tissue compartment is not clearly known. Studies in non-human primates using either CD8 depletion or passive immunization suggest that both arms of the immune system may be required. Though both T and B cell responses may be critical very little is known about the nature and magnitude of these responses that may be required to either prevent or contain infection. We are using the non-human primate model of HIV infection to address this question either using T/B cell depletion or specific vaccination strategies. We hope to get better insights into the role played by T and B cells in protecting the mucosal CD4 T cell compartment. This will allow us to design better vaccination and therapeutic approaches to control HIV.

Developing vaccine based strategies to protect the mucosal CD4 T cell compartment

Previous studies have shown that systemic vaccination induced immune responses in mucosal tissues of non-human primates that were challenged with pathogenic SIV (Simian immunodeficiency virus). However these responses were lower than those induced in the peripheral tissues. As a consequence the extent of protection was dramatically lower in mucosal tissues than in the periphery. One of our major objectives to explore the concept of mucosal immunizations to induce immune responses in the mucosa that are potent and larger in magnitude, and clarify the role these responses play in protection following challenge.

Understanding why CD8 T cells fail to control viral infection

HIV and SIV infections are associated with an early and massive loss of memory CD4 T cells. Our studies and others have shown these early changes were accompanied by a loss of SIV specific CD4 T cells. Studies in small animal models have demonstrated that CD4 T cell help is critical for the development and maintenance of adaptive CD8 T and B cell responses to viral infections. We hypothesize that the early loss of HIV specific CD4 T cells leads to the generation of defective T and B cell responses in HIV infection. These defects eventually lead to a failure of immune responses to control HIV infection. We are addressing this question in SIV infected non-human primates by using strategies that can protect their SIV specific CD4 T cells. By evaluating the effect of protecting SIV specific CD4 T cells on the phenotype and differentiation of CD8 T cells we hope to better understand the role of CD4 T cell help in anti-viral control of HIV infection.

Developing strategies aimed at reducing the viral reservoir in mucosal and other lymphoid tissues

HIV latency is associated with the generation of viral reservoir that appears to play a critical role in viral persistence. The inability to reduce the viral reservoir has been a major reason for the failure of anti-retroviral therapy. A critical requirement for developing strategies aimed at reducing the viral reservoir it to first clarify and understand where and how the viral reservoir evolves over time. We propose to develop SIV that has been fluorescently tagged to address this question. Using this approach we hope to be able to study the effects of infection, therapy and vaccination on the viral reservoir burden in the mucosa and other tissues.

Understanding the mechanisms underlying the reactivation of secondary viral infections such as EBV in mucosal tissues during immunodeficiency

HIV infection causes an extensive loss of CD4 T cells and severely disables the capacity of the immune system to mount secondary immune responses to previously encountered pathogens. This progressive loss of CD4 T cells leads to immunodeficiency and AIDS, and is accompanied by the reemergence of various opportunistic viral infections in the mucosal tissues such as EBV and CMV. The exact mechanisms leading to the reactivation of these opportunistic viral infections are not known. It is possible the both immune and non-immune related mechanisms may be playing a role in this process. Research is currently underway to delineate the mechanisms at a cellular and molecular level using the non-human primate model for HIV infection. These studies will provide valuable insights into the mechanisms of EBV and CMV reactivation that will aid in the development of novel therapeutic intervention strategies to control HIV infection associated opportunistic viral infections.

Selected Publications:

Peer Reviewed Publications

Taylor, B. C., Mattapallil, J., Scibienski, R and Stott, J. L. 1994. Characterization of a novel bovine cell-cell adhesion protein. Tissue Antigens. 44: 252-60.

Smit-McBride, Z., Mattapallil, J., Villinger, F., Ansari, A. A and Dandekar, S. 1998. Intracellular cytokine expression in the CD4+ and CD8+ T cells from intestinal mucosa of simian immunodeficiency virus infected macaques. J. Med. Primatol. 27: 129-140.

Smit-McBride, Z., Mattapallil, J., D. A. Ferrick and Dandekar, S. 1998. Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4 (+) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes. J. Virol. 72: 6646-56.

Mattapallil, J., Smit-McBride, Z., McChesney, M and Dandekar, S. 1998. Intestinal intraepithelial lymphocytes are primed for gamma interferon and MIP-1beta expression and display antiviral cytotoxic activity despite severe CD4 (+) T-cell depletion in primary simian immunodeficiency virus infection. J. Virol. 72: 6421-9.

Mattapallil, J., Smit-McBride, Z and Dandekar, S. 1999. Intestinal epithelium is an early extra-thymic site for the increased prevalence of CD34+ progenitor cells during primary SIV infection. J. Virol. 73: 4518-23.

Mattapallil, J., Smit-McBride, Z., Dailey, P and Dandekar, S. 1999. Activated memory CD4+ T cells repopulate the intestinal mucosa early after anti-retroviral therapy but exhibit a decreased potential to produce IL-2. J. Virol: 73. 6661-9.

Mattapallil, J., Dandekar, S., Canfield, D. R and Solnick, J. V. 2000. A predominant Th-1 type of immune response is induced early during acute H.pylori infection in rhesus macaques. Gastroenterology. 118. 307-315.

Mattapallil, J., Reay, E and Dandekar, S. 2000. Early expansion of CD8ab+ T cells is observed in the intestinal epithelium in contrast to the depletion CD8aa+ T cells during primary simian immunodeficiency virus infection of rhesus macaques. AIDS. 14: 637-46.

Mattapallil, J., Letvin, N. L and Roederer, M. 2004. T cell dynamics in acute SIV infection. AIDS. 18: 13-23.

Song, K., Rabin, R. L., Hill, B. J., De Rosa, S., Perfetto, S. P., Zhang, H. H., Foley, J. F., Reiner, J. S., Liu, J., Mattapallil, J., Douek, D. C., Roederer, M and Farber, J. M. 2005. Novel subsets of CD4+ memory T cells reveal early-branched pathways of T cell differentiation in humans. PNAS. 102: 7916-21.

Mattapallil, J., Douek, D. C., Hill, B., Nishimura, Y., Martin, M. A and Roederer, M. 2005. Massive infection and loss of memory CD4 T-cells in multiple tissues during acute SIV Infection. Nature. 434: 1093-97.

Nishimura, Y., Brown, C. R., Mattapallil, J., Igarashi, T., Buckler-White, A., Lafont, B., Hirsch, V., Roederer, M and Martin, M. A. 2005. Resting na?ve CD4+ T cells are massively infected and eliminated by X4-tropic SHIVs in Macaques. PNAS. 102: 8000-5.

Wille, U., Flynn, B. J., Lore, K., Koup, R. A., Kedl, R. M., Mattapallil, J., Nason, M., Roederer, M., Weiss, W. R and Seder, R. A. 2005. HIV Gag protein conjugated to a TLR7/8 agonist elicits Th1 and CD8+ T cell responses in monkeys. PNAS 102. 15190-4.

Kuwata, T., Dehghani, H., Plishka, R., Buckler-White, A., Igarashi, T., Mattapallil, J., Roederer, M and Hirsch, V. M. 2005. Characterization of Infectious Molecular Clones from a Simian Immunodeficiency Virus-infected Rapid Progressor (RP) Macaque: Differential Selection of RP-Specific Envelope Mutations in vitro and in vivo. J. Virol. 80: 1463-75.

Wille, U., Flynn, B. J., Lore, K., Koup, R. A., Miles, A.P., Saul, A., Kedl, R. M., Mattapallil, J., Weiss, W.R., Roederer, M and Seder, R. A. 2006. TLR agonists influence the magnitude and quality of Th1 and CD8+ T cell responses following prime-boost immunization in non-human primates. J. Exp. Med. 203: 1249-58.

Mattapallil, J., Douek, D.C., Buckler-White, A., Montefiori, D., Letvin, N. L., Nabel, G. J and Roederer, M. 2006. Vaccination prevents the destruction of CD4 memory T cells during acute SIV infection. J. Exp. Med. 203: 1533-41.

Mattapallil, J., Hill, B., Douek, D.C and Roederer, M. 2006. Systemic vaccination prevents the total destruction of mucosal CD4 T cells during acute SIV challenge. J. Med. Primatol. 35: 217-24.

Seggewiss, R., Lore, K., Guenaga, F.J., Pittaluga, S., Mattapallil, J., Chow, C. K., Koupm, R. A., Camphausen, K., Nason, M. C., Meier-Schellersheim, M., Donahue, R. E., Blazar, B. R., Dunbar, C. E and Douek, D. C. 2007. Keratinocyte growth factor augments immune reconstitution after autologous hematopoietic progenitor cell transplantation in rhesus macaques. Blood. 110: 441-9.

Petrovas, C., Price, D. A., Mattapallil, J., Ambrozak, D. R., Geldmacher, C., Cecchinato, V., Vaccari, M., Tryniszewska, E., Gostick, E., Roederer, M., Douek, D. C., Morgan, S. H., Davis, S. J., Franchini, G and Koup, R. A. 2007. SIV-specific CD8+T-cells express high levels of PD1 and cytokines but have impaired proliferative capacity in acute and chronic SIVmac251 infection. Blood. 110: 928-36.

Wilson, D.P., Mattapallil, J., Zhang, L., Roederer, M and Davenport, M. P. 2007. Estimating the infectivity of CCR5-tropic SIVmac251 in the gut: implications for HIV vaccination. J. Virol. 81: 8025-9.

Mattapallil, M. J., Augello, A., Cheadle, C., Teichberg, D., Becker, K., Chan, C. C., Mattapallil, J., Pennesi, G and Caspi, R. R. 2008. Differentially expressed genes in MHC-compatible rat strains that are susceptible or resistant to experimental autoimmune uveitis. Invest Ophthalmol Vis Sci. 49:1957-70.

Vaccari, M., Mattapallil, J., Song, K., Tsai, W.P., Hryniewicz, A., Venzon, D., Zanetti, M., Reimann, K.A., Roederer, M and Franchini, G. 2008. Reduced protection from SIVmac251 afforded by memory CD8+ T-cells induced by vaccination in condition of CD4+ T-cell deficiency. J. Virol. 82: 9629-38.

Kader, M., Hassan, W., Eberly, M., Piatak, M., Lifson, J., Roederer, M and Mattapallil, J. J. 2008. Anti-retroviral therapy prior to acute viral replication preserves CD4 T cells in the periphery but not in the rectal mucosa during acute simian immunodeficiency virus infection. J. Virol. 82: 11467-71. (Faculty of 1000 Medicine citation in 2008).

Petravic, J., Ribeiro, R. M., Casimiro, D. R., Mattapallil, J., Roederer. M., Shiver, J. W and Davenport, M. P. 2008. Estimating the impact of vaccination on acute simian-human immunodeficiency virus/simian immunodeficiency virus infections. J. Virol. 82: 11589-98.

Eberly, M. D., Kader, M., Hassan, W., Rogers, K. A., Zhou, J., Mueller, Y.M., Mattapallil, M. J., Piatak, M., Lifson, J. D., Katsikis, P. D., Roederer, M., Villinger, F and Mattapallil, J.J. 2009. Increased IL-15 production is associated with higher susceptibility of memory CD4 T cells to SIV during acute infection. J. Immunology. 182: 1439-48.

Nishimura, Y., Sadjadpour, R., Mattapallil, J., Igarashi, T., Lee, W., Buckler-White, A., Roederer, M., Chun, T. W and Martin, M. A. 2009. High frequencies of resting CD4+ T cells containing integrated viral DNA are found in rhesus macaques during acute lentivirus infection. PNAS: 106: 8015-20.

Kader, M., Wang, X., Piatak, M., Lifson, J., Roederer, M., Veazy, R and Mattapallil, J. J. 2009. a4+?7hiCD4+ memory T cells harbor most Th-17 cells and are preferentially infected during acute SIV infection. Mucosal Immunology: 2: 439-49. (Faculty of 1000 Medicine citation in 2010).

Kader, M., Bixler, S., Piatak, M., Lifson, J. D. and Mattapallil, J. 2009. Antiretroviral therapy fails to restore the severe Th-17: Tc-17 imbalance observed in peripheral blood during simian immunodeficiency virus infection. J Medical Primatol: 38: 24-31.

Kader, M., Bixler, S., Roederer, M., Veazey, R. and Mattapallil, J. 2009. CD4 T cell subsets in the mucosa are CD28+Ki-67?HLA-DR?CD69+ but show differential infection based on a4b7 receptor expression during acute SIV infection. J Medical Primatol: 38: 32-38.

Mueller, Y. M, Do, D. H., Boyer, J. D., Kader, M., Mattapallil, J., Lewis, M. G., Weiner, D. B., and Katsikis, P. D. 2009. CD8-depletion of SIV-infected macaques induces CD4+ T cell proliferation that contributes to increased viral loads. J. Immunology: 183: 5006-12.

Geisbert, T. W., Daddario-DiCaprio, K. M., Hickey, A. A., Smith, M. A., Chan, Y., Wang, L., Mattapallil, J.,Geisbert, J. B., Bossart, K. N., and Broder, C. C. 2010. Development of an acute highly pathogenic nonhuman primate model of Nipah virus infection. PLOS One: 18: e10690.

Song, K., Bolton, D., Wilson, R., Bao, S., Mattapallil, J., Andrews, C., Sadoff, G., Goudsmit, J., Pau, M., Seder, R. A., Kozolowski, P., Nabel, G. J., Roederer, M., and Rao, S. S. 2010. Potent local and systemic immunogenicity of finely -aerosolized adenoviral vaccines. PNAS: 107: 22213-8.

Grorge, J., Cofano, E., Lybarger, E., Louder, M., Lafont, B. A., Mascola, J. R., Robert-Guroff, M and Mattapallil, J. 2011. Alterations in Homeostatic Balance of CD4 and CD8+FoxP3+ T cells is Associated with Higher Viral Loads in SIV Infection. AIDS Research & Hum Retroviruses: 27: 763-75.

Teran, R., Mitre, E., Vaca, M., Erazo, S., Oviedo, G., Hubner, M. P., Quinzo, I., Chico, M. E., Mattapallil, J., Bickle, Q., Rodrigues, L. C., and Cooper, P. J. 2011. Immune system development during early childhood in tropical Latin America: evidence for the age-dependent down regulation of the innate immune response. Clinical Immunology: 138: 299-310.

Uchida, N., Bonifacino, A., Krouse, A. E., Metzger, M. E., Csako, G., Fasano, R. M., Leitman, S. F., Mattapallil, J. Hsieh, M. M., Tisdale, J. F and Donahue, R. E. 2011. Long-Term Reconstitution of Transduced Rhesus CD34+ Cells Mobilized by G-CSF and Plerixafor. Experimental Hematology: 39: 795-805.

Mattapallil, M. J., Silver, P. B., Mattapallil, J., Horai, R., Karabekian, Z., McDowell, H., Chan, C., James, E. A., Kwok, W. W., Sen, N., Nussenblatt., R. B., David, C, S., and Caspi, R. R. 2011. Uveitis-associated epitopes of retinal antigens are pathogenic in the humanized mouse model of EAU and identify autoagressive cells. Journal of Immunology: 187: 1977-85.

Kean, L. S., Sen, S., Singh, K., Robertson, J., Onabajo, O. O., Bonifacino, A. C., Metzger, M. E., Promislow, D. E. L., Mattapallil, J and Donahue, R. E. 2011. Significant mobilization of both conventional and regulatory T cells with AMD3100. Blood: 118: 6580-90.

Moore, A. C., Bixler, S. L., Lewis, M. G., Verthelyi, D and, Mattapallil, J. 2011. Mucosal and Peripheral Lin?HLA-DR+CD11c/123?CD14? Mononuclear cells are preferentially infected very early during simian immunodeficiency virus infection. J. Virology: 86: 1069-78.

Review Articles

Mattapallil, J., and Roederer, M. 2006. Acute HIV pathogenesis: it takes more than guts. Current opinion in HIV/AIDS. 1(1): 10-15.

Mattapallil, J., and Roederer, M. 2006. HIV Vaccines: can mucosal CD4 T cells be preserved? Current opinion in HIV/AIDS. 1(4): 272-276.

Roederer M and Mattapallil, J. 2007. CCR5 vs HIV: the less the better!. Blood. 109 (3): 854.

Mattapallil, J and Roederer M. 2008. The mucosa and vaccine induced immune protection in nonhuman primates. Current Opinion in HIV/AIDS. 3:387-92.

Mattapallil, J and Roederer M. 2011. Commentary on ?Massive infection and loss of memory CD4 T cells in multiple tissues during acute SIV infection. HIV Global HIV Vaccine Enterprise electronic resource for HIV vaccine research community

Profile

• 1988-1992 - B.S. Biology magna cum laude, 1992, Lyon College, Batesville, Arkansas
• 1994-1996 M.S. Microbiology, 1996, University of Arkansas, Fayetteville, Arkansas, Advisor: Mack Ivey
• 1996-2001 Ph.D. Molecular Biology and Microbiology, 2001, Tufts University School of Medicine, Boston, Massachusetts, Advisor: Andrew Camilli
• 2001-2004 Postdoctoral Fellow, Stanford School of Medicine, Advisor: Stanley Falkow

Research

Research in the Merrell lab focuses on 4 main areas:

1. Fur-regulation and iron-dependent gene expression.
2.  H. pylori therapeutics.
3. Virulence factor polymorphisms and epidemiology.
4. Metagenomics of infectious disease.

Fur-regulation and Iron-dependent Gene Expression

Iron is a critical nutrient for the vast majority of living organisms on the planet and bacteria are no exception. However, there is a fine line between too little and too much iron. One way that iron homeostasis is maintained in bacteria is through the use of the Ferric Uptake Regulator (Fur). Fur typically functions by repressing genes whose functions are usually related to iron uptake and storage. This occurs under conditions of iron abundance when the protein is bound by its ferrous iron cofactor. While this is by far the most common type of Fur regulation, iron-bound Fur has also been shown to function as an activator of some genes. In Helicobacter pylori, Fur has the ability to repress and activate genes in its iron-bound form, as well as in its apo form (i.e. in the absence of its ferrous iron cofactor). apo-Fur regulation has not been definitively shown to occur in any other bacterial species, making the study of Fur regulation of particular interest in H. pylori. Additionally, it is clear from transcriptional analyses that Fur controls expression of genes with a broad array of functions. In H. pylori, it has been demonstrated that Fur regulates genes involved in iron uptake and storage, genes that mediate oxidative, pH, and osmotic stress, genes involved in metabolism, and even genes that encode virulence factors. Numerous animal studies have also shown that Fur play a clear role in pathogenesis as animals infected with fur mutant strains show less pathology and slower disease progression than their wild-type infected counter parts. It is clear that Fur is crucial to the overall success of H. pylori as a pathogen.

 H. pylori Fur regulation is currently being explored in the lab from many different perspectives. First we are interested in understanding the structure - function relationships that allow for both iron-bound and apo-Fur regulation. These studies involve the characterization of both site specific fur mutants as well as a mutant library carrying random mutations in the fur coding region. In addition, we are interested in dissecting the role of iron-bound versus apo-Fur regulation in pathogenesis and stress adaptation. As there is not a well-defined DNA binding sequence for H. pylori Fur, ongoing studies seek to better define these binding regions, which are called "Fur boxes". Other areas of interest include expanding the known iron-bound and apo-Fur regulons. Going hand in hand with the Fur related work, other projects in the lab are geared towards further exploring and defining the iron-uptake systems utilized by H. pylori.

The goal of these projects is to better understand the contributions of Fur and iron regulation in H. pylori pathogenesis as well as to broaden our understanding of the role Fur plays in bacterial survival.

 H. pylori Therapeutics

There are three broad categories of potential treatment strategies for eradication of Helicobacter pylori infection: vaccination, antimicrobials, and naturopathic therapies. Currently there are no licensed vaccines or approved naturopathic therapies for H. pylori, so antibiotics have been used for treatment of the infection. However, the chronic nature of H. pylori colonization and eradication difficulties are responsible for the evolution of H. pylori antibiotic treatment strategies from mono to dual to triple therapies, and more recently to quadruple, sequential and rescue therapies. Metronidazole (MTZ), clarithromycin (CLR), tetracycline (TET) and amoxicillin (AMX) are the three antibiotics most currently used for treatment, which typically involve a combination of two or three antibiotics and a proton pump inhibitor. However, H. pylori is increasingly resistant to MTZ and CLR, and to a lesser extent to AMX. As a result, there has been an increasing rate of treatment failure, a need for more combinations of antibiotics, an increasing length of treatment period and an overall increased cost of therapy.

Although H. pylori is generally viewed as an extracellular pathogen, a growing body of evidence has established that a subset of the infecting H. pylori population becomes intracellular. It is evident that conventional antibiotics used to treat H. pylori infection likely do not reach suitable concentrations inside the host cell to sterilize the environment. Thus, there is an emerging belief that the ability of H. pylori to survive inside host cells may be partially responsible for treatment failure, and in turn, the spread of drug-resistant strains. To this end, our laboratory is involved in the development and study of novel antibiotics with the ability to traverse the host cell membrane and kill intracellular bacteria.

Virulence Factor Polymorphisms and Epidemiology

As a species, H. pylori is known to show an amazing level of genetic variability across strains. This diversity includes changes in overall gene content as well as polymorphism in gene products. Included among the list of genes that show variation are many that encode virulence factors. Numerous studies suggest that polymorphism in some of these factors may be partially responsible for the wide variety of possible disease outcomes that result from H. pylori infection; certain polymorphic forms of particular factors may be more virulent and cause more disease. We are investigating this possibility by conducting molecular epidemiological studies that attempt to correlate virulence factor genotype with clinical outcome of infection. Furthermore, we are conducting molecular studies that are focused specifically on the CagA toxin. CagA is a type IV secreted effector protein that is injected directly into host cells and causes dramatic alterations in host cell signaling. Studies in transgenic animals have shown that CagA is an oncoprotein; expression of CagA is sufficient to cause cancer in the transgenic animals. Based on the importance of the toxin in cancer development and our epidemiologic data that indicate that particular variants of the toxin are more toxic, we are currently utilizing isogenic H. pylori strains that differ only in the form of the toxin that they express to determine how toxin variation differentially affects various host signaling pathways.

Metagenomics of Infectious Disease

It is estimated that the bacterial cells found in and on the human body actually outnumber the human cells 100 to 1. These bacteria, which are known as our normal microflora, serve as the "first line of defense" against microbial pathogens, and are necessary for many essential processes. Recent advances in sequencing technology have begun to allow researchers to define members of the normal human microflora in both healthy and diseased states. As a result, it has become increasingly clear that the composition of the microbial community plays a significant role in maintaining the delicate balance between health and disease states.

Using 16S rRNA amplicon based studies, we are currently involved in collaborative efforts to 1) analyze bacterial population structures and 2) determine whether differences within these populations are associated with the pathogenesis of human disease. By defining the community members found in both healthy and diseased states, we hope to advance our current knowledge of how the presence or absence of particular community members protects, or fails to protect, against infection. In addition, identification of "keystone" members of the community may provide therapeutic or probiotic targets for disease treatment.

Examples of ongoing projects include analysis of microbial communities associated with tumor growth, as well as poly-microbial and multi-drug resistant bacterial infections.

 

Research Interests

Filaria Immunology

Our lab studies the immune response to filariae, tissue-invasive roundworms transmitted by arthropods. Filariae are parasitic organisms that cause devastating diseases throughout the tropics. Pathogenic human filariae include Wuchereria bancrofti and Brugia malayi, which cause lymphatic filariasis, Onchocerca volvulus, the causative agent of river blindness, and Loa loa, which causes African eyeworm. Lymphatic filariasis alone infects approximately 100 million people worldwide and causes painful and disfiguring manifestations such as genital swelling and lymphedema in over 40 million of these individuals. Onchocerciasis infects between 30 and 40 million people, causing skin disease in most and blindness or severe visual impairment in almost one million, mostly in sub-Saharan Africa. Because these diseases maim, but do not kill, they cause lifelong suffering and are a leading cause of morbidity worldwide.

The immune response to filariae is markedly different than that to most viral, bacterial, and fungal infections. Like other helminths, filariae induce a type 2 immune response characterized by eosinophilia, elevated serum levels of Ag-specific and polyclonal IgE, and increases in T-cell production of IL-4, IL-5, and IL-13. When a person is infected for a long time, however, the immune response to filarial worms diminishes, though the rest of the immune system continues to function against other infections. While it is clear that IL-4 plays a central role in driving type 2 responses, the exact factors responsible for the initiation, maintenance, and eventual diminution of the Th 2 immune response in filarial infections remain unknown. Recent studies from our group have demonstrated that basophils function to amplify type 2 immune responses and that the immunoregulation induced by chronic filarial infection does not impair the host's ability to control tuberculosis.

By studying the immune response to filarial worms, our lab hopes to develop novel methods to prevent and treat these infections.

 

Basic Parasitology

We conduct studies in basic parasitology to increase our knowledge of filaria biology, enhance our ability to work with these organisms, and gain insights into novel anti-filarial drug and vaccine discovery. Studies in this area include proteomic analyses of different anatomical structures of filariae, development of model systems of filariasis, enhancement of in vitro cultivation of filarial worms, and identification of the nutritional requirements of filariae.

 

Protection against Allergy and Autoimmunity

In addition to conducting research on filarial diseases, we also study the beneficial effects of parasitic worms on allergy and autoimmunity. By increasing our understanding of how worms protect against these inflammatory conditions, we hope to develop insights into novel approaches to treat these diseases. Diseases studied in the lab include type 1 diabetes, lupus, immediate cutaneous hypersensitivity, and immune complex-mediated (Type III) hypersensitivity. Recently, we demonstrated that worms induce protection against type 1 diabetes by increasing host production of IL-10, a downregulatory cytokine. We have also shown in both mouse models and humans that chronic helminth infections make it more difficult to fully activate basophils, which are important allergy-effector cells, through their IgE receptors. Finally, we actively work to develop new therapies for allergic and autoimmune diseases by identifying mechanisms to replicate the immune responses induced by worms without using live parasites.

 

Ongoing research projects

• Establishing a small mammal model of lymphatic filariasis
• Development of an in vitro culture system for filarial worms
• Determining protective mechanisms of immunity against filarial infections
• Elucidating the immunologic mechanisms responsible for immunoregulation in helminth infections and allergy immunotherapy
• Elucidating the mechanisms by which nematodes protect against autoimmune diseases and allergy
• Developing novel, helminth-derived therapies for allergy and autoimmune diseases

Research: Molecular Mechanisms of Bacterial Pathogenesis

The objective of the major program in the laboratory is to define the molecular mechanisms by which enterohemorrhagic Escherichia coli (EHEC) cause hemorrhagic colitis and the hemolytic uremic syndrome. EHEC are food-borne pathogens that cause outbreaks of disease associated with ingestion of undercooked hamburgers or raw milk.  Such an outbreak occurred in 1993 in the Pacific Northwest. EHEC are characterized by the production of Shiga toxins (Stxs) and the capacity to adhere avidly to the large bowel epithelium.  Our studies on the virulence mechanisms of EHEC include: creation of molecular tools (monoclonal antibodies and DNA probes) for detecting toxin, investigation of the molecular genetics and regulation of toxin synthesis, purification and characterization of toxins, development of small animal models to further clarify pathogenic traits of EHEC, and analysis of the molecular mechanisms by which EHEC adhere to epithelial cells.

On the left is a fluorescent image of HEp-2 cells infected with EHEC O157:H7. Polymerized actin (red), Tir (blue), and nucleolin (green) appear to be closely associated with the adherent bacteria. On the right is a phase contrast image of the same cell. (courtesy of Dr. James Sinclair).

Another project in my laboratory investigates the virulence factors of and host response to enteroaggregative E. coli strains that were isolated from deployed military personnel with travelers' diarrhea.  This is a collaborative research effort with Dr. David Tribble and CAPT Mark Riddle in PMB and IDCRP and Dr. Stephen Davies in MIC.

My laboratory also studies the roles of toxins and capsules in the pathogenesis of the highly pathogenic Bacillus cereus G9241 strain. B. cereus G9241 caused anthrax-like lung disease and produces the anthrax toxins.  It also makes two distinct polysaccharide capsules and a unique ADP-ribosyltransferase called Certhrax.

 

Selected Publications:

 

1.        Scheutz, F., L.D. Teel, L. Beutin, D. Piérard, G. Buvens, H. Karch, A. Melmann, A. Caprioli, R. Tozzoli, A.R. Melton-Celsa, M. Sanchez, S. Persson, N.A. Strockbine and A.D. O’Brien.  2012. Multi-center evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J. Clin. Microbiol.  50:2951-2963.  PMID: 22760050.

2.        Cote, C.K., L. Kaatz, J. Reinhardt, J. Bozue, S.A. Tobery, A.D. Bassett, P. Sanz, S.C. Darnell, F. Alem, A.D. O’Brien, and S.L. Welkos.  2012.  Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxemia.  J. Med. Microbiol.  61:1380-1392.  PMID: 22767539.

3.        Mallick, E.M., M.E. McBee, V.K. Vanguri, A.R. Melton-Celsa, K. Schlieper, B.J. Karalius, A.D. O’Brien, J. Butterton, J. Leong, and D. Schauer.  2012.  A novel murine model for Shiga toxin-producing Escherichia coli infection.  J. Clin. Invest. 122: 4012-4024.  PMID: 23041631.

4.        Garcia, T.A., C.L. Ventura, M.A. Smith, D.S. Merrell, and A.D. O’Brien.  2013.  Cytotoxic necrotizing factor 1 and hemolysin from uropathogenic Escherichia coli elicit different host responses in the murine bladder.  Infect. Immun. 81:99-109.  PMID: 23090961.

5.        Vergis, J.M., Cote, C.K., Bozue, J., Alem F., Ventura, C.L., Welkos, S.L., O’Brien A.D. 2013. Immunization of mice with formalin-inactivated spores from avirulent Bacillus cereus strains provides significant protection from challenge with Bacillus anthracis Ames.  Clin Vaccine Immunol. 20:56-65.  PMID: 23114705.

6.        Simon, N.C., J.M. Vergis, A.V. Ebrahimi, C.L. Ventura, A.D. O'Brien, and J.T. Barbieri.  2013.  Host cell cytotoxicity and cytoskeleton disruption by CerADPr, an ADP-ribosyltransferase of Bacillus cereus G9241.  Biochem.  52:2309-18.  PMID: 22934824.

7.        Zangari, T., Melton-Celsa, A.R., Panda, A., Boisen, N., Smith, M. A., Taratov, I., Detolla, L., Nataro, J., O’Brien, A.D. 2013. Virulence of the Shiga toxin type 2-expressing Escherichia coli O104:H4 German outbreak isolate in two animal models. Infec. Immun. 81(5):1562-74.  PMID: 23439303.

8.        Flora, A. D., Teel, L.D., Smith, M.A., Sinclair, J.F., Melton-Celsa, A.R., O’Brien, A.D. 2013. Ricin crosses polarized human intestinal cells and intestines of ricin-gavaged mice without evident damage and then disseminates to mouse kidneys.  PLoS One. 8(7):e69706.  PMID: 23874986.

9.        Zumbrun, S.D., Melton-Celsa, A.R., Smith, M.A., Gilbreath, J.J., Merrell, D.S., O’Brien A.D. 2013. Dietary choice affects Shiga toxin-producing Escherichia coli (STEC) O157:H7 colonization and disease. Proc. Natl. Acad. Sci. USA. 110(23):E2126-33.  PMID: 23690602.

10.     Bunger, J.C., A.R. Melton-Celsa, and A.D. O’Brien.  2013.  Shiga toxin type 2dact displays increased binding to globotriaosylceramide in vitro and increased lethality in mice after activation by elastase.  Toxins.  5:2074-2092.  PMID: 24217397.

11.     Russo, L.M., A.R. Melton-Celsa, and A.D. O’Brien.  2014.  Oral intoxication of mice with Shiga toxin type 2a (Stx2a) and protection by anti-Stx2a monoclonal antibody 11E10.  Infect. Immun.  82(3):1213-21.  PMID: 24379294.

12.     Russo, L.M., A.R. Melton-Celsa, M.J. Smith, and A.D. O’Brien.  2014.  Comparisons of native Shiga toxins (Stxs) type 1 and 2 with chimeric toxins indicate that the source of the binding subunit dictates degree of toxicity.  PLoS One.  9(3):e93463.  PMID: 24671194.

13.     Boisen, N., A.-M. Hansen, A.R. Melton-Celsa, T. Zangari, N.P. Mortensen, J.B. Kaper, A.D. O’Brien, and J.P. Nataro.  2014.  The presence of the pAA plasmid in the German O104:H4 Shiga toxin (Stx) type 2a-producing enteroaggregative Escherichia coli strain promotes the translocation of Stx2a across an epithelial cell monolayer.  J. Infect. Dis.  210(12):1909-19.  PMID: 25038258.

14.     Zangari, T., A.R. Melton-Celsa, M.A. Smith, and A.D. O’Brien.  2014.  Enhanced virulence of the Escherichia coli O157:H7 spinach-associated outbreak strain in two animal models is associated with higher levels of Stx2 production after induction with ciprofloxacin.  Infect Immun.  82(12):4968-77.  PMID: 25225244.

15.     O’Neill, K.M., A.M. Schilthuis, C.A. Leiter, K.M. Neihaus, N.A. Judge, E. Twiddy, A.D. O’Brien, and W.R. Curtis.  2015.  Scale-up of transgenic tobacco cells that express intimin of enterohemorrhagic Escherichia coli O157:H7 for use as an oral vaccine in cattle.  In Vitro Cell Develop Biol – Plant.  In Press.

16.     Smith, M.A., R.A. Weingarten, L.M. Russo, C.L. Ventura, and A.D. O’Brien.  2015.  Antibodies against Hemolysin and Cytotoxic Necrotizing Factor type 1 (CNF1) reduce bladder inflammation in a mouse model of urinary tract infection with toxigenic uropathogenic Escherichia coli.  Infect Immun.  83(4):1661-73.  PMID: 25667267.

17.     Melton-Celsa, A.R., H.M. Carvalho, C. Thuning-Roberson, and A.D. O’Brien.  2015.  Protective efficacy and pharmacokinetics of human/mouse chimeric anti-Stx1 and anti-Stx2 antibodies in mice.  Clin Vacc Immunol.  22:448-55.  PMID: 25716230.

18.     Pock, A.R., M. Ottolini, L.N. Pangaro, W.R. Gilliland, B.V. Reamy, P.A. Hemmer, M. Stephens, A. O’Brien, and L. Laughlin.  2015.  Academic change management: leadership lessons from curricular reform.  Mil Med.  180(4):160-162.  PMID: 25850145.

19.     Melton-Celsa, A.R., A.D. O’Brien, and P.C.H. Feng.  2015.  Virulence potential of Shiga toxin 2dact-producing Escherichia coli isolated from fresh produce.  J Food Protect.  78:2085-2088. PMID: 26555533.

20.     Bunger, J.C., A.R. Melton-Celsa, E.L. Maynard, and A.D. O’Brien.  2015.  Reduced toxicity of Shiga toxin (stx) Type 2c in mice compared to Stx2d is associated with instability of Stx2x holotoxin.  Toxins.  7:2306-2320.   PMID: 26110507. 

21.     Russo, L.M., N.F. Abdeltawab, A.D. O’Brien, M. Kotb, and A.R. Melton-Celsa.  2015.  Mapping of genetic loci that modulate differential colonization by Escherichia coli O157:H7 TUV86-2 in advanced recombinant inbred BXD mice.  BMC Genomics.  16:947.  PMID: 26573818.

22.     Russo, L.M., A.R. Melton-Celsa, and A.D. O’Brien.  2015.  Shiga toxin type 1a (Stx1a) reduces the oral toxicity of Stx2a.  J. Infect. Dis.  213(8):1271-1279.  PMID: 26743841

23.     Scarff, J.M., M.J. Raynor, Y.I. Seldina, C.L. Ventura, T.M. Koehler, and A.D. O’Brien.  2016.  The roles of AtxA orthologs in virulence of anthrax-like Bacillus cereus G9241.  Mol Microbiol.  doi: 10.1111/mmi.13478. PMID: 27490458

 

 

 

Research:  Mechanisms of leukocyte activation in response to infectious agents and cancer

Our research is focused on investigating signaling events that regulate lymphocyte activation.  Although we have a longstanding interest in T cell receptor (TCR) activation of NF-κB, our work has more recently diversified to include studies of the innate response to a variety of pathogens and cancer.  Our experimental approach combines cutting-edge imaging technologies with biochemistry, cell biology, and in vivo models of infection and tumorigenesis. We are currently focusing on the following major projects:

 

 

 Figure 1. The TCR to NF-κB pathway. Following TCR activation, PKCθ is recruited to the immunological synapse. Activated PKCθ phosphorylates CARMA1, resulting in formation of the CARMA1, BCL10, MALT1 (CBM) complex. The CBM complex transmits activating signals that ultimately result in ubiquitination (U) and degradation of the NF-κB inhibitor, IκBα. Following IκBα proteolysis, NF-κB translocates to the nucleus and activates transcription of genes required for T cell proliferation and differentiation.

 

 

1. Eucidating the molecular mechanisms and subcellular organization of T cell receptor-regulated NF-κB signaling intermediates.

Our published studies (Schaefer et al PNAS 2004; Rossman et al MBC 2006; Paul et al Immunity 2012; Paul et al., Science Signaling 2014) have documented that TCR stimulation results in the de novo formation of a cytoplasmic signaling structure that we have named the POLKADOTS signalosome. Both signal transmission to NF-κB and limitation of signal transmission by selective autophagy of the signaling adaptor, Bcl10, occur at this site. Currently, we are using “super-resolution” microscopy and other cutting-edge techniques to define nanoscale features of this complex, and to relate those features mechanistically to regulation of signaling.  This NIGMS-funded work is being conducted collaboratively with the group of Dr. Wolgang Losert at the University of Maryland.  Dr. Losert’s group is using advanced mathematical methods to quantify changes in structural features of this signalosome, to suggest novel hypotheses regarding mechanisms of signal transmission. 

Figure 2. PKCθ and Bcl10 redistribution after TCR stimulation. Conalbumin-loaded antigen presenting cells (APC) stimulate D10 T cells, triggering PKCθ (red) translocation to the immunological synapse and Bcl10 (green) clustering in the cytoplasm of T cells, forming the POLKADOTS signalosome.

2. Investigation of the biology of lyssavirus infection.

In collaboration with our Departmental colleague, Dr. Christopher Broder, we are investigating lyssavirus pathogenesis at the cellular and organism level, to identify weaknesses in the viral life cycle that can be exploited to enable an effective host response to these deadly pathogens. The long term goal of this work is to develop strategies for novel therapeutics, which would have efficacy following establishment of infection in the central nervous system. This work is funded by a USU Program Project grant.

Figure 3. TCR-activated CD4 T cell with cytoplasmic Bcl10 clusters that co-localize with LC3+ autophagosomes. Bcl10 (green) and LC3 (red) signals combine to produce yellow at the region of overlap. Blue is cell surface anti-CD4. The fluorescence image is overlayed on a grayscale DIC image.

 

3. Defining the role of macrophages in immunosuppression in lung cancer.

In this project, our studies are directed toward better defining the role of macrophages in lung cancer-associated immunosuppression. Our long-term goal is to provide data suggesting novel immunotherapy approaches, which may be more broadly successful in lung cancer than current available therapies.  This translational work is a collaborative effort with our USU colleague, Dr. Clifton Dalgard (Anatomy, Physiology, and Genetics) and Murtha Cancer Center/WRNNMC physicians Dr. Corey Carter and Dr. Karen Zeman. This project is funded by a grant from the Murtha Cancer Center.

Beyond the above funded work, we are also pursuing funding for several additional projects related to mechanistic studies of immunity and cancer.

Selected Publications

Paul S and Schaefer BC. Visualizing TCR-Induced POLKADOTS Formation and NF-κB Activation in the D10 T-Cell Clone and Mouse Primary Effector T Cells. NF-κB: Methods and Protocols, Methods in Molecular Biology, Michael J. May (ed.), 2015; 1280:219-38.

Paul S, Traver MK, Kashyap AK, Washington MA, Latoche JR, and Schaefer BC. T cell receptor signals to NF-κB are transmitted by a cytosolic p62-Bcl10-Malt1-IKK signalosome. Science Signaling. 2014; 7:ra45

Paul S, Kashyap AK, Jia W, He YW, Schaefer BC. Selective Autophagy of the Adaptor Protein Bcl10 Modulates T Cell Receptor Activation of NF-κB. Immunity. 2012; 36:947-58.

Lagraoui M, Latoche JR, Cartwright NG, Sukumar G, Dalgard CL, Schaefer BC. Controlled cortical impact and craniotomy induce strikingly similar profiles of inflammatory gene expression, but with distinct kinetics. Front Neurol. 2012; 3:155

Cartwright NG, Kashyap AK, Schaefer BC. An active kinase domain is required for retention of PKCθ at the T cell immunological synapse. Mol Biol Cell. 2011; 22:3491-7.

Kingeter LM, Paul S, Maynard SK, Cartwright NG, Schaefer BC. Cutting edge: TCR ligation triggers digital activation of NF-κB. J Immunol. 2010; 185:4520-4.

Kingeter LM and Schaefer BC. Malt1 and cIAP2-Malt1 as effectors of NF-κB activation: Kissing cousins or distant relatives? Cell Signal. 2010; 22:9-22.

Kingeter LM and Schaefer BC. Expanding the multicolor capabilities of basic confocal microscopes by employing red and near-infrared quantum dot conjugates. BMC Biotech. 2009; 9:49.

Langel FD, Jain NA, Rossman JS, Kingeter LM, Kashyap AK, and Schaefer BC. Multiple protein domains mediate interaction between Bcl10 and MALT1. J. Biol. Chem. 2008; 283: 32419-31.

Kingeter LM and Schaefer BC.  Loss of PKCθ, Bcl10, or Malt1 selectively impairs proliferation and NF-κB activation in the CD4+ T cell subset. J. Immunol. 2008; 181:6244-54.

Rossman JS, Stoicheva NG, Langel FD, Patterson GH, Lippincott-Schwartz J, and Schaefer BC. POLKADOTS are foci of functional interactions between cytosolic intermediates in T cell receptor-induced activation of NF-κB. Mol. Biol. Cell 2006; 17:2166-76.

Research:  Malaria Immunity and Transmission

Malaria continues to be a critical world health problem resulting in 100-200 million clinical cases a year with over 0.5 million deaths, mainly children under the age of five. In the past decade the introduction of insecticide-treated bed nets and Artemisinin combination therapy have reversed the increase in mortality caused by the spread of chloroquine-resistant parasites and led to renewed hope for malaria eradication. Key to eradication efforts is the development of new strategies to control parasite transmission, which is the focus the Williamson lab.

The Plasmodium parasite that causes malaria is transmitted from person to person by a mosquito (Fig. 1). Parasites are introduced into the blood stream by the bite of an infected mosquito and then travel to the liver. After development in liver cells, parasites are re-released to the blood stream where they now invade red blood cells and replicate, producing 16-32 new parasites every two days.  Instead of continuing to replicate, some parasites differentiate into either male or female gametocytes over the course of 12 days. Gametocytes, after being taken up in a blood meal by a mosquito, are stimulated to fertilize and begin a developmental cascade in the mosquito that leads to the production of sporozoites, which are infectious to humans. Sporozoites are stored in the salivary gland of a mosquito until release during another blood meal, initiating a new infection.

The complex life cycle allows the parasite to avoid many standard defenses used by the human immune system, but also provides multiple points for interventions. Ongoing projects in the Williamson lab are directed toward: 1) Malaria immunity/systems biology, 2) In vitro and in vivo molecular signaling of sexual differentiation, and 3) Identification of gametocytocidal drugs.

1) Malaria immunity/systems biology: The host specificity of P. falciparum makes direct investigation of the infection in humans critical to the development of effective interventions. Over 50 years ago human passive transfer experiments (Cohen et al. 61) clearly demonstrated a key role for naturally acquired antibodies that protect against malaria. However, reproducing this immunity by vaccination has proven difficult due to antigenic variation, genetic polymorphism, limited immunogenicity of recombinant proteins, and difficulties producing recombinant proteins with native conformation. Utilizing advances in high throughput sequencing and multi-dimensional data analysis we are taking a systems biology approach to understand the human response to repetitive challenges with P. falciparum infected-mosquitoes as happens in the field. Insights into the cellular and antibody responses and their association with clinical symptoms will be used to develop alternative vaccinology approaches to induce protective immunity. This strategy bypasses the use of recombinant antigens produced in heterologous organisms for screening and seeks to identify the protective responses that have evolved in the human host, including the initial innate response and the maturation of the Ig variable region following infection. Ultimately, Ig variable region sequences associated with protection will be generated recombinantly to confirm anti-P.falciparum activity. Target antigens will also be identified and used in conjunction with the insights gained into the early innate and cellular immune responses to develop vaccine strategies that mimic the protective human response.

2) In vitro and in vivo molecular signaling of sexual differentiation: For malaria to be transmitted from human to human via a mosquito, an intraerythrocytic parasite must undergo sexual differentiation into either a male or female gametocyte. We identified the first Plasmodium falciparum gene found to be required for gametocyte development, Pfgdv1 and theset of genes differentially expressed in early gametocytes (Pfge genes)(Eski et al 2012). Localization studies

Figure 2: Localization of PfGDV1

Fig. 2) Immunofluorescence assay of P. falciparum parasites expressing PfGDV1 tagged with GFP that have been probed with anti-PfMCM2 antibodies and DAPI DNA stain. The image includes two parasites: one in the focal plane (1) and one out of the focal plane (2). The hemozoin crystal in the food vacuole (H) is the dense structure in the bright field (BF) image.

demonstrate that in parasites undergoing gametocytogenesis PfGDV1 is located at the nuclear periphery adjacent to the replication origin protein MCM2 and histone modifying enzyme PfSir2 (Fig.2). This location could suggest that Pfgdv1 plays a role regulating gene expression during early gametocytogenesis. Currently, proteins and nucleic acids associated with this Pfgdv1 are being identified by chromatin and protein immunoprecipitation, as well as fluorescence in situ hybridization (FISH), and will be tested for a role in the regulation of Pfge genes. Pfge gene function is also being evaluated by targeted gene disruption and other molecular techniques adapted for P. falciparum by our laboratory (Ikadai et al 2013, Eksi et al. 2011, Morahan et al 2011, López‑Barragán et al. 2011). Combining these approaches will begin to define mechanisms the parasite uses to initiate sexual differentiation and develop into mature gametocytes. Moreover, field studies are being initiated to evaluate sexual differentiation in vivo by expression profiling and to determine if genetic polymorphisms in the Pfgdv1-related transcriptome correlate with transmission rates (Joice, R et al.2013, 2014). Such molecular relationships will be used to develop genetic or expression signatures of high transmission capacity to direct future control malaria control efforts.

3) Identification of gametocytocidal drugs: A sensitive, fluorescence-based, gametocytocidal assay has been developed in my laboratory to facilitate screening molecules for activity against sexual stage parasites (Tanaka and Williamson 2011). The miniaturization of this viability assay (Tanaka et al 2013) allowed the screening of more than 6000 bioactive compounds for gametocytocidal activity (Wei et al. 2014, Tanaka et al. 2014). Seven compounds had IC₅₀s of <50 nM against late stage (III-V) P. falciparum gametocytes, including Torin 2 (IC₅₀ = 8 nM). This compound is >1,000 times less effective against mammalian HepG2 cells and completely blocks P. berghei oocyst production in a mouse model of transmission. Interestingly, a closely related derivative, Torin 1, which has similar activity as Torin 2 against the mammalian target, mTOR, was 200 times less effective against gametocytes. This finding coupled with the lack of mTOR homologs in Plasmodium led us to hypothesize that the human and P. falciparum Torin 2 targets are distinct and that this difference will allow the development of an effective P. falciparum specific drug. Additional Torin 2 analogs are being developed by collaborators at the National Center for Advancing Translational Science (NCATS) at NIH and this project has been selected by the Therapeutics for Rare and Neglected Diseases Program at NCATS for further drug development efforts.