Phone: (804) 628-3382 (office), 628-3376 (lab)
Dept. Fax: (804) 828-9946
e-mail: jacarlyon@vcu.edu

Address:
Department of Microbiology & Immunology
Virginia Commonwealth University
PO Box 980678
1217 E. Marshall St., Kontos Medical Sciences Building 215 
Richmond, VA 23298-0678

Professional Experience

• B.S., 1993, Virginia Commonwealth University
• Ph.D., 1999, Virginia Commonwealth University School of Medicine
• 1999-2003, Postdoctoral Scholar, Yale University
• 2003-2004, Associate Research Scientist, Yale University
• 2004-2007, Assistant Professor, Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine.
• 2007-Present, Assistant Professor, Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine.

Research Interests:

Molecular pathogenesis of Anaplasma phagocytophilum

Intracellular pathogens are fascinating organisms for study and present wonderful opportunities to examine the complexities of the pathogen-host interface.  Residing within mammalian host cells affords intracellular pathogens with distinct survival advantages.  They are sequestered from antibody- and complement-mediated attack while being privy to a nutrient-rich environment.  My laboratory studies adhesion, invasion, and intracellular survival strategies of the obligate intracellular bacterium Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA; formerly human granulocytic ehrlichiosis).  HGA is an emerging and potentially fatal infectious disease that is gaining increased recognition in the United States, Europe, and Asia and is the second most common tick-transmitted disease in the U. S.  A. phagocytophilum is unique in that it persists within its mammalian host by colonizing neutrophils.  Neutrophils are key effectors in innate immunity that eradicate microbial invaders by ingesting and subjecting them to powerful oxidative and proteolytic killing mechanisms.  A. phagocytophilum invasion of the primary effector cell of microbial killing presents a striking paradox and raises questions as to why it chooses such a formidable host cell and how it evades and subverts neutrophil killing pathways.  We are seeking to answer these questions through multiple research projects.

The first case of HGA was documented in 1994, making this a field ripe for investigation.  Because it is an intracellular pathogen, studying A. phagocytophilum entails a multidisciplinary approach involving aspects of microbiology, cell and molecular biology, immunology, and biochemistry. Deciphering the adherence, invasion, and intracellular survival strategies of this unique organism will further our understanding of A. phagocytophilum pathogenesis and that of other intracellular pathogens and offers a novel approach to studying neutrophil biology and defense mechanisms.  There are many exciting research projects available.  If interested, please do not hesitate to contact me.

Project 1 - A. phagocytophilum adhesins that facilitate attachment to neutrophils.  The initial host-pathogen interaction preceding bacterial colonization is mediated by bacterial factors called adhesins.  These surface proteins have specific structural requirements for targeting their ligands, which results in colonization being restricted to certain cell populations that display the optimal receptors.  The remarkable tropism of A. phagocytophilum for neutrophils is attributable to its usage of P-selectin glycoprotein ligand-1 (PSGL-1) and sialylated and alpha 1,3-fucosylated glycans on neutrophil surfaces for cellular adhesion.  We, in collaboration with Dr. Rodger P. McEver of the Oklahoma Medical Research Foundation and Dr. Richard D. Cummings of Emory University, further defined the A. phagocytophilum cytoadherence mechanism by identifying two key molecular features of PSGL-1 to which the organism binds:  (1) a primary amino acid sequence found in the N-terminus of human PSGL-1 and (2) sialyl Lewis x (sLe^x), a sialylated and fucosylated tetrasaccharide that caps PSGL-1 and other selectin ligands.  The bacterial protein(s) that mediate these interactions are unknown, which represents a considerable gap in our knowledge.  We hypothesize that A. phagocytophilum adherence to neutrophils is dependent on multiple adhesins that cooperatively target PSGL-1 N-terminal peptide and sLe^x (Figure 1).  Identifying the cognate adhesins will be integral to developing strategies for disrupting the interaction of A. phagocytophilum with the surface of its host cell.  The objectives of this project are to identify and characterize the A. phagocytophilum adhesin(s) that mediate cytoadherence to human neutrophils.  This will involve: (1) isolating putative adhesins based on their affinities for PSGL-1 and sLe^x; (2) identifying, cloning, and expressing them using proteomic and molecular methods; (3) functionally characterizing the adhesin candidates using glycoconjugate and cellular binding assays; and (4) assessing whether disrupting their adhesion activities can inhibit infection in vitro and in vivo.  Accomplishing these goals will further our knowledge of A. phagocytophilum pathogenesis and bacterial adhesion strategies.  It will identify targets for potentially treating or preventing HGA and may foster development of new pharmacologic inhibitors of cellular adhesion events associated with inflammatory disorders.  This work is funded by R01 AI072683 from NIH/ National Institute of Allergy and Infectious Diseases.

Figure 1.  A. phagocytophilum (Ap) adhesin model.  We propose that Ap uses multiple adhesins that cooperatively recognize alpha 2,3-sialic acid and alpha a1,3-fucose of sLe^x and human PSGL-1 N-terminal peptide presented on myeloid cell surfaces.  Our evidence demonstrates that Ap uses >1 additional adhesin that recognizes >1 PSGL-1/ sLe^x-independent receptor.  We propose that this adhesin(s) is upregulated or differentially post-translationally modified in NCH-1A and NCH-1A2 populations, which lessens bacterial dependence on sLe^x –modified PSGL-1.

Project 2 – Identify novel A. phagocytophilum adhesins and receptors.  Though sLe^x-modified PSGL-1 is a confirmed A. phagocytophilum receptor, other unknown receptors are also utilized by the bacterium.  Indeed, by culturing A. phagocytophilum strain NCH-1 in host myeloid cell lines (HL-60) that are unable to construct sLe^x because they are incapable of sialylation (HL-60 sLe^{x -/low}) and/ or alpha 1,3-fucosylation (HL-60 A2) we have selected for sub-populations of bacteria that exhibit considerably lessened dependencies on sLe^x and PSGL-1 for binding and invasion.  These enriched populations are called NCH-1 and NCH-1A2, respectively.  A. phagocytophilum engagement of PSGL-1 sparks a signaling cascade that activates the host kinase Syk, which in turn activates ROCK1 to promote bacterial entry.  Yet, NCH-1A2 invades host cells in which Syk has been inactivated.  Therefore, we hypothesize that A. phagocytophilum utilizes >1 separate adhesin to bind to >1 PSGL-1/ sLe^x-independent receptor to mediate invasion.  We further propose that the PSGL-1/ sLe^x-independent adhesin is either upregulated or differentially post-translationally modified in NCH-1 and NCH-1A2 (Figure 1).  We will utilize NCH-1A and NCH-1A2 to (1) identify the Anaplasma adhesin(s) that mediate PSGL-1/ sLe^x-independent binding; (2) identify their cognate receptor(s); and (3) determine host cell signaling pathway induced by these interactions.  Realizing these goals will advance our knowledge of bacterial adhesion strategies and tick-transmitted pathogens and will identify novel targets for treating or preventing HGA.  This work is supported by a grant from the National Research Fund for Tick-Borne Diseases.

Project 3 – Characterization of the A. phagocytophilum inclusion membrane (AIM). Diverse intracellular pathogens hijack membrane traffic and actively modify the host-derived vacuoles in which they reside to construct safe havens within their host cells.  The resulting parasitophorous vacuole is developmentally arrested and sequestered outside the normal endocytic continuum.  We are barely beginning to understand the molecular basis of such mechanisms for only a select few microbes, which represents a considerable deficiency in our knowledge of how intracellular pathogens escape destruction.  Anaplasma phagocytophilum protects itself from neutrophil killing machinery by residing within a host cell-derived inclusion that fails to mature along the endosomal pathway, remains non-fusogenic with lysosomes, and excludes NADPH oxidase (the multi-component enzyme necessary for oxidative killing).  How A. phagocytophilum manipulates host cell intracellular traffic is unknown.  We have identified A. phagocytophilum proteins that are only expressed within host cells and

Figure 2.  The A. phagocytophilum inclusion membrane (AIM) is modified by A. phagocytophilum-encoded proteins.  A. phagocytophilum-infected HL-60 cells were screened using immunofluorescence microscopy.  Colonies of A. phagocytophilum organisms (green) are enclosed within host cell-derived vacuoles that are positive for A. phagocytophilum-encoded proteins (red).  We propose that these and other bacterial-encoded proteins are responsible for the AIM’s altered fusogenic properties and are therefore crucial for A. phagocytophilum intracellular survival.

localize to AIM (Figure 2).  We hypothesize that these A. phagocytophilum-encoded proteins are crucial for regulating host cell intracellular traffic and postulate that additional bacterial AIM proteins contribute to this process.  We will (1) determine which host membrane traffic regulatory proteins (Rabs, SNAREs, adaptor proteins) are recruited to the AIM and (2) which interact with the bacterial AIM-specific proteins.  We will (3) dissect the AIM using proteomics to identify the entirety of bacterial and host proteins comprising this unique vacuolar membrane.  Achieving these goals will shed much-needed light on our understanding of how A. phagocytophilum hijacks the primary effector cell of bacterial killing and will serve as a model for studying a paradigm exemplified by many intracellular pathogens – subversion of host membrane traffic to establish a protective niche.

Selected Publications:

1. D. V. Reneer, M. J. Troese, B. Huang, S. A. Kearns, and J. A. Carlyon. 2008 A. phagocytophilum PSGL-1-independent infection does not require Syk and leads to less-efficient AnkA delivery. Cell Microbiol. In press.

2. M. Sarkar, M. J. Troese, S. A. Kearns, T. Yang, D. V. Reneer, J. A. Carlyon. 2008. Anaplasma phagocytophilum MSP2 (P44)-18 predominates and is modified into multiple isoforms in human myeloid cells. Infect Immun. 76: 2090-98.

3. R. Feferman, D. V. Reneer, J. A. Carlyon, D. Borjesson. 2008. Anaplasma phagocytophilum infects cells of the megakaryocytic lineage through sialylated ligands but fails to alter platelet production. J Med Microbiol. 57: 416-23

4. M. Sarkar, D.V. Reneer, J. A. Carlyon. 2007. Sialyl Lewis x- and P-selectin glycoprotein ligand-1-independent infection by Anaplasma phagocytophilum strains HZ and HGE1. Infect Immun. 75: 5720-25.

5. D.V. Reneer, S. A. Kearns, J. Sims, T. Yago, R. D. Cummings, R. P. McEver, and J. A. Carlyon. 2006. Identification of a sialic acid- and PSGL-1-independent adhesion activity in the granulocytotropic bacterium Anaplasma phagocytophilum. Cell Microbio. 8, 1972-84.

6. Carlyon, J. A. and E. Fikrig. 2006 Mechanisms of evasion of neutrophil killing by Anaplasma phagocytophilum. Curr Opin Hematol Rev. 13: 28-33.

7. Carlyon, J. A. 2006. Laboratory maintenance of A. phagocytophilum. In: Current Protocols in Microbiology. R. Coico, T. F. Kowalik, J. M. Quarles, B. Stevenson, and R. Taylor (eds.) J. Wiley and Sons, Hoboken, N.J.

8. Sukumaran, B., J. A. Carlyon, J. Cai, N. Berliner, and E. Fikrig. 2005. Early transcriptional response of human neutrophils to Anaplasma phagocytohilum infection. Infect. Immun. 73, 8089-8099.

9. Carlyon, J. A., D. Ryan, K. Archer, E. Fikrig. 2005. The effects of Anaplasma phagocytophilum on host cell ferritin mRNA and protein levels. Infect Immun. 73: 7629-36.

10. Pedra, Joao H. F., B. Sukumaran, J. A. Carlyon, N. Berliner, E. Fikrig. 2005. Modulation of NB4 promyelocytic leukemic cell machinery by Anaplasma phagocytophilum. Genomics. 86: 365-77.

11. Carlyon Jason A. and Fikrig E.  2004.  Pathogenic strategies of Anaplasma phagocytophilum, a unique bacterium that colonizes neutrophils. In:  Sixty-third Symposium of the Society for General Microbiology. Microbe-Vector Interactions in Vector-Borne Diseases.  M. Scourfield, ed. University of Bath: Cambridge University Press; 301-329.

12. Carlyon, Jason A., D. Abdel Latif, M. Pypaert, P. Lacy, E. Fikrig.  2004.  Anaplasma phagocytophilum utilizes multiple host evasion mechanisms to thwart NADPH oxidase-mediated killing during neutrophil infection.  Infect Immun.  72: 4772-83.

13. Carlyon, Jason A. and E. Fikrig.  2003. Invasion and survival strategies of Anaplasma phagocytophilum.  Cell Microbio. 5: 743-54.

14. Carlyon, Jason A., M. Akkoyunlu, L. Xia, T. Yago, R. Cummings, R. P. McEver, and E. Fikrig.  2003.  Murine neutrophils require µ1,3-fucosylation but not PSGL-1 for productive Anaplasma phagocytophilum infection.  Blood.  102: 3387-95.

15. Yago, Tadayuki, A. Leppanen, J. A. Carlyon, M. Akkoyunlu, S. Karmakar, E. Fikrig, R. D. Cummings, and R. P. McEver. 2003. Structurally distinct requirements for binding of P-selectin glycoprotein ligand-1 and sialyl Lewis x to Anaplasma phagocytophilum and P-selectin.  J Biol Chem. 278: 37987-97.

16. Carlyon, Jason A., W. Chan, J. Galán, D. Roos, E. Fikrig. 2002. Repression of rac2 mRNA expression by Anaplasma phagocytophila is essential to the inhibition of superoxide production and bacterial proliferation.  J Immunol.  169: 7009-18.

17. De Martino, Sylvie J., J. A. Carlyon, E. Fikrig. 2001.  Coinfection with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis.  New Eng J Med.  345: 150-51.

18. Carlyon, Jason A., D. M. Roberts, M. Theisen, C. Sadler, and R. T. Marconi.  2000.  Molecular and immunological analyses of the Borrelia turicatae Bdr protein family:  a polymorphic, linear plasmid-carried, paralogous gene family.  Infect Immun.  68: 2369-73.

19. Roberts, David M., J. A. Carlyon, M. Theisen, and R. T. Marconi.  2000.  The bdr gene families of the Lyme disease and relapsing fever spirochetes: influence on biology, pathogenesis, and evolution.  Emerg Inf Dis.  6: 110-22.

20. Sung, Shian-Ying, J. McDowell, J. A. Carlyon, and R. T. Marconi.  2000.  Mutation and recombination in the upstream homology box-flanked ospE-related genes of the Lyme disease spirochetes result in the development of new antigenic variants during infection.  Infect Immun.  68: 1319-27.

21. Carlyon, Jason A., D. M. Roberts, and R. T. Marconi.  2000.  Evolutionary and molecular analyses of the Borrelia bdr super gene family:  delineation of distinct sub-families and demonstration of the genus wide conservation of putative functional domains, structural properties, and repeat motifs.  Microb Pathog.  28: 89-105.

22. Sung, Shian-Ying, C. P. LaVoie, J. A. Carlyon, and R. T. Marconi.  1998.  Genetic divergence and evolutionary instability in ospE related members of the UHB gene family in Borrelia burgdorferi sensu lato complex isolates.  Infect Immun.  66: 4656-4668.

23. Carlyon, Jason A. and R. T. Marconi.  1998.  Cloning and molecular characterization of a multicopy, linear plasmid-carried, repeat motif-containing gene from Borrelia turicatae, a causative agent of relapsing fever.  J Bacteriol.  180: 4974-4981.

24. Carlyon, Jason A., C. P. LaVoie, S. Y. Sung, and R. T. Marconi.  1998.  Analysis of the organization of multicopy linear- and circular-plasmid-carried open reading frames in Borrelia burgdorferi sensu lato isolates.  Infect Immun. 66: 1149-1158.

25. Marconi, Richard T., S. Y. Sung, C. A. Norton Hughes, J. A. Carlyon.  1996.  Molecular and evolutionary analyses of a variable series of genes in Borrelia burgdorferi that are related to ospE and ospF, constitute a gene family, and share a common upstream homology box.  J Bacteriol. 178: 5615-5626.

Radio Interview for the Impact of Glycomics:

Dr. Carlyon was a guest on “The Impact of Glycomics”, a radio show dedicated to advancing our overall understanding of the importance of glycobiology in biomedical research. Dr. Carlyon discussed with host Joseph McKenna how A. phagocytophilum interactions with the glycoprotein receptor PSGL-1 on the neutrophil surface promotes bacterial invasion. The interview was conducted on April 23, 2008. To learn more about The Impact of Glycomics, please visit www.impactofglycomics.com. To listen to a podcast of the interview, please click here.

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Date Last Modified: 07/10/2008
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