Robert A. Heinzen, Ph.D.
Chief, Coxiella Pathogenesis Section
Laboratory of Intracellular Parasites
Dr. Heinzen received his Ph.D. in Microbiology from Washington State University in 1991. After completing an Intramural Research Training Award fellowship in the Laboratory of Intracellular Parasites at the NIH in 1996, Dr. Heinzen joined the faculty of the University of Wyoming as an assistant professor of Molecular Biology where he was awarded tenure and promoted to associate professor in 2002. Dr. Heinzen was recruited to the NIH in 2003 as head of the new Coxiella Pathogenesis Section in the Laboratory of Intracellular Parasites. Dr. Heinzen has served on the executive council for the American Society Rickettsiology and is a past recipient of extramural NIH funding for his rickettsial work. He has served on numerous grant study sections and journal reviews and is internationally recognized for his studies on Coxiella and Rickettsia pathogenesis.
Description of Research Program
We are investigating the host-pathogen relationship of Coxiella burnetii, the causative agent of a disabling flu-like illness called Q fever. Coxiella has historically been considered an obligate intracellular bacterium and is a recognized category B biothreat. The organism targets mononuclear phagocytes where it directs the synthesis of a lysosome-like parasitophorous vacuole (PV) for replication. The mature PV is highly fusogenic, encompasses nearly the entire host cell cytoplasm, and contains hundreds of organisms in the absence of obvious cytopathic effect. Coxiella’s resistance to the harsh conditions of its PV also correlates with remarkable extracellular stability. Because PV biogenesis, host cell maintenance, and generation of developmental forms adapted to intracellular replication and extracellular resistance are central to Coxiella pathogenesis, we are conducting studies to better understand the molecular and cellular biology of these processes. Moreover, we are investigating the extent and relevance of Coxiella strain diversity and developing genetic methods to dissect the virulence of this refractory pathogen.
Major Areas of Research
Host interactions
Coxiella has the extraordinary ability to replicate within a PV with lysosomal characteristics. Our results indicate that Coxiella proteins remodel the vacuole to make it amenable for growth and that the pathogen exerts potent anti-apoptotic effects that sustain the host cell. Using a variety of contemporary cell biology techniques, we are characterizing the Coxiella PV to define both Coxiella and host factors that mediate its formation. Indeed, employing Legionella pneumophila as a surrogate host, we have identified several proteins that are secreted into the host cytosol by a specialized Dot/Icm type IV secretion system (T4SS). Functional characterization of theses effector molecules and their cellular targets will provide important insight into Coxiella virulence mechanisms.
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From left to right: Pseudocolored scanning electron micrograph of a cryo-prepared Vero cell (orange) containing a PV filled with Coxiella (green). Coxiella-infected HeLa cell showing localization of ectopically-expressed AnkG, a Dot/Icm T4SS substrate, to host cell microtubules. |
Developmental Biology
We have described a Coxiella biphasic developmental cycle wherein environmentally-resistant, non-replicative SCV morphologically differentiate into environmentally-fragile, replicative LCV. The programmed gene expression driving Coxiella development is unknown. Furthermore, how the pathogen transcriptionally responds to hostile elements of its lysosome-like PV is a mystery. To gain insight into morphological differentiation and intracellular survival, transcriptome analysis is being performed on Coxiella replicating in macrophages and within a defined host cell-free (axenic) medium. This comparative analysis is possible due to our recent breakthrough development of Acidified Citrate Cysteine Medium (ACCM), a medium that supports axenic growth and morphological differentiation of Coxiella. Cultivation in ACCM will allow identification of the global Coxiella infection-specific transcriptome and also the transcriptional responses to stresses specifically encountered in the PV. Along with previous proteome analyses, these efforts may identify cell form-specific antigens that provide the basis of rationally designed subunit vaccines and new diagnostics.
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| From left to right: Transmission electron micrograph showing a PV harboring Coxiella developmental forms. Purified SCV and LCV with characteristic condensed and dispersed chromatin, respectively. |
Genomics and Genetic Systems
Considerable genetic heterogeneity exists among Coxiella strains isolated from environmental sources and human acute or chronic disease patients. Using genome sequencing and high-density DNA arrays, we are conducting a comparative genomics study to better understand the degree and significance of Coxiella genetic variation. These efforts are aimed at identifying correlations between genetic polymorphisms and human disease potential. To date, we have discovered genetic lesions associated with Coxiella LPS phase variation, the minimal plasmid content of the organism, mechanisms of genome plasticity, and possible pathogenetic determinants of virulence. Identification of Coxiella virulence factors has been thwarted because the organism lacks a system of genetic manipulation. To this end, we are using a multifaceted approach to develop robust methods for genetic transformation and allelic exchange.
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| Mauve alignment of Coxiella Nine Mile and K isolate chromosomes showing rearranged syntenic chromosomal blocks. Recombination between abundant insertion sequences (black vertical lines with triangle) contributes to Coxiella genome plasticity. |
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| From left to right: Vero cells infected with genetically transformed Coxiella expressing mCherry red fluorescent protein. Transformants were generated by Himar1 transposon mutagenesis. Pseudocolored scanning electron micrograph of the first characterized mutant of Coxiella generated by genetic transformation: a filamentous Himar1 ftsZ mutant arranged in a “Q” shape. |
Research Group Members
Pictured left to right: Bob Heinzen, Dale Howe, Diane Cockrell, Anders Omsland, Paul Beare, Stacey Gilk.
Selected Publications
View list in PubMed.
Shannon, J. G., Howe D., and Heinzen, R. A.: Virulent Coxiella burnetii does not activate human dendritic cells: Role of lipopolysaccharide as a shielding molecule. Proc. Natl. Acad. Sci. USA 102: 8722-8727, 2005.
Howe, D., and Heinzen, R. A.: Coxiella burnetii inhabits a cholesterol-rich vacuole and influences cellular cholesterol metabolism. Cell. Microbiol. 8: 496-507, 2006.
Voth, D. E., Howe, D., and Heinzen, R. A.: Coxiella burnetii inhibits apoptosis in human THP-1 cells and monkey primary alveolar macrophages. Infect. Immun. 75: 4263-4271, 2007.
Beare, P. A., Howe, D., Cockrell, D. C., Omsland, A., Hansen, B., and Heinzen, R. A.: Characterization of Coxiella burnetii ftsZ mutant generated by Himar1 transposon mutagenesis. J. Bacteriol. 191: 1369-1381, 2009.
Omsland, A., Cockrell, D. C., Howe, D., Fischer, E. R., Virtaneva, K., Sturdevant, D. E., Porcella, S. F., and Heinzen, R. A.: Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc. Natl. Acad. Sci. USA 106: 4430-4434, 2009.
Voth, D. E., Howe, D., Beare, P. A.,Vogel, J. P., Unsworth, N., Samuel. J. E., and Heinzen, R. A.: The Coxiella burnetii ankyrin repeat domain-containing protein family is heterogeneous with C-terminal truncations that influence Dot/Icm-mediated secretion. J. Bacteriol. 191: 4232-4242, 2009.
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