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Robert A. Heinzen, Ph.D.
Rocky Mountain Laboratories
Bldg 6, Room 6210
903 South 4th Street
Hamilton, MT 59840-2932
Phone: 406-375-9695
Fax: 301-480-5725
rheinzen@niaid.nih.gov

Laboratory of Intracellular Parasites

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Robert A. Heinzen, Ph.D.

Photo of Robert A. Heinzen, Ph.D.

Chief, Coxiella Pathogenesis Section, LICP

Major Areas of Research

  • Genomics and genetic systems
  • Developmental biology
  • Host interactions
 

Program Description

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 biognesis 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.

Genomics and genetic systems

Considerable genetic heterogeneity exists among Coxiella strains isolated from environmental sources and human acute or chronic disease patients. We conducted a comparative genomics study, using genome sequencing and high-density DNA arrays, to better understand the degree and significance of Coxiella genetic variation and to identify potential pathogenetic determinates of human disease potential. 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 were revealed by this work. The former obligate intracellular nature of C. burnetii significantly impeded development of genetic tools to characterize suspected virulence genes. Metabolic pathway reconstructions based on genome data helped us define a medium that supports robust host cell-free (axenic) growth of C. burnetii under microaerobic conditions. Axenic growth of the organism has enabled our development of shuttle vector, transposon, inducible gene expression, and targeted gene inactivation technologies that are now allowing fulfillment of molecular Koch’s postulates for suspected C. burnetii virulence genes.

Lineup of Nine Mile and K isolate genomes
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.
 
Red fluorescent Coxiella in Vero cells
Filamentous red fluorescent Coxiella in Q shape
 
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.
 

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 also exert a potent pro-survival effect that sustains the host cell. Using contemporary cell biology techniques, we are characterizing the Coxiella PV to define both bacterial and host factors that mediate its formation. Moreover, using new genetic tools we are characterizing proteins that are secreted into the host cytosol by a specialized Dot/Icm type IVB secretion system. Functional characterization of these effector proteins and their cellular targets will provide important insight into Coxiella virulence mechanisms.

 
Electron micrograph of Coxiella
Confocal fluorescence micrograph showing that co-infection of Vero cells with Leishmania amazonensis (green) rescues intracellular growth of a Coxiella icmD
 
From left to right: Pseudocolored scanning electron micrograph of a cryo-prepared Vero cell (orange) containing a PV filled with Coxiella (green). Confocal fluorescence micrograph showing that co-infection of Vero cells with Leishmania amazonensis (green) rescues intracellular growth of a Coxiella icmD::Tnmutant (red) LAMP-3 positive (blue) PV.
 

Developmental biology

We have described a Coxiella biphasic developmental cycle wherein environmentally resistant, non-replicative small cell variants (SCV) morphologically differentiate into environmentally fragile, replicative large cell variants (LCV). The programmed gene expression driving Coxiella development is unknown. Furthermore, how the pathogen responds to hostile elements of its lysosome-like PV is a mystery. To gain insight into morphological differentiation and intracellular survival, transcriptome analyses are being performed on Coxiella replicating in macrophages and within axenic medium. Genes suspected of promoting development and/or resistance are being inactivated and mutant strains phenotyped. These studies complement previous proteome analyses and may identify cell form-specific antigens that provide the basis of rationally designed subunit vaccines and new diagnostics.

 
Coxiella in vacuole
Coxiella SCV and LCV
 
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.
 

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Biography

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 National Institutes of Health (NIH) in 1996, Dr. Heinzen joined the faculty of the molecular biology department at the University of Wyoming, where he was awarded tenure in 2002. Dr. Heinzen was recruited to NIH in 2003 as head of the new Coxiella Pathogenesis Section, where he was awarded tenure in 2010 and promoted to senior investigator. Dr. Heinzen has served on the executive council for the American Society for Rickettsiology. In 2011, Dr. Heinzen was elected fellow of the American Academy of Microbiology in recognition of his studies on Coxiella and Rickettsia pathogenesis.

Research Group

Coxiella pathogenesis section group members.
Pictured left to right: Bob Heinzen, Chris Stead, Dale Howe, Stacey Gilk, Charlie Larson Diane Cockrell, Paul Beare, Kelsi Sandoz.

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Selected Publications

Beare PA, Larson CL, Gilk SD, Heinzen RA. Two systems for targeted gene deletion in Coxiella burnetii. Appl Environ Microbiol. 2012 Jul;78(13):4580-9.

Beare PA, Gilk SD, Larson CL, Hill J, Stead CM, Omsland A, Cockrell DC, Howe D, Voth DE, Heinzen RA. Dot/Icm type IVB secretion system requirements for Coxiella burnetii growth in human macrophages. MBio. 2011 Sep 1;2(4):e00175-11.

Voth DE, Beare PA, Howe D, Sharma UM, Samoilis G, Cockrell DC, Omsland A, Heinzen RA. The Coxiella burnetii cryptic plasmid is enriched in genes encoding type IV secretion system substrates. J Bacteriol. 2011 Apr;193(7):1493-503.

Howe D, Shannon JG, Winfree S, Dorward DW, Heinzen RA. Coxiella burnetii phase I and II variants replicate with similar kinetics in degradative phagolysosome-like compartments of human macrophages. Infect Immun. 2010 Aug;78(8):3465-74.

Omsland A, Cockrell DC, Howe D, Fischer ER, Virtaneva K, Sturdevant DE, Porcella SF, Heinzen RA. Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc Natl Acad Sci U S A. 2009 Mar 17;106(11):4430-4.

Shannon JG, Howe D, Heinzen RA. Virulent Coxiella burnetii does not activate human dendritic cells: role of lipopolysaccharide as a shielding molecule. Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8722-7.

Visit PubMed for a complete publication listing.

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Last Updated November 01, 2012