We are investigating Coxiella burnetii, a bacterium that causes a severe flu-like illness called Q fever. Humans typically acquire the pathogen through inhalation of contaminated material generated by infected domestic livestock. Coxiella is also a recognized category B biothreat with potential for illegitimate use. During infection, the organism invades macrophages, where it directs formation of a lysosome-like parasitophorous vacuole (PV) in which to replicate. A mature PV containing hundreds of organisms can occupy nearly the entire host cell cytoplasm in the absence of obvious cytopathic effect. Coxiella’s resistance to the harsh conditions of its PV also correlates with spore-like environmental 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. In addition to providing needed information on pathogen biology, our research goals are aimed at development of new Coxiella countermeasures, such as rationally designed subunit vaccines and tools for molecular epidemiology.
Genomics and genetic systems
Our comparative genomics studies revealed genetic heterogeneity among Coxiella strains and predicted strain-specific virulence factors. Moreover, metabolic pathway reconstructions based on genome data helped us develop a medium that supports robust host cell-free (axenic) growth of Coxiella. Rescue of Coxiella from an obligate intracellular lifestyle has enabled our development of complete set of genetic tools are now allowing fulfillment of molecular Koch’s postulates for suspected Coxiella virulence genes.
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.
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.
Coxiella has the extraordinary ability to replicate within a vacuole with lysosomal characteristics. Our results indicate that modulation of host cell functions by Coxiella proteins is required for PV formation and pathogen growth. 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 are being employed to characterize 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.
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.
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). Transcriptome, proteome, and lipid analyses have revealed determinants of development. Genes suspected of promoting development and/or resistance are being inactivated and mutant strains phenotyped.
Transmission electron micrograph showing a PV harboring Coxiella developmental forms.
Purified SCV and LCV with characteristic condensed and dispersed chromatin, respectively.
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.
Pictured left to right: Diane Cockrell, Charlie Larson, Kelsi Sandoz, Bob Heinzen, Anna Llewellyn, Dale Howe, Chris Stead, Paul Beare.