Jane E Koehler, MD
Professor
Research in my laboratory is focused on defining the clinical spectrum, microbiology, and molecular pathogenesis of the emerging pathogen Bartonella. Our research encompasses both translational and basic investigation. B. henselae and B. quintana are fastidious, gram-negative bacteria that cause bacillary angiomatosis lesions, which are unique, potentially lethal, vascular proliferative lesions affecting HIV-infected patients. Relapsing and/or persistent bloodstream infection is a more frequent manifestation of Bartonella infection. We are investigating the molecular mechanisms associated with two virulence phenotypes of Bartonella: relapsing and persistent bloodstream infection; and hemin (iron)-regulation of gene expression. Finally, we are characterizing the mechanisms involved in differential transcription of virulence genes in the two environmental niches encountered by Bartonella, e.g., in the gut of the poikilothermic body louse vector, and in the bloodstream of the homeothermic mammalian host.
1) Mechanisms of Persistent Bloodstream Infection
Bartonellae can persist in the bloodstream of some mammalian hosts indefinitely (even years in humans) and reach levels of 106 cfu/ml blood, often with minimal apparent systemic effects. Our animal model made possible the analysis of serial Bartonella bloodstream isolates, to detect sequential alteration(s) in surface structural components of Bartonella that confer the ability to adapt and persist in the host. We evaluated outer membrane proteins (OMP) from blood isolates over time as infection persisted/relapsed, and observed a family of three OMP that were present in the inoculum strain, but which were absent in later bloodstream isolates from the same animal.
We determined that these variably-expressed OMP (Vomp) are chromosomally encoded by four tandemly arranged, highly homologous genes that undergo phase variation, gene duplication or deletion, and high frequency recombination. The Vomp are members of the newly described family of trimeric autotransporter adhesins and are associated with several critical virulence properties, including host cell adhesion and autoaggregation. Each Vomp binds a different, unique substrate; binding specificity is determined by the major variable sequence at the tip of each Vomp adhesin. We are currently investigating the Vomp architecture (homotrimeric vs. heterotrimeric) on the surface of Bartonella, and the mechanisms involved in transport of the Vomp to the bacterial surface. The Vomp are essential virulence factors (the vomp null mutant is avirulent in vivo), and understanding the function and structure of the Vomp will provide important clues about the interactions between the host and Bartonella, including the mechanisms of persistence in the human bloodstream.
2) Regulation of Virulence Gene Expression by Environmental Cues
A second research emphasis in my laboratory is the regulation of virulence gene expression by environmental cues. BQ is a Gram-negative bacillus that occupies two very disparate niches: the bloodstream of the homeothermic human reservoir (37°C, very low hemin/Fe level), and the gut of the poikilothermic body louse arthropod vector (28°C, toxic hemin level). The ability of BQ to survive in the body louse is essential for transmission from the body louse vector to humans, the only known host. We identified genes that are up regulated only at the lower temperature of the body louse, including several genes that control iron acquisition by Bartonella.
a) Virtually all bacteria protect themselves from environmental stressors by activating a general stress response (GSR). After BQ is taken up during a blood meal, the bacterium utilizes a unique system of transcriptional regulation to survive the decreased temperature (28°C) and heme-toxic (10 mM) gut in the body louse. We identified an alternative RNA polymerase sigma subunit that is central to the Bartonella GSR that is elicited at body louse temperature. The mechanism of GSR activation has been studied at the molecular level in a collaboration between the SSGCID at U WA. In collaboration with Dr. John Clark at UMass, we also began studying the regulation of BQ gene expression in wild type lice from a colony that can be fed a blood meal spiked with BQ wild type and mutant strains. This has enabled us to identify the signal cascade that activates the GSR after uptake of BQ into the body louse, as well as to identify genes that are critical for BQ survival in the arthropod host. Studying BQ virulence gene expression and regulation in the body louse enables us to define critical interactions between arthropod vector and bacterium that ultimately could provide targets for interrupting the transmission cycle to humans.
b) Iron is an essential growth factor for virtually all bacteria, and the ability to acquire iron from the environment governs potential virulence and pathogenicity in many bacterial organisms (e.g., iron influences the expression of the diphtheria toxin by Corynebacterium diphtheriae). Acquisition of iron and expression of many virulence factors are under transcriptional regulation by the fur gene product, the ferric uptake regulation (Fur) protein, and its homodimeric complex. At sufficient intracellular iron levels, the co-repressors Fur and Fe2+ bind a consensus sequence in the promoter region of genes regulated by Fur, controlling expression of genes encoding iron-scavenging proteins and toxins. Bartonella is a hemophilic bacterium that is not free-living but is restricted to two alternate niches: the iron-rich gut of obligately hematophagous arthropods, or the severely iron-restricted bloodstream of mammals. Iron availability provides a signal to Bartonella indicating which niche (arthropod vector or mammalian reservoir) it occupies, and it is probable that the Fur protein is responsible for global regulation of genes required in each specific host environment. These studies will enable us to characterize the vector- and host-induced responses that facilitate the infection and persistence of Bartonella in humans.
1) Mechanisms of Persistent Bloodstream Infection
Bartonellae can persist in the bloodstream of some mammalian hosts indefinitely (even years in humans) and reach levels of 106 cfu/ml blood, often with minimal apparent systemic effects. Our animal model made possible the analysis of serial Bartonella bloodstream isolates, to detect sequential alteration(s) in surface structural components of Bartonella that confer the ability to adapt and persist in the host. We evaluated outer membrane proteins (OMP) from blood isolates over time as infection persisted/relapsed, and observed a family of three OMP that were present in the inoculum strain, but which were absent in later bloodstream isolates from the same animal.
We determined that these variably-expressed OMP (Vomp) are chromosomally encoded by four tandemly arranged, highly homologous genes that undergo phase variation, gene duplication or deletion, and high frequency recombination. The Vomp are members of the newly described family of trimeric autotransporter adhesins and are associated with several critical virulence properties, including host cell adhesion and autoaggregation. Each Vomp binds a different, unique substrate; binding specificity is determined by the major variable sequence at the tip of each Vomp adhesin. We are currently investigating the Vomp architecture (homotrimeric vs. heterotrimeric) on the surface of Bartonella, and the mechanisms involved in transport of the Vomp to the bacterial surface. The Vomp are essential virulence factors (the vomp null mutant is avirulent in vivo), and understanding the function and structure of the Vomp will provide important clues about the interactions between the host and Bartonella, including the mechanisms of persistence in the human bloodstream.
2) Regulation of Virulence Gene Expression by Environmental Cues
A second research emphasis in my laboratory is the regulation of virulence gene expression by environmental cues. BQ is a Gram-negative bacillus that occupies two very disparate niches: the bloodstream of the homeothermic human reservoir (37°C, very low hemin/Fe level), and the gut of the poikilothermic body louse arthropod vector (28°C, toxic hemin level). The ability of BQ to survive in the body louse is essential for transmission from the body louse vector to humans, the only known host. We identified genes that are up regulated only at the lower temperature of the body louse, including several genes that control iron acquisition by Bartonella.
a) Virtually all bacteria protect themselves from environmental stressors by activating a general stress response (GSR). After BQ is taken up during a blood meal, the bacterium utilizes a unique system of transcriptional regulation to survive the decreased temperature (28°C) and heme-toxic (10 mM) gut in the body louse. We identified an alternative RNA polymerase sigma subunit that is central to the Bartonella GSR that is elicited at body louse temperature. The mechanism of GSR activation has been studied at the molecular level in a collaboration between the SSGCID at U WA. In collaboration with Dr. John Clark at UMass, we also began studying the regulation of BQ gene expression in wild type lice from a colony that can be fed a blood meal spiked with BQ wild type and mutant strains. This has enabled us to identify the signal cascade that activates the GSR after uptake of BQ into the body louse, as well as to identify genes that are critical for BQ survival in the arthropod host. Studying BQ virulence gene expression and regulation in the body louse enables us to define critical interactions between arthropod vector and bacterium that ultimately could provide targets for interrupting the transmission cycle to humans.
b) Iron is an essential growth factor for virtually all bacteria, and the ability to acquire iron from the environment governs potential virulence and pathogenicity in many bacterial organisms (e.g., iron influences the expression of the diphtheria toxin by Corynebacterium diphtheriae). Acquisition of iron and expression of many virulence factors are under transcriptional regulation by the fur gene product, the ferric uptake regulation (Fur) protein, and its homodimeric complex. At sufficient intracellular iron levels, the co-repressors Fur and Fe2+ bind a consensus sequence in the promoter region of genes regulated by Fur, controlling expression of genes encoding iron-scavenging proteins and toxins. Bartonella is a hemophilic bacterium that is not free-living but is restricted to two alternate niches: the iron-rich gut of obligately hematophagous arthropods, or the severely iron-restricted bloodstream of mammals. Iron availability provides a signal to Bartonella indicating which niche (arthropod vector or mammalian reservoir) it occupies, and it is probable that the Fur protein is responsible for global regulation of genes required in each specific host environment. These studies will enable us to characterize the vector- and host-induced responses that facilitate the infection and persistence of Bartonella in humans.