Transcriptional Regulation of the Heme Binding Protein Gene Family of Bartonella Quintana is Accomplished by a Novel Promoter Element and Iron Response Regulator

The Bartonellaceae are members of the order Rhizobiales, a group of Alphaproteobacteria responsible for a variety of mammalian (bartonellosis, brucellosis) and plant (crown gall) diseases. Bartonella quintana infection of humans results in an acute febrile syndrome called trench fever, and chronic manifestations of persistent infection include endocarditis, bacillary angiomatosis, and bacillary peliosis (29, 40). B. quintana has affected millions of people during war and is presently reemerging in inner cities and in AIDS patients throughout the world (11, 20, 37, 48). Although the majority of human diseases caused by the Rhizobiales are considered zoonoses, maintenance of B. quintana in nature is thought to be restricted to humans and body lice (Pediculus humanus corporis). Humans are infected when body louse fecal matter, or a crushed louse containing the bacterium, is introduced into the bloodstream through breaches in the integument concurrent with the itching caused by louse infestation. Overcrowded, unhygienic conditions disseminate infected lice and can quickly result in an epidemic. Living between the clothing and skin, body lice normally take several blood meals per day and acquire B. quintana by imbibing the blood of a bacteremic human (12). To generate disease, B. quintana must survive and proliferate throughout the human-louse-human cycle and must respond to the disparate environments of the human and body louse. An environmental change of particular interest is the concentration of heme, which is used as a prosthetic subunit in a large number of proteins and consists of an iron atom contained in the center of porphyrin. (The Fe3+ oxidation product of heme is called hemin.) Free heme is quite rare in humans (5), whereas potentially toxic levels are generated in the louse gut during blood meals (26, 49, 65), which can occur several times daily (12). B. quintana has the greatest known bacterial requirement for exogenous heme (42, 43, 67), and it is generally accepted that this extraordinary supplement is needed by all members of the Bartonellaceae, because erythrocytes, hemoglobin, or hemin (20 to 40 μg/ml of medium) is essential for in vitro cultivation (8). Since combinations of iron and porphyrin cannot substitute for heme in cultivation, researchers have hypothesized that high levels are necessary for one or more of the following: a source of iron (14, 59) or porphyrin (43), a hydrogen peroxide-detoxifying agent (42), or a means to establish a microaerobic intracellular environment (7) (as leghemoglobin functions for nitrogen-fixing rhizobia [2]). Considering the fluctuations in heme levels throughout the human-louse-human cycle and the extraordinary concentration required, it is obvious that heme acquisition mechanisms are essential for the replication and ultimately the pathogenesis of B. quintana. Previously, we discovered a family of hemin-binding proteins (HbpA to HbpE) synthesized by B. quintana that serve as outer membrane hemin receptors yet share no similarity to known bacterial heme binding proteins (14, 41). Hbp orthologues are found throughout the Rhizobiales. In fact, the Brucella outer membrane protein (omp) family (16, 58, 68) has become a major focus of vaccine development (10, 22, 27), and recently it was demonstrated that Omp31 is also a hemin-binding protein (17). The obvious similarity between the Bartonella hbp family and the Brucella omp family was the impetus for the inclusion of batR transcript analysis in the present study (see below), since BvrR (the Brucella orthologue) has been implicated in the regulation of Brucella abortus Omp25 synthesis (23, 61). Orthologues of Hbp's are termed Rop's in the rhizobia and have been identified and partially characterized (18, 54, 55). Finally, the prediction of a beta-barrel porin-like structure for Hbp's (as well as its orthologues) suggests that Hbp's may function to bind heme and subsequently transport some portion of it (15, 73). Recently (7), we showed that environmental signals that simulate oxygen, heme, and temperature conditions encountered by B. quintana in the human bloodstream (37°C, 5% O2, low hemin) and the insect vector (30°C, high hemin) significantly influence the expression of the hbp family in a coordinated and differential manner. These environmental stimuli generated transcript profiles that were either “louse-like” (relatively high for hbpC and hbpB [subgroup I] and low for hbpA, hbpD, and hbpE) or “bloodstream-like” (relatively high for hbpA, hbpD, and hbpE [subgroup II] and apparently repressed for hbpC and hbpB). The most dramatic change in the transcript profile of the hbp family occurred in response to a “louse-like” temperature of 30°C (34, 39), where a >100-fold increase in the hbpC mRNA transcript level was demonstrated relative to the level at the human bloodstream temperature (37°C). We proposed that subgroup I (HbpC and, to a lesser extent, HbpB) is preferentially synthesized in the louse and that subgroup II proteins (HbpA, HbpD, and HbpE) are employed for heme acquisition in humans. Accordingly, it was recently shown that HbpE is a dominant immunoreactive surface antigen in humans infected with B. quintana (9). Finally, we demonstrated the existence of a cis-acting regulatory element located in the hbpA promoter region (7). The goal of the present study was to elucidate the coordinated and differential regulation of the hbp family, with a focus on identification of a transcriptional regulator responsible for the apparent repression of hbpC observed at 37°C compared to 30°C (>100-fold increase).