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

Abstract

We previously identified a five-member family of hemin-binding proteins (Hbp's) of Bartonella quintana that bind hemin on the outer surface but share no homology with known bacterial heme receptors. Subsequently, we demonstrated that expression of the hbp family is significantly influenced by oxygen, heme, and temperature conditions encountered by the pathogen in the human host and the body louse vector; e.g., we observed a dramatic (>100-fold) increase in hbpC transcript levels in response to the "louse-like" temperature of 30°C. The goal of the present study was to identify a transcription factor(s) involved in the coordinated and differential regulation of the hbp family. First, we used quantitative real-time PCR (qRT-PCR) to show that the same environmental conditions generate parallels in the transcript profiles of four candidate transcriptional regulators (Irr, Fur, RirA, and BatR) described in the order Rhizobiales, with the greatest overall change in the transcription of irr (a >5-fold decrease) at a "louse-like" temperature, suggesting that Irr may function as an hbpC repressor. Second, a B. quintana strain hyperexpressing Irr was constructed; it exhibits a "bloodstreamlike" hbp transcript profile in the absence of an environmental stimulus (i.e., hbpC is repressed and hbpA, hbpD, and hbpE mRNAs are relatively abundant). Furthermore, when this strain is grown at a "louse-like" temperature, an inversion of the transcript profile occurs, where derepression of hbpC and repression of hbpA, hbpD, and hbpE are readily evident, strongly suggesting that Irr and temperature influence hbp family expression. Third, electrophoretic mobility shift analyses show that recombinant Irr binds specifically to the hbpC promoter region at a sequence that is highly conserved in Bartonella hbp genes, which we designated the hbp family box, or "H-box." Fourth, we used the H-box to search the B. quintana genome and discovered a number of intriguing open reading frames, e.g., five members of a six-member family of cohemolysin autotransporters. Finally, qRT-PCR data regarding the effects of Fur and RirA overexpression on the hbp family are provided; they show that Fur's effect on the hbp family is relatively minor but RirA generates a "bloodstream-like" hbp transcript profile in the absence of an environmental stimulus, as observed for the Irr-hyperexpressing strain.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 ...

Intruders below the Radar: Molecular Pathogenesis of Bartonella spp

“…Several iba autotransporters were confirmed to be upregulated during in vitro infections with B. henselae in a second study and were found to be essential for virulence in the two STM screens (e.g., Cfa) and immunogenic in infections (347,374,427,432). Furthermore, representatives of this group in B. quintana were shown to contain an H box in their promoters, which indicates regulation in response to the transcription factor Irr together with other virulencerelated genes, like those encoding the hemin binding proteins (26).…”

“…Similarly, the filA gene just upstream of ctpA has also been suspected to encode a virulence factor, but no experimental findings that would confirm or discard this assumption have been published (293). It was, however, shown that the promoter of filA contains an H box that is indicative of transcriptional regulation by Irr together with other virulence factors such as the hemin binding proteins or OMP43 (26).…”

“…Comprehensive theories explaining the contribution of Bartonella Hbps to pathogenesis are lacking, although a plethora of experimental data are available and various (not mutually exclusive) hypotheses have been proposed. In short, the Hbps were suggested to be (part of) a heme uptake system, to form an antioxidant coat, to maintain a microaerobic intracellular environment, or to participate directly in host cell manipulation (26).…”

TheBradyrhizobium japonicumIrr Protein Is a Transcriptional Repressor with High-Affinity DNA-Binding Activity

“…However, the affinity of Irr for those DNA elements is very weak in vitro (21). In fact, in vitro binding studies of Irr from several organisms all require micromolar concentrations of protein to detect DNA binding (3,14,21,27). Herein, we show that Irr is a transcriptional repressor with high affinity for target DNA and that Irr occupies the promoters of target genes in vivo.…”

HmuP Is a Coactivator of Irr-Dependent Expression of Heme Utilization Genes in Bradyrhizobium japonicum

“…However, genetic regulation of heme utilization and iron homeostasis in general differs substantially in the Alphaproteobacteria from that in Escherichia coli or other wellstudied model organisms. Whereas Fur is the primary global regulator of the iron stimulon in numerous bacterial species, this role is carried out by Irr, RirA, or both in the Alphaproteobacteria (3,5,6,11,23,27,28,30,34).…”