Michael F. Minnick

Sequestration and Scavenging of Iron in Infection

ABSTRACT The proliferative capability of many invasive pathogens is limited by the bioavailability of iron. Pathogens have thus developed strategies to obtain iron from their host organisms. In turn, host defense strategies have evolved to sequester iron from invasive pathogens. This review explores the mechanisms employed by bacterial pathogens to gain access to host iron sources, the role of iron in bacterial virulence, and iron-related genes required for the establishment or maintenance of infection. Host defenses to limit iron availability for bacterial growth during the acute-phase response and the consequences of iron overload conditions on susceptibility to bacterial infection are also examined. The evidence summarized herein demonstrates the importance of iron bioavailability in influencing the risk of infection and the ability of the host to clear the pathogen.

Mitogenic Effect of Bartonella Bacilliformis on Human Vascular Endothelial Cells and Involvement of GroEL

ABSTRACT Bartonellae are bacterial pathogens for a wide variety of mammals. In humans, bartonellosis can result in angioproliferative lesions that are potentially life threatening to the patient, including bacillary angiomatosis, bacillary peliosis, and verruga peruana. The results of this study show that Bartonella bacilliformis, the agent of Oroya fever and verruga peruana, produces a proteinaceous mitogen for human vascular endothelial cells (HUVECs) that acts in a dose-dependent fashion in vitro with maximal activity at ≥72 h of exposure and results in a 6- to 20-fold increase in cell numbers relative to controls. The mitogen increases bromodeoxyuridine (BrdU) incorporation into HUVECs by almost twofold relative to controls. The mitogen is sensitive to heat and trypsin but is not affected by the lipopolysaccharide inhibitor polymyxin B. The mitogen does not affect caspase 3 activity in HUVECs undergoing serum starvation-induced apoptosis. The Bartonella mitogen was found in bacterial culture supernatants, the soluble cell lysate fraction, and, to a lesser degree, in insoluble cell fractions of the bacterium. In contrast, soluble cell lysate fractions from closely related B. henselae, although possessing significant mitogenicity for HUVECs, resulted in only about a twofold increase in cell numbers. Biochemical and immunological analyses identified GroEL as a participant in the observed HUVEC mitogenicity. A B. bacilliformis strain containing the intact groES-groEL operon on a multicopy plasmid was generated and used to demonstrate a correlation between HUVEC mitogenicity and GroEL levels in the lysate (r2 = 0.85). Antiserum to GroEL significantly inhibited mitogenicity of the lysate. Data also show that GroEL is located in the soluble and insoluble fractions (including inner and outer membranes) of the cell and is actively secreted by B. bacilliformis.

Hemin-binding surface protein from Bartonella quintana.

ABSTRACT Bartonella quintana, the agent of trench fever and a cause of endocarditis and bacillary angiomatosis in humans, has the highest reported in vitro hemin requirement for any bacterium. We determined that eight membrane-associated proteins from B. quintana bind hemin and that a ∼25-kDa protein (HbpA) was the dominant hemin-binding protein. Like many outer membrane proteins, HbpA partitions to the detergent phase of a Triton X-114 extract of the cell and is heat modifiable, displaying an apparent molecular mass shift from approximately 25 to 30 kDa when solubilized at 100°C. Immunoblots of purified outer and inner membranes and immunoelectron microscopy with whole cells show that HbpA is strictly located in the outer membrane and surface exposed, respectively. The N-terminal sequence of mature HbpA was determined and used to clone the HbpA-encoding gene (hbpA) from a lambda genomic library. The hbpA gene is 816 bp in length, encoding a predicted immature protein of approximately 29.3 kDa and a mature protein of 27.1 kDa. A Fur box homolog with 53% identity to the Escherichia coli Fur consensus is located upstream of hbpA and may be involved in regulating expression. BLAST searches indicate that the closest homologs to HbpA include the Bartonella henselae phage-associated membrane protein, Pap31 (58.4% identity), and the OMP31 porin from Brucella melitensis(31.7% identity). High-stringency Southern blots indicate that all five pathogenic Bartonella spp. possess hbpAhomologs. Recombinant HbpA can bind hemin in vitro; however, it does not confer a hemin-binding phenotype upon E. coli. IntactB. quintana treated with purified anti-HbpA Fab fragments show a significant (P < 0.004) dose-dependent decrease in hemin binding relative to controls, suggesting that HbpA plays an active role in hemin acquisition and therefore pathogenesis. HbpA is the first potential virulence determinant characterized fromB. quintana.

Five-Member Gene Family of Bartonella quintana

ABSTRACT Bartonella quintana, the agent of trench fever and an etiologic agent of bacillary angiomatosis, has an extraordinarily high hemin requirement for growth compared to other bacterial pathogens. We previously identified the major hemin receptor of the pathogen as a 30-kDa surface protein, termed HbpA. This report describes four additional homologues that share approximately 48% amino acid sequence identity with hbpA. Three of the genes form a paralagous cluster, termed hbpCAB, whereas the other members, hbpD and hbpE, are unlinked. Secondary structure predictions and other evidence suggest that Hbp family members are β-barrels located in the outer membrane and contain eight transmembrane domains plus four extracellular loops. Homologs from a variety of gram-negative pathogens were identified, including Bartonella henselae Pap31, Brucella Omp31, Agrobacterium tumefaciens Omp25, and neisserial opacity proteins (Opa). Family members expressed in vitro-synthesized proteins ranging from ca. 26.5 to 35.1 kDa, with the exception of HbpB, an ∼55.9-kDa protein whose respective gene has been disrupted by a ∼510 GC-rich element containing variable-number tandem repeats. Transcription analysis by quantitative reverse transcriptase-PCR (RT-PCR) indicates that all family members are expressed under normal culture conditions, with hbpD and hbpB transcripts being the most abundant and the rarest, respectively. Mutagenesis of hbpA by allelic exchange produced a strain that exhibited an enhanced hemin-binding phenotype relative to the parental strain, and analysis by quantitative RT-PCR showed elevated transcript levels for the other hbp family members, suggesting that compensatory expression occurs.

Experimental Model of Human Body Louse Infection Using Green Fluorescent Protein-Expressing Bartonella quintana

ABSTRACT A laboratory colony of human body lice was experimentally infected by feeding on rabbits made artificially bacteremic with a green fluorescent protein-expressing Bartonella quintana. B. quintana was detected in the gut and feces until death but not in the eggs. The life span of the lice was not modified. The rabbit model should provide valuable clues to the role of lice in the transmission of B. quintana.

Proteomic and immunoblot analyses of Bartonella quintana total membrane proteins identify antigens recognized by sera from infected patients

ABSTRACT Bartonella quintana is a fastidious, gram-negative, rod-shaped bacterium that causes prolonged bacteremia in immunocompetent humans and severe infections in immunocompromised individuals. We sought to define the outer membrane subproteome of B. quintana in order to obtain insight into the biology and pathogenesis of this emerging pathogen and to identify the predominant B. quintana antigens targeted by the human immune system during infection. We isolated the total membrane proteins of B. quintana and identified 60 proteins by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mass fingerprinting. Using the newly constructed proteome map, we then utilized two-dimensional immunoblotting with sera from 21 B. quintana-infected patients to identify 24 consistently recognized, immunoreactive B. quintana antigens that have potential relevance for pathogenesis and diagnosis. Among the outer membrane proteins, the variably expressed outer membrane protein adhesins (VompA and VompB), peptidyl-prolyl cis-trans-isomerase (PpI), and hemin-binding protein E (HbpE) were recognized most frequently by sera from patients, which is consistent with surface expression of these virulence factors during human infection.

Establishing a direct role for the Bartonella bacilliformis invasion-associated locus B (IalB) protein in human erythrocyte parasitism.

ABSTRACT The invasion-associated locus A and B genes (ialAB) ofBartonella bacilliformis were previously shown to confer an erythrocyte-invasive phenotype upon Escherichia coli, indirectly implicating their role in virulence. We report the first direct demonstration of a role for ialB as a virulence factor in B. bacilliformis. The presence of a secretory signal sequence and amino acid sequence similarity to two known outer membrane proteins involved in virulence suggested that IalB was an outer membrane protein. To develop an antiserum for protein localization, the ialB gene was cloned in frame into an expression vector with a six-histidine tag and under control of thelacZ promoter. The IalB fusion protein was purified by nickel affinity chromatography and used to raise polyclonal antibodies. IalB was initially localized to the bacterial membrane fraction. To further localize IalB, B. bacilliformis inner and outer membranes were fractionated by sucrose density gradient centrifugation and identified by appearance, buoyant density (ρ), and cytochromeb content. Inner and outer membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and IalB was positively identified by Western blot. Contrary to expectations, IalB was localized to the inner membrane of the pathogen. To directly demonstrate a role for IalB in erythrocyte parasitism, the B. bacilliformis ialB gene was disrupted by insertional mutagenesis. The resulting ialB mutant strain was complemented in trans with a replicative plasmid encoding the full-length ialB gene. PCR and high-stringency DNA hybridization confirmed mutagenesis and transcomplementation events. Abrogation and restoration of ialB expression was verified by SDS-PAGE and immunoblotting. In vitro virulence assays showed that mutagenesis of ialB decreased bacterial association and invasion of human erythrocytes by 47 to 53% relative to controls. Transcomplementation of ialB restored erythrocyte association and invasion rates to levels observed in the parental strain. These data provide direct evidence for IalBs role in erythrocyte parasitism and represent the first demonstration of molecular Kochs postulates for a Bartonella species.

Cell entry and the pathogenesis of Bartonella infections

Erythrocyte parasitism, or hemotrophy, is not a common strategy for bacteria. However, Bartonella species are elegantly adapted to parasitize a variety of cell types including red blood cells. Bartonella bacilliformis, a useful model organism for the genus, has been used to study virulence determinants involved in colonization, attachment and invasion of host cells.

Oroya Fever and Verruga Peruana: Bartonelloses Unique to South America

Bartonella bacilliformis is the bacterial agent of Carrións disease and is presumed to be transmitted between humans by phlebotomine sand flies. Carrións disease is endemic to high-altitude valleys of the South American Andes, and the first reported outbreak (1871) resulted in over 4,000 casualties. Since then, numerous outbreaks have been documented in endemic regions, and over the last two decades, outbreaks have occurred at atypical elevations, strongly suggesting that the area of endemicity is expanding. Approximately 1.7 million South Americans are estimated to be at risk in an area covering roughly 145,000 km2 of Ecuador, Colombia, and Peru. Although disease manifestations vary, two disparate syndromes can occur independently or sequentially. The first, Oroya fever, occurs approximately 60 days following the bite of an infected sand fly, in which infection of nearly all erythrocytes results in an acute hemolytic anemia with attendant symptoms of fever, jaundice, and myalgia. This phase of Carrións disease often includes secondary infections and is fatal in up to 88% of patients without antimicrobial intervention. The second syndrome, referred to as verruga peruana, describes the endothelialcell-derived, blood-filled tumors that develop on the surface of the skin. Verrugae are rarely fatal, but can bleed and scar the patient. Moreover, these persistently infected humans provide a reservoir for infecting sand flies and thus maintaining B. bacilliformis in nature. Here, we discuss the current state of knowledge regarding this life-threatening, neglected bacterial pathogen and review its host-cell parasitism, molecular pathogenesis, phylogeny, sand fly vectors, diagnostics, and prospects for control.

Environmental Signals Generate a Differential and Coordinated Expression of the Heme Receptor Gene Family of Bartonella Quintana

ABSTRACT Of all bacteria, Bartonella quintana has the highest reported in vitro hemin requirement, yet an explanation for this remains elusive. To produce diseases such as trench fever, endocarditis, and bacillary angiomatosis, B. quintana must survive and replicate in the disparate environments of the Pediculus humanus corporis (body louse) gut and the human vasculature. We previously identified a five-member family of hemin binding proteins (Hbps) synthesized by B. quintana that bind hemin on the outer surface but share no similarity to known bacterial heme receptors. In the present study, we examine the transcription, regulation, and synthesis of this virulence factor family by cultivation of the bacterium in environments that simulate natural heme, oxygen, and temperature conditions encountered in the host and insect vector. First, quantitative real-time PCR data show that hbpC expression is regulated by temperature, where a >100-fold increase in transcript quantity was seen at 30°C relative to 37°C, suggesting that HbpC synthesis would be greatest in the cooler temperature of the louse. Second, cultivation at human bloodstream oxygen concentration (5% relative to 21% atmospheric) significantly decreases the transcript quantity of all hbp genes, indicating that expression is influenced by O2 and/or reactive oxygen species. Third, a differential expression pattern within the hbp family is revealed when B. quintana is grown in a range of hemin concentrations: subgroup I (hbpC and hbpB) predominates in a simulated louse environment (high heme), and subgroup II (hbpA, hbpD, and hbpE) is preferentially expressed in a simulated human background (low heme). By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and matrix-assisted laser desorption ionization—time of flight mass spectrometry fingerprinting, we demonstrate that synthesis of HbpA correlates with hbpA transcript increases observed at low hemin concentrations. Finally, an hbpA promoter-lacZ reporter construct in B. quintana demonstrates that a transcriptional regulator(s) is controlling the expression of hbpA through a cis-acting regulatory element located in the hbpA promoter region.