Heme Binding Proteins of Bartonella henselae Are Required when Undergoing Oxidative Stress During Cell and Flea Invasion

Bartonella are hemotropic bacteria responsible for emerging zoonoses. These heme auxotroph alphaproteobacteria must import heme for their growth, since they cannot synthesize it. To import exogenous heme, Bartonella genomes encode for a complete heme uptake system enabling transportation of this compound into the cytoplasm and degrading it to release iron. In addition, these bacteria encode for four or five outer membrane heme binding proteins (Hbps). The structural genes of these highly homologous proteins are expressed differently depending on oxygen, temperature and heme concentrations. These proteins were hypothesized as being involved in various cellular processes according to their ability to bind heme and their regulation profile. In this report, we investigated the roles of the four Hbps of Bartonella henselae, responsible for cat scratch disease. We show that Hbps can bind heme in vitro. They are able to enhance the efficiency of heme uptake when co-expressed with a heme transporter in Escherichia coli. Using B. henselae Hbp knockdown mutants, we show that these proteins are involved in defense against the oxidative stress, colonization of human endothelial cell and survival in the flea.     

Hemin binding,functional expression,and complementation analysis of Pap 31 from Bartonella henselae

Growth of Bartonella henselae is strongly heme dependent, and B. henselae is unable to synthesize heme itself. At least five outer membrane-associated proteins from B. henselae bind hemin, including the 31-kDa protein designated Pap31. The gene of this protein was heterologously expressed in Escherichia coli M15(pREP4) and detected with monoclonal antibodies in the outer membrane fraction. Complementation of the hemA-deficient mutant E. coli K-12 EB53 (aroB tsx malT hemA) with pap31 demonstrated that this protein is involved in heme acquisition and may be an important virulence factor in the pathogenesis of B. henselae.     

An induced fit conformational change underlies the binding mechanism of the heme transport proteobacteria-protein HemS

Bacteria rely on their environment and/or host to acquire iron and have evolved specialized systems to sequester and transport heme. The heme uptake system HemRSTUV is common to proteobacteria, and a major challenge is to understand the molecular mechanism of heme binding and transfer between the protein molecules that underlie this heme transport relay process. In the Gram-negative pathogen Yersinia enterocolitica, the HemRSTUV system culminates with the cytoplasmic recipient HemS, which stores and delivers heme for cellular needs. HemS belongs to a family of proteins essential and unique to proteobacteria. Here we report on the binding mechanism of HemS based on structural data from its apo- and ligand-loaded forms. This heme carrier protein associates with its cargo through a novel, partly preformed binding pocket, formed between a large beta-sheet dome and a three-helix subdomain. In addition to a histidine interacting with the iron, the complex is stabilized by a distal non-coordinating arginine that packs along the porphyrin plane and extensive electrostatic contacts that firmly anchor the heme propionate groups within the protein. Comparison of apo- and ligand-bound HemS crystal structures reveals striking conformational changes that underlie a "heme-induced fit" binding mechanism. Local shifts in amino acid positions combine with global, rigid body-like domain movements, and together, these bring about a switch from an open, apo-form to a closed, bound state. This is the first report in which both liganded and unliganded forms of a heme transport protein are described, thus providing penetrating insights into its mechanism of heme binding and release.     

Crystal structure of AGR_C_4470p from Agrobacterium tumefaciens

We report here the crystal structure at 2.0 A resolution of the AGR_C_4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR_C_4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR_C_4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR_C_4470p in E. coli, in addition to the ChuS protein.     

Crystal structure of hemoglobin protease, a heme binding autotransporter protein from pathogenic Escherichia coli

The acquisition of iron is essential for the survival of pathogenic bacteria, which have consequently evolved a wide variety of uptake systems to extract iron and heme from host proteins such as hemoglobin. Hemoglobin protease (Hbp) was discovered as a factor involved in the symbiosis of pathogenic Escherichia coli and Bacteroides fragilis, which cause intra-abdominal abscesses. Released from E. coli, this serine protease autotransporter degrades hemoglobin and delivers heme to both bacterial species. The crystal structure of the complete passenger domain of Hbp (110 kDa) is presented, which is the first structure from this class of serine proteases and the largest parallel beta-helical structure yet solved.     

细菌血红素降解蛋白结构特点及作用机制

铁离子是几乎所有生物包括细菌生存必需的营养元素. 在宿主体内,绝大多数的铁离子均以血红素的形式存在于各种血红素结合蛋白,如血红蛋白、肌红蛋白等. 当致病菌感染宿主后,血红素将成为某些致病菌主要的铁离子来源. 致病菌编码血红素转运系统,并利用该系统将血红素转运至胞浆,在胞浆血红素被细菌的血红素降解蛋白降解,释放铁离子供细菌利用. 在致病菌中,目前至少有两种血红素降解酶被发现和鉴定. 第一种为经典的血红素氧化酶(heme oxygenase, HO),它催化血红素氧化形成胆绿素、一氧化碳(CO)和Fe2+;第二种非经典降解酶,包括金黄色葡萄球菌的IsdG/IsdI蛋白及其同系物MhuD蛋白,催化血红素分别产生staphylobilin和mycobilin. 另外,部分细菌内存在其它血红素降解因子,其与前两种血红素降解酶无结构同源性,但在血红素降解实验中可产生胆绿素(biliverdin)或CO,因而被鉴定为“血红素降解蛋白”. 对细菌血红素降解蛋白分子结构解析及作用机制的深入理解,将有助于新的血红素降解蛋白的发现和鉴定.     

Hemin uptake system of Yersinia enterocolitica: similarities with other TonB-dependent systems in gram-negative bacteria.

The hemin receptor HemR of Yersinia enterocolitica was identified as a 78 kDa iron regulated outer membrane protein. Cells devoid of the HemR receptor as well as cells mutated in the tonB gene were unable to take up hemin as an iron source. The hemin uptake operon from Y. enterocolitica was cloned in Escherichia coli K12 and was shown to encode four proteins: HemP (6.5 kDa), HemR (78 kDa), HemS (42 kDa) and HemT (27 kDa). When expressed in E.coli hemA aroB, a plasmid carrying genes for HemP and HemR allowed growth on hemin as a porphyrin source. Presence of genes for HemP, HemR and HemS was necessary to allow E.coli hemA aroB cells to use hemin as an iron source. The nucleotide sequence of the hemR gene and its promoter region was determined and the amino acid sequence of the HemR receptor deduced. HemR has a signal peptide of 28 amino acids and a typical TonB box at its amino-terminus. Upstream of the first gene in the operon (hemP), a well conserved Fur box was found which is in accordance with the iron-regulated expression of HemR.     

Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7.

Shigella species can use heme as the sole source of iron. In this work, the heme utilization locus of Shigella dysenteriae was cloned and characterized. A cosmid bank of S. dysenteriae serotype 1 DNA was constructed in an Escherichia coli siderophore synthesis mutant incapable of heme transport. A recombinant clone, pSHU12, carrying the heme utilization system of S. dysenteriae was isolated by screening on iron-poor medium supplemented with hemin. Transposon insertional mutagenesis and subcloning identified the region of DNA in pSHU12 responsible for the phenotype of heme utilization. Minicell analysis indicated that a 70-kDa protein encoded by this region was sufficient to allow heme utilization in E. coli. Synthesis of this protein, designated Shu (Shigella heme uptake), was induced by iron limitation. The 70-kDa protein is located in the outer membrane and binds heme, suggesting it is the S. dysenteriae heme receptor. Heme iron uptake was found to be TonB dependent in E. coli. Transformation of an E. coli hemA mutant with the heme utilization subclone, pSHU262, showed that heme could serve as a source of porphyrin as well as iron, indicating that the entire heme molecule is transported into the bacterial cell. DNA sequences homologous to shu were detected in strains of S. dysenteriae serotype 1 and E. coli O157:H7.     

Human bartonellosis: seroepidemiological and clinical features with an emphasis on data from Brazil - a review

Bartonellae are fastidious Gram-negative bacteria that are widespread in nature with several animal reservoirs (mainly cats, dogs, and rodents) and insect vectors (mainly fleas, sandflies, and human lice). Thirteen species or subspecies of Bartonella have been recognized as agents causing human disease, including B. bacilliformis, B. quintana, B. vinsonii berkhoffii, B. henselae, B. elizabethae, B. grahamii, B. washoensis, B. koehlerae, B. rocha-limaea, and B. tamiae. The clinical spectrum of infection includes lymphadenopathy, fever of unknown origin, endocarditis, neurological and ophthalmological syndromes, Carrion's disease, and others. This review provides updated information on clinical manifestations and seroepidemiological studies with an emphasis on data available from Brazil.     

Functional differences between heme permeases: Serratia marcescens HemTUV permease exhibits a narrower substrate specificity (restricted to heme) than the Escherichia coli DppABCDF peptide-heme permease

Serratia marcescens hemTUV genes encoding a potential heme permease were cloned in Escherichia coli recombinant mutant FB827 dppF::Km(pAM 238-hasR). This strain, which expresses HasR, a foreign heme outer membrane receptor, is potentially capable of using heme as an iron source. However, this process is invalidated due to a dppF::Km mutation which inactivates the Dpp heme/peptide permease responsible for heme, dipeptide, and delta-aminolevulinic (ALA) transport through the E. coli inner membrane. We show here that hemTUV genes complement the Dpp permease for heme utilization as an iron source and thus are functional in E. coli. However, hemTUV genes do not complement the Dpp permease for ALA uptake, indicating that the HemTUV permease does not transport ALA. Peptides do not inhibit heme uptake in vivo, indicating that, unlike Dpp permease, HemTUV permease does not transport peptides. HemT, the periplasmic binding protein, binds heme. Heme binding is saturable and not inhibited by peptides that inhibit heme uptake by the Dpp system. Thus, the S. marcescens HemTUV permease and, most likely, HemTUV orthologs present in many gram-negative pathogens form a class of heme-specific permeases different from the Dpp peptide/heme permease characterized in E. coli.     

A new role for heme, facilitating release of iron from the bacterioferritin iron biomineral

Bacterioferritin (BFR) from Escherichia coli is a member of the ferritin family of iron storage proteins and has the capacity to store very large amounts of iron as an Fe(3+) mineral inside its central cavity. The ability of organisms to tap into their cellular stores in times of iron deprivation requires that iron must be released from ferritin mineral stores. Currently, relatively little is known about the mechanisms by which this occurs, particularly in prokaryotic ferritins. Here we show that the bis-Met-coordinated heme groups of E. coli BFR, which are not found in other members of the ferritin family, play an important role in iron release from the BFR iron biomineral: kinetic iron release experiments revealed that the transfer of electrons into the internal cavity is the rate-limiting step of the release reaction and that the rate and extent of iron release were significantly increased in the presence of heme. Despite previous reports that a high affinity Fe(2+) chelator is required for iron release, we show that a large proportion of BFR core iron is released in the absence of such a chelator and further that chelators are not passive participants in iron release reactions. Finally, we show that the catalytic ferroxidase center, which is central to the mechanism of mineralization, is not involved in iron release; thus, core mineralization and release processes utilize distinct pathways.     

Identification and cloning of a fur regulatory gene in Yersinia pestis.

Yersinia pestis is one of many microorganisms responding to environmental iron concentrations by regulating the synthesis of proteins and an iron transport system(s). In a number of bacteria, expression of iron uptake systems and other virulence determinants is controlled by the Fur regulatory protein. DNA hybridization analysis revealed that both pigmented and nonpigmented cells of Y. pestis possess a DNA locus homologous to the Escherichia coli fur gene. Introduction of a Fur-regulated beta-galactosidase reporter gene into Y. pestis KIM resulted in iron-responsive beta-galactosidase activity, indicating that Y. pestis KIM expresses a functional Fur regulatory protein. A cloned 1.9-kb ClaI fragment of Y. pestis chromosomal DNA hybridized specifically to the fur gene of E. coli. The coding region of the E. coli fur gene hybridized to a 1.1-kb region at one end of the cloned Y. pestis fragment. The failure of this clone to complement an E. coli fur mutant suggests that the 1.9-kb clone does not contain a functional promoter. Subcloning of this fragment into an inducible expression vector restored Fur regulation in an E. coli fur mutant. In addition, a larger 4.8-kb Y. pestis clone containing the putative promoter region complemented the Fur- phenotype. These results suggest that Y. pestis possesses a functional Fur regulatory protein capable of interacting with the E. coli Fur system. In Y. pestis Fur may regulate the expression of iron transport systems and other virulence factors in response to iron limitation in the environment. Possible candidates for Fur regulation in Y. pestis include genes involved in ferric iron transport as well as hemin, heme/hemopexin, heme/albumin, ferritin, hemoglobin, and hemoglobin/haptoglobin utilization.     

The theft of host heme by Gram-positive pathogenic bacteria

The element iron is essential for bacteria and plays a key role in the virulence and pathology of bacterial diseases. The largest reservoir of iron within the human body is in protoporphyrin IX, the compound commonly referred to as heme and bound by hemoglobin. For many years, the study of heme uptake in bacteria was restricted to Gram-negative organisms. However, recent studies have shed light on how bacteria containing a thick peptidoglycan, such as Gram-positive bacteria, acquire and transport heme. This review summarizes old and new research covering the acquisition, transport, and utilization of heme in Gram-positive bacterial pathogens.     

Interaction of bacteria of the genus Bartonella with the host: inhibition of apoptosis, induction of proliferation, and formation of tumors

The bacteria of the genus Bartonella are the causative agents for earlier not diagnosed or re-emergent diseases of the humans, danger of which increases in relation with increasing number of persons with the disturbed immune status. Bartonellae are intracellular parasites, the places of their habitation in the humans and animals are the endothelial cells of blood vessels and erythrocytes. The modern data concerning major factors of the Bartonellae virulence and host-bacteria interactions were considered and discussed in this article. The induction of the type IV secretion system, effector protein transmission, inhibition of the endothelial cells apoptosis, and induction of their proliferation lead to formation of new blood vessels and tumors.     

Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli

EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.     

Staining of Bartonella henselae with carboxyfluorescein diacetate succinimidyl ester for tracking infection in erythrocytes and epithelial cells

Bartonella infection (Bartonella henselae in particular) is responsible for a widening spectrum of human diseases. The persistent colonization of erythrocytes is a feature of Bartonella infection. Endothelial and epithelial cells are also widely used to study the pathogenesis of bartonellosis in vitro. Exploring a convenient method for visualizing the bacillus without affecting infectivity would be very interesting. Carboxyfluorescein diacetate succinimidyl ester (CFSE) has been previously used for staining several bacterial species to study their adhesion to host cells. The present study demonstrated the efficiency and safety of using CFSE in staining B. henselae. The staining of bacillus-invaded erythrocytes and epithelial cells in vitro successfully allowed for flow cytometry and confocol microscopy analyses. Parallel tests using untreated bacteria confirmed that CFSE staining did not result in side effects on the infectivity of B. henselae. Labeling Bartonella with CFSE is a valuable method for studying the bacteria-host interaction.     

Dynamic ligation properties of the Escherichia coli heme chaperone CcmE to non-covalently bound heme

The cytochrome c maturation protein CcmE is an essential membrane-anchored heme chaperone involved in the post-translational covalent attachment of heme to c-type cytochromes in Gram-negative bacteria such as Escherichia coli. Previous in vitro studies have shown that CcmE can bind heme both covalently (via a histidine residue) and non-covalently. In this work we present results on the latter form of heme binding to a soluble form of CcmE. Examination of a number of site-directed mutants of E. coli CcmE by resonance Raman spectroscopy has identified ligands of the heme iron and provided insight into the initial steps of heme binding by CcmE before it binds the heme covalently. The heme binding histidine (His-130) appears to ligate the heme iron in the ferric oxidation state, but two other residues ligate the iron in the ferrous form, thereby freeing His-130 to undergo covalent attachment to a heme vinyl group. It appears that the heme ligation in the non-covalent form is different from that in the holo-form, suggesting that a change in ligation could act as a trigger for the formation of the covalent bond and showing the dynamic and oxidation state-sensitive ligation properties of CcmE.     

Structure and heme binding properties of Escherichia coli O157:H7 ChuX

For many pathogenic microorganisms, iron acquisition from host heme sources stimulates growth, multiplication, ultimately enabling successful survival and colonization. In gram‐negative Escherichia coli O157:H7, Shigella dysenteriae and Yersinia enterocolitica the genes encoded within the heme utilization operon enable the effective uptake and utilization of heme as an iron source. While the complement of proteins responsible for heme internalization has been determined in these organisms, the fate of heme once it has reached the cytoplasm has only recently begun to be resolved. Here we report the first crystal structure of ChuX, a member of the conserved heme utilization operon from pathogenic E. coli O157:H7 determined at 2.05 Å resolution. ChuX forms a dimer which remarkably given low sequence homology, displays a very similar fold to the monomer structure of ChuS and HemS, two other heme utilization proteins. Absorption spectral analysis of heme reconstituted ChuX demonstrates that ChuX binds heme in a 1:1 manner implying that each ChuX homodimer has the potential to coordinate two heme molecules in contrast to ChuS and HemS where only one heme molecule is bound. Resonance Raman spectroscopy indicates that the heme of ferric ChuX is composed of a mixture of coordination states: 5‐coordinate and high‐spin, 6‐coordinate and low‐spin, and 6‐coordinate and high‐spin. In contrast, the reduced ferrous form displays mainly a 5‐coordinate and high‐spin state with a minor contribution from a 6‐coordinate and low‐spin state. The ν_Fe‐CO and ν_C‐O frequencies of ChuX‐bound CO fall on the correlation line expected for histidine‐coordinated hemoproteins indicating that the fifth axial ligand of the ferrous heme is the imidazole ring of a histidine residue. Based on sequence and structural comparisons, we designed a number of site‐directed mutations in ChuX to probe the heme binding sites and dimer interface. Spectral analysis of ChuX and mutants suggests involvement of H65 and H98 in heme coordination as mutations of both residues were required to abolish the formation of the hexacoordination state of heme‐bound ChuX.     

The crystal structure of HasA, a hemophore secreted by Serratia marcescens.

Free iron availability is strongly limited in vertebrate hosts, making the iron acquisition by siderophores inappropriate. Pathogenic bacteria have developed various ways to use the host's iron from iron-containing proteins. Serratia marcescens can use the iron from hemoglobin through the secretion of a hemophore called HasA, which takes up the heme from hemoglobin and shuttles it to the receptor HasR, which in turn, releases heme into the bacterium. We report here the first crystal structure of such a hemophore, bound to a heme group at two different pH values and at a resolution of 1.9 A. The structure reveals a new original fold and suggests a hypothetical mechanism for both heme uptake and release.