Delineating the regions of human transferrin involved in interactions with transferrin binding protein B from Neisseria meningitidis

Pathogenic bacteria in the Neisseriaceae possess a surface receptor mediating iron acquisition from human transferrin (hTf) that consists of a transmembrane iron transporter (TbpA) and a surface‐exposed lipoprotein (TbpB). In this study, we used hydrogen/deuterium exchange coupled to mass spectrometry (H/DX‐MS) to elucidate the effects on hTf by interaction with TbpB or derivatives of TbpB. An overall conserved interaction was observed between hTf and full‐length or N‐lobe TbpB from Neisseria meningitidis strains B16B6 or M982 that represent two distinct subtypes of TbpB. Changes were observed exclusively in the C‐lobe of hTf and were caused by the interaction with the N‐lobe of TbpB. Regions localized to the ‘lip’ of the C1 and C2 domains that flank the interdomain cleft represent sites of direct contact with TbpB whereas the peptides within the interdomain cleft that encompass iron binding ligands are inaccessible in the closed (holo) conformation. Although substantial domain separation upon binding TbpB cannot be excluded by the H/DX‐MS data, the preferred model of interaction involves binding hTf C‐lobe in the closed conformation. Alternate explanations are provided for the substantial protection from deuteration of the peptides encompassing iron binding ligands within the interdomain cleft but cannot be differentiated by the H/DX‐MS data.     

Identification of human transferrin-binding sites within meningococcal transferrin-binding protein B.

Transferrin-binding protein B (TbpB) from Neisseria meningitidis binds human transferrin (hTf) at the surface of the bacterial cell as part of the iron uptake process. To identify hTf binding sites within the meningococcal TbpB, defined regions of the molecule were produced in Escherichia coli by a translational fusion expression system and the ability of the recombinant proteins (rTbpB) to bind peroxidase-conjugated hTf was characterized by Western blot and dot blot assays. Both the N-terminal domain (amino acids [aa] 2 to 351) and the C-terminal domain (aa 352 to 691) were able to bind hTf, and by a peptide spot synthesis approach, two and five hTf binding sites were identified in the N- and C-terminal domains, respectively. The hTf binding activity of three rTbpB deletion variants constructed within the central region (aa 346 to 543) highlighted the importance of a specific peptide (aa 377 to 394) in the ligand interaction. Taken together, the results indicated that the N- and C-terminal domains bound hTf approximately 10 and 1000 times less, respectively, than the full-length rTbpB (aa 2 to 691), while the central region (aa 346 to 543) had a binding avidity in the same order of magnitude as the C-terminal domain. In contrast with the hTf binding in the N-terminal domain, which was mediated by conformational epitopes, linear determinants seemed to be involved in the hTf binding in the C-terminal domain. The host specificity for transferrin appeared to be mediated by the N-terminal domain of the meningococcal rTbpB rather than the C-terminal domain, since we report that murine Tf binds to the C-terminal domain. Antisera raised to both N- and C-terminal domains were bactericidal for the parent strain, indicating that both domains are accessible at the bacterial surface. We have thus identified hTf binding sites within each domain of the TbpB from N. meningitidis and propose that the N- and C-terminal domains together contribute to the efficient binding of TbpB to hTf with their respective affinities and specificities for determinants of their ligand.     

Identification of sequences in human transferrin that bind to the bacterial receptor protein, transferrin-binding protein B

Alignment of amino-acid sequences from the N-terminal and C-terminal halves of transferrin-binding protein B revealed an underlying bilobed nature with several regions of identity. Based on this analysis, purified recombinant fusion proteins of maltose-binding protein (Mbp) with intact TbpB, its N-terminal half or C-terminal half from the human pathogens Neisseria meningitidis and Moraxella catarrhalis were produced. Solid-phase binding assays and affinity isolation assays demonstrated that the N-terminal and C-terminal halves of TbpB could bind independently to human transferrin (hTf). A solid-phase overlapping synthetic peptide library representing the amino-acid sequence of hTf was probed with soluble, labelled Mbp-TbpB fusions to localize TbpB-binding regions on hTf. An essentially identical series of peptides from domains within both lobes of hTf was recognized by intact TbpB from both organisms, demonstrating a conserved TbpB-hTf interaction. Both halves of TbpB from N. meningitidis bound the same series of peptides, which included peptides from equivalent regions on the two hTf lobes, indicating that TbpB interacts with each lobe of hTf in a similar manner. Mapping of the peptide-binding regions on a molecular model of hTf revealed a series of nearly adjacent surface regions that nearly encircled each lobe. Binding studies with chimeric hTf/bTf transferrins demonstrated that regions in the C-lobe of hTf were preferentially recognized by the N-terminal half of TbpB. Collectively, these results provide evidence that TbpB consists of two lobes, each with distinct yet homologous Tf-binding regions.     

Anchor peptide of transferrin-binding protein B is required for interaction with transferrin-binding protein A

Gram-negative bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae, and Neisseriaceae families rely on an iron acquisition system that acquires iron directly from host transferrin (Tf). The process is mediated by a surface receptor composed of transferrin-binding proteins A and B (TbpA and TbpB). TbpA is an integral outer membrane protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is a surface-exposed lipoprotein that facilitates the iron uptake process. In this study, we demonstrate that the region encompassing amino acids 7-40 of Actinobacillus pleuropneumoniae TbpB is required for forming a complex with TbpA and that the formation of the complex requires the presence of porcine Tf. These results are consistent with a model in which TbpB is responsible for the initial capture of iron-loaded Tf and subsequently interacts with TbpA through the anchor peptide. We propose that TonB binding to TbpA initiates the formation of the TbpB-TbpA complex and transfer of Tf to TbpA.     

The Negatively Charged Regions of Lactoferrin Binding Protein B,an Adaptation against Anti-Microbial Peptides

Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein’s C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides.     

Conserved interaction between transferrin and transferrin-binding proteins from porcine pathogens

Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.     

Transferrin-binding protein B of Neisseria meningitidis: sequence-based identification of the transferrin-Binding site confirmed by site-directed mutagenesis

A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.     

Pyrophosphate-Mediated Iron Acquisition from Transferrin in Neisseria meningitidis Does Not Require TonB Activity

The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival.     

Insight into the structure and function of the transferrin receptor from Neisseria meningitidis using microcalorimetric techniques

The transferrin receptor of Neisseria meningitidis is composed of the transmembrane protein TbpA and the outer membrane protein TbpB. Both receptor proteins have the capacity to independently bind their ligand human transferrin (htf). To elucidate the specific role of these proteins in receptor function, isothermal titration calorimetry was used to study the interaction between purified TbpA, TbpB or the entire receptor (TbpA + TbpB) with holo- and apo-htf. The entire receptor was shown to contain a single high affinity htf-binding site on TbpA and approximately two lower affinity binding sites on TbpB. The binding sites appear to be independent. Purified TbpA was shown to have strong ligand preference for apo-htf, whereas TbpA in the receptor complex with TbpB preferentially binds the holo form of htf. The orientation of the ligand specificity of TbpA toward holo-htf is proposed to be the physiological function of TbpB. Furthermore, the thermodynamic mode of htf binding by TbpB of isotypes I and II was shown to be different. A protocol for the generation of active, histidine-tagged TbpB as well as its individual N- and C-terminal domains is presented. Both domains are shown to strongly interact with each other, and isothermal titration calorimetry and circular dichroism experiments provide clear evidence for this interaction causing conformational changes. The N-terminal domain of TbpB was shown to be the site of htf binding, whereas the C-terminal domain is not involved in binding. Furthermore, the interactions between TbpA and the different domains of TbpB have been demonstrated.     

Plant‐derived recombinant human serum transferrin demonstrates multiple functions

Human serum transferrin (hTf) is the major iron‐binding protein in human plasma, having a vital role in iron transport. Additionally, hTf has many other uses including antimicrobial functions and growth factor effects on mammalian cell proliferation and differentiation. The multitask nature of hTf makes it highly valuable for different therapeutic and commercial applications. However, the success of hTf in these applications is critically dependent on the availability of high‐quality hTf in large amounts. In this study, we have developed plants as a novel platform for the production of recombinant (r)hTf. We show here that transgenic plants are an efficient system for rhTf production, with a maximum accumulation of 0.25% total soluble protein (TSP) (or up to 33.5 μg/g fresh leaf weight). Furthermore, plant‐derived rhTf retains many of the biological activities synonymous with native hTf. In particular, rhTf reversibly binds iron in vitro, exhibits bacteriostatic activity, supports cell proliferation in serum‐free medium and can be internalized into mammalian cells in vitro. The success of this study validates the future application of plant rhTf in a variety of fields. Of particular interest is the use of plant rhTf as a novel carrier for cell‐specific or oral delivery of protein/peptide drugs for the treatment of human diseases such as diabetes. To demonstrate this hypothesis, we have additionally expressed an hTf fusion protein containing glucagon‐like peptide 1 (GLP‐1) or its derivative in plants. Here, we show that plant‐derived hTf‐GLP‐1 fusion proteins retain the ability to be internalized by mammalian cells when added to culture medium in vitro.     

Kinetic analysis of ligand interaction with the gonococcal transferrin-iron acquisition system

The transferrin iron acquisition system of Neisseria gonorrhoeae consists of two dissimilar transferrin binding proteins (Tbp) A and B. TbpA is a TonB dependent transporter while TbpB is a lipoprotein that makes iron acquisition from transferrin (Tf) more efficient. In an attempt to further define the individual roles of these receptors in the process of Tf-iron acquisition, the kinetics of the receptor proteins in regards to ligand association and dissociation were evaluated. Tf association with TbpB was rapid as compared to TbpA. Tf dissociation from the wild-type receptor occurred in a biphasic manner; an initial rapid release was followed by a slower dissociation over time. Both TbpA and TbpB demonstrated a two-phase release pattern; however, TbpA required both TonB and TbpB for efficient Tf dissociation from the cell surface. The roles of TbpA and TbpB in Tf dissociation were further examined, utilizing previously created HA fusion proteins. Using a Tf-utilization deficient TbpA-HA mutant, we concluded that the slower rate of ligand dissociation demonstrated by the wild-type transporter was a function of successful iron internalization. Insertion into the C-terminus of TbpB decreased the rate of Tf dissociation, while insertion into the N-terminus had no effect on this process. From these studies, we propose that TbpA and TbpB function synergistically during the process of Tf iron acquisition and that TbpB makes the process of Tf-iron acquisition more efficient at least in part by affecting association and dissociation of Tf from the cell surface.     

Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis

Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf. This receptor consists of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a surface lipoprotein, lactoferrin binding protein B (LbpB). The N-lobe of the homologous transferrin binding protein B, TbpB, has been shown to facilitate transferrin binding in the process of iron acquisition. Currently there is little known about the role of LbpB in iron acquisition or how Lf interacts with the bacterial receptor proteins. No structural information on any LbpB or domain is available. In this study, we express and purify from Escherichia coli the full-length LbpB and the N-lobe of LbpB from the bovine pathogen Moraxella bovis for crystallization trials. We demonstrate that M. bovis LbpB binds to bovine but not human Lf. We also report the crystal structure of the N-terminal lobe of LbpB from M. bovis and compare it with the published structures of TbpB to speculate on the process of Lf mediated iron acquisition.     

A dynamic model of the meningococcal transferrin receptor.

Iron is an essential nutrient for all organisms and consequently, the ability to bind transferrin and sequester iron from his source constitutes a distinct advantage to a blood-borne bacterial pathogen. Levels of free iron are strictly limited in human serum, largely through the action of the iron-binding protein transferrin. The acquisition of trasferrin-iron is coincident with pathogenicity among Neisseria species and a limited number of other pathogens of human and veterinary significance. In Neisseria meningitidis, transferrin binding relies on two co-expressed, outer membrane proteins distinct in aspects of both structure and function. These proteins are independently and simultaneously capable of binding human transferrin and both are required for the optimal uptake of iron from this source. It has been established that transferrin-binding proteins (designated TbpA and TbpB) form a discrete, specific complex which may be composed of a transmembrane species (composed of the TbpA dimer) associated with a single surface-exposed lipoprotein (TbpB). This more exposed protein is capable of selectively binding iron-saturated transferrin and the receptor complex has ligand-binding properties which are distinct from either of its components. Previous in vivo analyses of N. gonorrhoeae, which utilizes a closely related transferrin-iron uptake system, indicated that this receptor exists in several conformations influenced in part by the presence (or absence) of transferrin.Here we propose a dynamic model of the meningococcal transferrin receptor which is fully consistent with the current data concerning this subject. We suggest that TbpB serves as the initial binding site for iron-saturated transferrin and brings this ligand close to the associated transmembrane dimer, enabling additional binding events and orientating transferrin over the dual TbpA pores. The antagonistic association of these receptor proteins with a single ligand molecule may also induce conformational change in transferrin, thereby favouring the release of iron. As, in vivo, transferrin may have iron in one or both lobes, this dynamic molecular arrangement would enable iron uptake from either iron-binding site. In addition, the predicted molecular dimensions of the putative TbpA dimer and hTf are fully consistent with these proposals. Given the diverse data used in the formulation of this model and the consistent characteristics of transferrin binding among several significant Gram-negative pathogens, we speculate that such receptor-ligand interactions may be, at least in part, conserved between species. Consequently, this model may be applicable to bacteria other than N. meningitidis.     

Steric and allosteric factors prevent simultaneous binding of transferrin-binding proteins A and B to transferrin

The ability to acquire iron directly from host Tf (transferrin) is an adaptation common to important bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae and Neisseriaceae families. A surface receptor comprising an integral outer membrane protein, TbpA (Tf-binding protein A), and a surface-exposed lipoprotein, TbpB (Tf-binding protein B), mediates the iron acquisition process. TbpB is thought to extend from the cell surface for capture of Tf to initiate the process and deliver Tf to TbpA. TbpA functions as a gated channel for the passage of iron into the periplasm. In the present study we have mapped the effect of TbpA from Actinobacillus pleuropneumoniae on pTf (porcine Tf) using H/DX-MS (hydrogen/deuterium exchange coupled to MS) and compare it with a previously determined binding site for TbpB. The proposed TbpA footprint is adjacent to and potentially overlapping the TbpB-binding site, and induces a structural instability in the TbpB site. This suggests that simultaneous binding to pTf by both receptors would be hindered. We demonstrate that a recombinant TbpB lacking a portion of its anchor peptide is unable to form a stable ternary TbpA-pTf-TbpB complex. This truncated TbpB does not bind to a preformed Tf-TbpA complex, and TbpA removes pTf from a preformed Tf-TbpB complex. Thus the results of the present study support a model whereby TbpB 'hands-off' pTf to TbpA, which completes the iron removal and transport process.     

Discrimination of Psychrotrophic and Mesophilic Strains of the Bacillus cereus Group by PCR Targeting of Major Cold Shock Protein Genes

Detection of psychrotrophic strains (those able to grow at or below 7°C) of the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides) in food products is at present extremely slow with conventional microbiology. This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7°C) by means other than growth at low temperature, which takes 5 to 10 days for detection. Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the B. cereus group and discriminating between psychrotrophic and mesophilic strains. It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the B. cereus group.     

Preparation and Characterization of Neisseria meningitidis Mutants Deficient in Production of the Human Lactoferrin-Binding Proteins LbpA and LbpB

Pathogenic members of the family Neisseriaceae produce specific receptors facilitating iron acquisition from transferrin (Tf) and lactoferrin (Lf) of their mammalian host. Tf receptors are composed of two outer membrane proteins, Tf-binding proteins A and B (TbpA and TbpB; formerly designated Tbp1 and Tbp2, respectively). Although only a single Lf-binding protein, LbpA (formerly designated Lbp1), had previously been recognized, we recently identified additional bacterial Lf-binding proteins in the human pathogens Neisseria meningitidis and Moraxella catarrhalis and the bovine pathogen Moraxella bovis by a modified affinity isolation technique (R. A. Bonnah, R.-H. Yu, and A. B. Schryvers, Microb. Pathog. 19:285–297, 1995). In this report, we characterize an open reading frame (ORF) located immediately upstream of the N. meningitidis B16B6 lbpA gene. Amino acid sequence comparisons of various TbpBs with the product of the translated DNA sequence from the upstream ORF suggests that the region encodes the Lf-binding protein B homolog (LbpB). The LbpB from strain B16B6 has two large stretches of negatively charged amino acids that are not present in the various transferrin receptor homologs (TbpBs). Expression of the recombinant LbpB protein as a fusion with maltose binding protein demonstrated functional Lf-binding activity. Studies with N. meningitidis isogenic mutants in which the lbpA gene and the ORF immediately upstream of lbpA (putative lbpB gene) were insertionally inactivated demonstrated that LbpA, but not LbpB, is essential for iron acquisition from Lf in vitro.     

Molecular characterization of hybrid Tbp2 proteins from Neisseria meningitides

Transferrin-binding protein 2 (Tbp2) from Neisseria is an outer membrane-associated extracellular lipoprotein that is involved in iron capture within the infected host. The analysis of tbp2 clones isolated from various bacterial strains revealed extensive divergences throughout the open reading frame (ORF), with predicted amino acid (aa) sequences displaying 47% to 83% identity. Such a variability is likely to have resulted from the selective pressure exerted by the host immune system, but raises questions regarding the existing constraints for conservation of protein function. Indeed, the neisserial Tbp2s include a large structured domain, extending throughout the N-terminal half of the protein (～270–290 aa), which is extremely stable and whose conformational integrity is required for efficient binding to human transferrin (hTf). In this work, a functional study of Tbp2s encoded by hybrid genes constructed by reassorting highly divergent tbp2 sequences in the region of the ORF encoding this structured domain was performed. The data demonstrate that the determinant intramolecular interactions allowing formation of a stable Tbp2 structure able to interact efficiently with hTf or/and that the Tbp2 residues involved in the interaction with hTf are not well conserved. However, a number of rearrangements appeared to generate genes encoding proteins which have retained structural stability and hTf-binding capacity. This suggested that despite the extreme aa sequence divergence and the conformational constraints, horizontal genetic exchanges, which are known to occur in neisserial populations, may have contributed significantly to the generation of sequence variation within tbp2 ORFs. The analysis of two tbp2clones characterized in this work supports this hypothesis.     

Raman Spectroscopy and Chemometrics for Identification and Strain Discrimination of the Wine Spoilage Yeasts Saccharomyces cerevisiae,Zygosaccharomyces bailii,and Brettanomyces bruxellensis

The yeasts Zygosaccharomyces bailii, Dekkera bruxellensis (anamorph, Brettanomyces bruxellensis), and Saccharomyces cerevisiae are the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeasts Z. bailii, B. bruxellensis, and S. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% for Z. bailii, 92.3% for B. bruxellensis, and 98.6% for S. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%.     

Full genome SNP-based phylogenetic analysis reveals the origin and global spread of Brucella melitensis

Background

Brucellosis is an important zoonotic disease that affects both humans and animals. We sequenced the full genome and characterised the genetic diversity of two Brucella melitensis isolates from Malaysia and the Philippines. In addition, we performed a comparative whole-genome single nucleotide polymorphism (SNP) analysis of B. melitensis strains collected from around the world, to investigate the potential origin and the history of the global spread of B. melitensis.

Results

Single sequencing runs of each genome resulted in draft genome sequences of MY1483/09 and Phil1136/12, which covered 99.85% and 99.92% of the complete genome sequences, respectively. The B. melitensis genome sequences, and two B. abortus strains used as the outgroup strains, yielded a total of 13,728 SNP sites. Phylogenetic analysis using whole-genome SNPs and geographical distribution of the isolates revealed spatial clustering of the B. melitensis isolates into five genotypes, I, II, III, IV and V. The Mediterranean strains, identified as genotype I, occupied the basal node of the phylogenetic tree, suggesting that B. melitensis may have originated from the Mediterranean regions. All of the Asian B. melitensis strains clustered into genotype II with the SEA strains, including the two isolates sequenced in this study, forming a distinct clade denoted here as genotype IId. Genotypes III, IV and V of B. melitensis demonstrated a restricted geographical distribution, with genotype III representing the African lineage, genotype IV representing the European lineage and genotype V representing the American lineage.

Conclusion

We showed that SNPs retrieved from the B. melitensis draft full genomes were sufficient to resolve the interspecies relationships between B. melitensis strains and to discriminate between the vaccine and endemic strains. Phylogeographic reconstruction of the history of B. melitensis global spread at a finer scale by using whole-genome SNP analyses supported the origin of all B. melitensis strains from the Mediterranean region. The possible global distribution of B. melitensis following the ancient trade routes was also consistent with whole-genome SNP phylogeny. The whole genome SNP phylogenetics analysis, hence is a powerful tool for intraspecies discrimination of closely related species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1294-x) contains supplementary material, which is available to authorized users.