Iron Acquisition Mechanisms and Their Role in the Virulence of Burkholderia Species

Abstract

Burkholderia is a genus within the β-Proteobacteriaceae that contains at least 90 validly named species which can be found in a diverse range of environments. A number of pathogenic species occur within the genus. These include Burkholderia cenocepacia and Burkholderia multivorans, opportunistic pathogens that can infect the lungs of patients with cystic fibrosis, and are members of the Burkholderia cepacia complex (Bcc). Burkholderia pseudomallei is also an opportunistic pathogen, but in contrast to Bcc species it causes the tropical human disease melioidosis, while its close relative Burkholderia mallei is the causative agent of glanders in horses. For these pathogens to survive within a host and cause disease they must be able to acquire iron. This chemical element is essential for nearly all living organisms due to its important role in many enzymes and metabolic processes. In the mammalian host, the amount of accessible free iron is negligible due to the low solubility of the metal ion in its higher oxidation state and the tight binding of this element by host proteins such as ferritin and lactoferrin. As with other pathogenic bacteria, Burkholderia species have evolved an array of iron acquisition mechanisms with which to capture iron from the host environment. These mechanisms include the production and utilization of siderophores and the possession of a haem uptake system. Here, we summarize the known mechanisms of iron acquisition in pathogenic Burkholderia species and discuss the evidence for their importance in the context of virulence and the establishment of infection in the host. We have also carried out an extensive bioinformatic analysis to identify which siderophores are produced by each Burkholderia species that is pathogenic to humans.

Keywords: Burkholderia; cystic fibrosis; haem uptake; iron; melioidosis; siderophores.

Figure 1

The major groups of Burkholderia species. Groups are based on the 16S rRNA-based phylogenetic tree (see, for example, Depoorter et al., 2016). Species most commonly associated with infections in humans are shown in red font (for a full list of the Bcc species see Table 1). Alternative classification schemes involving the proposed new genera Paraburkholderia and Caballeronia are also indicated (see text for details). Recently, it has been proposed that B. rhizoxinica should be transferred to a new, as yet unnamed genus (Beukes et al., 2017).

Figure 2

Structure of siderophores produced by Burkholderia species. (A) Ornibactins contain an N-terminal ornithine that is acylated with a C4, C6, or C8 β-hydroxycarboxylic acid on the δ-amino nitrogen atom, giving rise to ornibactin-C4, -C6, or -C8. The δ-amino nitrogen atom is also hydroxylated. The other three amino acids in the tetrapeptide are D-hydroxyaspartate, L-serine, and the C-terminal ornithine that is formylated and hydroxylated on the δ-amino nitrogen atom and the carboxyl group is conjugated to putrescine. As with the malleobactins, they contain two bidentate hydroxamate ligands and a single bidentate α-hydroxycarboxylate ligand. (B) Malleobactin E, the siderophore-active malleobactin congener of B. thailandensis. (C) The siderophore-active malleobactin congener of B. xenovorans, tentatively referred to here as “malleobactin X.” (D) Cepaciachelin contains two 2,3-DHBA groups that form amide linkages with the two amino groups of lysine, which in turn is conjugated to a molecule of putrescine (1,4-diaminobutane) on its α-carboxyl group. (E) Pyochelin contains two less commonly occurring bidentate iron-chelating groups (2-hydroxyphenyl thiazoline and N-methylthiazolidine-4-carboxylate). (F) Cepabactin, a cyclic hydroxamate bidentate siderophore. Chemical groups that distinguish the ornibactins and malleobactins are indicated in red circles or ellipses.

Figure 3

Organization of ornibactin, malleobactin, and phymabactin biosynthesis and utilization genes in pathogenic Burkholderia and related species. Genes are represented by block arrows and are color coded as indicated in the figure [the precise role of the MbtH-like OrbH/MbaG/PhmF proteins is unknown but they are assumed to be required for biosynthesis of the siderophore based on the requirement for other MbtH-like proteins for NRPS-mediated biosynthesis of some peptides; (Wolpert et al., ; Baltz, 2011)]. The numbering of each gene cluster corresponds to the numbering system for the species listed at the bottom of the figure. The font color used for each species name corresponds to the siderophore produced as follows: black, ornibactin; red, malleobactins; green, phymabactin. NRPSpredictor2 (Rottig et al., 2011) was used to predict the siderophore product based on the substrates accepted by the four adenylation domains present in OrbI/OrbJ, MbaA/MbaB, and PhmA/PhmB for each species. For systems known to specify ornibactin, the first and last (N- and C-terminal) adenylation domains of the OrbI-OrbJ NRPS pair are both predicted to accept leucine with highest probability, reflecting the presence of N⁵-3-hydroxyacyl-N⁵-hydroxyornithine and N⁵-formyl-N⁵-hydroxyornithine, respectively, at these positions in the tetrapeptide product. For malleobactin, the predicted specificity of the N-terminal adenylation domain changes to β-hydroxytyrosine although the accepted substrate is N⁵-formyl-N⁵-hydroxyornithine. In some species, such as B. thailandensis, the N-terminal N⁵-formyl-N⁵-hydroxyornithine is formylated on the N²-amino group upon formation of the tetrapeptide to generate malleobactin E (Franke et al., 2015), whereas in B. xenovorans the N-terminal N⁵-formyl-N⁵-hydroxyornithine does not appear to undergo such a tailoring reaction (Vargas-Straube et al., 2016) and so we tentatively refer to this siderophore as “malleobactin X.” The N- and C-terminal adenylation domains of PhmA-PhmB are predicted to accept aspartate and cysteine, respectively, but the structure of the product, phymabactin, is unknown, although it is predicted to have siderophore activity based on its genomic context (Esmaeel et al., 2016). In all cases, the second and third adenylation domains are predicted to accept aspartate and serine, respectively, which correspond to β-hydroxy-D-aspartate and L-serine in the final product. The nomenclature proposed for each gene is shown below the gene clusters, and the gene designations are color coded as follows: ornibactin, black (Agnoli et al., 2006); the two systems for malleobactin, red (upper, Alice et al., ; lower, Franke et al., 2013); the two systems for phymabactin, green (upper, Esmaeel et al., ; lower, this study). Genes indicated by a single letter have the same prefix as the gene name at the extreme left. Dashes indicate the absence of a gene. Question mark indicates where a gene name has not been proposed. The initial annotation of the phymabactin gene cluster (Esmaeel et al., 2016) did not include the third and last genes in the cluster or the fact that a TBDR gene occurs in other species bearing this gene cluster (upper annotation in green font). Therefore, we have proposed an alternative nomenclature based on the ornibactin gene cluster (lower annotation in green font). Scale bar refers to gene lengths and not intergenic regions, which in some cases have been exaggerated to permit alignment of each gene cluster. Species marked with an asterisk belong to a subclade within the B. xenovorans group and have been reassigned to the new genus Paraburkholderia (Sawana et al., 2014). Collimonas is a genus within the Oxalobacteraceae, a family belonging to the order Burkholderiales. Member species of the Bcc are enclosed in a box (B. arboris is not listed as its genome sequence is not currently available). Gene loci are shown in Supplementary Table 1.

Figure 4

Organization of the cepaciachelin and pyochelin biosynthesis and utilization genes and the genes for the haem uptake system in Burkholderia species. (A) The cepaciachelin gene cluster in Burkholderia species encodes enzymes for the synthesis of the precursor 2,3-dihydroxybenzoic acid (DHBA) from chorismate and its assembly into the siderophore. Genes encoding possible cepaciachelin transport proteins are also present, including the TonB-dependent receptor, CpcG, and an MFS transporter, CpcH. Most of the genes have been previously annotated (Esmaeel et al., 2016) but the annotation has been extended here (genes labeled in red font) and includes components of a cytoplasmic membrane ABC transporter which may be involved in uptake of ferric cepaciachelin (CpcF, -I, and -J). EstA is homologous to the cytoplasmic enterobactin and salmochelin esterases Fes and IroD that are required for removal of iron from ferric-enterobactins following their uptake. However, it contains a putative Sec-dependent signal peptide and so may be periplasmically located like the C. jejuni enterobactin esterase, Cee (Zeng et al., 2013). This may suggest that the putative cepaciachelin receptor (CpcG) and the cytoplasmic membrane transporter also recognizes ferric-enterobactin. With the exception of B. ambifaria and B. pseudomultivorans the cepaciachelin gene cluster includes a gene encoding a DAHP synthase which catalyses the first step in the shikimate pathway that leads to the biosynthesis of chorismate from erythrose-4-phosphate and PEP. Note that in Esmaeel et al. (2016) cphA-cphC should be annotated as cpcA-cpcC, as shown here (V. Leclere, personal communication). -, gene not assigned a four letter name. Gene loci are shown in Supplementary Table 2. (B) The pyochelin gene cluster. Genes annotated with a single letter are designated with the prefix pch. Products of the biosynthetic genes generate the precursor salicylic acid from chorismate (PchAB), activate it (PchD) and assemble it into pyochelin along with two molecules of cysteine (PchCEFG). The pchHI and fptABCX genes encode membrane proteins, of which two (FptA and FptX) are involved in the transport of exogenous ferric-pyochelin across the outer and inner membranes, respectively. fptBC and pchHI appear not to be essential for export of pyochelin nor for uptake of iron-bound pyochelin (see Youard et al., for a review). Gene loci are shown in Supplementary Table 3. (C) Organization of the Burkholderia haem uptake genes, bhuRSTUV (Shalom et al., ; Thomas, 2007), also referred to as hmuRSTUV, huvA-hmuSTUV or omr-hmuSTUV (Yuhara et al., ; Kvitko et al., ; Tyrrell et al., 2015). Note that in B. stagnalis a VOC family protein is encoded between bhuU and bhuV, and in B. gladioli the bhu genes are organized into two operons present on separate chromosomes. Gene loci of representative species are given in Supplementary Table 4.

Figure 5

Proposed iron uptake pathways in the Burkholderia. (A) The ornibactin/malleobactin uptake system. Ferric-ornibactin is recognized by the OrbA/MbaD TBDR and is translocated into the periplasmic space through a conformational change in the plug domain of the TBDR that requires energy transduction by the TonB complex (TonB-ExbB-ExbD). The iron-siderophore complex is then transported across the cytoplasmic membrane by a periplasmic binding protein-dependent ABC transporter (OrbBCD/MbaLIJ). Oncethe ferric-siderophore complex has been internalized, iron is released from ornibactin through its reduction to the ferrous form by OrbF/MbaK. (B) Ferric-cepaciachelin is proposed to require the CpcG TBDR. Genes neighboring cpcG encode a periplasmic binding protein-dependent ABC transporter (CpcFHI) that may be involved in transport of the bis-catecholate complex across the cytoplasmic membrane. (C) Ferric-pyochelin uptake requires the FptA TBDR and the single subunit cytoplasmic membrane transporter, FptX. (D) Uptake of haem via the Bhu system. Haem uptake is proposed to follow an analogous pathway to that of ornibactin/malleobactin and cepaciachelin. Cytoplasmic haem is bound by the BhuS protein which is proposed to play a role in haem trafficking and haemostasis. If required, iron can be released form haem by haem oxygenases (not shown). The FtrABCD system is not shown. OM, outer membrane; CM, cytoplasmic membrane.