Ferritin-like family proteins in the anaerobe Bacteroides fragilis: when an oxygen storm is coming, take your iron to the shelter

Abstract

Bacteroides are gram-negative anaerobes and one of the most abundant members the lower GI tract microflora where they play an important role in normal intestinal physiology. Disruption of this commensal relationship has a great impact on human health and disease. Bacteroides spp. are significant opportunistic pathogens causing infections when the mucosal barrier integrity is disrupted following predisposing conditions such as GI surgery, perforated or gangrenous appendicitis, perforated ulcer, diverticulitis, trauma and inflammatory bowel diseases. B. fragilis accounts for 60-90 % of all anaerobic infections despite being a minor component of the genus (<1 % of the flora). Clinical strains of B. fragilis are among the most aerotolerant anaerobes. When shifted from anaerobic to aerobic conditions B. fragilis responds to oxidative stress by inducing the expression of an extensive set of genes involved in protection against oxygen derived radicals and iron homeostasis. In Bacteroides, little is known about the metal/oxidative stress interactions and the mobilization of intra-cellular non-heme iron during the oxidative stress response has been largely overlooked. Here we present an overview of the work carried out to demonstrate that both oxygen-detoxifying enzymes and iron-storage proteins are essential for B. fragilis to survive an adverse oxygen-rich environment. Some species of Bacteroides have acquired multiple homologues of the iron storage and detoxifying ferritin-like proteins but some species contain none. The proteins found in Bacteroides are classical mammalian H-type non-heme ferritin (FtnA), non-specific DNA binding and starvation protein (Dps) and the newly characterized bacterial Dps-Like miniferritin protein. The full contribution of ferritin-like proteins to pathophysiology of commensal and opportunistic pathogen Bacteroides spp. still remains to be elucidated.

Fig. 1

The genetic structure of the ftnA1 locus in the genomes of representative Bacteroides species. Strain designations are shown in each panel. Bf B. fragilis, Bt B. thetaiotaomicron, Bo B. ovatus, Bu B. uniformis and Bv B. vulgatus. The chromosomal nucleotide sequences flanking the ftnA1 genes were retrieved from public genome database (http://www.ncbi.nlm.nih.gov/) and used to create a DNA molecule map in Vector NTI Advance 11.5.2. Open reading frame (ORF) features were added to the molecule using the nucleotides position corresponding to the annotated protein locus_tag. The ftnA1 ORF and direction of transcription is depicted by a dark arrow. Open arrows depict ORFs and transcription orientation of the genes flanking ftnA1 chromosomal region. The locus_tag for each gene is shown below its respective ORF and gene assignment. AAT: aspartate aminotransferase superfamily; AK: aspartokinase III; ftsE: cell division ATP-binding protein; hisIE: histidine biosynthesis bifunctional protein; HK: Histidine kinase-like ATPases; HP: conserved hypothetical protein; lysA: diaminopimelate decarboxylase; nadB: NAD dependent nucleotide-diphosphate-sugar epimerase; OPT: oligopeptide transporter; S16: 30S ribosomal protein S16; tnp: transposase; xyl: xylanase

Fig. 2

Multiple alignments of the Bacteroides and Parabacteroides deduced amino acid sequences for FtnA, Dps, and DpsL protein homologues. The E. coli FtnA and Dps proteins were included for comparison purposes. Conserved amino acid residues (>50 % identity) are labeled with blue boxes. Semi-conserved amino acid substitutions are depicted by green boxes. Key ferroxidase center residues of FtnA and Dps (Andrews 1998, 2010) are depicted by one letter, respectively, below and above the sequence. For DpsL, the di-iron site ligands (Gauss et al. 2012) are depicted by circled one-letter code amino acid below the sequence. The third metal ligand sites of DpsL proteins are depicted by grey boxes. The conserved residues of Dps metal ligands motifs are not highlighted. Cysteine residues are labeled in yellow boxes. Alignment of protein sequences was performed using the AlignX program component of Vector NTI Advance 11.5.2 with the peptide scoring matrix default data file blosum62mt2 for the comparison of amino acids substitution

Fig. 2

Multiple alignments of the Bacteroides and Parabacteroides deduced amino acid sequences for FtnA, Dps, and DpsL protein homologues. The E. coli FtnA and Dps proteins were included for comparison purposes. Conserved amino acid residues (>50 % identity) are labeled with blue boxes. Semi-conserved amino acid substitutions are depicted by green boxes. Key ferroxidase center residues of FtnA and Dps (Andrews 1998, 2010) are depicted by one letter, respectively, below and above the sequence. For DpsL, the di-iron site ligands (Gauss et al. 2012) are depicted by circled one-letter code amino acid below the sequence. The third metal ligand sites of DpsL proteins are depicted by grey boxes. The conserved residues of Dps metal ligands motifs are not highlighted. Cysteine residues are labeled in yellow boxes. Alignment of protein sequences was performed using the AlignX program component of Vector NTI Advance 11.5.2 with the peptide scoring matrix default data file blosum62mt2 for the comparison of amino acids substitution

Fig. 3

Phylogenetic analysis of Bacteroides and Parabacteroides ferritin family of iron-storage proteins. The amino acid sequences of 57 FtnA, Dps and DpsL proteins were aligned using Multiple Sequence Comparison by Log- Expectation (MUSCLE). The tree was constructed in MEGA5 by the Maximum Likelihood method based on the JTT matrix-based model (Jones et al. 1992; Tamura et al. 2011). The E. coli FtnA, E. coli Dps, and S. sulfataricus DpsL proteins were included for comparison purposes. Branches corresponding to partitions reproduced in <50 % bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (50 replicates) are shown next to the branches (Felsenstein 1985). Initial trees for the heuristic search were obtained automatically as follows. When the number of common sites was <100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. All positions containing gaps were eliminated. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Asterisks depict the Dps protein homologous containing conserved cysteine residues

Fig. 4

Circular map showing the chromosomal locations of FtnA, Dps, and DpsL proteins in Bacteroides and Parabacteroides species. The protein sequences of B. fragilis 638R FtnA (BF638R_2891), Dps (Bf638R_1333, and DpsL (BF638R_3305) were, respectively, used to identify the homologous proteins and respective locus_tag number in complete ungapped genomes of B. thetaiotaomicron VPI 5482 (NC_004663), B. vulgatus ATCC 8482 (CP000139), and P. distasonis ATCC 8503 (CP000140). The genomic nucleotide sequences were used to create circular maps in Vetor NTI Advance 11.5.2. The gene locus positions were inserted in the map using respective annotated nucleotide sequence positions for each locus_tag. The genome length is depicted in each panel below the respective strain name

Fig. 5

The genetic structure of the ftnA2 and ftn3 loci in the genomes of Bacteroides species. Strain designations are shown in each panel. Bt B. thetaiotaomicron, Bu B. uniformis, Bv B. vulgatus and Bd B. dorei. The chromosomal nucleotide sequence flanking the ftnA2 gene was retrieved from public genome database (http://www.ncbi.nlm.nih.gov/) and used to create a DNA molecule map in Vector NTI Advance 11.5.2. Open reading frame (ORF) features were added to the molecule using the nucleotides position corresponding to the annotated protein locus_tag. The ftnA2 ORF and direction of transcription is depicted by a dark grey arrow while ftnA3 is depicted by silver grey arrows respectively. Open arrows depict ORFs and transcription orientation of the genes flanking ftnA2 and ftnA3 chromosomal region. The locus_tag for each gene is shown below its respective ORF and gene assignment. ECF: RNA polymerase ECF-type sigma factor; fbaB: fructose-bisphosphate aldolase; glgA: alpha-glucan phosphorylase; gpmA: phosphoglyceromutase; sHSP: small heat shock protein; marC: hypothetical protein; mgsA: methylglyoxal synthase; pfkA: 6-phosphofructokinase; speB: arginase; trkA: sodium/proton antiporter

Fig. 6

The genetic structure of the dpsL (formerly bfr-related gene) locus in the genomes of representative Bacteroides species. Strain designations are shown in each panel. Bf B. fragilis, Bt B. thetaiotaomicron, Bo B. ovatus, Bv B. vulgatus and Bd B. dorei. The chromosomal nucleotide sequence flanking the dpsL gene was retrieved from public genome database (http://www.ncbi.nlm.nih.gov/) and used to create a DNA molecule map in Vector NTI Advance 11.5.2. Open reading frame (ORF) features were added to the molecule using the nucleotides position corresponding to the annotated protein locus_tag. The dpsL ORF and direction of transcription is depicted by a dark grey arrow. Open arrows depict ORFs and transcription orientation of the genes flanking dpsL chromosomal region. The locus_tag for each gene is shown below respective ORF and gene assignment. acrR: transcriptional regulator TetR; crp: transcriptional regulator; dagK: lipid kinase; ftsW: rod shape-determining protein; gapA: glyceralde-hyde 3-phosphate dehydrogenase; gldH: gliding motility-associated lipoprotein; glnA: glutamine synthetase; guaA: GMP synthase; holB: DNA polymerase III, delta subunit; HP: conserved hypothetical protein; kdsA: 2-dehydro-3-deoxypho-sphooctonate aldolase; lpxK: tetraacyldisaccharide kinase; miaA: tRNA delta(2)-isopentenylpyrophosphate; metF: 5,10-methylenetetrahydrofolate reductase; mscL: large-conductance mechanosensitive channel; OEP: outer membrane efflux protein; pbp2_mrdA: penicillin-binding protein 2; pnp: purine nucleoside phosphorylase I; sspA: protease IV; thiL: thiamine monophosphate kinase; TM: putative transmembrane protein; tnp: transposase

Fig. 7

Schematic model of the interplay between oxidative stress response and intra-cellular iron mobilization in B. fragilis. Intra-cellular superoxide anions are predominantly generated from fumarate reductase complex (Frd) and membrane oxidases when B. fragilis is exposed to oxygen. Superoxide anions attack iron-sulfur clusters, cause disassociation and release of ferrous iron. Superoxide dismutase eliminates superoxide anions by dismutation with generation of H₂O₂. Increased intra-cellular levels of H₂O₂ activate the peroxide response regulator OxyR which induces the expression of the peroxide detoxifying enzymes, catalase (KatB), alkyl hydroperoxidase (AhpCF), thioredoxin peroxidase (Tpx) and rubrerythrin (Rbr1). Induction of FtnA, Dps, and DpsL expression is controlled in part by an OxyR-dependent and an unidentified OxyR-independent oxygen-dependent mechanism. Ferrous ion may enter the cell through a putative ferrous iron transporter (FeoAB). Heme is transported into the cell is also a source of ferrous iron when iron is removed from heme with release of intact protoporphyrin IX (PpIX). The enzyme(s) involved in Bacteroides heme demetalase activity is yet to be identified. Heme transport systems and ferrous iron transport in B. fragilis are empirically represented in the diagram based on putative homologues in its chromosome. The mobilization of free ferrous iron inside anaerobic bacteria cells is not known but it is assumed to be mobilized and loaded into the iron-storage proteins in a non-toxic ferric form following oxidative stress. These mechanisms will prevent the reaction of H₂O₂ with ferrous iron (Fenton reaction) and avoid the formation of toxic hydroxyl radicals (HO^•). Model compiled and adapted from; Rocha et al. (2000, , Imlay (2002), Rocha and Smith (2004, , Meehan and Malamy (2012)