Anson C. K. Chan

Structure and Function of P19, a High-Affinity Iron Transporter of the Human Pathogen Campylobacter jejuni.

Campylobacter jejuni, a major cause of acute bacterial diarrhea in humans, expresses numerous proteins to import diverse forms of essential iron. The expression of p19 and an adjacent iron transporter homologue (ftr1) is strongly induced upon iron limitation, suggesting a function in iron acquisition. Here, we show that the loss of P19 alone is detrimental to growth on iron-restricted media. Furthermore, metal binding analysis demonstrates that recombinant P19 has distinct copper and iron binding sites. Crystal structures of P19 have been solved to 1.41 A resolution, revealing an immunoglobulin-like fold. A P19 homodimer in which both monomers contribute ligands to two equivalent copper sites located adjacent to methionine-rich patches is observed. Copper coordination occurs via three histidine residues (His42, His95, and His132) and Met88. A solvent channel lined with conserved acidic residues leads to the copper site. Soaking crystals with a solution of manganese as iron analog reveals a second metal binding site in this solvent channel (metal-metal distance, 7.7 A). Glu44 lies between the metal sites and displays multiple conformations in the crystal structures, suggesting a role in regulating metal-metal interaction. Dimerization is shown to be metal dependent in vitro and is detected in vivo by cross-linking.

Characterization of a dipartite iron-uptake system from uropathogenic Escherichia coli strain F11

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His44, Met90, His97, and His127, and CuB, a second degenerate octahedral geometry with the addition of Glu46. The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.

Helical Shape of Helicobacter pylori Requires an Atypical Glutamine as a Zinc Ligand in the Carboxypeptidase Csd4

Background: Csd4 is required for the helical shape of Helicobacter pylori. Results: Csd4 activity relies on a Gln-zinc ligand to cleave cell wall tripeptides and produce helical shape. Conclusion: Carboxypeptidase activity can be achieved with a Gln, His, and Glu zinc coordination. Significance: Csd4 represents a new subfamily of carboxypeptidases. Peptidoglycan modifying carboxypeptidases (CPs) are important determinants of bacterial cell shape. Here, we report crystal structures of Csd4, a three-domain protein from the human gastric pathogen Helicobacter pylori. The catalytic zinc in Csd4 is coordinated by a rare His-Glu-Gln configuration that is conserved among most Csd4 homologs, which form a distinct subfamily of CPs. Substitution of the glutamine to histidine, the residue found in prototypical zinc carboxypeptidases, resulted in decreased enzyme activity and inhibition by phosphate. Expression of the histidine variant at the native locus in a H. pylori csd4 deletion strain did not restore the wild-type helical morphology. Biochemical assays show that Csd4 can cleave a tripeptide peptidoglycan substrate analog to release m-DAP. Structures of Csd4 with this substrate analog or product bound at the active site reveal determinants of peptidoglycan specificity and the mechanism to cleave an isopeptide bond to release m-DAP. Our data suggest that Csd4 is the archetype of a new CP subfamily with a domain scheme that differs from this large family of peptide-cleaving enzymes.

The bacterial meta-cleavage hydrolase LigY belongs to the amidohydrolase superfamily, not to the α/β-hydrolase superfamily

Strain SYK-6 of the bacterium Sphingobium sp. catabolizes lignin-derived biphenyl via a meta-cleavage pathway. In this pathway, LigY is proposed to catalyze the hydrolysis of the meta-cleavage product (MCP) 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate. Here, we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2,4-dienoate as the products and determined the kcat and kcat/Km values as 9.3 ± 0.6 s−1 and 2.5 ± 0.2 × 107 m−1 s−1, respectively. Sequence analyses and a 1.9 Å resolution crystal structure established that LigY belongs to the amidohydrolase superfamily, unlike previously characterized MCP hydrolases, which are serine-dependent enzymes of the α/β-hydrolase superfamily. The active-site architecture of LigY resembled that of α-amino-β-carboxymuconic-ϵ-semialdehyde decarboxylase, a class III amidohydrolase, with a single zinc ion coordinated by His-6, His-8, His-179, and Glu-282. Interestingly, we found that LigY lacks the acidic residue proposed to activate water for hydrolysis in other class III amidohydrolases. Moreover, substitution of His-223, a conserved residue proposed to activate water in other amidohydrolases, reduced the kcat to a much lesser extent than what has been reported for other amidohydrolases, suggesting that His-223 has a different role in LigY. Substitution of Arg-72, Tyr-190, Arg-234, or Glu-282 reduced LigY activity over 100-fold. On the basis of these results, we propose a catalytic mechanism involving substrate tautomerization, substrate-assisted activation of water for hydrolysis, and formation of a gem-diol intermediate. This last step diverges from what occurs in serine-dependent MCP hydrolases. This study provides insight into C–C–hydrolyzing enzymes and expands the known range of reactions catalyzed by the amidohydrolase superfamily.

Structure-function analyses reveal key features in Staphylococcus aureus IsdB-associated unfolding of the heme-binding pocket of human hemoglobin

IsdB is a receptor on the surface of the bacterial pathogen Staphylococcus aureus that extracts heme from hemoglobin (Hb) to enable growth on Hb as a sole iron source. IsdB is critically important both for in vitro growth on Hb and in infection models and is also highly up-regulated in blood, serum, and tissue infection models, indicating a key role of this receptor in bacterial virulence. However, structural information for IsdB is limited. We present here a crystal structure of a complex between human Hb and IsdB. In this complex, the α subunits of Hb are refolded with the heme displaced to the interface with IsdB. We also observe that atypical residues of Hb, His58 and His89 of αHb, coordinate to the heme iron, which is poised for transfer into the heme-binding pocket of IsdB. Moreover, the porphyrin ring interacts with IsdB residues Tyr440 and Tyr444. Previously, Tyr440 was observed to coordinate heme iron in an IsdB·heme complex structure. A Y440F/Y444F IsdB variant we produced was defective in heme transfer yet formed a stable complex with Hb (Kd = 6 ± 2 μm) in solution with spectroscopic features of the bis-His species observed in the crystal structure. Haptoglobin binds to a distinct site on Hb to inhibit heme transfer to IsdB and growth of S. aureus, and a ternary complex of IsdB·Hb·Hp was observed. We propose a model for IsdB heme transfer from Hb that involves unfolding of Hb and heme iron ligand exchange.

Surface-layer protein from Caulobacter crescentus: expression, purification and X-ray crystallographic analysis.

Protein surface layers are self-assembling, paracrystalline lattices on the surface of many prokaryotes. Surface-layer proteins have not benefited from widespread structural analysis owing to their resistance to crystallization. Here, the successful expression of a truncated version of RsaA, the surface-layer protein from Caulobacter crescentus, from a Caulobacter protein-expression system is reported. The purification, crystallization and initial X-ray diffraction analysis of the truncated RsaA, the largest surface-layer protein studied to date and the first from a Gram-negative bacterium, are also reported.

Investigating the Campylobacter jejuni Transcriptional Response to Host Intestinal Extracts Reveals the Involvement of a Widely Conserved Iron Uptake System

ABSTRACT Campylobacter jejuni is a pathogenic bacterium that causes gastroenteritis in humans yet is a widespread commensal in wild and domestic animals, particularly poultry. Using RNA sequencing, we assessed C. jejuni transcriptional responses to medium supplemented with human fecal versus chicken cecal extracts and in extract-supplemented medium versus medium alone. C. jejuni exposed to extracts had altered expression of 40 genes related to iron uptake, metabolism, chemotaxis, energy production, and osmotic stress response. In human fecal versus chicken cecal extracts, C. jejuni displayed higher expression of genes involved in respiration (fdhTU) and in known or putative iron uptake systems (cfbpA, ceuB, chuC, and CJJ81176_1649–1655 [here designated 1649–1655]). The 1649–1655 genes and downstream overlapping gene 1656 were investigated further. Uncharacterized homologues of this system were identified in 33 diverse bacterial species representing 6 different phyla, 21 of which are associated with human disease. The 1649 and 1650 (p19) genes encode an iron transporter and a periplasmic iron binding protein, respectively; however, the role of the downstream 1651–1656 genes was unknown. A Δ1651–1656 deletion strain had an iron-sensitive phenotype, consistent with a previously characterized Δp19 mutant, and showed reduced growth in acidic medium, increased sensitivity to streptomycin, and higher resistance to H2O2 stress. In iron-restricted medium, the 1651–1656 and p19 genes were required for optimal growth when using human fecal extracts as an iron source. Collectively, this implicates a function for the 1649–1656 gene cluster in C. jejuni iron scavenging and stress survival in the human intestinal environment. IMPORTANCE Direct comparative studies of C. jejuni infection of a zoonotic commensal host and a disease-susceptible host are crucial to understanding the causes of infection outcome in humans. These studies are hampered by the lack of a disease-susceptible animal model reliably displaying a similar pathology to human campylobacteriosis. In this work, we compared the phenotypic and transcriptional responses of C. jejuni to intestinal compositions of humans (disease-susceptible host) and chickens (zoonotic host) by using human fecal and chicken cecal extracts. The mammalian gut is a complex and dynamic system containing thousands of metabolites that contribute to host health and modulate pathogen activity. We identified C. jejuni genes more highly expressed during exposure to human fecal extracts in comparison to chicken cecal extracts and differentially expressed in extracts compared with medium alone, and targeted one specific iron uptake system for further molecular, genetic, and phenotypic study. IMPORTANCE Direct comparative studies of C. jejuni infection of a zoonotic commensal host and a disease-susceptible host are crucial to understanding the causes of infection outcome in humans. These studies are hampered by the lack of a disease-susceptible animal model reliably displaying a similar pathology to human campylobacteriosis. In this work, we compared the phenotypic and transcriptional responses of C. jejuni to intestinal compositions of humans (disease-susceptible host) and chickens (zoonotic host) by using human fecal and chicken cecal extracts. The mammalian gut is a complex and dynamic system containing thousands of metabolites that contribute to host health and modulate pathogen activity. We identified C. jejuni genes more highly expressed during exposure to human fecal extracts in comparison to chicken cecal extracts and differentially expressed in extracts compared with medium alone, and targeted one specific iron uptake system for further molecular, genetic, and phenotypic study.