Alan Riboldi-Tunnicliffe

Innate immune defense against pneumococcal pneumonia requires pulmonary complement component C3.

ABSTRACT Complement is known to be involved in protection against systemic infection with Streptococcus pneumoniae. However, less is known about effects of complement within the lungs during pneumococcal pneumonia. By intranasally infecting transgenic mice unable to express complement C3, we investigated the role of complement in pulmonary defenses against S. pneumoniae. It was demonstrated that within the lungs, there is a requirement for C3 during the initial hours of infection. It was found that within 1 h of infection, bacterial loads decreased within lung airways of control mice as C3 protein increased. The lack of C3 resulted in the inability to control growth of wild-type or attenuated pneumococci within the lungs and bloodstream, resulting in an overwhelming inflammatory response and shorter survival times. Our results show that during the initial hours of infection with S. pneumoniae, C3 is protective within the lungs and subsequently plays an important role systemically.

1.2 Å crystal structure of the S. pneumoniae PhtA histidine triad domain a novel zinc binding fold

The recently described pneumococcal histidine triad protein family has been shown to be highly conserved within the pneumococcus. As part of our structural genomics effort on proteins from Streptococcus pneumoniae, we have expressed, crystallised and solved the structure of PhtA‐166–220 at 1.2 Å using remote SAD with zinc. The structure of PhtA‐166–220 shows no similarity to any protein structure. The overall fold contains 3β‐strands and a single short α‐helix. The structure appears to contain a novel zinc binding motif. The remaining 4 histidine triad repeats from PhtA have been modelled based on the crystal structure of the PhtA histidine triad repeat 2. From this modelling work, we speculate that only three of the five histidine triad repeats contain the residues in the correct geometry to allow the binding of a zinc ion.

Crystal structure of the response regulator 02 receiver domain, the essential YycF two-component system of Streptococcus pneumoniae in both complexed and native states.

A variety of bacterial cellular responses to environmental signals are mediated by two-component signal transduction systems comprising a membrane-associated histidine protein kinase and a cytoplasmic response regulator (RR), which interpret specific stimuli and produce a measured physiological response. In RR activation, transient phosphorylation of a highly conserved aspartic acid residue drives the conformation changes needed for full activation of the protein. Sequence homology reveals that RR02 from Streptococcus pneumoniae belongs to the OmpR subfamily of RRs. The structures of the receiver domains from four members of this family, DrrB and DrrD from Thermotoga maritima, PhoB from Escherichia coli, and PhoP from Bacillus subtilis, have been elucidated. These domains are globally very similar in that they are composed of a doubly wound alpha(5)beta(5); however, they differ remarkably in the fine detail of the beta4-alpha4 and alpha4 regions. The structures presented here reveal a further difference of the geometry in this region. RR02 is has been shown to be the essential RR in the gram-positive bacterium S. pneumoniae R. Lange, C. Wagner, A. de Saizieu, N. Flint, J. Molnos, M. Stieger, P. Caspers, M. Kamber, W. Keck, and K. E. Amrein, Gene 237:223-234, 1999; J. P. Throup, K. K. Koretke, A. P. Bryant, K. A. Ingraham, A. F. Chalker, Y. Ge, A. Marra, N. G. Wallis, J. R. Brown, D. J. Holmes, M. Rosenberg, and M. K. Burnham, Mol. Microbiol. 35:566-576, 2000). RR02 functions as part of a phosphotransfer system that ultimately controls the levels of competence within the bacteria. Here we report the native structure of the receiver domain of RR02 from serotype 4 S. pneumoniae (as well as acetate- and phosphate-bound forms) at different pH levels. Two native structures at 2.3 A, phased by single-wavelength anomalous diffraction (xenon SAD), and 1.85 A and a third structure at pH 5.9 revealed the presence of a phosphate ion outside the active site. The fourth structure revealed the presence of an acetate molecule in the active site.

The PPIase active site of Legionella pneumophila Mip protein is involved in the infection of eukaryotic host cells.

Abstract We analysed eight monoclonal antibodies (mAbs) directed against the Mip (macrophage infectivity potentiator) protein, a virulence factor of the intracellular pathogen Legionella pneumophila. Mip belongs to the FK506-binding proteins (FKBPs) and exhibits peptidyl prolyl cis/trans isomerase (PPIase) activity. Five of the mAbs recognised epitopes in the Cterminal, FKBP-homologous domain of Mip, which is highly conserved among all Legionella species. Upon immunological binding to Mip, all but one of these mAbs caused inhibition of the PPIase activity in vitro. mAb binding to the N-terminal domain of Mip did not influence its enzymatic activity. All but one of the PPIase inhibiting mAbs were able to significantly inhibit the early establishment and initiation of an intracellular infection of the bacteria in Acanthamoeba castellanii, the natural host, and in the human phagocytic cell line U937. These data demonstrate for the first time that for the virulence-enhancing property of the L. pneumophila Mip protein, an intact active site of the enzyme is an essential requirement.

Nanomolar Competitive Inhibitors of Mycobacterium tuberculosis and Streptomyces coelicolor Type II Dehydroquinase

Isomeric nitrophenyl and heterocyclic analogues of the known inhibitor (1S,3R,4R)‐1,3,4‐trihydroxy‐5‐cyclohexene‐1‐carboxylic acid have been synthesized and tested as inhibitors of M. tuberculosis and S. coelicolor type II dehydroquinase, the third enzyme of the shikimic acid pathway. The target compounds were synthesized by a combination of Suzuki and Sonogashira cross‐coupling and copper(I)‐catalyzed 2,3‐dipolar cycloaddition reactions from a common vinyl triflate intermediate. These studies showed that a para‐nitrophenyl derivative is almost 20‐fold more potent as a competitive inhibitor against the S. coelicolor enzyme than that of M. tuberculosis. The opposite results were obtained with the meta isomer. Five of the bicyclic analogues reported herein proved to be potent competitive inhibitors of S. coelicolor dehydroquinase, with inhibition constants in the low nanomolar range (4–30 nM). These derivatives are also competitive inhibitors of the M. tuberculosis enzyme, but with lower affinities. The most potent inhibitor against the S. coelicolor enzyme, a 6‐benzothiophenyl derivative, has a Ki value of 4 nM—over 2000‐fold more potent than the best previously known inhibitor, (1R,4R,5R)‐1,5‐dihydroxy‐4‐(2‐nitrophenyl)cyclohex‐2‐en‐1‐carboxylic acid (8 μM), making it the most potent known inhibitor against any dehydroquinase. The binding modes of the analogues in the active site of the S. coelicolor enzyme (GOLD 3.0.1), suggest a key π‐stacking interaction between the aromatic rings and Tyr 28, a residue that has been identified as essential for enzyme activity.

Design, Synthesis, and Structural Studies on Potent Biaryl Inhibitors of Type II Dehydroquinases

Filling the pocket: Biaryl inhibitors of type II dehydroquinase are described, designed to bind to both the active site and a subsidiary pocket. Compounds exhibited nanomolar inhibition of the enzyme, which is rationalised by molecular docking and protein crystallographic studies.

Structure of myelin P2 protein from equine spinal cord

Equine P2 protein has been isolated from horse spinal cord and its structure determined to 2.1 A. Since equine myelin is a viable alternative to bovine tissue for large-scale preparations, characterization of the proteins from equine spinal cord myelin has been initiated. There is an unusually high amount of P2 protein in equine CNS myelin compared with other species. The structure was determined by molecular replacement and subsequently refined to an R value of 0.187 (Rfree=0.233). The structure contains a molecule of the detergent LDAO and HEPES buffer in the binding cavity and is otherwise analogous to other cellular retinol-binding proteins.

Outer Membrane Protein A of Bovine and Ovine Isolates of Mannheimia haemolytica Is Surface Exposed and Contains Host Species-Specific Epitopes

ABSTRACT Mannheimia haemolytica is the etiological agent of pneumonic pasteurellosis of cattle and sheep; two different OmpA subclasses, OmpA1 and OmpA2, are associated with bovine and ovine isolates, respectively. These proteins differ at the distal ends of four external loops, are involved in adherence, and are likely to play important roles in host adaptation. M. haemolytica is surrounded by a polysaccharide capsule, and the degree of OmpA surface exposure is unknown. To investigate surface exposure and immune specificity of OmpA among bovine and ovine M. haemolytica isolates, recombinant proteins representing the transmembrane domain of OmpA from a bovine serotype A1 isolate (rOmpA1) and an ovine serotype A2 isolate (rOmpA2) were overexpressed, purified, and used to generate anti-rOmpA1 and anti-rOmpA2 antibodies, respectively. Immunogold electron microscopy and immunofluorescence techniques demonstrated that OmpA1 and OmpA2 are surface exposed, and are not masked by the polysaccharide capsule, in a selection of M. haemolytica isolates of various serotypes and grown under different growth conditions. To explore epitope specificity, anti-rOmpA1 and anti-rOmpA2 antibodies were cross-absorbed with the heterologous isolate to remove cross-reacting antibodies. These cross-absorbed antibodies were highly specific and recognized only the OmpA protein of the homologous isolate in Western blot assays. A wider examination of the binding specificities of these antibodies for M. haemolytica isolates representing different OmpA subclasses revealed that cross-absorbed anti-rOmpA1 antibodies recognized OmpA1-type proteins but not OmpA2-type proteins; conversely, cross-absorbed anti-rOmpA2 antibodies recognized OmpA2-type proteins but not OmpA1-type proteins. Our results demonstrate that OmpA1 and OmpA2 are surface exposed and could potentially bind to different receptors in cattle and sheep.

Expression, purification and X-ray characterization of residues 18–230 from the pneumococcal histidine triad protein A (PhtA) from Streptococcus pneumoniae

A fragment of the Streptococcus pneumoniae PhtA gene product (residues 18-230) was cloned and overexpressed in Escherichia coli. The purified protein was crystallized using the sitting-drop vapour-diffusion technique. Crystals belong to the monoclinic space group C2, with unit-cell parameters a = 62.19, b = 35.9, c = 72.54 A, beta = 90.01 degrees. The crystals diffract X-rays to beyond 1.2 A resolution.

Purification, crystallization and preliminary X-ray diffraction analysis of RafE, a sugar-binding lipoprotein from Streptococcus pneumoniae

The mature form of RafE has been expressed, purified and crystallized. X-ray diffraction data have been collected to 3.65 and 2.90 Å resolution from native and selenomethionine-derivative crystals, respectively.