Biomedical subjects

David L Gordon

Publications and source records attributed to David L Gordon.

13 recordsLinked to original sources

PubMed2006

Molecular analyses of the interaction of Borrelia hermsii FhbA with the complement regulatory proteins factor H and factor H-like protein 1.

Borrelia hermsii, the primary etiological agent of tick-borne relapsing fever in North America, binds the complement regulatory protein factor H (FH) as a means of evading opsonophagocytosis and the alternative complement pathway. The ability of FH-binding protein A (FhbA) to bind FH-like protein 1 (FHL-1) has not been assessed previously. In this study, using a whole-cell absorption assay, we demonstrated that B. hermsii absorbs both FH and FHL-1 from human serum. Consistent with this, affinity ligand binding immunoblot analyses revealed that FH constructs spanning short consensus repeats 1 to 7 and 16 to 20 bind to FhbA. To investigate the molecular basis of the interaction of FhbA with FH/FHL-1, recombinant FhbA truncated proteins were generated and tested for FH/FHL-1 binding. Binding required determinants located in both the N- and C-terminal domains of FhbA, suggesting that long-range intramolecular interactions are involved in the formation and presentation of the FH/FHL-1-binding pocket. To identify specific FhbA residues involved in binding, random mutagenesis was performed. These analyses identified a loop region of FhbA that may serve as a contact point for FH/FHL-1. The data presented here expand our understanding of the pathogenic mechanisms of the relapsing fever spirochetes and of the molecular nature of the interaction between FH/FHL-1 and FhbA.

Amino Acid Sequence↗

PubMed2005

Localization of the third heparin-binding site in the human complement regulator factor H1.

Complement factor H (fH) plays a pivotal role in regulating the alternative pathway, allowing complement activation to proceed on foreign surfaces, whilst protecting surrounding host cell surfaces from complement-mediated damage. Host cell recognition is mediated by polyanions such as sialic acid and glycosaminoglycans (GAGs), which promote a high affinity interaction between fH and C3b deposited on host cell surfaces. Factor H is composed of 20 short consensus repeats (SCRs); two heparin-binding sites have been identified within SCR 7 and SCR 20 and a third site is thought to exist within or near SCR 13. Using an extensive series of recombinant fH fragments and heparin affinity chromatography, we have localized the third heparin-binding domain to SCR 9. A recombinant fH fragment containing both SCR 7 and SCR 9 exhibited higher affinity for heparin than SCR 7 alone, suggesting that the individual heparin-binding sites interact simultaneously with heparin to create a higher avidity interaction. Recombinant fragments containing SCR 9 bound to endothelial cells, indicating that this domain is capable of interacting with polyanions within a physiologically relevant environment. In addition, the three heparin-binding sites exhibited differences in their specificity for certain GAGs, suggesting that the individual binding domains may possess separate GAG recognition functions.

Amino Acid Sequence↗

PubMed2005

Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein.

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5-7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5-7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.

Blood Proteins↗

PubMed2005

A new focus of Rickettsia honei spotted fever in South Australia.

We recently diagnosed rickettsial spotted fever in four patients from the south-eastern coastal region of South Australia near Adelaide, an area not known to be endemic for this infection. All infections were acquired within the geographic range of Aponomma hydrosauri, the tick vector of Rickettsia honei. Infection by R. honei was confirmed in two patients. This extension of the known geographic range of R. honei infection may be explained, in part, by alterations in host-parasite ecology.

Adult↗

PubMed2005

Binding of complement factor H to endothelial cells is mediated by the carboxy-terminal glycosaminoglycan binding site.

Factor H (FH), the major fluid phase regulator of the alternative complement pathway, mediates protection of plasma-exposed host structures. It has recently been shown that short consensus repeats 19 to 20 of FH are mutational hot spots associated with atypical hemolytic uremic syndrome (aHUS), a disease with endothelial cell damage. Domain 20 of FH contains binding sites for heparin, C3b, and the cleavage product C3d. To study the role of these binding sites in target recognition, we performed site-directed mutagenesis in domain 20 and assayed the resulting recombinant proteins. The mutant FH15-20A (substitutions R1203E, R1206E, and R1210S) bound neither heparin nor endothelial cells. Similarly, an aHUS-derived mutant FH protein (E1172Stop, lacking domain 20) failed to bind endothelial cells and showed impaired binding to heparin. Binding of FH to endothelial cells was inhibited by heparin and a specific monoclonal antibody that inhibited heparin but not C3d binding, demonstrating that the heparin site on domains 19 to 20 mediates interaction of FH to endothelial cells. Binding of FH15-20 to heparin was inhibited by several cell surface- and basement membrane-associated glycosaminoglycans, suggesting that binding site specificity is not restricted to heparin. Thus, defective heparin/glycosaminoglycan-binding site on domains 19 to 20 of FH most probably mediates complement-induced endothelial cell damage in aHUS.

Amino Acid Substitution↗

PubMed2005

Demonstration of factor H-like protein 1 binding to Treponema denticola, a pathogen associated with periodontal disease in humans.

Treponema denticola is an important contributor to periodontal disease. In this study we investigated the ability of T. denticola to bind the complement regulatory proteins factor H and factor H-like protein 1 (FHL-1). The binding of these proteins has been demonstrated to facilitate evasion of the alternative complement cascade and/or to play a role in adherence and invasion. Here we demonstrate that T. denticola specifically binds FHL-1 via a 14-kDa, surface-exposed protein that we designated FhbB. Consistent with its FHL-1 binding specificity, FhbB binds only to factor H recombinant fragments spanning short consensus repeats (SCRs) 1 to 7 (H7 construct) and not to SCR constructs spanning SCRs 8 to 15 and 16 to 20. Binding of H7 to FhbB was inhibited by heparin. The specific involvement of SCR 7 in the interaction was demonstrated using an H7 mutant (H7AB) in which specific charged residues in SCR 7 were replaced by alanine. This construct lost FhbB binding ability. Analyses of the ability of FHL-1 bound to the surface of T. denticola to serve as a cofactor for factor I-mediated cleavage of C3b revealed that C3b is cleaved in an FHL-1/factor I-independent manner, perhaps by an unidentified protease. Based on the data presented here, we hypothesize that the primary function of FHL-1 binding by T. denticola might be to facilitate adherence to FHL-1 present on anchorage-dependent cells and in the extracellular matrix.

Bacterial Proteins↗

PubMed2005

Loss of complement activation and leukocyte adherence as Nippostrongylus brasiliensis develops within the murine host.

Complement activation and C3 deposition on the surface of parasitic helminths may be important for recruitment of leukocytes and for damage to the target organism via cell-mediated mechanisms. Inhibition of complement activation would therefore be advantageous to parasites, minimizing damage and enhancing migration through tissues. The aim of this study was to determine ex vivo if complement activation by, and leukocyte adherence to, the nematode Nippostrongylus brasiliensis change as the parasite matures and migrates through the murine host. Pathways of activation of complement and the mechanism of adherence of leukocytes were also defined using sera from mice genetically deficient in either C1q, factor B, C1q and factor B, C3, or C4. Substantive deposition of C3 and adherence of eosinophil-rich leukocytes were seen with infective-stage (L3) but not with lung-stage (L4) larvae. Adult intestinal worms had low to intermediate levels of both C3 and leukocyte binding. For L3 and adult worms, complement deposition was principally dependent on the alternative pathway. For lung-stage larvae, the small amount of C3 detected was dependent to similar degrees on both the lectin and alternative pathways. The classical pathway was not involved for any of the life stages of the parasite. These results suggest that in primary infections, the infective stage of N. brasiliensis is vulnerable to complement-dependent attack by leukocytes. However, within the first 24 h of infection, N. brasiliensis acquires the ability to largely avoid complement-dependent immune responses.

Animals↗

PubMed2004

Paradoxical inflammatory reaction during treatment of Cryptococcus neoformans var. gattii meningitis in an HIV-seronegative woman.

A human immunodeficiency virus (HIV)-seronegative woman was admitted to the hospital with postpartum onset Cryptococcus neoformans var. gattii meningitis and markedly increased intracranial pressure. A poor initial response to antifungal therapy was followed, 2 months after hospital admission, by severe acute meningeal and cerebral inflammation and clearance of yeast cells from cerebrospinal fluid. This first reported case of paradoxical inflammatory reaction to C. neoformans illustrates important aspects of the host-pathogen interaction and highlights possible effects of immunomodulatory therapies.

Adolescent↗

PubMed2004

Quantitation of complement and leukocyte binding to a parasitic helminth species.

Methods used to quantify complement deposition and cell adherence to parasitic helminths usually involve subjective visual comparisons of immunofluorescence or time-consuming manual counting of bound cells. Such targets are relatively large and, generally, few individual organisms can be analysed. More objective and efficient radiometric assays are available, but these also have considerable disadvantages. We have developed an improved immunofluorescence-based method for quantitation of complement deposition on viable third-stage larvae of the nematode Nippostrongylus brasiliensis (L3). A similar strategy was also applied to measuring leukocyte adherence to the parasite. Fluorescein isothiocyanate (FITC)-conjugated antibodies were used to detect complement on serum-treated larvae. The adherence of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled mouse leukocytes to larvae was investigated using the same basic approach. Images of fluorescent larvae or fluorescent cells attached to larvae were generated with a Bio-Rad Molecular Imager FX and fluorescence intensity was quantified. Hundreds of larvae can be analysed simultaneously in multiple samples, and these strategies allow rapid and sensitive quantitation that is directly proportional to the amount of protein or the number of leukocytes added to cultures. These techniques may also be applicable to other large objects, organisms or biological surfaces.

Animals↗

PubMed2003

A common site within factor H SCR 7 responsible for binding heparin, C-reactive protein and streptococcal M protein.

The complement inhibitor factor H (fH) interacts via its seventh short consensus repeat (SCR) domain with multiple ligands including heparin, streptococcal M protein and C-reactive protein (CRP). The aim of this study was to localize the residues in SCR 7 required for these interactions. We initially built a homology model of fH SCR 6-7 using the averaged NMR structures of fH SCR 15-16 and vaccinia control protein SCR 3-4 as templates. Electrostatic potentials of the model's surface demonstrated a co-localization of three clusters of positively charged residues on SCR 7, labeled site A (R369 and K370), site B (R386 and K387) and site C (K392). These residues, localized to the linker region preceding SCR 7 and to the end of a "hypervariable loop" in SCR 7, were systematically replaced with uncharged alanine residues in an fH construct containing SCR 1-7. The resulting proteins were expressed in the methylotrophic yeast, Pichia pastoris. By ELISA analysis we demonstrated: first, that substituting site A inhibited heparin and CRP binding; secondly, that substituting site B inhibited binding to heparin, CRP and M protein; and thirdly, that substituting site C clearly inhibited only heparin binding.

Amino Acid Substitution↗

PubMed2003

MR findings in Murray Valley encephalitis.

Murray Valley encephalitis (MVE) is caused by a flavivirus related to West Nile and St. Louis encephalitis viruses. We report a case of MVE resulting in quadriplegia and respiratory failure. MR imaging demonstrated thalamic hyperintensity on T2-weighted images, with similar involvement of the red nucleus, substantia nigra, and cervical cord. These findings preceded serologic diagnosis and are similar to those of Japanese encephalitis. In the appropriate setting, thalamic T2 hyperintensity is suggestive of flavivirus infection.

Diagnosis, Differential↗

PubMed2002

Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46).

Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.

Amino Acid Sequence↗

PubMed2002

The human complement regulator factor H binds pneumococcal surface protein PspC via short consensus repeats 13 to 15.

The innate ability of Streptococcus pneumoniae to resist complement activation and complement-mediated phagocytosis may be a direct consequence of the ability of the bacteria to bind components of the complement regulatory system. One such component, factor H (fH), is a crucial fluid-phase negative regulator of the alternative pathway of complement and is utilized by a number of pathogenic organisms to resist complement attack. The pneumococcal surface protein C (PspC [also known as CbpA] and SpsA) has been shown to bind fH, although the exact binding site within one or more of the 20 short consensus repeats (SCRs) of the molecule is not known. The purpose of the current study was to map specific SCRs on fH responsible for this binding. Initial experiments utilizing type 2 pneumococcal strain D39 and its isogenic PspC-negative derivative (D39/pspC mutant) showed that fH binding was PspC dependent. A purified recombinant protein derivative of PspC that lacked the proline-rich region (PspCDeltaPro) had a reduced binding efficiency for fH, thereby directly showing the importance of this region for the fH interaction. We have specifically shown by inhibition experiments that SCRs responsible for heparin and C3b binding of fH are not involved in binding PspC and the interaction between fH and PspC is largely hydrophobic, since no inhibition was observed in the presence of high concentrations of NaCl. Construction of SCR proteins encompassing the whole fH molecule showed that SCRs 8 to 15 (SCR 8-15) mediated binding to PspC. Further localization experiments revealed that SCR 13 and SCR 15 were required for full binding, although partial binding was retained when either SCR was removed.

Bacterial Proteins↗