Mark A. Hancock

Isolation and characterization of α-enolase, a novel fibronectin-binding protein from Streptococcus suis

Streptococcus suis is an important swine pathogen that causes meningitis, endocarditis, arthritis and septicaemia. As a zoonotic agent, S. suis also causes similar diseases in humans. Binding of pathogenic bacteria to extracellular matrix components enhances their adhesion to and invasion of host cells. In the present study we isolated and identified a novel fibronectin-binding protein from S. suis. The native protein (designated SsEno) possessed not only high homology with other bacterial enolases but also enolase activity. We cloned, expressed and purified SsEno and showed that it is ubiquitously expressed by all S. suis serotypes and we identified its surface localization using immunoelectron microscopy. ELISA demonstrated that SsEno binds specifically to fibronectin and plasminogen in a lysine-dependent manner. Additional surface plasmon resonance assays demonstrated that SsEno binds to fibronectin or plasminogen with low nanomolar affinity. Inhibition experiments with anti-SsEno antibodies also showed that bacterial SsEno is important for the adhesion to and invasion of brain microvascular endothelial cells by S. suis. Overall, the present work is the first study, to our knowledge, to demonstrate a fibronectin-binding activity of a bacterial enolase, and shows that, similar to other bacterial fibronectin-binding proteins, SsEno may contribute to the virulence of S. suis.

Interactions between TonB from Escherichia coli and the Periplasmic Protein FhuD

For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent outer membrane receptor of Escherichia coli, is required. The periplasmic protein FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated permease FhuB/C. We exploited phage display to map protein-protein interactions in the E. coli cell envelope that contribute to ferrichrome transport. By panning random phage libraries against TonB and against FhuD, we identified interaction surfaces on each of these two proteins. Their interactions were detected in vitro by dynamic light scattering and indicated a 1:1 TonB-FhuD complex. FhuD residue Thr-181, located within the siderophorebinding site and mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD T181C was reacted with two thiol-specific fluorescent probes; addition of the siderophore ferricrocin quenched fluorescence emissions of these conjugates. Similarly, quenching of fluorescence from both probes confirmed binding of TonB and established an apparent KD of ∼300 nm. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with KD values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore.

Anti-myelin antibodies modulate clinical expression of childhood multiple sclerosis

Anti-myelin basic protein (MBP) antibodies in pediatric-onset MS and controls were characterized. Serum samples were obtained from 94 children with MS and 106 controls. Paired CSF and serum were obtained from 25 children with MS at time of their initial episode of acute demyelinating syndrome (ADS). Complementary assays were applied across samples to evaluate the presence, and the physical binding properties, of anti-MBP antibodies. While the prevalence and titers of serum anti-MBP antibodies against both immature and mature forms of MBP were similar in children with MS and in controls, binding characteristics and formal Surface Plasmon Resonance (SPR) studies indicated surprisingly high binding affinities of all pediatric anti-MBP antibodies. Serum levels of anti-MBP antibodies correlated significantly with their CSF levels, and their presence in children with MS was associated with significantly increased risk of an acute disseminated encephalomyelitis-like initial clinical presentation. While antibodies to both immature and mature forms of MBP can be present as part of the normal pediatric humoral repertoire, these anti-myelin antibodies are of surprisingly high affinity, can access the CNS during inflammation, and have the capacity to modulate disease expression. Our findings identify an immune mechanism that could contribute to the observed heterogeneity in spectrum of clinical presentations in early-onset MS.

Mutation in the LPS outer core biosynthesis gene, galU, affects LPS interaction with the RTX toxins ApxI and ApxII and cytolytic activity of Actinobacillus pleuropneumoniae serotype 1

Lipopolysaccharides (LPS) and Apx toxins are major virulence factors of Actinobacillus pleuropneumoniae, a pathogen of the respiratory tract of pigs. Here, we evaluated the effect of LPS core truncation in haemolytic and cytotoxic activities of this microorganism. We previously generated a highly attenuated galU mutant of A. pleuropneumoniae serotype 1 that has an LPS molecule lacking the GalNAc‐Gal II‐Gal I outer core residues. Our results demonstrate that this mutant exhibits wild‐type haemolytic activity but is significantly less cytotoxic to porcine alveolar macrophages. However, no differences were found in gene expression and secretion of the haemolytic and cytotoxic toxins ApxI and ApxII, both secreted by A. pleuropneumoniae serotype 1. This suggests that the outer core truncation mediated by the galU mutation affects the toxins in their cytotoxic activities. Using both ELISA and surface plasmon resonance binding assays, we demonstrate a novel interaction between LPS and the ApxI and ApxII toxins via the core oligosaccharide. Our results indicate that the GalNAc‐Gal II‐Gal I trisaccharide of the outer core is fundamental to mediating LPS/Apx interactions. The present study suggests that a lack of binding between LPS and ApxI/II affects the cytotoxicity and virulence of A. pleuropneumoniae.

Interaction between MMACHC and MMADHC, two human proteins participating in intracellular vitamin B12 metabolism

The identification of eight genes involved in inherited cobalamin (Cbl) disorders has provided insight into the complexity of the vitamin B₁₂ trafficking pathway. Detailed knowledge about the structure, interaction, and physiological functions for many of the gene products, including the MMACHC and MMADHC proteins, is lacking. Having cloned, expressed, and purified MMACHC in Escherichia coli, we demonstrated its monodispersity by dynamic light scattering and measured its hydrodynamic radius, either alone or in complex with each of four vitamin B₁₂ derivatives. Using solution-phase intrinsic fluorescence and label-free, real-time surface plasmon resonance (SPR), MMACHC bound cyanocobalamin and hydroxycobalamin with similar low micromolar affinities (K(D) 6.4 and 9.8 μM, respectively); adenosylcobalamin and methylcobalamin also shared similar binding affinities for MMACHC (K(D) 1.7 and 1.4 μM, respectively). To predict specific regions of interaction between MMACHC and the proposed partner protein MMADHC, MMACHC was subjected to phage display. Five putative MMACHC-binding sites were identified. Finally, MMADHC was confirmed as a binding partner for MMACHC both in vitro (SPR) and in vivo (bacterial two-hybrid system).

Conformation Change in a Self-recognizing Autotransporter Modulates Bacterial Cell-Cell Interaction

Bacteria mostly live as multicellular communities, although they are unicellular organisms, yet the mechanisms that tie individual bacteria together are often poorly understood. The adhesin involved in diffuse adherence (AIDA-I) is an adhesin of diarrheagenic Escherichia coli strains. AIDA-I also mediates bacterial auto-aggregation and biofilm formation and thus could be important for the organization of communities of pathogens. Using purified protein and whole bacteria, we provide direct evidence that AIDA-I promotes auto-aggregation by interacting with itself. Using various biophysical and biochemical techniques, we observed a conformational change in the protein during AIDA-AIDA interactions, strengthening the notion that this is a highly specific interaction. The self-association of AIDA-I is of high affinity but can be modulated by sodium chloride. We observe that a bile salt, sodium deoxycholate, also prevents AIDA-I oligomerization and bacterial auto-aggregation. Thus, we propose that AIDA-I, and most likely other similar autotransporters such as antigen 43 (Ag43) and TibA, organize bacterial communities of pathogens through a self-recognition mechanism that is sensitive to the environment. This could permit bacteria to switch between multicellular and unicellular lifestyles to complete their infection.

An Immunogenic Peptide in the A-box of HMGB1 Protein Reverses Apoptosis-induced Tolerance through RAGE Receptor

Background: The role of caspase-1 in regulating the immunogenic properties of HMGB1 has not been previously reported. Results: We have mapped a peptide in the A-box of HMGB1 that reverses tolerance through RAGE. Conclusion: Inflammasome signaling regulates the immunogenic activity of HMGB1. Significance: Immunogenic peptides within the HMGB1 A-box may be exploited to reverse immune tolerance in sepsis patients. Apoptotic cells trigger immune tolerance in engulfing phagocytes. This poorly understood process is believed to contribute to the severe immunosuppression and increased susceptibility to nosocomial infections observed in critically ill sepsis patients. Extracellular high mobility group box 1 (HMGB1) is an important mediator of both sepsis lethality and the induction of immune tolerance by apoptotic cells. We have found that HMGB1 is sensitive to processing by caspase-1, resulting in the production of a fragment within its N-terminal DNA-binding domain (the A-box) that signals through the receptor for advanced glycation end products (RAGE) to reverse apoptosis-induced tolerance. In a two-hit mouse model of sepsis, we show that tolerance to a secondary infection and its associated mortality were effectively reversed by active immunization with dendritic cells treated with HMGB1 or the A-box fragment, but not a noncleavable form of HMGB1. These findings represent a novel link between caspase-1 and HMGB1, with potential therapeutic implications in infectious and inflammatory diseases.

TonB Interacts with BtuF, the Escherichia coli Periplasmic Binding Protein for Cyanocobalamin

By its direct contact with outer membrane receptor BtuB, the cytoplasmic membrane transducer TonB delivers energy that mediates cyanocobalamin uptake in Escherichia coli. This activity has been generally proposed to be the role of TonB in cyanocobalamin uptake. We now report the discovery and characterization of interactions between TonB and periplasmic binding protein BtuF. Phage display experiments predicted interaction between TonB and BtuF, identifying potential binding residues on each protein. Dynamic light scattering experiments measured a complex of 55 kDa, consistent with a TonB-BtuF heterodimer. The hydrodynamic radius of the complex was unchanged in the presence of cyanocobalamin. Surface plasmon resonance measured TonB-BtuF interaction kinetics that were independent of cyanocobalamin and that deviated from a simple binding model. Binding isotherms from intrinsic fluorescence suggested a multifaceted interaction that was independent of cyanocobalamin. In addition, the presence of TonB did not abrogate subsequent binding of cyanocobalamin by BtuF. Taken together, these data support a previously proposed model wherein TonB serves as a scaffold to optimally position BtuF for initial binding of cyanocobalamin and for its subsequent release. These results substantiate a diverse role for TonB with its multiple protein-protein interactions in bacterial nutrient uptake systems.

Structural features of recombinant MMADHC isoforms and their interactions with MMACHC, proteins of mammalian vitamin B12 metabolism

The genes MMACHC and MMADHC encode critical proteins involved in the intracellular metabolism of cobalamin. Two clinical features, homocystinuria and methylmalonic aciduria, define inborn errors of these genes. Based on disease phenotypes, MMADHC acts at a branch point for cobalamin delivery, apparently exerting its function through interaction with MMACHC that demonstrates dealkylase and decyanase activities. Here we present biophysical analyses of MMADHC to identify structural features and to further characterize its interaction with MMACHC. Two recombinant tag-less isoforms of MMADHC (MMADHCΔ1-12 and MMADHCΔ1-61) were expressed and purified. Full length MMACHC and full length MMADHC were detected in whole cell lysates of human cells; by Western blotting, their molecular masses corresponded to purified recombinant proteins. By clear-native PAGE and by dynamic light scattering, recombinant MMADHCs were stable and monodisperse. Both species were monomeric, adopting extended conformations in solution. Circular dichroism and secondary structure predictions correlated with significant regions of disorder within the N-terminal domain of MMADHC. We found no evidence that MMADHC binds cobalamin. Phage panning against MMADHC predicted four binding regions on MMACHC, two of which overlap with predicted sites on MMACHC at which it may self-associate. Specific, concentration-dependent responses were observed for MMACHC binding to itself and to both MMADHC constructs. As estimated in the sub-micromolar range, the binding of MMACHC to itself was weaker compared to its interaction with either of the MMADHC isoforms. We propose that the function of MMADHC is exerted through its structured C-terminal domain via interactions with MMACHC.

TonB induces conformational changes in surface-exposed loops of FhuA, outer membrane receptor of Escherichia coli.

FhuA, outer membrane receptor of Escherichia coli, transports hydroxamate‐type siderophores into the periplasm. Cytoplasmic membrane–anchored TonB transduces energy to FhuA to facilitate siderophore transport. Because the N‐terminal cork domain of FhuA occludes the C‐terminal β‐barrel lumen, conformational changes must occur to enable siderophore passage. To localize conformational changes at an early stage of the siderophore transport cycle, four anti‐FhuA monoclonal antibodies (mAbs) were purified to homogeneity, and the epitopes that they recognize were determined by phage display. We mapped continuous and discontinuous epitopes to outer surface‐exposed loops 3, 4, and 5 and to β‐barrel strand 14. To probe for conformational changes of FhuA, surface plasmon resonance measured mAb binding to FhuA in its apo‐ and siderophore‐bound states. Changes in binding kinetics were observed for mAbs whose epitopes were mapped to outer surface‐exposed loops. Further, we measured mAb binding in the absence and presence of TonB. After forming immobilized FhuA–TonB complexes, changes in kinetics of mAb binding to FhuA were even more pronounced compared with kinetics of binding in the absence of TonB. Measurement of extrinsic fluorescence of the dye MDCC conjugated to residue 336 in outer surface‐exposed loop 4 revealed 33% fluorescence quenching upon ferricrocin binding and up to 56% quenching upon TonB binding. Binding of mAbs to apo‐ and ferricrocin‐bound FhuA complemented by fluorescence spectroscopy studies showed that their cognate epitopes on loops 3, 4, and 5 undergo conformational changes upon siderophore binding. Further, our data demonstrate that TonB binding promotes conformational changes in outer surface‐exposed loops of FhuA.