Structural determination of the complement inhibitory domain of Borrelia burgdorferi BBK32 provides insight into classical pathway complement evasion by Lyme disease spirochetes

The carboxy-terminal domain of the BBK32 protein from Borrelia burgdorferi sensu stricto, termed BBK32-C, binds and inhibits the initiating serine protease of the human classical complement pathway, C1r. In this study we investigated the function of BBK32 orthologues of the Lyme-associated Borrelia burgdorferi sensu lato complex, designated BAD16 from B. afzelii strain PGau and BGD19 from B. garinii strain IP90. Our data show that B. afzelii BAD16-C exhibits BBK32-C-like activities in all assays tested, including high-affinity binding to purified C1r protease and C1 complex, and potent inhibition of the classical complement pathway. Recombinant B. garinii BGD19-C also bound C1 and C1r with high-affinity yet exhibited significantly reduced in vitro complement inhibitory activities relative to BBK32-C or BAD16-C. Interestingly, natively produced BGD19 weakly recognized C1r relative to BBK32 and BAD16 and, unlike these proteins, BGD19 did not confer significant protection from serum killing. Site-directed mutagenesis was performed to convert BBK32-C to resemble BGD19-C at three residue positions that are identical between BBK32 and BAD16 but different in BGD19. The resulting chimeric protein was designated BXK32-C and this BBK32-C variant mimicked the properties observed for BGD19-C. To query the disparate complement inhibitory activities of BBK32 orthologues, the crystal structure of BBK32-C was solved to 1.7Ã… limiting resolution. BBK32-C adopts an anti-parallel four-helix bundle fold with a fifth alpha-helix protruding from the helical core. The structure revealed that the three residues targeted in the BXK32-C chimera are surface-exposed, further supporting their potential relevance in C1r binding and inhibition. Additional binding assays showed that BBK32-C only recognized C1r fragments containing the serine protease domain. The structure-function studies reported here improve our understanding of how BBK32 recognizes and inhibits C1r and provide new insight into complement evasion mechanisms of Lyme-associated spirochetes of the B. burgdorferi sensu lato complex

Symmetry breaking during homodimeric assembly activates an E3 ubiquitin ligase

Citation: Ye, Z., Needham, P. G., Estabrooks, S. K., Whitaker, S. K., Garcia, B. L., Misra, S., . . . Camacho, C. J. (2017). Symmetry breaking during homodimeric assembly activates an E3 ubiquitin ligase. Scientific Reports, 7(1). doi:10.1038/s41598-017-01880-4C-terminus of Hsc/p70-Interacting Protein (CHIP) is a homodimeric E3 ubiquitin ligase.Each CHIP monomer consists of a tetratricopeptide-repeat (TPR), helix-turn-helix (HH), and U-box domain.In contrast to nearly all homodimeric proteins, CHIP is asymmetric.To uncover the origins of asymmetry, we performed molecular dynamics simulations of dimer assembly.We determined that a CHIP monomer is most stable when the HH domain has an extended helix that supports intra-monomer TPR-U-box interaction, blocking the E2-binding surface of the U-box.We also discovered that monomers first dimerize symmetrically through their HH domains, which then triggers U-box dimerization.This brings the extended helices into close proximity, including a repulsive stretch of positively charged residues.Unable to smoothly unwind, this conflict bends the helices until the helix of one protomer breaks to relieve the repulsion.The abrupt snapping of the helix forces the C-terminal residues of the other protomer to disrupt that protomer's TPR-U-box tight binding interface, swiftly exposing and activating one of the E2 binding sites.Mutagenesis and biochemical experiments confirm that C-terminal residues are necessary both to maintain CHIP stability and function.This novel mechanism indicates how a ubiquitin ligase maintains an inactive monomeric form that rapidly activates only after asymmetric assembly. © 2017 The Author(s)

Outer Surface Lipoproteins from the Lyme Sisease Spirochete Exploit the Molecular Switch Mechanism of the Complement Protease C1s

Proteolytic cascades comprise several important physiological systems, including a primary arm of innate immunity called the complement cascade. To safeguard against complement-mediated attack, the etiologic agent of Lyme disease, Borreliella burgdorferi, produces numerous outer surface–localized lipoproteins that contribute to successful complement evasion. Recently, we discovered a pair of B. burgdorferi surface lipoproteins of the OspEF-related protein family—termed ElpB and ElpQ—that inhibit antibody-mediated complement activation. In this study, we investigate the molecular mechanism of ElpB and ElpQ complement inhibition using an array of biochemical and biophysical approaches. In vitro assays of complement activation show that an independently folded homologous C-terminal domain of each Elp protein maintains full complement inhibitory activity and selectively inhibits the classical pathway. Using binding assays and complement component C1s enzyme assays, we show that binding of Elp proteins to activated C1s blocks complement component C4 cleavage by competing with C1s–C4 binding without occluding the active site. C1s-mediated C4 cleavage is dependent on activation-induced binding sites, termed exosites. To test whether these exosites are involved in Elp–C1s binding, we performed site-directed mutagenesis, which showed that ElpB and ElpQ binding require C1s residues in the anion-binding exosite located on the serine protease domain of C1s. Based on these results, we propose a model whereby ElpB and ElpQ exploit activation-induced conformational changes that are normally important for C1s-mediated C4 cleavage. Our study expands the known complement evasion mechanisms of microbial pathogens and reveals a novel molecular mechanism for selective C1s inhibition by Lyme disease spirochetes

Host-specialized fibrinogen-binding by a bacterial surface protein promotes biofilm formation and innate immune evasion

Fibrinogen is an essential part of the blood coagulation cascade and a major component of the extracellular matrix in mammals. The interface between fibrinogen and bacterial pathogens is an important determinant of the outcome of infection. Here, we demonstrate that a canine host-restricted skin pathogen, Staphylococcus pseudintermedius, produces a cell wall-associated protein (SpsL) that has evolved the capacity for high strength binding to canine fibrinogen, with reduced binding to fibrinogen of other mammalian species including humans. Binding occurs via the surface-expressed N2N3 subdomains, of the SpsL A-domain, to multiple sites in the fibrinogen α-chain C-domain by a mechanism analogous to the classical dock, lock, and latch binding model. Host-specific binding is dependent on a tandem repeat region of the fibrinogen α-chain, a region highly divergent between mammals. Of note, we discovered that the tandem repeat region is also polymorphic in different canine breeds suggesting a potential influence on canine host susceptibility to S. pseudintermedius infection. Importantly, the strong host-specific fibrinogen-binding interaction of SpsL to canine fibrinogen is essential for bacterial aggregation and biofilm formation, and promotes resistance to neutrophil phagocytosis, suggesting a key role for the interaction during pathogenesis. Taken together, we have dissected a bacterial surface protein-ligand interaction resulting from the co-evolution of host and pathogen that promotes host-specific innate immune evasion and may contribute to its host-restricted ecology

MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne’s Disease

The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that con- tains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne’s disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were ex- pressed and evaluated with sera from cattle with Johne’s disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a dis- tinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis

Eradicating Infecting Bacteria while Maintaining Tissue Integration on Photothermal Nanoparticle-Coated Titanium Surfaces

Photothermal nanoparticles locally release heat when irradiated by near-infrared (NIR). Clinical applications initially involved tumor treatment, but currently extend toward bacterial infection control. Applications toward much smaller, micrometer-sized bacterial infections, however, bear the risk of collateral damage by dissipating heat into tissues surrounding an infection site. This can become a complication when photothermal nanoparticle coatings are clinically applied on biomaterial surfaces requiring tissue integration, such as titanium-made, bone-Anchored dental implants. Dental implants can fail due to infection in the pocket formed between the implant screw and the surrounding soft tissue ("peri-implantitis"). We address the hitherto neglected potential complication of collateral tissue damage by evaluating photothermal, polydopamine nanoparticle (PDA-NP) coatings on titanium surfaces in different coculture models. NIR irradiation of PDA-NP-coated (200 μg/cm2) titanium surfaces with adhering Staphylococcus aureus killed staphylococci within an irradiation time window of around 3 min. Alternatively, when covered with human gingival fibroblasts, this irradiation time window maintained surface coverage by fibroblasts. Contaminating staphylococci on PDA-NP-coated titanium surfaces, as can be per-operatively introduced, reduced surface coverage by fibroblasts, and this could be prevented by NIR irradiation for 5 min or longer prior to allowing fibroblasts to adhere and grow. Negative impacts of early postoperative staphylococcal challenges to an existing fibroblast layer covering a coated surface were maximally prevented by 3 min NIR irradiation. Longer irradiation times caused collateral fibroblast damage. Late postoperative staphylococcal challenges to a protective keratinocyte layer covering a fibroblast layer required 10 min NIR irradiation for adverting a staphylococcal challenge. This is longer than foreseen from monoculture studies because of additional heat uptake by the keratinocyte layer. Summarizing, photothermal treatment of biomaterial-Associated infection requires precise timing of NIR irradiation to prevent collateral damage to tissues surrounding the infection site

MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne’s Disease

The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that con- tains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne’s disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were ex- pressed and evaluated with sera from cattle with Johne’s disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a dis- tinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis