Glycoepitopes of Staphylococcal Wall Teichoic Acid Govern Complement-mediated Opsonophagocytosis via Human Serum Antibody and Mannose-binding Lectin

Serum antibodies and mannose-binding lectin (MBL) are important host defense factors for host adaptive and innate immunity, respectively. Antibodies and MBL also initiate the classical and lectin complement pathways, respectively, leading to opsonophagocytosis. We have shown previously that Staphylococcus aureus wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with α- or β-O-N-acetyl-d-glucosamine (GlcNAc) and d-alanine, is recognized by MBL and serum anti-WTA IgG. However, the exact antigenic determinants to which anti-WTA antibodies or MBL bind have not been determined. To answer this question, several S. aureus mutants, such as α-GlcNAc glycosyltransferase-deficient S. aureus ΔtarM, β-GlcNAc glycosyltransferase-deficient ΔtarS, and ΔtarMS double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant S. aureus strain. Here, we describe the unexpected finding that β-GlcNAc WTA-deficient ΔtarS mutant cells (which have intact α-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas α-GlcNAc WTA-deficient ΔtarM mutant cells (which have intact β-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in S. aureus cells was lost in the ΔtarMS double mutant but not in either single mutant. When we determined the serum concentrations of the anti-α- or anti-β-GlcNAc-specific WTA IgGs, anti-β-GlcNAc WTA-IgG was dominant in pooled human IgG fractions and in the intact sera of healthy adults and infants. These data demonstrate the importance of the WTA sugar conformation for human innate and adaptive immunity against S. aureus infection.     

Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of membrane attack complex (MAC)

The complement membrane attack complex (MAC) is formed by the sequential assembly of C5b with four homologous proteins as follows: one copy each of C6, C7, and C8 and 12-14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MAC-perforin domain flanked by 4-9 "auxiliary" domains. Here, we report the crystal structure of C6, the first and longest of the pore proteins to be recruited by C5b. Comparisons with the structures of the C8αβγ heterodimer and perforin show that the central domain of C6 adopts a "closed" (perforin-like) state that is distinct from the "open" conformations in C8. We further show that C6, C8α, and C8β contain three homologous subdomains ("upper," "lower," and "regulatory") related by rotations about two hinge points. In C6, the regulatory segment includes four auxiliary domains that stabilize the closed conformation, inhibiting release of membrane-inserting elements. In C8β, rotation of the regulatory segment is linked to an opening of the central β-sheet of its clockwise partner, C8α. Based on these observations, we propose a model for initiation and unidirectional propagation of the MAC in which the auxiliary domains play key roles: in the assembly of the C5b-8 initiation complex; in driving and regulating the opening of the β-sheet of the MAC-performin domain of each new recruit as it adds to the growing pore; and in stabilizing the final pore. Our model of the assembled pore resembles those of the cholesterol-dependent cytolysins but is distinct from that recently proposed for perforin.     

Microtubule-associated Proteins 1 (MAP1) Promote Human Immunodeficiency Virus Type I (HIV-1) Intracytoplasmic Routing to the Nucleus

After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules.     

Glycerol Monomycolate Is a Novel Ligand for the Human,but Not Mouse Macrophage Inducible C-type Lectin,Mincle

An array of lipidic compounds that constitute the cell wall of mycobacteria is recognized by host receptors. Examples include trehalose dimycolate (TDM), which is a major surface-exposed glycolipid of mycobacteria, that interacts with the macrophage inducible C-type lectin, Mincle, and exerts its highly potent adjuvant functions. Recent evidence has suggested that glycerol monomycolate (GroMM), another mycolate-containing lipid species produced by mycobacteria, can stimulate innate immune cells; however, its specific host receptors have yet to be identified. We here demonstrated that cell transfectants expressing human Mincle (hMincle) reacted to both TDM and GroMM, while those expressing mouse Mincle (mMincle) only reacted to TDM and failed to recognize GroMM. Studies using domain swap chimeras confirmed that the ectodomain of hMincle, but not that of mMincle, interacted with GroMM, and site-directed mutagenesis analyses revealed that short stretches of amino acid residues at positions 174–176 and 195–196 were involved in GroMM recognition. To further substantiate the differential recognition of GroMM by hMincle and mMincle, hMincle transgenic/mMincle knock-out mice (i.e. hMincle⁺ mice) were established and compared with non-transgenic mice (i.e. mMincle⁺ mice). We showed that macrophages derived from hMincle⁺ mice were activated by GroMM and produced inflammatory cytokines, whereas those derived from mMincle⁺ mice did not exhibit any reactivity to GroMM. Furthermore, local inflammatory responses were elicited in the GroMM-injected skin of hMincle⁺, but not mMincle⁺ mice. These results demonstrated that GroMM is a unique ligand for hMincle that is not recognized by mMincle.     

Heterologous Src Homology 4 Domains Support Membrane Anchoring and Biological Activity of HIV-1 Nef

The HIV-1 pathogenicity factor Nef enhances viral replication by modulation of multiple host cell transport and signaling pathways. Nef associates with membranes via an N-terminal Src homology 4 (SH4) domain, and membrane association is believed to be essential for its biological functions. At which subcellular site(s) Nef exerts its different functions and how kinetics of membrane interactions contribute to its biological activity are unknown. To address how specific characteristics of Nef membrane association affect its biological properties, the SH4 domain of Nef was replaced by heterologous membrane targeting domains. The use of a panel of heterologous SH4 domains resulted in chimeric Nef proteins with distinct steady state subcellular localization, membrane association efficiency, and anterograde transport routes. Irrespective of these modifications, cardinal Nef functions affecting host cell vesicular transport and actin dynamics were fully preserved. In contrast, stable targeting of Nef to the surface of mitochondria, peroxisomes, or the Golgi apparatus, and thus prevention of plasma membrane delivery, caused potent and broad loss of Nef activity. These results support the concept that Nef adopts its active conformation in the membrane-associated state but exclude that membrane-associated Nef simply acts by recruiting soluble factors independently of its local microenvironment. Rather than its steady state subcellular localization or membrane affinity, the ability to undergo dynamic anterograde and internalization cycles appear to determine Nef function. These results reveal that functional membrane interactions of Nef underlie critical spatiotemporal regulation and suggest that delivery to distinct subcellular sites via such transport cycles provides the basis for the multifunctionality of Nef.     

Novel epidithiodiketopiperazines as anti-viral zinc ejectors of the Feline Immunodeficiency Virus (FIV) nucleocapsid protein as a model for HIV infection

Focused libraries of multi-substituted epidithiodiketopiperazines (ETP) were prepared and evaluated for efficacy of inhibiting the nucleocapsid protein function of the Feline Immunodeficiency Virus (FIV) as a model for HIV. This activity was compared and contrasted to observed toxicity utilising an in-vitro cell culture approach. This resulted in the identification of several promising lead compounds with nanomolar potency in cells with low toxicity and a favorable therapeutic index.     

Crystal structure of C5b-6 suggests structural basis for priming assembly of the membrane attack complex

The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12–18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly.     

Structure function analysis of an ADP-ribosyltransferase type III effector and its RNA-binding target in plant immunity

The Pseudomonas syringae type III effector HopU1 is a mono-ADP-ribosyltransferase that is injected into plant cells by the type III protein secretion system. Inside the plant cell it suppresses immunity by modifying RNA-binding proteins including the glycine-rich RNA-binding protein GRP7. The crystal structure of HopU1 at 2.7-Å resolution reveals two unique protruding loops, L1 and L4, not found in other mono-ADP-ribosyltransferases. Site-directed mutagenesis demonstrates that these loops are essential for substrate recognition and enzymatic activity. HopU1 ADP-ribosylates the conserved arginine 49 of GRP7, and this reduces the ability of GRP7 to bind RNA in vitro. In vivo, expression of GRP7 with Arg-49 replaced with lysine does not complement the reduced immune responses of the Arabidopsis thaliana grp7-1 mutant demonstrating the importance of this residue for GRP7 function. These data provide mechanistic details how HopU1 recognizes this novel type of substrate and highlights the role of GRP7 in plant immunity.     

Negative Regulation of Interferon-induced Transmembrane Protein 3 by SET7-mediated Lysine Monomethylation

Although lysine methylation is classically known to regulate histone function, its role in modulating antiviral restriction factor activity remains uncharacterized. Interferon-induced transmembrane protein 3 (IFITM3) was found monomethylated on its lysine 88 residue (IFITM3-K88me1) to reduce its antiviral activity, mediated by the lysine methyltransferase SET7. Vesicular stomatitis virus and influenza A virus infection increased IFITM3-K88me1 levels by promoting the interaction between IFITM3 and SET7, suggesting that this pathway could be hijacked to support infection; conversely, IFN-α reduced IFITM3-K88me1 levels. These findings may have important implications in the design of therapeutics targeting protein methylation against infectious diseases.     

Cytoplasmic Body Component TRIM5α Requires Lipid-enriched Microdomains for Efficient HIV-1 Restriction

TRIM5α is a member of the tripartite motif (TRIM) family of proteins and affects both early and late phases of the retroviral life cycle. Although TRIM5α multimerizes to form cytoplasmic bodies, which are thought to play an important role in viral restriction, the identity of TRIM5α-containing cytoplasmic bodies remains elusive. To better understand TRIM5α cytoplasmic body constituents and the cellular proteins that could be involved in the TRIM5α-mediated antiviral activities, we sought TRIM5α-binding factors. We identified a lipid microdomain protein flotillin-1/Reggie-2 as an interacting partner of TRIM5α via co-immunoprecipitation. Immunohistochemistry studies confirmed the co-localization of rhesus monkey TRIM5α (TRIM5αrh) cytoplasmic bodies with flotillin-1/Reggie-2. Caveolin-1, another lipid microdomain-associated protein, also co-localized with TRIM5α cytoplasmic bodies. Intriguingly, disruption of cellular cholesterol by cyclodextrin perturbed TRIM5α cytoplasmic body formation. Furthermore, lipid starvation partially relieved the endogenous post-entry restriction of HIV-1 infection, which could be subsequently restored by lipid repletion. These observations indicate the involvement of cellular lipids in TRIM5α-mediated antiviral activities. Given that many viruses utilize cellular lipid microdomains for viral entry and assembly, it is plausible that lipid-enriched domains provide microenvironments where TRIM5α recognizes retroviral components.     

BBA70 of Borrelia burgdorferi Is a Novel Plasminogen-binding Protein

The Lyme disease spirochete Borrelia burgdorferi lacks endogenous, surface-exposed proteases. In order to efficiently disseminate throughout the host and penetrate tissue barriers, borreliae rely on recruitment of host proteases, such as plasmin(ogen). Here we report the identification of a novel plasminogen-binding protein, BBA70. Binding of plasminogen is dose-dependent and is affected by ionic strength. The BBA70-plasminogen interaction is mediated by lysine residues, primarily located in a putative C-terminal α-helix of BBA70. These lysine residues appear to interact with the lysine-binding sites in plasminogen kringle domain 4 because a deletion mutant of plasminogen lacking that domain was unable to bind to BBA70. Bound to BBA70, plasminogen activated by urokinase-type plasminogen activator was able to degrade both a synthetic chromogenic substrate and the natural substrate fibrinogen. Furthermore, BBA70-bound plasmin was able to degrade the central complement proteins C3b and C5 and inhibited the bacteriolytic effects of complement. Consistent with these functional activities, BBA70 is located on the borrelial outer surface. Additionally, serological evidence demonstrated that BBA70 is produced during mammalian infection. Taken together, recruitment and activation of plasminogen could play a beneficial role in dissemination of B. burgdorferi in the human host and may possibly aid the spirochete in escaping the defense mechanisms of innate immunity.