Pasteurella pneumotropica Evades the Human Complement System by Acquisition of the Complement Regulators Factor H and C4BP

Pasteurella pneumotropica is an opportunist Gram negative bacterium responsible for rodent pasteurellosis that affects upper respiratory, reproductive and digestive tracts of mammals. In animal care facilities the presence of P. pneumotropica causes severe to lethal infection in immunodeficient mice, being also a potential source for human contamination. Indeed, occupational exposure is one of the main causes of human infection by P. pneumotropica. The clinical presentation of the disease includes subcutaneous abscesses, respiratory tract colonization and systemic infections. Given the ability of P. pneumotropica to fully disseminate in the organism, it is quite relevant to study the role of the complement system to control the infection as well as the possible evasion mechanisms involved in bacterial survival. Here, we show for the first time that P. pneumotropica is able to survive the bactericidal activity of the human complement system. We observed that host regulatory complement C4BP and Factor H bind to the surface of P. pneumotropica, controlling the activation pathways regulating the formation and maintenance of C3-convertases. These results show that P. pneumotropica has evolved mechanisms to evade the human complement system that may increase the efficiency by which this pathogen is able to gain access to and colonize inner tissues where it may cause severe infections.     

Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation

Background

Trichinella spiralis expresses paramyosin (Ts-Pmy) as a defense mechanism. Ts-Pmy is a functional protein with binding activity to human complement C8 and C9 and thus plays a role in evading the attack of the host’s immune system. In the present study, the binding activity of Ts-Pmy to human complement C1q and its ability to inhibit classical complement activation were investigated.

Methods and Findings

The binding of recombinant and natural Ts-Pmy to human C1q were determined by ELISA, Far Western blotting and immunoprecipitation, respectively. Binding of recombinant Ts-Pmy (rTs-Pmy) to C1q inhibited C1q binding to IgM and consequently inhibited C3 deposition. The lysis of antibody-sensitized erythrocytes (EAs) elicited by the classical complement pathway was also inhibited in the presence of rTs-Pmy. In addition to inhibiting classical complement activation, rTs-Pmy also suppressed C1q binding to THP-1-derived macrophages, thereby reducing C1q-induced macrophages migration.

Conclusion

Our results suggest that T. spiralis paramyosin plays an important role in immune evasion by interfering with complement activation through binding to C1q in addition to C8 and C9.     

Fine Mapping of the Interaction between C4b-Binding Protein and Outer Membrane Proteins LigA and LigB of Pathogenic Leptospira interrogans

The complement system consists of more than 40 proteins that participate in the inflammatory response and in pathogen killing. Complement inhibitors are necessary to avoid the excessive consumption and activation of this system on host cells. Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Pathogenic leptospires are able to escape from complement activation by binding to host complement inhibitors Factor H [FH] and C4b-binding protein (C4BP) while non-pathogenic leptospires are rapidly killed in the presence of fresh serum. In this study, we demonstrate that complement control protein domains (CCP) 7 and 8 of C4BP α-chain interact with the outer membrane proteins LcpA, LigA and LigB from the pathogenic leptospire L. interrogans. The interaction between C4BP and LcpA, LigA and LigB is sensitive to ionic strength and inhibited by heparin. We fine mapped the LigA and LigB domains involved in its binding to C4BP and heparin and found that both interactions are mediated through the bacterial immunoglobulin-like (Big) domains 7 and 8 (LigA7-8 and LigB7-8) of both LigA and LigB and also through LigB9-10. Therefore, C4BP and heparin may share the same binding sites on Lig proteins.     

Borrelia valaisiana Resist Complement-Mediated Killing Independently of the Recruitment of Immune Regulators and Inactivation of Complement Components

Spirochetes belonging to the Borrelia (B.) burgdorferi sensu lato complex differ in their resistance to complement-mediated killing, particularly in regard to human serum. In the present study, we elucidate the serum and complement susceptibility of B. valaisiana, a genospecies with the potential to cause Lyme disease in Europe as well as in Asia. Among the investigated isolates, growth of ZWU3 Ny3 was not affected while growth of VS116 and Bv9 was strongly inhibited in the presence of 50% human serum. Analyzing complement activation, complement components C3, C4 and C6 were deposited on the surface of isolates VS116 and Bv9, and similarly the membrane attack complex was formed on their surface. In contrast, no surface-deposited components and no aberrations in cell morphology were detected for serum-resistant ZWU3 Ny3. While further investigating the protective role of bound complement regulators in mediating complement resistance, we discovered that none of the B. valaisiana isolates analyzed bound complement regulators Factor H, Factor H-like protein 1, C4b binding protein or C1 esterase inhibitor. In addition, B. valaisiana also lacked intrinsic proteolytic activity to degrade complement components C3, C3b, C4, C4b, and C5. Taken together, these findings suggest that certain B. valaisiana isolates differ in their capability to resist complement-mediating killing by human serum. The molecular mechanism utilized by B. valaisiana to inhibit bacteriolysis appears not to involve binding of the key host complement regulators of the alternative, classical, and lectin pathways as already known for serum-resistant Lyme disease or relapsing fever borreliae.     

Complement Protein C1q Forms a Complex with Cytotoxic Prion Protein Oligomers

A growing number of studies have investigated the interaction between C1q and PrP, but the oligomeric form of PrP involved in this interaction remains to be determined. Aggregation of recombinant full-length murine PrP in the presence of 100 mm NaCl allowed us to isolate three different types of oligomers by size-exclusion chromatography. In contrast to PrP monomers and fibrils, these oligomers activate the classical complement pathway, the smallest species containing 8–15 PrP protomers being the most efficient. We used Thioflavine T fluorescence to monitor PrP aggregation and showed that, when added to the reaction, C1q has a cooperative effect on PrP aggregation and leads to the formation of C1q-PrP complexes. In these complexes, C1q interacts through its globular domains preferentially with the smallest oligomers, as shown by electron microscopy, and retains the ability to activate the classical complement pathway. Using two cell lines, we also provide evidence that C1q inhibits the cytotoxicity induced by the smallest PrP oligomers. The cooperative interaction between C1q and PrP could represent an early step in the disease, where it prevents elimination of the prion seed, leading to further aggregation.     

Myotubularin-related Proteins 3 and 4 Interact with Polo-like Kinase 1 and Centrosomal Protein of 55 kDa to Ensure Proper Abscission

The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein–protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to endosomal sorting complex required for transport-I components. Disruption of any of these interactions results in abscission failure, by disrupting the proper recruitment of CEP55, and subsequently, of endosomal sorting complex required for transport-I, to the midbody. Our data suggest that myotubularin-related protein 3 and myotubularin-related protein 4 may act as a bridge between CEP55 and polo-like kinase 1, ensuring proper CEP55 phosphorylation and regulating CEP55 recruitment to the midbody. This work provides a novel role for myotubularin-related protein 3/4 heterodimers, and highlights the temporal and spatial complexity of the regulation of cytokinesis.The myotubularins are a subfamily of protein tyrosine phosphatases (PTPs)¹, consisting of sixteen conserved proteins. Despite containing the conserved C(X)₅R catalytic motif found in all protein tyrosine phosphatases, myotubularins harbor active sites that do not dephosphorylate tyrosine, but instead catalyze the conversion of the phosphatidylinositol-type lipids phosphatylinositol 3 phosphate (PI3P) and phosphatylinositol 3,5 phosphate (PI3,5P) to phosphatidylinositol (PI) and phosphatylinositol 5 phosphate (PI5P), respectively (1). Phosphatidylinositols are important molecules in a variety of processes, and as enzymatic regulators, myotubularins may function in cell proliferation, differentiation, survival, and cytoskeletal and junctional dynamics (1, 2). Of the sixteen myotubularins, only nine are active enzymes (supplemental Fig. S1A), as several lack catalytic cysteine residues (3). Myotubularins interact extensively with each other, and interactions between active and inactive pairs are frequent (4). It is thought that inactive myotubularins regulate the activity, substrate binding, and/or localization of their active binding partners (2).Several myotubularins are linked to human disease. Myotubularin (MTM1), the first reported family member, is mutated in X-linked centronuclear myopathy (5), and Myotubularin related protein 14 (MTMR14) is mutated in autosomal centronuclear myopathy (6). Mutations in the active MTMR2 or its inactive binding partner, SET binding factor (SBF)2 (MTMR13), cause Charcot-Marie-Tooth diseases CMT4A and CMT4B, respectively (7–9). MTMR7 and MTMR9 have been associated with metabolic syndrome and obesity (MTMR9) (10, 11), epilepsy (MTMR7/9) (12), and Creutzfeldt-Jakob disease (MTMR7) (13). In addition, misregulation of the active phosphatase MTMR3 contributes to susceptibility to gastric and colon carcinomas (14), oral cancer (15), and lung cancer (16), and contributes to metastasis (15, 17). Aberrant expression of the inactive MTMR11 has been observed in acute myeloid leukemia (18), acute lymphocytic leukemia (19), and Her2-positive breast cancer (20). Generally, myotubularins are thought to integrate different cellular pathways, through both phosphatidylinositol regulation and protein–protein interactions (2). Despite their proposed involvement in a variety of cellular processes as well as disease states, many myotubularins remain poorly characterized, with their precise cellular functions not yet elucidated, and the pathological significance of those functions still unknown.To gain insight on the biological functions of myotubularin family phosphatases, we have used affinity purification coupled to mass spectrometry (AP-MS) to identify protein–protein interactions for each myotubularin. The results expand upon the known repertoire of intra-myotubularin interactions, and, critically, identify specific novel interactions for individual myotubularins, providing valuable clues toward their respective functions. Further investigation revealed an unexpected role for MTMR3 and MTMR4 in abscission (21), the fission event at the end of cytokinesis that severs the final membrane link between divided daughter cells. Future studies of additional identified protein–protein interactions will undoubtedly illuminate the cellular roles of myotubularin family phosphatases.     

Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex

Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems.     

The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β₂-glycoprotein I and interferes with innate immune recognition.     

Shigella flexneri 3a Outer Membrane Protein C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity

Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections.     

Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T₀ transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T₂ generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.     

Identification of a Novel HIV-1 Inhibitor Targeting Vif-dependent Degradation of Human APOBEC3G Protein

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(−) T cells and had an IC₅₀ as low as 8.4 μm and a TC₅₀ of >100 μm when tested against HIV-1_Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC₅₀ as low as 4.2 μm). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.     

Binding of Complement Inhibitor C4b-binding Protein to a Highly Virulent Streptococcus pyogenes M1 Strain Is Mediated by Protein H and Enhances Adhesion to and Invasion of Endothelial Cells

Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of ¹²⁵I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92–109, which specifically bound C4BP. Biacore was used to determine K_D = 6 × 10⁻⁷ m between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.     

Characterization of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

Background

It was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined.

Methodology/Principal Findings

We expressed the Rv0045c protein to high levels in E. coli and purified the protein to high purity. We confirmed that the prepared protein was the Rv0045c protein by mass spectrometry analysis. Circular dichroism spectroscopy analysis showed that the protein possessed abundant β-sheet secondary structure, and confirmed that its conformation was stable in the range pH 6.0–10.0 and at temperatures ≤40°C. Enzyme activity analysis indicated that the Rv0045c protein could efficiently hydrolyze short chain p-nitrophenyl esters (C₂–C₈), and its suitable substrate was p-nitrophenyl caproate (C₆) with optimal catalytic conditions of 39°C and pH 8.0.

Conclusions/Significance

Our results demonstrated that the Rv0045c protein is a novel esterase. These experiments will be helpful in understanding ester/lipid metabolism related to M. tuberculosis.     

A Molecular Switch Governs the Interaction between the Human Complement Protease C1s and Its Substrate,Complement C4

The complement system is an ancient innate immune defense pathway that plays a front line role in eliminating microbial pathogens. Recognition of foreign targets by antibodies drives sequential activation of two serine proteases, C1r and C1s, which reside within the complement Component 1 (C1) complex. Active C1s propagates the immune response through its ability to bind and cleave the effector molecule complement Component 4 (C4). Currently, the precise structural and biochemical basis for the control of the interaction between C1s and C4 is unclear. Here, using surface plasmon resonance, we show that the transition of the C1s zymogen to the active form is essential for C1s binding to C4. To understand this, we determined the crystal structure of a zymogen C1s construct (comprising two complement control protein (CCP) domains and the serine protease (SP) domain). These data reveal that two loops (492–499 and 573–580) in the zymogen serine protease domain adopt a conformation that would be predicted to sterically abrogate C4 binding. The transition from zymogen to active C1s repositions both loops such that they would be able to interact with sulfotyrosine residues on C4. The structure also shows the junction of the CCP1 and CCP2 domains of C1s for the first time, yielding valuable information about the exosite for C4 binding located at this position. Together, these data provide a structural explanation for the control of the interaction with C1s and C4 and, furthermore, point to alternative strategies for developing therapeutic approaches for controlling activation of the complement cascade.     

Zebrafish scube1 (Signal Peptide-CUB (Complement Protein C1r/C1s,Uegf, and Bmp1)-EGF (Epidermal Growth Factor) Domain-containing Protein 1) Is Involved in Primitive Hematopoiesis

scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis.     

Identification of a gp41 Core-Binding Molecule with Homologous Sequence of Human TNNI3K-Like Protein as a Novel Human Immunodeficiency Virus Type 1 Entry Inhibitor

Human immunodeficiency virus type 1 (HIV-1) gp41 plays a critical role in the viral fusion process, and its N- and C-terminal heptad repeat domains serve as important targets for developing anti-HIV-1 drugs, like T-20 (generic name, enfuvirtide; brand name, Fuzeon). Here, we conducted a yeast two-hybrid screening on a human bone marrow cDNA library using the recombinant soluble gp41 ectodomain as the bait and identified a novel gp41 core-binding molecule, designated P20. P20 showed no homology with a current HIV fusion inhibitor, T-20, but had sequence homology to a human protein, troponin I type 3 interacting kinase (TNNI3K)-like protein. While it could bind to the six-helix bundle core structure formed by the N- and C-terminal heptad repeats, P20 did not interrupt the formation of the six-helix bundle. P20 was effective in blocking HIV-1 Env-mediated syncytium formation and inhibiting infection by a broad spectrum of HIV-1 strains with distinct subtypes and coreceptor tropism, while it was ineffective against other enveloped viruses, such as vesicular stomatitis virus and influenza A virus. P20 exhibited no significant cytotoxicity to the CD4⁺ cells that were used for testing antiviral activity. Among the 11 P20 mutants, four analogous peptides with a common motif (WGRLEGRRT) exhibited significantly reduced anti-HIV-1 activity, suggesting that this region is the critical active site of P20. Therefore, this peptide can be used as a lead for developing novel HIV fusion inhibitors and as a probe for studying the membrane-fusogenic mechanism of HIV.Human immunodeficiency virus type 1 (HIV-1) is an enveloped virus, and its envelope protein (Env) complex controls the key processes by which HIV-1 delivers its replicative material into target cells. Specifically, the Env surface subunit, gp120, binds the cellular receptor CD4 and a coreceptor, CCR5 or CXCR4, which triggers conformational changes of the transmembrane subunit, gp41 (8). The N-terminal heptad repeat (NHR) in the gp41 ectodomain interacts with its C-terminal heptad repeat (CHR) to form a trimer of hairpins, or six-helix bundle (6-HB; also known as the gp41 fusion core) (38, 51), which brings the viral and target cell membranes into close proximity and promotes membrane fusion (3, 51). Therefore, the gp41 6-HB core plays an important role in viral fusion and may serve as an attractive target for the development of HIV fusion/entry inhibitors (20).In the early 1990s, a number of peptides derived from the gp41 NHR and CHR regions were discovered to exhibit highly potent anti-HIV-1 activity by binding to the corresponding region of gp41 at the fusion-intermediate state (22, 23, 38, 52, 53) and blocking gp41 6-HB core formation (4, 9, 32, 47). One of the CHR-peptides, T-20 (generic name, enfuvirtide; brand name, Fuzeon), was licensed by the FDA as the first member of a new class of anti-HIV drugs, the HIV fusion inhibitors (33, 53). Although T-20 is very effective in inhibiting infection by a broad spectrum of HIV-1 strains, especially those resistant to current antiretroviral therapies (26), T-20 itself also can easily induce drug resistance in T-20-treated patients, resulting in virologic failure (36, 46, 50, 55). Therefore, it is essential to identify and develop novel HIV-1 fusion inhibitors having a mechanism of action or target different from that for T-20 and with improved drug resistance profiles.Here, we sought to screen a human bone marrow cDNA library in a yeast two-hybrid screening assay using the recombinant soluble gp41 ectodomain (rsgp41e) as the bait in hopes of identifying a novel HIV fusion inhibitor with sequence homology to a human protein and low immunogenicity to humans to avoid its rapid clearance by specific human antibodies (1). We identified a 32-mer peptide, designated P20, with sequence homology to human troponin I type 3 interacting kinase (TNNI3K)-like protein. P20 could specifically bind to the gp41 6-HB core and strongly blocked HIV-1 Env-mediated membrane fusion. It potently inhibited infection by a number of laboratory-adapted HIV-1 strains, including T-20-resistant variants, and a broad spectrum of primary HIV-1 isolates. These results suggest that P20 has the potential to be developed further as a novel anti-HIV-1 therapeutic and can be used as a probe to study the role of the HIV-1 gp41 6-HB core in the membrane fusion process.     

TgICMAP1 Is a Novel Microtubule Binding Protein in Toxoplasma gondii

The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction.