Identification of novel pig and human immunoglobulin G-binding proteins and characterization of the binding regions of enolase from Streptococcus suis serotype 2

Streptococcus suis, a major emerging pathogen in swine and humans, expresses immunoglobulin G (IgG)-binding proteins (IBPs), which contribute to the ability of organism to evasion of host immune system. The objective of this study was to identify novel pig IgG (pIgG) and human IgG (hIgG)-binding proteins and characterize the binding regions of enolase from Streptococcus suis serotype 2 (S. suis 2). Here, four pIgG-binding proteins (pIBPs) and five hIgG-binding proteins (hIBPs) were identified from S. suis 2 surface proteins by 2D-Far-western blot assays. All the newly captured proteins were expressed and further confirmed their binding activity to pIgG or hIgG by Far-western blot and dot blot. In addition to previously identified factor H, fibronectin, collagen, fibrinogen, plasminogen and laminin, we also found that both pIgG and hIgG can specifically interact with enolase. Binding assays indicated that interactions of S. suis 2 enolase with pIgG and hIgG is primarily mediated by the enolase C-terminal portion (Enolase-C, a.a. 142-432). We found that hIgG exhibited stronger binding ability to Enolase-C than pIgG. Further analysis of the C-terminal regions of enolase (Enolase-C1 and Enolase-C2) suggested that the C-terminus possessed two different binding domains with distinct host IgG proteins. Strikingly, we confirmed that pIgG interacted with the Enolase-C1 (a.a. 142-271) and hIgG interacted with the Enolase-C2 (a.a. 271-432). These observations of enolase provide interesting insights in the pathogenesis of S. suis infection.

Identification of novel pig and human immunoglobulin G-binding proteins and characterization of the binding regions of enolase from Streptococcus suis serotype 2

Streptococcus suis, a major emerging pathogen in swine and humans, expresses immunoglobulin G (IgG)-binding proteins (IBPs), which contribute to the ability of organism to evasion of host immune system. The objective of this study was to identify novel pig IgG (pIgG) and human IgG (hIgG)-binding proteins and characterize the binding regions of enolase from Streptococcus suis serotype 2 (S. suis 2). Here, four pIgG-binding proteins (pIBPs) and five hIgG-binding proteins (hIBPs) were identified from S. suis 2 surface proteins by 2D-Far-western blot assays. All the newly captured proteins were expressed and further confirmed their binding activity to pIgG or hIgG by Far-western blot and dot blot. In addition to previously identified factor H, fibronectin, collagen, fibrinogen, plasminogen and laminin, we also found that both pIgG and hIgG can specifically interact with enolase. Binding assays indicated that interactions of S. suis 2 enolase with pIgG and hIgG is primarily mediated by the enolase C-terminal portion (Enolase-C, a.a. 142-432). We found that hIgG exhibited stronger binding ability to Enolase-C than pIgG. Further analysis of the C-terminal regions of enolase (Enolase-C1 and Enolase-C2) suggested that the C-terminus possessed two different binding domains with distinct host IgG proteins. Strikingly, we confirmed that pIgG interacted with the Enolase-C1 (a.a. 142-271) and hIgG interacted with the Enolase-C2 (a.a. 271-432). These observations of enolase provide interesting insights in the pathogenesis of S. suis infection.

C-Type Lectin-20 Interacts with ALP1 Receptor to Reduce Cry Toxicity in Aedes aegypti

Aedes aegypti is a crucial vector for human diseases, such as yellow fever, dengue, chikungunya, and Zika viruses. Today, a major challenge throughout the globe is the insufficient availability of antiviral drugs and vaccines against arboviruses, and toxins produced by Bacillus thuringiensis (Bt) are still used as biological agents for mosquito control. The use of Cry toxins to kill insects mainly depends on the interaction between Cry toxins and important toxin receptors, such as alkaline phosphatase (ALP). In this study, we investigated the function of A. aegypti C-type lectin-20 (CTL-20) in the tolerance of Cry toxins. We showed that recombinant CTL-20 protein interacted with both Cry11Aa and ALP1 by the Far-Western blot and ELISA methods, and CTL-20 bound to A. aegypti larval brush border membrane vesicles (BBMVs). Binding affinity of CTL-20 to ALP1 was higher than that of Cry11Aa to ALP1. Furthermore, the survival rate of A. aegypti larvae fed with Cry11Aa toxin mixed with recombinant CTL-20 fusion protein was significantly increased compared with that of the control larvae fed with Cry11Aa mixed with thioredoxin. Our novel results suggest that midgut proteins like CTLs may interfere with interactions between Cry toxins and toxin receptors by binding to both Cry toxins and receptors to alter Cry toxicity.

PmpI antibody reduces the inhibitory effect of Vp1 on Chlamydia trachomatis infectivity

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infections. The effect of antibiotic treatment is not satisfactory, and there is currently no vaccine to prevent C. trachomatis infection. Our results showed that Chlamydia virus CPG1 capsid protein Vp1 treatment significantly inhibited C. trachomatis growth in cell culture, and the inclusion numbers of different C. trachomatis serotypes were decreased. In addition, we conducted a preliminary investigation of the possible mechanisms behind the Vp1 inhibition effects and the C. trachomatis molecules targeted by Vp1. Using far-western blot and GST pull-down assay, we found that purified Vp1 can bind to the C. trachomatis outer membrane protein PmpI. PmpI polyclonal antibody treatment markedly reduced the inhibitory effect of Vp1 on C. trachomatis infectivity. On the basis of these experimental results, we infer that PmpI participates in the inhibitory effect of Vp1 and may be a potential receptor of Vp1 in the outer membrane of C. trachomatis. Our research provides clues regarding the molecular mechanisms underlying the interactions between chlamydia virus and chlamydia.

Trypanosoma cruzi Bromodomain Factor 3 Binds Acetylated α-Tubulin and Concentrates in the Flagellum during Metacyclogenesis

ABSTRACTBromodomains are highly conserved acetyl-lysine binding domains found mainly in proteins associated with chromatin and nuclear acetyltransferases. TheTrypanosoma cruzigenome encodes at least four bromodomain factors (TcBDFs). We describe here bromodomain factor 3 (TcBDF3), a bromodomain-containing protein localized in the cytoplasm.TcBDF3 cytolocalization was determined, using purified antibodies, by Western blot and immunofluorescence analyses in all life cycle stages ofT. cruzi. In epimastigotes and amastigotes, it was detected in the cytoplasm, the flagellum, and the flagellar pocket, and in trypomastigotes only in the flagellum. Subcellular localization ofTcBDF3 was also determined by digitonin extraction, ultrastructural immunocytochemistry, and expression ofTcBDF3 fused to cyan fluorescent protein (CFP). Tubulin can acquire different posttranslational modifications, which modulate microtubule functions. Acetylated α-tubulin has been found in the axonemes of flagella and cilia, as well as in the subpellicular microtubules of trypanosomatids.TcBDF3 and acetylated α-tubulin partially colocalized in isolated cytoskeletons and flagella fromT. cruziepimastigotes and trypomastigotes. Interaction between the two proteins was confirmed by coimmunoprecipitation and far-Western blot assays with synthetic acetylated α-tubulin peptides and recombinantTcBDF3.

Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example

Entamoeba histolytica, the causative agent of amoebiasis, possesses the dithiol-containing redox proteins Trx (thioredoxin) and TrxR (Trx reductase). Both proteins were found to be covalently modified and inactivated by metronidazole, a 5-nitroimidazole drug that is commonly used to treat infections with microaerophilic protozoan parasites in humans. Currently, very little is known about enzymes and other proteins participating in the Trx-dependent redox network of the parasite that could be indirectly affected by metronidazole treatment. On the basis of the disulfide/dithiol-exchange mechanism we constructed an active-site mutant of Trx, capable of binding interacting proteins as a stable mixed disulfide intermediate to screen the target proteome of Trx in E. histolytica. By applying Trx affinity chromatography, two-dimensional gel electrophoresis and MS, peroxiredoxin and 15 further potentially redox-regulated proteins were identified. Among them, EhSat1 (E. histolytica serine acetyltransferase-1), an enzyme involved in the L-cysteine biosynthetic pathway, was selected for detailed analysis. Binding of Trx to EhSat1 was verified by Far-Western blot analysis. Trx was able to restore the activity of the oxidatively damaged EhSat1 suggesting that the TrxR/Trx system protects sensitive proteins against oxidative stress in E. histolytica. Furthermore, the activity of peroxiredoxin, which is dependent on a functioning TrxR/Trx system, was strongly reduced in metronidazole-treated parasites.