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Several spore-forming strains of Bacillus are marketed as probiotics due to their ability to survive harsh gastrointestinal conditions and confer health benefits to the host. We report the complete genomes of two commercially available probiotics, Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, and compare them with the genomes of other Bacillus and Lactobacillus. The taxonomic position of both organisms was established with a maximum-likelihood tree based on twenty six housekeeping proteins. Analysis of all probiotic strains of Bacillus and Lactobacillus reveal that the essential sporulation proteins are conserved in all Bacillus probiotic strains while they are absent in Lactobacillus spp. We identified various antibiotic resistance, stress-related, and adhesion-related domains in these organisms, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival during antibiotic treatment and harsh conditions.

F-type lectins are fucolectins with characteristic fucose and calcium-binding sequence motifs and a unique lectin fold (the "F-type" fold). F-type lectins are phylogenetically widespread with selective distribution. Several eukaryotic F-type lectins have been biochemically and structurally characterized, and the F-type lectin domain (FLD) has also been studied in the bacterial proteins, Streptococcus mitis lectinolysin and Streptococcus pneumoniae SP2159. However, there is little knowledge about the extent of occurrence of FLDs and their domain organization, especially, in bacteria. We have now mined the extensive genomic sequence information available in the public databases with sensitive sequence search techniques in order to exhaustively survey prokaryotic and eukaryotic FLDs. We report 437 FLD sequence clusters (clustered at 80% sequence identity) from eukaryotic, eubacterial and viral proteins. Domain architectures are diverse but mostly conserved in closely related organisms, and domain organizations of bacterial FLD-containing proteins are very different from their eukaryotic counterparts, suggesting unique specialization of FLDs to suit different requirements. Several atypical phylogenetic associations hint at lateral transfer. Among eukaryotes, we observe an expansion of FLDs in terms of occurrence and domain organization diversity in the taxa Mollusca, Hemichordata and Branchiostomi, perhaps coinciding with greater emphasis on innate immune strategies in these organisms. The naturally occurring FLDs with diverse domain organizations that we have identified here will be useful for future studies aimed at creating designer molecular platforms for directing desired biological activities to fucosylated glycoconjugates in target niches., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In this paper, we present two complementary strategies for enrichment of glycoproteins on living cells that combine the desirable attributes of "robust enrichment" afforded by covalent-labeling techniques and "specificity for glycoproteins" typically provided by lectin or antibody affinity reagents. Our strategy involves the selective introduction of aldehydes either into sialic acids by periodate oxidation (periodate oxidation and aniline-catalyzed oxime ligation (PAL)) or into terminal galactose and N-acetylgalactosamine residues by galactose oxidase (galactose oxidase and aniline-catalyzed oxime ligation (GAL)), followed by aniline-catalyzed oxime ligation with aminooxy-biotin to biotinylate the glycans of glycoprotein subpopulations with high efficiency and cell viability. As expected, the two methods exhibit reciprocal tagging efficiencies when applied to fully sialylated cells compared with sialic acid-deficient cells. To assess the utility of these labeling methods for glycoproteomics, we enriched the PAL- and GAL-labeled (biotinylated) glycoproteome by adsorption onto immobilized streptavidin. Glycoprotein identities (IDs) and N-glycosylation site information were then obtained by liquid chromatography-tandem mass spectrometry on total tryptic peptides and on peptides subsequently released from N-glycans still bound to the beads using peptide N-glycosidase F. A total of 175 unique N-glycosylation sites were identified, belonging to 108 nonredundant glycoproteins. Of the 108 glycoproteins, 48 were identified by both methods of labeling and the remainder was identified using PAL on sialylated cells (40) or GAL on sialic acid-deficient cells (20). Our results demonstrate that PAL and GAL can be employed as complementary methods of chemical tagging for targeted proteomics of glycoprotein subpopulations and identification of glycosylation sites of proteins on cells with an altered sialylation status.

CD22, a regulator of B-cell signaling, is a siglec that recognizes the sequence NeuAcalpha2-6Gal on glycoprotein glycans as ligands. CD22 interactions with glycoproteins on the same cell (in cis) and apposing cells (in trans) modulate its activity in B-cell receptor signaling. Although CD22 predominantly recognizes neighboring CD22 molecules as cis ligands on B-cells, little is known about the trans ligands on apposing cells. We conducted a proteomics scale study to identify candidate trans ligands of CD22 on B-cells by UV photocross-linking CD22-Fc chimera bound to B-cell glycoproteins engineered to carry sialic acids with a 9-aryl azide moiety. Using mass spectrometry-based quantitative proteomics to analyze the cross-linked products, 27 glycoproteins were identified as candidate trans ligands. Next, CD22 expressed on the surface of one cell was photocross-linked to glycoproteins on apposing B-cells followed by immunochemical analysis of the products with antibodies to the candidate ligands. Of the many candidate ligands, only the B-cell receptor IgM was found to be a major in situ trans ligand of CD22 that is selectively redistributed to the site of cell contact upon interaction with CD22 on the apposing cell.

We describe a simple method for efficiently labeling cell-surface sialic acid-containing glycans on living animal cells. The method uses mild periodate oxidation to generate an aldehyde on sialic acids, followed by aniline-catalyzed oxime ligation with a suitable tag. Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell-surface sialylated glycoproteins while maintaining high cell viability.

15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2alpha. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process.

DSG (15-deoxyspergualin), an immunosuppressant with tumoricidal properties, binds potently to the regulatory C-terminal 'EEVD' motif of Hsps (heat-shock proteins). In the present study we demonstrate that DSG inhibits eukaryotic protein synthesis by sequestering Hsp70 which is required for maintaining HRI (haem-regulated inhibitor), a kinase of the eIF2alpha (eukaryotic initiation factor 2alpha), inactive. DSG stalled initiation of protein synthesis through phosphorylation of HRI and eIF2alpha. Addition of a recombinant eIF2alpha (S51A) protein, which lacks the phosphorylation site, lowered the inhibitory potential of DSG in reticulocyte lysate. The inhibitory effect of DSG was also attenuated in HRI knockdown cells. Moreover, exogenous addition of Hsp70 or the peptide 'EEVD' reversed the inhibitory effect of DSG. Interestingly, the inhibitory effect of DSG in different mammalian cancer cells was found to negatively correlate with the amount of Hsp70 expressed in the cells, emphasizing the link with Hsp70 in DSG inhibition of eukaryotic translation.

Targeting of apicoplast replication and protein synthesis in the apicomplexan Toxoplasma gondii has conventionally been associated with the typical "delayed death" phenotype, characterized by the death of parasites only in the generation following drug intervention. We demonstrate that antibiotics like clindamycin, chloramphenicol, and tetracycline, inhibitors of prokaryotic protein synthesis, invoke the delayed death phenotype in Plasmodium falciparum, too, as evident from a specific reduction of apicoplast genome copy number. Interestingly, however, molecules like triclosan, cerulenin, fops, and NAS-91, inhibitors of the recently discovered fatty acid synthesis pathway, and succinyl acetone, an inhibitor of heme biosynthesis that operates in the apicoplast of the parasite, display rapid and striking parasiticidal effects. Our results draw a clear distinction between apicoplast functions per se and the apicoplast as the site of metabolic pathways, which are required for parasite survival, and thus subserve the development of novel antimalarial therapy.

We designed some novel diphenyl ethers and determined their binding energies for Enoyl-Acyl Carrier Protein Reductase (ENR) of Plasmodium falciparum using Autodock. Out of these, we synthesized the promising compounds and tested them for their inhibitory activity against ENRs of P. falciparum as well as Escherichia coli. Some of these compounds show nanomolar inhibition of PfENR and low micromolar inhibition of EcENR. They also exhibit low micromolar potency against in vitro cultures of P. falciparum and E. coli. The study of structure-activity relationship of these compounds paves the way for further improvements in the design of novel diphenyl ethers with improved activity against purified enzyme and the pathogens.

Heat shock proteins are essential for the survival of all cells. The C-terminal EEVD motif of Hsp70 has previously been implicated in binding 15-deoxyspergualin (DSG), an immunosuppressant with antimalarial activity whose mechanism of action is uncertain. We report the cloning, overexpression, and characterization of three members of the heat shock family, PfHsp70-1 (an Hsp70 protein with a C-terminal EEVD motif), PfHsp70-2 (an Hsp70 protein without the EEVD motif), and PfHsp70 interacting protein. The chaperone activity of PfHsp70-1, and PfHsp70-2 was enhanced by ATP and by PfHip. Interestingly, while binding of protein substrates to PfHsp70-1, PfHsp70-2 and PfHip was unaffected in the presence of DSG, the ATP enhanced chaperone activity of PfHsp70-1 but not PfHsp70-2 was stimulated further by DSG. Our finding suggests that the binding partner of DSG in the parasite cellular milieu is PfHsp70-1 and paves the way for the elucidation of the mechanism of antimalarial action of DSG.

We demonstrate, for the first time, a functional polyamine biosynthetic pathway in the malaria parasite Plasmodium falciparum that culminates in the synthesis of spermine. Additionally, we also report putrescine and spermidine salvage in the malaria parasite. Putrescine and spermidine transport in P. falciparum infected red blood cells is a highly specific, carrier mediated and active process, mediated by new transporters that differ from the transporters of uninfected red blood cells in their kinetic parameters, Vmax and km, as well as in their activation energy.

Glycosylation, particularly N-linked glycosylation, profoundly affects protein folding, oligomerization and stability. The increased efficiency of folding of glycosylated proteins could be due to the chaperone-like activity of glycans, which is observed even when the glycan is not attached to the protein. Covalently linked glycans could also facilitate oligomerization by mediating inter-subunit interactions in the protein or stabilizing the oligomer in other ways. Glycosylation also affects the rate of fibril formation in prion proteins: N-glycans reduce the rate of fibril formation, and O-glycans affect the rate either way depending on factors such as position and orientation. It has yet to be determined whether there is any correlation among the sites of glycosylation and the ensuing effect in multiply glycosylated proteins. It is also not apparent whether there is a common pattern in the conservation of glycans in a related family of glycoproteins, but it is evident that glycosylation is a multifaceted post-translational modification. Indeed, glycosylation serves to "outfit" proteins for fold-function balance.

Plasmepsin group of enzymes are key enzymes in the life cycle of malarial parasites. As inhibition of plasmepsins leads to the parasite's death, these enzymes can be utilized as potential drug targets. Although many drugs are available, it has been observed that Plasmodium falciparum, the species that causes most of the malarial infections and subsequent death, has developed resistance against most of the drugs. Based on the cleavage sites of hemglobin, the substrate for plasmepsins, we have designed two compounds (p-nitrobenzoyl-leucine-beta-alanine and p-nitrobenzoyl-leucine-isonipecotic acid), synthesized them, solved their crystal structures and studied their inhibitory effect using experimental and theoretical (docking) methods. In this paper, we discuss the synthesis, crystal structures and inhibitory nature of these two compounds which have a potential to inhibit plasmepsins.

Malaria, a tropical disease caused by Plasmodium sp., has been haunting mankind for ages. Unsuccessful attempts to develop a vaccine, the emergence of resistance against the existing drugs and the increasing mortality rate all call for immediate strategies to treat it. Intense attempts are underway to develop potent analogues of the current antimalarials, as well as a search for novel drug targets in the parasite. The indispensability of apicoplast (plastid) to the survival of the parasite has attracted a lot of attention in the recent past. The present review describes the origin and the essentiality of this relict organelle to the parasite. We also show that among the apicoplast specific pathways, the fatty acid biosynthesis system is an attractive target, because its inhibition decimates the parasite swiftly unlike the 'delayed death' phenotype exhibited by the inhibition of the other apicoplast processes. As the enzymes of the fatty acid biosynthesis system are present as discrete entities, unlike those of the host, they are amenable to inhibition without impairing the operation of the host-specific pathway. The present review describes the role of these enzymes, the status of their molecular characterization and the current advancements in the area of developing inhibitors against each of the enzymes of the pathway.

Until recently, it has only been possible to grow crystals of peanut lectin when complexed with sugar ligands. It is now shown that it is possible to grow peanut lectin crystals at acidic pH in the presence of oligopeptides corresponding to a loop in the lectin molecule. Crystals have also been prepared in the presence of these peptides as well as lactose. Low-pH crystal forms of the lectin-lactose complex similar to those obtained at neutral pH have also been grown. Thus, crystals of peanut lectin grown under different environmental conditions, at two pH values with and without sugar bound to the lectin, are now available. They have been used to explore the plasticity and hydration of the molecule. A detailed comparison between different structures shows that the lectin molecule is sturdy and that the effect of changes in pH, ligand binding and environment on it is small. The region involving the curved front beta-sheet and the loops around the second hydrophobic core is comparatively rigid. The back beta-sheet involved in quaternary association, which exhibits considerable variability, is substantially flexible, as is the sugar-binding region. The numbers of invariant water molecules in the hydration shell are small and they are mainly involved in metal coordination or in stabilizing unusual structural features. Small consistent movements occur in the combining site upon sugar binding, although the site is essentially preformed.

The upsurge of multiple-drug-resistant microbes warrants the development and/or use of effective antibiotics. Triclosan, though used in cosmetic and dermatological preparations for several decades, has not been used as a systemic antibacterial agent due to problems of drug administration. Here we report the striking efficacy of triclosan in a mouse model of acute systemic bacterial infection. Triclosan not only significantly extends the survival time of the infected mice, it also restores blood parameters and checks liver damage induced by the bacterial infection. We believe that the excellent safety track record of triclosan in topical use coupled with our findings qualifies triclosan as a candidate drug or lead compound for exploring its potential in experimental systems for treating systemic bacterial infections.

The emergence of drug-resistant forms of Plasmodium falciparum emphasizes the need to develop new antimalarials. In this context, the fatty acid biosynthesis (FAS) pathway of the malarial parasite has recently received a lot of attention. Due to differences in the fatty acid biosynthesis systems of Plasmodium and man, this pathway is a good target for the development of new and selective therapeutic drugs directed against malaria. In continuation of these efforts we report cloning and overexpression of P. falciparum beta-hydroxyacyl-acyl carrier protein (ACP) dehydratase (PffabZ) gene that codes for a 17-kDa protein. The enzyme catalyzes the dehydration of beta-hydroxyacyl-ACP to trans-2-acyl-ACP, the third step in the elongation phase of the FAS cycle. It has a Km of 199 microM and kcat/Km of 80.4 m-1 s-1 for the substrate analog beta-hydroxybutyryl-CoA but utilizes crotonoyl-CoA, the product of the reaction, more efficiently (Km = 86 microM, kcat/Km = 220 m-1 s-1). More importantly, we also identify inhibitors (NAS-91 and NAS-21) for the enzyme. Both the inhibitors prevented the binding of crotonoyl-CoA to PfFabZ in a competitive fashion. Indeed these inhibitors compromised the growth of P. falciparum in cultures and inhibited the parasite fatty acid synthesis pathway both in cell-free extracts as well as in situ. We modeled the structure of PfFabZ using Escherichia coli beta-hydroxydecanoyl thioester dehydratase (EcFabA) as a template. We also modeled the inhibitor complexes of PfFabZ to elucidate the mode of binding of these compounds to FabZ. The discovery of the inhibitors of FabZ, reported for the first time against any member of this family of enzymes, essential to the type II FAS pathway opens up new avenues for treating a number of infectious diseases including malaria.

Thermodynamic parameters associated with the unfolding of the legume lectin, WBA II, were determined by isothermal denaturation. The analysis of isothermal denaturation data provided values for conformational stability and heat capacity for WBA II unfolding. To explore the role of intersubunit contact in stability, we carried out similar studies under identical conditions on Concanavalin A, a legume lectin of nearly similar size, buried hydrophobic surface area and tertiary structure to that of WBA II but with a different oligomerization pattern. Both proteins showed a reversible two-state unfolding with guanidine hydrochloride. As expected, the change in heat capacity upon unfolding was similar for both proteins at 3.5 and 3.7 kcal mol(-1) K(-1) for Concanavalin A and WBA II, respectively. Although the deltaG(H20) at the maximum stability of both proteins is around 16 kcal/mol, Concanavalin A exhibits greater stability at higher temperatures. The T(g) obtained for Concanavalin A and WBA II were 21 degrees C apart at 87.2 and 66.6 degrees C, respectively. The higher conformational stability at higher temperatures and the T(g) of Concanavalin A as compared to that of WBA II are largely due to substantial differences in the degree of subunit contact in these dimeric proteins. Ionic interactions and hydrogen bonding between the monomers of the two proteins also seem to play a significant role in the observed stability differences between these two proteins.