Glycopeptidolipids: Immuno-modulators in greasy mycobacterial cell envelope

Species of opportunistic mycobacteria are the major causative agent for disseminating pulmonary infections in immuno-compromised individuals. These naturally resistant strains recruit a unique type of glycolipid known as glycopeptidolipids (GPLs), noncovalently attached to the outer surface of their thick lipid rich cell envelope. Species specific GPLs constitute the chemical determinants of most nontuberculous mycobacterial serotypes, and their absence from the cell surface confers altered colony morphology, hydrophobicity, and inability to grow as biofilms. The objective of this review is to present a comprehensive account and highlight the renewed interest on this much neglected group of pleiotropic molecules with respect to their structural diversity and biosynthesis. In addition, the role of GPLs in mycobacterial survival, both intracellular and in the environment is also discussed. It also explores the possibility of identifying new targets for intervening Mycobacterium avium complex-related infections. These antigenic molecules have been considered to play a pivotal role in immune suppression and can also induce various cytokine mediated innate immune responses, the molecular mechanism of which remains obscure. (c) 2012 IUBMB IUBMB Life, 2012

Infection of Human Endothelial Cells by Japanese Encephalitis Virus: Increased Expression and Release of Soluble HLA-E

Japanese encephalitis virus (JEV) is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-beta and TNF-alpha production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E) from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP). In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-alpha as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-alpha and IFN-beta as well as the dsRNA analog, poly (I:C). Both IFN-beta and TNF-alpha further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.

Sequestration of the abrin A chain to the nucleus by BASP1 increases the resistance of cells to abrin toxicity

Abrin, a type II ribosome-inactivating protein, comprises A and B subunits wherein the A subunit harbours toxin activity and the B subunit has a galactose-specific lectin activity. The entry of the protein inside the cell is through the binding of the B chain to cell surface glycoproteins followed by receptor-mediated endocytosis and retrograde transport. A previous study from our laboratory showed that different cell lines exhibited differences of as great as similar to 200-fold in abrin toxicity, prompting the present study to compare the trafficking of the toxin within cells. Observations made in this regard revealed that the abrin A chain, after being released into the cytosol, is sequestered into the nucleus through interaction with a cellular protein of similar to 25 kDa, BASP1 (brain acid-soluble protein 1). The nuclear localization of the A chain is seen predominantly in cells that are less sensitive to abrin toxicity and dependent on the levels of BASP1 in cells. The sequestration by BASP1 renders cells increasingly resistant to the inhibition of protein synthesis by abrin and the nucleus act as a sink to overcome cellular stress induced

Stabilizing the Native Trimer of HIV-1 Env by Destabilizing the Heterodimeric Interface of the gp41 Postfusion Six-Helix Bundle

The HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, prefusion trimeric Env exists in a metastable conformation and attains a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives the fusion of viral and cellular membranes. We attempted to stabilize native Env trimers by incorporation of mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The mutations V570D and I573D stabilize native Env of the HIV-1 JRFL strain and occlude nonneutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6-HB. The mutations prevent soluble-CD4 (sCD4)-induced gp120 shedding and 6-HB formation. In the context of cell surface-expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin coexpression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the postfusion 6-HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format. IMPORTANCE The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/coreceptor binding and gp41 in the fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native trimeric Env exists in a metastable conformation. gp41 readily forms a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers that drives fusion of viral and cellular membranes. While it is expected that native Env is a good immunogen, its metastability results in exposure of immunodominant nonneutralizing epitopes. In the present study, we stabilize native Env trimers by incorporation of a number of different mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The stabilized constructs described here can be incorporated into DNA vaccine candidates.

Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells

Productive infection of human endothelial cells with Japanese encephalitis virus (JEV), a single stranded RNA virus induces shedding of sHLA-E. We show here that sHLA-E that is released upon infection with this flavivirus can inhibit IL-2 and PMA mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. Virus infected or IFN-gamma treated cell culture supernatants containing sHLA-E were found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. It was also found that sHLA-E could inhibit IL-2 induced H-3]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit NK cell responses. Hence JEV-induced shedding of sHLA-E needs further investigation to better understand immune responses in JEV infections since it may have a role in viral evasion of NK cell responses. (C) 2014 Elsevier B.V. All rights reserved.

Novel anti IGFBP2 single chain variable fragment inhibits glioma cell migration and invasion

Insulin like growth factor binding protein 2 (IGFBP2) is highly up regulated in glioblastoma (GBM) tissues and has been one of the prognostic indicators. There are compelling evidences suggesting important roles for IGFBP2 in glioma cell proliferation, migration and invasion. Extracellular IGFBP2 through its carboxy terminal arginine glycine aspartate (RGD) motif can bind to cell surface alpha 5 beta 1 integrins and activate pathways downstream to integrin signaling. This IGFBP2 activated integrin signaling is known to play a crucial role in IGFBP2 mediated invasion of glioma cells. Hence a molecular inhibitor of carboxy terminal domain of IGFBP2 which can inhibit IGFBP2-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of IGFBP2, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I (Library size 1.47 x 10(8)) and Tomlinson J (Library size 1.37 x 10(8)) using human recombinant IGFBP2. After screening we obtained three IGFBP2 specific binders out of which one scFv B7J showed better binding to IGFBP2 at its carboxy terminal domain, blocked IGFBP2-cell surface association, reduced activity of matrix metalloprotease 2 in the conditioned medium of glioma cells and inhibited IGFBP2 induced migration and invasion of glioma cells. We demonstrate for the first time that in vitro inhibition of extracellular IGFBP2 activity by using human scFv results in significant reduction of glioma cell migration and invasion. Therefore, the inhibition of IGFBP2 can serve as a potential therapeutic strategy in the management of GBM.

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p) ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel(Msm) and DcpA, encoded by rel(Msm) and dcpA genes, respectively. We have previously shown that the Delta rel(Msm) and Delta dcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p) ppGpp and c-di-GMP in mycobacteria. We have shown that both (p) ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Delta rel(Msm) and Delta dcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p) ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p) ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p) ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Delta rel(Msm) and Delta dcpA strains are defective in biofilm formation. In this work, the overproduction of (p) ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p) ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p) ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.

Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal beta 1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) crossreacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.

Soft-particle model analysis of effect of LPS on electrophoretic softness of Acidithiobacillus ferrooxidans grown in presence of different metal ions

The effect of Surface lipopolysaccharides (LPS) on the electrophoretic softness and fixed charge density in the ion-penetrable layer of Acidithiobacillus ferrooxidans cells grown in presence of copper or arsenic ions have been discussed, The electrophoretic mobility data were analyzed using the soft-particle electrophoresis theory. Cell surface potentials of all the strains based on soft-particle theory were lower than those estimated using the conventional Smoluchowski theory, Exposure to metal ions increased the Surface electrophoretic softness with decrease in the fixed charge density. Effect of cell surface lipopolysaccharides on the model parameters are investigated and discussed.

sopB of Salmonella enterica serovar Typhimurium is a potential DNA vaccine candidate in conjugation with live attenuated bacteria

The immune response against Salmonella is multi-faceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify a protective T cell epitope(s) of Salmonella, as cell mediated immunity conferred by CD8+ T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the genbank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of Salmonella enterica serovar Typhimurium (S. typhimurium) and S. enterica serovar Typhi (S. typhi). They were subjected to BIMAS and SYFPEITHI analysis to map MHC-I and MHC-II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire SopB and SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5-fold on day 4 and day 8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone.

Probing into the role of conserved N-glycosylation sites in the Tyrosinase glycoprotein family

N-linked glycosylation has a profound effect on the proper folding, oligomerization and stability of glycoproteins. These glycans impart many properties to proteins that may be important for their proper functioning, besides having a tendency to exert a chaperone-like effect on them. Certain glycosylation sites in a protein however, are more important than other sites for their function and stability. It has been observed that some N-glycosylation sites are conserved over families of glycoproteins over evolution, one such being the tyrosinase related protein family. The role of these conserved N-glycosylation sites in their trafficking, sorting, stability and activity has been examined here. By scrutinizing the different glycosylation sites on this family of glycoproteins it was inferred that different sites in the same family of polypeptides can perform distinct functions and conserved sites across the paralogues may perform diverse functions.

Virus-Specific Cytolytic Antibodies to Nonstructural Protein 1 of Japanese Encephalitis Virus Effect Reduction of Virus Output from Infected Cells

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.

Proteomics and mass spectrometric studies reveal planktonic growth of Mycobacterium smegmatis in biofilm cultures in the absence of rpoZ

Mycobacterium smegmatis is known to form biofilms and many cell surface molecules like core glycopeptidolipids and short-chain mycolates appear to play important role in the process. However, the involvement of the cell surface molecules in mycobacteria towards complete maturation of biofilms is still not clear. This work demonstrates the importance of the glycopeptidolipid species with hydroxylated alkyl chain and the epoxylated mycolic acids, during the process of biofilm development. In our previous study, we reported the impairment of biofilm formation in rpoZ-deleted M. smegmatis, where rpoZ codes for the ω subunit of RNA polymerase (R. Mathew, R. Mukherjee, R. Balachandar, D. Chatterji, Microbiology 152 (2006) 1741). Here we report the occurrence of planktonic growth in a mc2155 strain which is devoid of rpoZ gene. This strain is deficient in selective incorporation of the hydroxylated glycopeptidolipids and the epoxy mycolates to their respective locations in the cell wall. Hence it forms a mutant biofilm defective in maturation, wherein the cells undertake various alternative metabolic pathways to survive in an environment where oxygen, the terminal electron acceptor, is limiting.

Microbially Induced Mineral Beneficiation

The divergent role of microbes in the field of mineral processing starting from mining and beneficiation to efficient waste disposal has been well recognized now. The roles of various microorganisms and bioreagents in the beneficiation of minerals are illustrated in this paper. Various types of microorganisms useful in bringing about selective flotation and flocculation of various oxide and sulfide minerals are illustrated. Interfacial phenomena governing microbe-mineral interactions are discussed with reference to bacterial cell wall architecture, cell surface hydrophobicity, electrokinetic data, and adsorption behavior on various minerals. Applications of microbially induced mineral beneficiation are demonstrated with respect to beneficiation of iron ores, bauxite, limestone, and complex multimetal sulfides.

Adhesion of Acidithiobacillus ferrooxidans to mineral surfaces

Direct contact mechanism in bioleaching implies prior mineral adhesion of Acidithiobacillus ferrooxidans and subsequent enzymatic attack.Prior bacterial adaptation to sulfide mineral substrates influences bacterial ferrous ion oxidation rates. It is highly beneficial to understand major biooxidation mechanisms with reference to solution- and mineral-grown cells in order to optimize bioleaching reactions. For A. ferrooxidans grown in the presence of solid substrates such as sulfur, pyrite and chalcopyrite, bacterial adhesion is required for its enzymatic machinery to come into close contact for mineral dissolution.But when grown in solution substrate such as ferrous ions and thiosulfate, such an adhesion machinery is not required for substrate utilization. Proteinaceous compounds were observed on the surface of sulfur-grown cells. Such an induction of relatively hydrophobic proteins and down regulation of exposed polysaccharides leads to changes in cell surface chemistry. Sulfur-grown and pyrite- and chalcopyrite-grown bacterial cells were found to be more efficient in the bioleaching of chalcopyrite than those grown in the presence of ferrous ions and thiosulfate. (C) 2010 Elsevier B.V. All rights reserved.