Non-invasive vaccine delivery in transfersomes, niosomes and liposomes: a comparative study.

Non-invasive vaccine delivery is a top priority for public health agencies because conventional immunization practices are unsafe and associated with numerous limitations. Recently, the skin has emerged as a potential alternative route for non-invasive delivery of vaccine. Topical immunization (TI), introduction of antigen through topical application onto the intact skin, has many practical merits compared to injectable routes of administration. One of the possibilities for increasing the penetration of bioactives through the skin is the use of vesicular systems. Specially designed lipid vesicles are attracting intense attention and can be used for non-invasive antigen delivery. In the present study, elastic vesicle transfersomes, non-ionic surfactant vesicles (niosomes) and liposomes were used to study their relative potential in non-invasive delivery of tetanus toxoid (TT). Transfersomes, niosomes and liposomes were prepared and characterized for shape, size and entrapment efficiency. These vesicles were extruded through polycarbonate filter (50-nm pore size) to assess the elasticity of the vesicles. The immune stimulating activity of transfersomes, niosomes and liposomes were studied by measuring the serum anti-TT IgG titre following topical immunization. The immune response elicited by topical immunization was compared with that elicited by same dose of alum-adsorbed tetanus toxoid (AATT) given intramuscularly. The results indicate that optimal formulations of transfersomes, niosomes and liposomes could entrap 72.7+/-3.4, 42.5+/-2.4 and 41.3+/-2.2% of antigen and their elasticity values were 124.4+/-4.2, 29.3+/-2.4 and 21.7+/-1.9, respectively. In vivo study revealed that topically given TT containing transfersomes, after secondary immunization, could elicit immune response (anti-TT-IgG) that was equivalent to one that produced following intramuscularly alum-adsorbed TT-based immunization. In comparison to transfersomes, niosomes and liposomes elicited weaker immune response. Thus transfersomes hold promise for effective non-invasive topical delivery of antigen(s).

Escherichia coli RNA polymerase subunit omega and its N-terminal domain bind full-length beta' to facilitate incorporation into the alpha2beta subassembly.

The omega subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (alpha2betabeta'). The presence of omega is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an omega-less strain of E. coli, GroEL appears to substitute for omega in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch on to beta' at its N-terminus and C-terminus. We show here that omega binds only the intact beta' subunit and not the beta' N-terminal domain or beta' C-terminal domain, implying that omega binding requires both these regions of beta'. We further show that omega can prevent the aggregation of beta' during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of omega is sufficient for binding and renaturation of beta'. CD and functional assays show that this N-terminal fragment retains the structure of native omega and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps alpha + alpha --> alpha2 + beta --> alpha2beta + beta' --> alpha2betabeta'. It is shown here that omega participates during the last stage of enzyme assembly when beta' associates with the alpha2beta subassembly.

Revisiting the stringent response, ppGpp and starvation signaling.

Microbial adaptation to environmental stress plays an important role in survival. It is necessary to understand the mechanisms underlying the survival of microbes under stress, as they may eventually aid in the successful control of the growth and persistence of these organisms. During nutrient starvation, Escherichia coli elicits a stringent response to conserve energy. The hallmark of the stringent response is the accumulation of guanosine tetra- (ppGpp) and pentaphosphates (pppGpp), which probably bind RNA polymerase to regulate gene expression at certain promoters. Recently, there has been renewed interest in the stringent responses of other microbes, with a view to correlating it with sporulation, virulence and long-term persistence.

Stabilizing interactions in the dimer interface of alpha-subunit in Escherichia coli RNA polymerase: a graph spectral and point mutation study.

The formation of alpha(2) dimer in Escherichia coli core RNA polymerase (RNAP) is thought to be the first step toward the assembly of the functional enzyme. A large number of evidences indicate that the alpha-subunit dimerizes through its N-terminal domain (NTD). The crystal structures of the alpha-subunit NTD and that of a homologous Thermus aquaticus core RNAP are known. To identify the stabilizing interactions in the dimer interface of the alpha-NTD of E. coli RNAP, we identified side-chain clusters by using the crystal structure coordinates of E. coli alpha-NTD. A graph spectral algorithm was used to identify side-chain clusters. This algorithm considers the global nonbonded side-chain interactions of the residues for the clustering procedure and is unique in identifying residues that make the largest number of interactions among the residues that form clusters in a very quantitative way. By using this algorithm, a nine-residue cluster consisting of polar and hydrophobic residues was identified in the subunit interface adjacent to the hydrophobic core. The residues forming the cluster are relatively rigid regions of the interface, as measured by the thermal factors of the residues. Most of the cluster residues in the E. coli enzyme were topologically and sequentially conserved in the T. aquaticus RNAP crystal structure. Residues 35F and 46I were predicted to be important in the stability of the alpha-dimer interface, with 35F forming the center of the cluster. The predictions were tested by isolating single-point mutants alpha-F35A and alpha-I46S on the dimer interface, which were found to disrupt dimerization. Thus, the identified cluster at the edge of the dimer interface seems to be a vital component in stabilizing the alpha-NTD.

Functional complementation between mutations at two distant positions in Escherichia coli RNA polymerase as revealed by second-site suppression.

Subunit-subunit interactions are critical for the assembly of the core of Escherichia coli RNA polymerase. The mutant alpha-subunit C131A is unable to complement the temperature-sensitive alpha-R45C mutant strain, which is defective for binding of the beta-subunit. In vitro reconstitution experiments, however, indicate that the alpha-C131A variant is able to form the intermediate alpha2beta, but is defective in contacting the beta'-subunit. We used this alpha-C131A mutant to isolate a suppressor mutation in the beta'-subunit. Genetic and biochemical characterization of the beta' suppressor indicates the allele-specific nature of its effect. Sequence analysis of the suppressor revealed a single substitution of Gly at position 333, an evolutionarily conserved position in the conserved region C of the beta'-subunit, by Asp. However, the crystal structure of the bacterial RNA polymerase indicates that the primary mutation (alpha-C131A) and its suppressor lie far apart. Thus, we propose that long-range interactions, as in this case, may play an important role in the functional assembly of E. coli RNA polymerase.

High intracellular level of guanosine tetraphosphate in Mycobacterium smegmatis changes the morphology of the bacterium.

Almost one-third of the world population today harbors the tubercle bacillus asymptomatically. It is postulated that the morphology and staining pattern of the long-term persistors are different from those of actively growing culture. Interestingly, it has been found that the morphology and staining pattern of the starved in vitro population of mycobacteria is similar to the persistors obtained from the lung lesions. In order to delineate the biochemical characteristics of starved mycobacteria, Mycobacteria smegmatis was grown in 0.2% glucose as a sole carbon source along with an enriched culture in 2% glucose. Accumulation of the stringent factor guanosine tetraphosphate (ppGpp) with a concomitant change in morphology was observed for M. smegmatis under carbon-deprived conditions. In addition, M. smegmatis assumed a coccoid morphology when ppGpp was ectopically produced by overexpressing Escherichia coli relA, even in an enriched medium. The Mycobacterium tuberculosis relA and spoT homologue, when induced in M. smegmatis, also resulted in the overproduction of ppGpp with a change in the bacterium's growth characteristics.

GroEL is involved in activation of Escherichia coli RNA polymerase devoid of the omega subunit in vivo.

Highly purified Escherichia coli RNA polymerase contains a small subunit termed omega that has a molecular mass of 10 105 Da and is comprised of 91 amino acids. E. coli strains lacking omega (omega-less) are viable, but exhibit a slow-growth phenotype. Renaturation of RNA polymerase isolated from an omega-less mutant, in the presence of omega, resulted in maximum recovery of activity. The omega-less RNA polymerase from omega-less strains recruits the chaperonin, GroEL (unlike the wild-type enzyme), suggesting a structural deformity of the mutant enzyme. The GroEL-containing core RNA polymerase interacts efficiently with sigma70 to generate the fully functional holoenzyme. However, when GroEL was removed, the enzyme was irreversibly nonfunctional and was unable to bind to sigma70. The damaged enzyme regained activity after going through a cycle of denaturation and reconstitution in the presence of omega or GroEL. GroES was found to have an inhibitory effect on the core-sigma70 association unlike the omega subunit. The omega subunit may therefore be needed for stabilization of the structure of RNA polymerase.

Detection of putative Zn(II) binding sites within Escherichia coli RNA polymerase: inconsistency between sequence-based prediction and 65Zn blotting.

The availability of repeating 'Cys' and/or 'His' units in a particular order prompts the prediction of Zn(II) finger motifs in a protein. Escherichia coli RNA polymerase has two tightly bound Zn(II) per molecule of the enzyme as detected by atomic absorption spectroscopy. One Zn(II) was identified to be at the beta subunit, whereas the other putative Zn(II) binding site has recently been predicted to be at the N-terminal half of the beta' subunit, from primary sequence analysis. We show here that the beta' subunit has no ability to bind 65Zn(II). On the other hand, the N-terminal domain of the alpha subunit has strong Zn(II) binding ability with no obvious functional implications.

DNA-dependent RNA polymerase II from Candida species is a multiple zinc-containing metalloenzyme.

We have purified DNA-dependent RNA polymerase II from Candida albicans, a human pathogenic yeast. The enzyme consists of 9 polypeptides that are unique to C. albicans, their mobility on SDS-PAGE being different from the mobility of the corresponding subunits of RNA polymerase II from Saccharomyces cerevisiae or C. utilis. In the present study we also demonstrate that RNA pol II from C. albican and C. utilis are metalloproteins containing approximately 5 mol of zinc per mole of enzyme. Although prolonged dialysis in 10 or 20 mM EDTA failed to remove Zn(II) from the C. albicans enzyme, in the C. utilis enzyme 3 Zn(II) ions could be removed and then reconstituted in the presence of excess Zn(II). o-Phenanthroline (5 mM) removed Zn(II) from C. albicans enzyme irreversibly in a time-dependent fashion with concomitant loss of enzyme activity. Circular dichroism studies revealed structural changes on removal of zinc, thus suggesting a role for Zn in maintenance of structural stability. Further, we demonstrate that the largest subunit of the C. utilis enzyme and the 3 large subunits of the C. albicans enzyme can bind radioactive zinc.

The differential effects of guanosine tetraphosphate on open complex formation at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1.

The effects of guanosine tetraphosphate (ppGpp) on inhibition of single-round in vitro transcription and on the kinetics of open complex formation were investigated at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1. The two promoters differ in their saturation characteristics and sensitivities to ppGpp. With a 10:1 molar ratio of RNA polymerase (RNAP) to DNA, saturation of transcription activity and weak inhibition (approximately 30%) are observed at rplJ, in contrast to the weak activity and strong inhibition (approximately 80%) at rpsA P1. In the absence of ppGpp, the two promoters show a threefold difference in the overall rate constants of association (ka) (6.5 x 10(7) M-1 s-1 at rplJ and 2.0 x 10(7) M-1 s-1 at rpsA P1), while the dissociation rate constants (kd) are similar (approximately 4.8 x 10(-5) s-1). The addition of ppGpp causes a twofold reduction in k2 (isomerisation constant) rplJ and a threefold decrease in KB (equilibrium constant of RNAP binding) at rpsA P1. There is a significant twofold increase in kd at rplJ, compared with smaller changes at rpsA P1 and at the non-stringent lacUV5 promoter. These results indicate that ppGpp affects the formation and stability of the open complex at the rplJ promoter, in contrast to the inhibition of RNAP binding to the rpsA P1 promoter.

Guanosine tetraphosphate-induced dissociation of open complexes at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1: nanosecond depolarization spectroscopic studies.

We have measured the fluorescence anisotropy decays of various transcription complexes formed between Escherichia coli RNA polymerase (RNAP) and the rplJ, rpsA P1 and lacUV5 promoters, where the sigma 70-subunit of RNAP is covalently labeled with the fluorescent probe 1,5-IAEDANS. The observed changes in the rotational correlation times (phi r) of the sigma 70-bound probe upon ppGpp or NTP addition to preformed open complexes, were used to directly infer the extent of association of the sigma-subunit with these transcription complexes. At the rplJ and rpsA P1 promoters, the addition of ppGpp (in the absence of heparin and nucleotides), results in the dissociation of RNAP from the binary complex. This is either accompanied by, or leads to the dissociation of a fraction of the holoenzyme-bound sigma 70. At the lacUV5 promoter, only a marginal dissociation of RNAP is observed. We propose a model where two types of ppGpp-bound RNAP interact with the ribosomal protein promoters. One is transcription-competent and releases sigma 70 upon elongation, while the other dissociates from the open complex. A fraction of the latter species releases the sigma 70 subunit and is unable to form a transcription-competent holoenzyme. Our data supports the mechanism of open complex-destabilization at stringent promoters by ppGpp.

The mediator for stringent control, ppGpp, binds to the beta-subunit of Escherichia coli RNA polymerase.

BACKGROUND: Inhibition of transcription of rRNA in Escherichia coli upon amino acid starvation is thought to be due to the binding of ppGpp to RNA polymerase. However, the nature of this interaction still remains obscure. RESULTS: Here, the azido-derivative of ppGpp was synthesized from azido-GDP and [gamma-32P]ATP by way of the phosphate transfer reaction of the RelA enzyme. The product was subsequently characterized by one and two-dimensional chromatography. The resulting compound [32P]azido-ppGpp, where the azido group is attached to the base moiety, was purified to homogeneity and was photo-crosslinked to Escherichia coli RNA polymerase. SDS-PAGE analysis of the azido-ppGpp-bound enzyme, tryptic digestion and Western blot analysis suggested that azido-ppGpp binds to the beta-subunit of RNA polymerase. CONCLUSION: It was observed that both the N-terminal and C-terminal domains of the beta-subunit were labelled with azido-ppGpp in the native enzyme. However, under denaturing conditions only the C-terminal part from amino acid residue 802 to residue 1211/1216/1223 was predominantly crosslinked to azido-ppGpp. The excess of unlabelled ppGpp competes with azido-ppGpp for binding to the enzyme. azido-ppGpp inhibits single-round transcription at the stringent promoter like rrnBP1. In addition, ribosomal protein genes were also found to be inhibited by N3ppGpp. On the other hand, transcription at the lac UV5 promoter remained unaffected upon the addition of azido-ppGpp.

Studies on the omega subunit of Escherichia coli RNA polymerase--its role in the recovery of denatured enzyme activity.

Highly purified Escherichia coli RNA polymerase contains a small subunit termed omega that has a molecular mass of 10,105 Da and is comprised of 91 amino acids. To elucidate the function of omega, whose role is as yet undefined, the subunit was purified to over 95% purity from an overproducing strain [BL21 (pGP1-2, pE3C-2)]. Purified omega was then reconstituted with RNA polymerase isolated from an omega-less mutant. Externally added omega inhibited promoter-specific transcriptional activity at all promoters tested. Renaturation of fully denatured omega-less RNA polymerase in the presence of excess omega yielded maximum recovery of activity suggesting a structural rather than functional role for omega.

A useful method for the synthesis of 8 azido-guanosine tri- and tetraphosphate: important substrates for many enzymatic reactions.

8-N3 GMP was synthesised from 8-BrGMP by the addition of LiN3. 8-N3GMP was then phosphorylated to N3-GDP and N3-GTP by controlled enzymatic reaction. 8-N3GDP can be converted to N3-ppGpp with crude Rel A, which phosphorylates the 3'-OH of GDP.

Interaction of bacteriophage T4 AsiA protein with Escherichia coli sigma70 and its variant.

Bacteriophage T4 produces a small protein AsiA, which inhibits transcription from sigma70-dependent promoters in E. coli by tightly binding to sigma70 and is therefore termed as anti-sigma factor. We observed that there was no inhibition of single round transcription at lac UV5 promoter when AsiA was added to preformed open complex between RNA polymerase and template DNA. However, transcription was found to proceed normally at 'extended -10' promoters in the presence of AsiA. It appears therefore that AsiA binds sigma70 at its 4.2-subdomain or in its close vicinity. Further experiments on immunoprecipitation of sigma70 and a mutant sigma70-V576G with AsiA seem to corroborate such conclusion.

The presence of two tightly bound Zn2+ ions is essential for the structural and functional integrity of yeast RNA polymerase II.

DNA-dependent RNA polymerases (RNApol) are Zn2+ metalloproteins where the Zn2+ ion plays both catalytic and structural roles. Although the ubiquitous presence of Zn2+ with the RNApol from eukaryotes had already been established, the exact stoichiometry of Zn2+ ion(s) per mole enzyme is not well documented, and its role in enzymatic function remains elusive. We show here that RNApolII from Saccharomyces cerevisiae has two Zn2+ ions tightly associated with it which are necessary for its transcriptional activity. Upon prolonged dialysis against 10 mM EDTA for 4-5 h, the enzyme loses one Zn2+, as well as partial activity. However, Zn2+ can be added back to the enzyme, but without recovering its total activity. 5 mM orthophenanthroline (OP) removes one Zn2+ within 2 h; the enzyme, however, cannot be reconstituted back with Zn2+. Circular dichroism (CD) studies showed that the conformation of the native enzyme is unique and cannot be reproduced with Zn2+-reconstituted RNApolII. Similarly, the rate of abortive synthesis of a dinucleotide product over a non-specific template is faster when catalyzed by two Zn2+-native enzymes. Zn2+-reconstituted RNApolII or one Zn2+-RNApolII showed a slower abortive synthesis rate. 65Zn2+-blotting experiments indicated that the removal of one Zn2+ from the enzyme destroys the Zn2+-binding ability of the larger subunits of yeast RNApolII. In order to check whether the presence of Zn2+ ions has any effect on substrate recognition, we followed the binding of (gamma-AmNS)UTP, a fluorescent substrate analog to RNApolII. It was observed that OP-treated enzyme showed non-specific substrate recognition, whereas two Zn2+-native RNApol binds substrate at a single site.

Mutations in the 1.1 subdomain of Escherichia coli sigma factor sigma70 and disruption of its overall structure.

Among various group I sigma factors, two amino acids, Val55 and Ala59 are the conserved amino acids in the 1.1 hydrophobic subdomain. These two sites have been mutated to generate variants designated as [Gly55]sigma70 and [Gly59]sigma70, where glycine replaces valine and alanine, respectively. The function of these sigma mutants is reported here. The molecular mass of these proteins determined on denaturing gels was 70 kDa, which is the expected calculated molecular mass; wild-type sigma70 has an apparent molecular mass of 87 kDa. However, [Gly434]sigma70, which contains a mutation at the DNA-binding rpoD box region, also migrates as a 70-kDa protein on SDS/PAGE. Circular dichroism spectral analysis indicated that both [Gly55]sigma70 and [Gly59]sigma70 have reduced helicity (20%) compared to wild-type sigma70 (50%). Binding of sigma factors with the hydrophobic, surface active probe 1-anilinonapthalene-8-sulphonate, has shown that more hydrophobic surfaces are available/exposed in [Gly55]sigma70, [Gly59]sigma70 as well as in [Gly434]sigma70 in comparison to wild-type sigma70. Time-resolved emission spectroscopic studies have suggested transient binding between these mutants and DNA. The different holoenzyme RNA polymerases generated upon reconstituting these mutants independently with core RNA polymerase (alpha2beta beta') have shown reduced transcriptional activity in comparison to the enzyme containing wild-type sigma factor. However, another mutation (Val-->Gly) in the hydrophobic subdomain 1.2 at position 83, which is designated as [Gly83]sigma70, has similar properties as the wild-type with respect to its mobility on denaturing gels, circular dichroism profile, and transcriptional activity when reconstituted with core RNA polymerase. It appears that the 1.1 subdomain in sigma70 may interact hydrophobically with the 2.3/2.4 DNA-binding region.

Recognition of promoter DNA by subdomain 4.2 of Escherichia coli sigma 70: a knowledge based model of -35 hexamer interaction with 4.2 helix-turn-helix motif.

In Escherichia coli, subdomains 2.4 and 4.2 of the primary transcription factor sigma 70 are the most highly conserved regions and are responsible for the recognition of -10 and -35 promoter elements respectively. Mutational studies provide evidence to this end and indicate that the side chains of subdomain 4.2 make specific contacts with the nucleotides at -35. Subdomain 4.2 is highly conserved among group-1 sigma factors and is strongly homologous to the classical helix-turn-helix (HTH) motif shared by bacteriophage lembda cl, Cro, the CAP protein and other homeodomain proteins, suggesting that sigma factor also belongs to the HTH class of proteins. In this study, a single point mutation of the conserved hydrophobic residue valine at position 576, in the 4.2 subdomain results in a mutant that is transcriptionally inefficient although conformationally similar to wild-type sigma. The mutant sigma, like wild-type, migrates as a 87 kDa protein on SDS gels and has 50% helicity. However, transcription at "extended -10 promoter' by RNA polymerase containing mutant sigma 70-V576G, synthesized appreciable amount of RNA product, when compared with that generated by sigma 70-W434G, a mutation in -10 DNA binding domain. A model of HTH motif for the conserved 20 residue region of 4.2 domain of E. coli sigma 70 as well as its mutant sigma 70-V576G and sigma 70-V576T were constructed based on five other homologous HTH motifs from DNA-protein complexes for which X-ray or NMR structure is available. A B-DNA structure was designed for -35 region using sequence dependent base pair parameters. The modeled HTH structure was docked into the major groove formed by the -35 hexamer DNA using the DNA-recognition rules and amino acid-nucleotide base contact information of homologous DNA-protein complexes. Analysis of the residue contact information of the model was tested and found to have good agreement with the experimental reports.

Time-resolved emission spectroscopy as a tool to follow nucleic acid-protein interaction.

Fluorescence spectroscopy is undoubtedly a useful tool to study the structural and functional aspects of nucleic acids-protein interactions as well as the catalytic functions of particular residues of multi-subunit enzyme complexes. The dynamic interaction of nucleic acids and proteins occurring at nanosecond time scale can now be monitored by making life-time measurements or by time-resolved emission spectroscopy. These measurements are made by exploiting the intrinsic fluorescent residues in proteins i.e. W or by the use of extrinsic fluorophores which are tagged on to particular residues and that are sensitive to the microenvironment changes. In this study we describe the use of time resolved emission spectroscopy to (a) analyse the transient binding between sigma 70 and DNA by monitoring the quenching of W residues and (b) monitor the various states which nucleosomes of active, inducible or inactive chromatin may adopt in vivo.