A candidate glycoconjugate vaccine induces protective antibodies in the serum and intestinal secretions, antibody recall response and memory T cells and protects against both typhoidal and non-typhoidal Salmonella serovars.

Human Salmonella infections pose significant public health challenges globally, primarily due to low diagnostic yield of systemic infections, emerging and expanding antibiotic resistance of both the typhoidal and non-typhoidal Salmonella strains and the development of asymptomatic carrier state that functions as a reservoir of infection in the community. The limited long-term efficacy of the currently licensed typhoid vaccines, especially in smaller children and non-availability of vaccines against other Salmonella serovars necessitate active research towards developing a multivalent vaccine with wider coverage of protection against pathogenic Salmonella serovars. We had earlier reported immunogenicity and protective efficacy of a subunit vaccine containing a recombinant outer membrane protein (T2544) of Salmonella Typhi in a mouse model. This was achieved through the robust induction of serum IgG, mucosal secretory IgA and Salmonella-specific cytotoxic T cells as well as memory B and T cell response. Here, we report the development of a glycoconjugate vaccine, containing high molecular weight complexes of Salmonella Typhimurium O-specific polysaccharide (OSP) and recombinant T2544 that conferred simultaneous protection against S. Typhi, S. Paratyphi, S. Typhimurium and cross-protection against S. enteritidis in mice. Our findings corroborate with the published studies that suggested the potential of Salmonella OSP as a vaccine antigen. The role of serum antibodies in vaccine-mediated protection is suggested by rapid seroconversion with high titers of serum IgG and IgA, persistently elevated titers after primary immunization along with a strong antibody recall response with higher avidity serum IgG against both OSP and T2544 and significantly raised SBA titers of both primary and secondary antibodies against different Salmonella serovars. Elevated intestinal secretory IgA and bacterial motility inhibition by the secretory antibodies supported their role as well in vaccine-induced protection. Finally, robust induction of T effector memory response indicates long term efficacy of the candidate vaccine. The above findings coupled with protection of vaccinated animals against multiple clinical isolates confirm the suitability of OSP-rT2544 as a broad-spectrum candidate subunit vaccine against human infection due to typhoidal and non-typhoidal Salmonella serovars.

A Peptide-PNA Hybrid Beacon for Sensitive Detection of Protein Biomarkers in Biological Fluids.

Specific and rapid detection of proteins in biological fluids poses a challenging problem. In biological fluids, many proteins are present at low concentrations, requiring high affinity and specificity of the beacon-protein interaction. We report the design of a peptide-PNA hybrid beacon that exploits the dimeric nature of a target protein, S100B, a biomarker for brain trauma, to enhance binding affinity and specificity. The complementary base-pairing of the PNA bases brings the two arms of the beacon, one carrying an Alexa tag and the other carrying a Dabcyl moiety, into proximity, thus quenching Alexa fluorescence. Each of the arms carries a sequence that binds to one of the subunits. Binding to the target separates the quencher from the probe lifting the quenching of fluorescence. Enhanced affinity and specificity resulting from simultaneously binding to two sites allowed specific detection of S100B at low-nanomolar concentrations in the presence of serum. The design can be easily adapted for the detection of proteins containing multiple binding sites and could prove useful for rapid and sensitive biomarker detection.

Genome scale patterns of supercoiling in a bacterial chromosome.

DNA in bacterial cells primarily exists in a negatively supercoiled state. The extent of supercoiling differs between regions of the chromosome, changes in response to external conditions and regulates gene expression. Here we report the use of trimethylpsoralen intercalation to map the extent of supercoiling across the Escherichia coli chromosome during exponential and stationary growth phases. We find that stationary phase E. coli cells display a gradient of negative supercoiling, with the terminus being more negatively supercoiled than the origin of replication, and that such a gradient is absent in exponentially growing cells. This stationary phase pattern is correlated with the binding of the nucleoid-associated protein HU, and we show that it is lost in an HU deletion strain. We suggest that HU establishes higher supercoiling near the terminus of the chromosome during stationary phase, whereas during exponential growth DNA gyrase and/or transcription equalizes supercoiling across the chromosome.

Simultaneous inhibition of key growth pathways in melanoma cells and tumor regression by a designed bidentate constrained helical peptide.

Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight-binding peptide, TRTK-12. The helical conformation of the peptide was constrained by the substitution of α-amino isobutyric acid--an amino acid having high helical propensity--in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell-penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild-type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development.

A genetic network that balances two outcomes utilizes asymmetric recognition of operator sites.

Stability and induction of the lysogenic state of bacteriophage λ are balanced by a complex regulatory network. A key feature of this network is the mutually exclusive cooperative binding of a repressor dimer (CI) to one of two pairs of binding sites, O(R)1-O(R)2 or O(R)2-O(R)3. The structural features that underpin the mutually exclusive binding mode are not well understood. Recent studies have demonstrated that CI is an asymmetric dimer. The functional importance of the asymmetry is not fully clear. Due to the asymmetric nature of the CI dimer as well as its binding sites, there are two possible bound orientations. By fluorescence resonance energy transfer measurements we showed that CI prefers one bound orientation. We also demonstrated that the relative configuration of the binding sites is important for CI dimer-dimer interactions and consequent cooperative binding. We proposed that the operator configuration dictates the orientations of the bound CI molecules, which in turn dictates CI cooperative interaction between the O(R)1-O(R)2 or O(R)2-O(R)3, but not both. Modeling suggests that the relative orientation of the C- and N-terminal domains may play an important role in the mutually exclusive nature of the cooperative binding. This work correlates unique structural features of a transcription regulatory protein with the functional properties of a gene regulatory network.

Antagonists of Hsp16.3, a low-molecular-weight mycobacterial chaperone and virulence factor, derived from phage-displayed peptide libraries.

The persistence of Mycobacterium tuberculosis is a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.

Ligand specificity and ligand-induced conformational change in gal repressor.

Gal repressor (GalR) binds D-galactose, which is responsible for lifting of repression of the gal operon. Proton T1 measurements of alpha- and beta-anomers of galactose as a function of gal repressor show preferential binding of the beta-anomer. The beta-anomer was isolated by high-performance liquid chromatography and was shown to bind tightly to GalR. Calorimetry was used to determine enthalpy changes at several temperatures. Heat capacity change was found to be positive, indicating that a significant amount of hydrophobic surface area was exposed upon galactose binding. Bis-ANS binding to GalR is significantly enhanced in the presence of a saturating amount of galactose, indicating additional exposure of hydrophobic surfaces. We propose that the galactose-induced conformational change involves the opening of the two subdomains, which may disrupt protein-protein interactions responsible for repression.