Fragment Screening to Identify Inhibitors Targeting Ribosome Binding of Shiga Toxin 2.

Shiga toxins are the main virulence factors of Shiga toxin producing E. coli (STEC) and S. dysenteriae. There is no effective therapy to counter the disease caused by these toxins. The A1 subunits of Shiga toxins bind the C-termini of ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. The ribosome binding site of Shiga toxin 2 has not been targeted by small molecules. We screened a fragment library against the A1 subunit of Shiga toxin 2 (Stx2A1) and identified a fragment, BTB13086, which bound at the ribosome binding site and mimicked the binding mode of the P-stalk proteins. We synthesized analogs of BTB13086 and identified a series of molecules with similar affinity and inhibitory activity. These are the first compounds that bind at the ribosome binding site of Stx2A1 and inhibit activity. These compounds hold great promise for further inhibitor development against STEC infection.

A fluorescence anisotropy-based competition assay to identify inhibitors against ricin and Shiga toxin ribosome interactions.

Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne pathogens. There is no effective therapy against ricin or STEC and there is an urgent need for inhibitors. Ricin toxin A subunit (RTA) and A1 subunit of Stx2a (Stx2A1) bind to the C-terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. Modulation of toxin-ribosome interactions has not been explored as a strategy for inhibition. Therefore, development of assays that detect inhibitors targeting toxin-ribosome interactions remains a critical need. Here we describe a fluorescence anisotropy (FA)-based competitive binding assay using a BODIPY-TMR labeled 11-mer peptide (P11) derived from the P-stalk CTD to measure the binding affinity of peptides ranging from 3 to 11 amino acids for the P-stalk pocket of RTA and Stx2A1. Comparison of the affinity with the surface plasmon resonance (SPR) assay indicated that although the rank order was the same by both methods, the FA assay could differentiate better between peptides that show nonspecific interactions by SPR. The FA assay detects only interactions that compete with the labeled P11 and can validate inhibitor specificity and mechanism of action.

Structure-based design and optimization of a new class of small molecule inhibitors targeting the P-stalk binding pocket of ricin.

Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No effective therapy exists for ricin or Stx intoxication. Ribosome binding sites of the toxins have not been targeted by small molecules. We previously identified CC10501, which inhibits toxin activity by binding the P-stalk pocket of ricin toxin A subunit (RTA) remote from the catalytic site. Here, we developed a fluorescence polarization assay and identified a new class of compounds, which bind P-stalk pocket of RTA with higher affinity and inhibit catalytic activity with submicromolar potency. A lead compound, RU-NT-206, bound P-stalk pocket of RTA with similar affinity as a five-fold larger P-stalk peptide and protected cells against ricin and Stx2 holotoxins for the first time. These results validate the P-stalk binding site of RTA as a critical target for allosteric inhibition of the active site.

Synthesis and antibacterial study of cell-penetrating peptide conjugated trifluoroacetyl and thioacetyl lysine modified peptides.

Substrate-based sirtuin inhibitors target bacterial genome and RNA and provide a promising approach to address bacterial resistance issues, if cellular internalisation can be achieved. We designed N-trifluoroacetyl lysine and N-thioacetyl lysine peptides (KP 13, KP 15 and KP 24) as inhibitors of bacterial sirtuins and their cell-penetrating peptide conjugates Tat KP 13, Tat KP 15 and Tat KP 24. The conjugated peptides were successfully internalised and showed signs of bacterial transcription inhibition resulting in enhanced antibacterial potency against model Gram negative and Gram positive pathogens. Synergistic activity in combination with streptomycin and polymyxin B has also been established. These peptides were effective in inhibiting biofilm formation and eradicating preformed biofilms. Morphological analysis using both SEM and TEM showed bacterial membrane disruption. Calcein dye leakage analysis established the selectivity of these peptides to bacterial membranes. This study documents the first report of the application of substrate-based sirtuin inhibitors as antimicrobial therapeutics.

Identification of genome-wide targets and DNA recognition sequence of the Arabidopsis HMG-box protein AtHMGB15 during cold stress response.

AtHMGB15 belongs to a group of ARID-HMG proteins which are plant specific. The presence of two known DNA binding domains: AT rich interacting domain (ARID) and High Mobility Group (HMG)-box, in one polypeptide, makes this protein intriguing. Although proteins containing individual HMG and ARID domains have been characterized, not much is known about the role of ARID-HMG proteins. Promoter analysis of AtHMGB15 showed the presence of various stress responsive cis regulatory elements along with MADS-box containing transcription factors. Our result shows that the expression of AtHMGB15 increased significantly upon application of cold stress. Using ChIP-chip approach, we have identified 6128 and 4689 significantly enriched loci having AtHMGB15 occupancy under control and cold stressed condition respectively. GO analysis shows genes belonging to abiotic stress response, cold response and root development were AtHMGB15 targets during cold stress. DNA binding and footprinting assays further identified A(A/C)--ATA---(A/T)(A/T) as AtHMGB15 binding motif. The enriched probe distribution in both control and cold condition shows a bias of AtHMGB15 binding towards the transcribed (gene body) region. Further, the expression of cold stress responsive genes decreased in athmgb15 knockout plants compared to wild-type. Taken together, binding enrichment of AtHMGB15 to the promoter and upstream to stress loci suggest an unexplored role of the protein in stress induced transcription regulation.

Evidence of Robustness in a Two-Component System Using a Synthetic Circuit.

Variation in the concentration of biological components is inescapable for any cell. Robustness in any biological circuit acts as a cushion against such variation and enables the cells to produce homogeneous output despite the fluctuation. The two-component system (TCS) with a bifunctional sensor kinase (that possesses both kinase and phosphatase activities) is proposed to be a robust circuit. Few theoretical models explain the robustness of a TCS, although the criteria and extent of robustness by these models differ. Here, we provide experimental evidence to validate the extent of the robustness of a TCS signaling pathway. We have designed a synthetic circuit in Escherichia coli using a representative TCS of Mycobacterium tuberculosis, MprAB, and monitored the in vivo output signal by systematically varying the concentration of either of the components or both. We observed that the output of the TCS is robust if the concentration of MprA is above a threshold value. This observation is further substantiated by two in vitro assays, in which we estimated the phosphorylated MprA pool or MprA-dependent transcription yield by varying either of the components of the TCS. This synthetic circuit could be used as a model system to analyze the relationship among different components of gene regulatory networks.IMPORTANCE Robustness in essential biological circuits is an important feature of the living organism. A few pieces of evidence support the existence of robustness in vivo in the two-component system (TCS) with a bifunctional sensor kinase (SK). The assays were done under physiological conditions in which the SK was much lower than the response regulator (RR). Here, using a synthetic circuit, we varied the concentrations of the SK and RR of a representative TCS to monitor output robustness in vivo. In vitro assays were also performed under conditions where the concentration of the SK was greater than that of the RR. Our results demonstrate the extent of output robustness in the TCS signaling pathway with respect to the concentrations of the two components.

Enhanced Silkworm Cecropin B Antimicrobial Activity against Pseudomonas aeruginosa from Single Amino Acid Variation.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen causing severe infections in hospitalized and immunosuppressed patients, particularly individuals affected by cystic fibrosis. Several clinically isolated P. aeruginosa strains were found to be resistant to three or more antimicrobial classes indicating the importance of identifying new antimicrobials active against this pathogen. Here, we characterized the antimicrobial activity and the action mechanisms against P. aeruginosa of two natural isoforms of the antimicrobial peptide cecropin B, both isolated from the silkworm Bombyx mori. These cecropin B isoforms differ in a single amino acid substitution within the active portion of the peptide, so that the glutamic acid of the E53 CecB variant is replaced by a glutamine in the Q53 CecB isoform. Both peptides showed a high antimicrobial and membranolytic activity against P. aeruginosa, with Q53 CecB displaying greater activity compared with the E53 CecB isoform. Biophysical analyses, live-cell NMR, and molecular-dynamic-simulation studies indicated that both peptides might act as membrane-interacting elements, which can disrupt outer-membrane organization, facilitating their translocation toward the inner membrane of the bacterial cell. Our data also suggest that the amino acid variation of the Q53 CecB isoform represents a critical factor in stabilizing the hydrophobic segment that interacts with the bacterial membrane, determining the highest antimicrobial activity of the whole peptide. Its high stability to pH and temperature variations, tolerance to high salt concentrations, and low toxicity against human cells make Q53 CecB a promising candidate in the development of CecB-derived compounds against P. aeruginosa.

A transcriptomic analysis of the mycobacteriophage D29 genome reveals the presence of novel stoperator-associated promoters in its right arm.

Mycobacteriophage D29 is a lytic phage that infects various species of Mycobacterium including M. tuberculosis. Its genome has 77 genes distributed almost evenly between two converging operons designated as left and right. Transcription of the phage genome is negatively regulated by multiple copies of an operator-like element known as stoperator that acts by binding the phage repressor Gp71. The function of the D29 genes and their expression status are poorly understood and therefore we undertook a transcriptome analysis approach to address these issues. The results indicate that the average transcript intensity of the right arm genes was higher than of those on the left, at the early stage of infection. Moreover, the fold increase from early to the late stage was found to be less for the right arm genes than for the left. Both observations support the prediction that the right arm genes are expressed early whereas the left arm ones are expressed late. The analysis further revealed a break in the continuity of the right arm operon between 89, the first gene in it, and 88, the next. Gene 88 was found to be expressed from a newly identified promoter located between 88 and 89. Another new promoter was found upstream of 89. Thus, the promoter Pleft, identified earlier, is not the only one that drives expression of the right arm genes. All these promoters overlap with stoperators, with which they share a conserved sequence motif, TTGACA, commonly known as the -35 promoter element. We demonstrate mutually exclusive binding of RNA polymerase and Gp71 to the stoperator-promoters and conclude that stoperators can function as -35 promoter elements and that they can control gene expression not only negatively as was believed earlier but in many cases positively as well.

Mode of Action of a Designed Antimicrobial Peptide: High Potency against Cryptococcus neoformans.

There is a significant need for developing compounds that kill Cryptococcus neoformans, the fungal pathogen that causes meningoencephalitis in immunocompromised individuals. Here, we report the mode of action of a designed antifungal peptide, VG16KRKP (VARGWKRKCPLFGKGG) against C. neoformans. It is shown that VG16KRKP kills fungal cells mainly through membrane compromise leading to efflux of ions and cell metabolites. Intracellular localization, inhibition of in vitro transcription, and DNA binding suggest a secondary mode of action for the peptide, hinting at possible intracellular targets. Atomistic structure of the peptide determined by NMR experiments on live C. neoformans cells reveals an amphipathic arrangement stabilized by hydrophobic interactions among A2, W5, and F12, a conventional folding pattern also known to play a major role in peptide-mediated Gram-negative bacterial killing, revealing the importance of this motif. These structural details in the context of live cell provide valuable insights into the design of potent peptides for effective treatment of human and plant fungal infections.

Deciphering the role of the AT-rich interaction domain and the HMG-box domain of ARID-HMG proteins of Arabidopsis thaliana.

ARID-HMG DNA-binding proteins represent a novel group of HMG-box containing protein family where the AT-rich interaction domain (ARID) is fused with the HMG-box domain in a single polypeptide chain. ARID-HMG proteins are highly plant specific with homologs found both in flowering plants as well as in moss such as Physcomitrella. The expression of these proteins is ubiquitous in plant tissues and primarily localises in the cell nucleus. HMGB proteins are involved in several nuclear processes, but the role of ARID-HMG proteins in plants remains poorly explored. Here, we performed DNA-protein interaction studies with Arabidopsis ARID-HMG protein HMGB11 (At1g55650) to understand the functionality of this protein and its individual domains. DNA binding assays revealed that AtHMGB11 can bind double-stranded DNA with a weaker affinity (Kd = 475 ± 17.9 nM) compared to Arabidopsis HMGB1 protein (Kd = 39.8 ± 2.68 nM). AtHMGB11 also prefers AT-rich DNA as a substrate and shows structural bias for supercoiled DNA. Molecular docking of the DNA-AtHMGB11 complex indicated that the protein interacts with the DNA major groove, mainly through its ARID domain and the junction region connecting the ARID and the HMG-box domain. Also, predicted by the docking model, mutation of Lys(85) from the ARID domain and Arg(199) & Lys(202) from the junction region affects the DNA binding affinity of AtHMGB11. In addition, AtHMGB11 and its truncated form containing the HMG-box domain can not only promote DNA mini-circle formation but are also capable of inducing negative supercoils into relaxed plasmid DNA suggesting the involvement of this protein in several nuclear events. Overall, the study signifies that both the ARID and the HMG-box domain contribute to the optimal functioning of ARID-HMG protein in vivo.