Twin-arginine Translocation Research Articles

Far-reaching cellular consequences of tat deletion in Escherichia coli revealed by comprehensive proteome analyses

In Escherichia coli, the Twin-arginine translocation (Tat) pathway secretes a set of folded proteins with important physiological functions to the periplasm and outer membrane. The loss of Tat secretion impairs outer membrane integrity and leads to decreased cell growth. Only recently, the Tat pathway has gained more attention due to its essential role in bacterial virulence and applications in the production of fully folded heterologous proteins. In this study, we investigated the influence of the deletion of all active Tat pathway components on the E. coli cells. The comprehensive proteomic analysis revealed activation of several stress responses and experimentally confirmed the dependence of certain proteins on the Tat system for export. We observed that a tat deletion triggers protein aggregation, membrane vesiculation, synthesis of colanic acid and biofilm formation. Furthermore, the mislocalization of Tat-dependent proteins disturbs iron and molybdenum homeostasis and impairs the cell envelope integrity. The results show that the functional Tat pathway is important for the physiological stability and that its dysfunction leads to a series of severe changes in E. coli cells.

Tat-exported peptidoglycan amidase-dependent cell division contributes to Salmonella Typhimurium fitness in the inflamed gut.

Salmonella enterica serovar Typhimurium (S. Tm) is a cause of food poisoning accompanied with gut inflammation. Although mucosal inflammation is generally thought to be protective against bacterial infection, S. Tm exploits the inflammation to compete with commensal microbiota, thereby growing up to high densities in the gut lumen and colonizing the gut continuously at high levels. However, the molecular mechanisms underlying the beneficial effect of gut inflammation on S. Tm competitive growth are poorly understood. Notably, the twin-arginine translocation (Tat) system, which enables the transport of folded proteins outside bacterial cytoplasm, is well conserved among many bacterial pathogens, with Tat substrates including virulence factors and virulence-associated proteins. Here, we show that Tat and Tat-exported peptidoglycan amidase, AmiA- and AmiC-dependent cell division contributes to S. Tm competitive fitness advantage in the inflamed gut. S. Tm tatC or amiA amiC mutants feature a gut colonization defect, wherein they display a chain form of cells. The chains are attributable to a cell division defect of these mutants and occur in inflamed but not in normal gut. We demonstrate that attenuated resistance to bile acids confers the colonization defect on the S. Tm amiA amiC mutant. In particular, S. Tm cell chains are highly sensitive to bile acids as compared to single or paired cells. Furthermore, we show that growth media containing high concentrations of NaCl and sublethal concentrations of antimicrobial peptides induce the S. Tm amiA amiC mutant chain form, suggesting that gut luminal conditions such as high osmolarity and the presence of antimicrobial peptides impose AmiA- and AmiC-dependent cell division on S. Tm. Together, our data indicate that Tat and the Tat-exported amidases, AmiA and AmiC, are required for S. Tm luminal fitness in the inflamed gut, suggesting that these proteins might comprise effective targets for novel antibacterial agents against infectious diarrhea.

Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates

In bacteria, the twin-arginine translocation (Tat) pathway allows the export of folded proteins through the inner membrane. Proteins targeted to this system are synthesized with N-terminal signal peptides bearing a conserved twin-arginine motif. The Tat pathway is critical for many bacterial processes including pathogenesis and virulence. However, the full set of Tat substrates is unknown in many bacteria, and the reliability of in silico prediction methods largely uncertain. In this work, we performed a combination of in silico analysis and experimental validation to identify a core set of Tat substrates in the opportunistic pathogen Pseudomonas aeruginosa. In silico analysis predicted 44 putative Tat signal peptides in the P. aeruginosa PA14 proteome. We developed an improved amidase-based Tat reporter assay to show that 33 of these are real Tat signal peptides. In addition, in silico analysis of the full translated genome revealed a Tat candidate with a missassigned start codon. We showed that it is a new periplasmic protein in P. aeruginosa. Altogether we discovered and validated 34 Tat substrates. These show little overlap with Escherichia coli Tat substrates, and functional analysis points to a general role for the P. aeruginosa Tat system in the colonization of environmental niches and pathogenicity.

Tat system is required for the virulence of Dickeya zeae on rice plants

The Gram-negative pathogenic bacterium Dickeya zeae causes soft rot in rice, corn and many other plants. The molecular mechanisms underlying D. zeae virulence are not clearly understood. In this study, we characterized the twin-arginine translocation (Tat) system of this pathogen. The mutants of tatA, tatB, tatC and tatE of D. zeae EC1 were generated through the use of the suicide vector pKNG101 and their phenotypic characters were determined. Our results showed that mutations in D. zeae Tat system have no effect on the bacterial growth. △tatA, △tatB and △tatE mutants reduced the motility distinctly, and could not form biofilm, while the △tatC mutant did not show a distinctive decrease on the motility and biofilm formation. Less production of proteases, pectate lyases and cellulases were shown in △tatA, △tatB, △tatC and △tatE mutants as compared with the wild type EC1, whereas the toxin in Tat mutants was reduced, as compared with the wild type. The Tat mutants △tatA, △tatB, △tatC and △tatE could cause soft rot in potato and Chinese cabbage and hypersensitive reactions (HR) in tobacco, that were not different significantly from those of the wild type EC1. However, △tatA, △tatB and △tatE were not significantly effective in the inhibition of rice seed germination compared to the wild type. Furthermore, the disease incidence of the Tat mutants decreased significantly when they were inoculated in rice plants. This reveals that the Tat system has a different role in the virulence of D. zeae in rice, potato and cabbage and it is required for the virulence of D. zeae in rice plants.

The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N-oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC.

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

The twin-arginine translocation (Tat) system that comprises the TatA, TatB, and TatC components transports folded proteins across energized membranes of prokaryotes and plant plastids. It is not known, however, how the transport of this protein cargo is achieved. Favored models suggest that the TatA component supports transport by weakening the membrane upon full translocon assembly. Using Escherichia coli as a model organism, we now demonstrate in vivo that the N terminus of TatA can indeed destabilize the membrane, resulting in a lowered membrane energization in growing cells. We found that in full-length TatA, this effect is counterbalanced by its amphipathic helix. Consistent with these observations, the TatA N terminus induced proton leakage in vitro, indicating membrane destabilization. Fluorescence quenching data revealed that substrate binding causes the TatA hinge region and the N-terminal part of the TatA amphipathic helix to move toward the membrane surface. In the presence of TatBC, substrate binding also reduced the exposure of a specific region in the amphipathic helix, indicating a participation of TatBC. Of note, the substrate-induced reorientation of the TatA amphipathic helix correlated with detectable membrane weakening. We therefore propose a two-state model in which membrane-destabilizing effects of the short TatA membrane anchor are compensated by the membrane-immersed N-terminal part of the amphipathic helix in a resting state. We conclude that substrate binding to TatABC complexes switches the position of the amphipathic helix, which locally weakens the membrane on demand to allow substrate translocation across the membrane.

Expression and characterization of novel laccase gene from Pandoraea sp. ISTKB and its application

In the present study, a non-blue laccase gene from previously reported lignin degrading bacterium, Pandoraea sp. ISTKB, was isolated, cloned and expressed in E. coli. Bioinformatics analysis of sequence discovered twin-arginine translocation signal sequence, copper binding motifs and presence of more random coil compare to helices and sheets in structure. The enzyme was found to be active on wide pH range and the pH optima was observed at pH 4 and 8 on substrate 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 2,6-Dimethoxyphenol respectively. This is a thermophilic enzyme with maximum activity around 50–70 °C. The enzyme was further characterized by spectroscopy, reaction kinetics and effect of metal ions and inhibitors were studied. Compared to laccase alone; the treatment of dyes with laccase plus mediator resulted in enhanced decolorization of crystal violet, methylene blue, azure B, carmine and Congo red but the effect of mediator was not observed on trypan blue. Laccase treatment triggered polymerization on vanillic acid (VA) and kraft lignin (KL). Laccase plus mediator treatment reversed the polymerization and resulted in transformation or degradation of VA and KL. This thermophilic and alkalophilic non-blue laccase from Pandoraea sp. ISTKB is promising with prospective biotechnological application.

RGD/TAT-functionalized chitosan-graft-PEI-PEG gene nanovector for sustained delivery of NT-3 for potential application in neural regeneration

In this study, we prepared a multifunctional gene delivery nanovector containing a chitosan (CS) backbone and polyethylenimine (PEI) arms with arginine-glycine-aspartate (RGD)/twin-arginine translocation (TAT) conjugated via polyethylene glycol (PEG). Branched PEI, with a molecular weight of 2000 Da, was used to achieve a balance between biocompatibility and transfection efficiency, whereas RGD/TAT peptides were conjugated for enhanced targeting ability and cellular uptake. Synthesis of the copolymers was confirmed by characterizing the chemical structure with 1H nuclear magnetic resonance and Fourier Transform Infrared Spectroscopy (FTIR). The nanovector was biocompatible with cells and showed excellent capability for DNA condensation; the resulting complexes with DNA were well-formed, and possessed small particle size and reasonable positive charge. Higher gene transfection efficiency, compared to that achieved with PEI (25 kDa), was confirmed in tumor (HeLa cells) and normal cells (293T and NIH 3T3 cells). More importantly, the cells transfected with the chitosan-graft-PEI-PEG/pCMV-EGFP-Ntf3 complex produced sustained neurotrophin-3 with a linear increase in cumulative concentration, which induced neuronal differentiation of neural stem cell and promoted neurite outgrowth. These findings suggested that our multifunctional copolymers might be ideal nanovectors for engineering cells via gene transfection, and could potentially be applied in tumor therapy and regenerative medicine. Statement of SignificanceWe successfully prepared a multifunctional gene delivery nanovector containing branched PEI with a molecular weight of 2000 Da to balance between biocompatibility and transfection efficiency, and RGD/TAT peptides for enhanced targeting ability and cellular uptake. The well-formed CPPP/DNA complexes of small particle size and reasonable positive charges potentially enhanced gene transfection in both tumor and normal cells. More importantly, the CPPP/pCMV-EGFP-Ntf3 complex-transfected 293T cells could produce sustained NT-3 with a constant ratio, which induced neuron differentiation of NSC and promoted neurite outgrowth. Therefore, our study provided an effective strategy for producing neurotrophins by engineering cells with gene delivery, which deserved wide investigation and potential application in regenerative medicine.

Precursor-Receptor Interactions in the Twin Arginine Protein Transport Pathway Probed with a New Receptor Complex Preparation.

The twin arginine translocation (Tat) system moves folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Signal peptide-bearing substrates of the Tat pathway (precursor proteins) are recognized at the membrane by the TatBC receptor complex. The only established preparation of the TatBC complex uses the detergent digitonin, rendering it unsuitable for biophysical analysis. Here we show that the detergent glyco-diosgenin (GDN) can be used in place of digitonin to isolate homogeneous TatBC complexes that bind precursor proteins with physiological specificity. We use this new preparation to quantitatively characterize TatBC–precursor interactions in a fully defined system. Additionally, we show that the GDN-solubilized TatBC complex co-purifies with substantial quantities of phospholipids.

Unanticipated functional diversity among the TatA-type components of the Tat protein translocase

Twin-arginine translocation (Tat) systems transport folded proteins that harbor a conserved arginine pair in their signal peptides. They assemble from hexahelical TatC-type and single-spanning TatA-type proteins. Many Tat systems comprise two functionally diverse, TatA-type proteins, denominated TatA and TatB. Some bacteria in addition express TatE, which thus far has been characterized as a functional surrogate of TatA. For the Tat system of Escherichia coli we demonstrate here that different from TatA but rather like TatB, TatE contacts a Tat signal peptide independently of the proton-motive force and restricts the premature processing of a Tat signal peptide. Furthermore, TatE embarks at the transmembrane helix five of TatC where it becomes so closely spaced to TatB that both proteins can be covalently linked by a zero-space cross-linker. Our results suggest that in addition to TatB and TatC, TatE is a further component of the Tat substrate receptor complex. Consistent with TatE being an autonomous TatAB-type protein, a bioinformatics analysis revealed a relatively broad distribution of the tatE gene in bacterial phyla and highlighted unique protein sequence features of TatE orthologs.

The Pentatricopeptide Repeat Protein MEF31 is Required for Editing at Site 581 of the Mitochondrial tatC Transcript and Indirectly Influences Editing at Site 586 of the Same Transcript.

Pentatricopeptide repeat (PPR) proteins constitute the largest family of proteins in angiosperms, and most members are predicted to play roles in the maturation of organellar RNAs. Here we describe the novel mitochondrial editing factor 31 (MEF31), an E-PPR protein involved in editing at two close sites in the same transcript encoding subunit C of the twin-arginine translocation (tat) pathway. MEF31 is essential for editing at site tatC-581 and application of the recently proposed amino acid code for RNA recognition by PPR proteins supports the view that MEF31 directly targets this site by recognizing its cis sequence. In contrast, editing at site tatC-586 five nucleotides downstream is only partially affected in plants lacking MEF31, being restored to wild-type levels in complemented plants. Application of the amino acid code and analysis of individual RNA molecules for editing at sites 581 and 586 suggest that MEF31 does not directly target site tatC-586, and only indirectly influences editing at this site. It is likely that editing at site tatC-581 improves recognition of the site tatC-586 cis sequence by a second unknown PPR protein.

Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide

Twin-arginine translocation (Tat) systems transport folded proteins across cellular membranes with the concerted action of mostly three membrane proteins: TatA, TatB, and TatC. Hetero-oligomers of TatB and TatC form circular substrate-receptor complexes with a central binding cavity for twin-arginine-containing signal peptides. After binding of the substrate, energy from an electro-chemical proton gradient is transduced into the recruitment of TatA oligomers and into the actual translocation event. We previously reported that Tat-dependent protein translocation into membrane vesicles of Escherichia coli is blocked by the compound N,N'-dicyclohexylcarbodiimide (DCCD, DCC). We have now identified a highly conserved glutamate residue in the transmembrane region of E. coli TatC, which when modified by DCCD interferes with the deep insertion of a Tat signal peptide into the TatBC receptor complex. Our findings are consistent with a hydrophobic binding cavity formed by TatB and TatC inside the lipid bilayer. Moreover, we found that DCCD mediates discrete intramolecular cross-links of E. coli TatC involving both its N- and C-tails. These results confirm the close proximity of two distant sequence sections of TatC proposed to concertedly function as the primary docking site for twin-arginine signal peptides.

Membrane binding of the insertion sequence of Proteus vulgaris L-amino acid deaminase stabilizes protein structure and increases catalytic activity

Proteus vulgaris L-amino acid deaminase (pvLAAD) belongs to a class of bacterial membrane-bound LAADs mainly express in genus Proteus, Providencia and Morganella. These LAADs employ a non-cleavable N-terminal twin-arginine translocation (Tat) peptide to transport across membrane and bind to bacterial surface. Recent studies revealed that a hydrophobic insertion sequence (INS) in these LAADs also interacts with bacterial membrane. However, the functional significance of INS-membrane interaction is not clear. In this study, we made site-directed mutagenesis on the surface-exposed hydrophobic residues of pvLAAD INS, and we found that these mutations impaired the INS-membrane interaction but did not affect pvLAAD activity in the solution. We further found that when cell membrane is present, the catalytic activity can be increased by 8~10 folds for wild-type but not INS-mutated pvLAAD, indicating that the INS-membrane interaction is necessary for increasing activity of pvLAAD. Molecular dynamic (MD) simulations suggested that INS is flexible in the solution, and its conformational dynamics could lead to substrate channel distortion. Circular dichroism (CD) spectroscopy experiments indicated that bacterial membrane was able to maintain the conformation of INS. Our study suggests the function of the membrane binding of INS is to stabilize pvLAAD structure and increase its catalytic activity.

Tat proteins as novel thylakoid membrane anchors organize a biosynthetic pathway in chloroplasts and increase product yield 5-fold

Photosynthesis drives the production of ATP and NADPH, and acts as a source of carbon for primary metabolism. NADPH is also used in the production of many natural bioactive compounds. These are usually synthesized in low quantities and are often difficult to produce by chemical synthesis due to their complex structures. Some of the crucial enzymes catalyzing their biosynthesis are the cytochromes P450 (P450s) situated in the endoplasmic reticulum (ER), powered by electron transfers from NADPH. Dhurrin is a cyanogenic glucoside and its biosynthesis involves a dynamic metabolon formed by two P450s, a UDP-glucosyltransferase (UGT) and a P450 oxidoreductase (POR). Its biosynthetic pathway has been relocated to the chloroplast where ferredoxin, reduced through the photosynthetic electron transport chain, serves as an efficient electron donor to the P450s, bypassing the involvement of POR. Nevertheless, translocation of the pathway from the ER to the chloroplast creates other difficulties, such as the loss of metabolon formation and intermediate diversion into other metabolic pathways. We show here that co-localization of these enzymes in the thylakoid membrane leads to a significant increase in product formation, with a concomitant decrease in off-pathway intermediates. This was achieved by exchanging the membrane anchors of the dhurrin pathway enzymes to components of the Twin-arginine translocation pathway, TatB and TatC, which have self-assembly properties. Consequently, we show 5-fold increased titers of dhurrin and a decrease in the amounts of intermediates and side products in Nicotiana benthamiana. Further, results suggest that targeting the UGT to the membrane is a key factor to achieve efficient substrate channeling.

Biosynthesis of 4-hydroxyphenylpyruvic acid from l-tyrosine using recombinant Escherichia coli cells expressing membrane bound l-amino acid deaminase

Abstract 4-Hydroxyphenylpyruvic acid (4-HPPA), a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to produce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound l -amino acid deaminase (m l -AAD), which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H2O2 production during the reaction. In this study, the m l -AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from l -tyrosine. The secretory efficiency of m l -AAD conducted by its own twin-arginine signal peptide (twin-arginine translocation pathway, Tat) and integrated pelB (the general secretory pathway, Sec)-Tat signal peptide was determined and compared firstly, using two pET systems (pET28a and pET20b). It was found that the Tat pathway (pET28a-mlaad) resulted in higher cell-associated m l -AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression hosts Bl21(DE3) and 0.05 mmol·L− 1 IPTG were found to be suitable for m l -AAD expression. The reaction conditions for m l -AAD were optimized and 72.72 mmol·L− 1 4-HPPA was obtained from 100 mmol·L− 1 tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.

Novel reductive dehalogenases from the marine sponge associated bacterium Desulfoluna spongiiphila.

Desulfoluna spongiiphila strain AA1 is an organohalide respiring bacterium, isolated from the marine sponge Aplysina aerophoba, that can use brominated and iodinated phenols, in addition to sulfate and thiosulfate as terminal electron acceptors. The genome of Desulfoluna spongiiphila strain AA1 is approximately 6.5 Mb. Three putative reductive dehalogenase (rdhA) genes involved in respiratory metabolism of organohalides were identified within the sequence. Conserved motifs found in respiratory reductive dehalogenases (a twin arginine translocation signal sequence and two iron-sulfur clusters) were present in all three putative AA1 rdhA genes. Transcription of one of the three rdhA genes was significantly upregulated during respiration of 2,6-dibromophenol and sponge extracts. Strain AA1 appears to have the ability to synthesize cobalamin, the key cofactor of most characterized reductive dehalogenase enzymes. The genome contains genes involved in cobalamin synthesis and uptake and can grow without cobalamin supplementation. Identification of this target gene associated with debromination lays the foundation for understanding how dehalogenating bacteria control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle. Inthe sponge environment, D. spongiiphila strain AA1 may thus take advantage of both brominated compounds and sulfate as electron acceptors for respiration.

Novel Insights into Tat Pathway in Xanthomonas oryzae pv. oryzae Stress Adaption and Virulence: Identification and Characterization of Tat-Dependent Translocation Proteins.

Xanthomonas oryzae pv. oryzae, an economically important bacterium, causes a serious disease in rice production worldwide called bacterial leaf blight. How X. oryzae pv. oryzae infects rice and causes symptoms remains incompletely understood. Our earlier works demonstrated that the twin-arginine translocation (Tat) pathway plays an vital role in X. oryzae pv. oryzae fitness and virulence but the underlying mechanism is unknown. In this study, we used strain PXO99A as a working model, and identified 15 potential Tat-dependent translocation proteins (TDTP) by using comparative proteomics and bioinformatics analyses. Combining systematic mutagenesis, phenotypic characterization, and gene expression, we found that multiple TDTP play key roles in X. oryzae pv. oryzae adaption or virulence. In particular, four TDTP (PXO_02203, PXO_03477, PXO_02523, and PXO_02951) were involved in virulence, three TDTP (PXO_02203, PXO_03477, and PXO_02523) contributed to colonization in planta, one TDTP (PXO_02671) had a key role in attachment to leaf surface, four TDTP (PXO_02523, PXO_02951, PXO_03132, and PXO_03841) were involved in tolerance to multiple stresses, and two TDTP (PXO_02523 and PXO_02671) were required for full swarming motility. These findings suggest that multiple TDTP may have differential contributions to involvement of the Tat pathway in X. oryzae pv. oryzae adaption, physiology, and pathogenicity.

Design and construction of a synthetic E. coli protease inhibitor detecting biomachine.

Protease inhibitors (PIs) have been used to treat various types of symptoms or diseases. However, current PIs block the protease activity by targeting the protease active site which has been shown to be sensitive to the off-target effect due to crossreactivity with protease homologues. An alternative approach to inhibiting protease activity is to target the substrate, specifically by blocking the substrate cleavage site. We propose to employ synthetic biology approach to create a synthetic E. coli to be used as a protease inhibitor detecting biomachine that can effectively isolate intrabodies, a new generation of protease inhibitor drug. The in vivo selection system, comprised of three biological devices, i.e., protease activity detector, protease generator and protease blocking devices, is based on the ability to transport folded protein of the E. coli twin-arginine translocation (Tat) pathway and antibiotic resistance of TEM-1 β-lactamase (Bla) using as reporter protein. By linking protease degradation to antibiotic resistance, we can isolate the suitable intrabodies simply by plating cells containing appropriate devices on solid agar containing β-lactam ring antibiotics. As a proof of concept, we applied a previously isolated HIV-1 p17 intrabody (scFvp17) that binds to the C-terminus of HIV-1 matrix protein (p17) to our synthetic E. coli. This work demonstrated that binding of scFvp17 to its epitope on p17 can physically interfere with HIV-1 protease activity and inhibit proteolytic cleavage at the p17Δp24 cleavage site when expressed in the designed format. The device was optimized by varying plating conditions such as incubation temperatures, induction levels, and Carbenicillin concentrations which was used as selection pressure. The feasibility of this assay has opened the door to protease inhibitor selection which can be used for various applications such as optimization of the current protease inhibitors and selection of new ones.

Probing the Mechanism of LAL-32, a Gold Nanoparticle-Based Antibiotic Discovered through Small Molecule Variable Ligand Display.

The unrelenting rise of antimicrobial-resistant bacteria has necessitated the search for novel antibiotic solutions. Herein we describe further mechanistic studies on a 2.0-nm-diameter gold nanoparticle-based antibiotic (designated LAL-32). This antibiotic exhibits bactericidal activity against the Gram-negative bacterium Escherichia coli at 1.0 μM, a concentration significantly lower than several clinically available antibiotics (such as ampicillin and gentamicin), and acute treatment with LAL-32 does not give rise to spontaneous resistant mutants. LAL-32 treatment inhibits cellular division, daughter cell separation, and twin-arginine translocation (Tat) pathway dependent shuttling of proteins to the periplasm. Furthermore, we have found that the cedA gene imparts increased resistance to LAL-32, and shown that an E. coli cedA transposon mutant exhibits increased susceptibility to LAL-32. Taken together, these studies further implicate cell division pathways as the target for this nanoparticle-based antibiotic and demonstrate that there may be inherently higher barriers for resistance evolution against nanoscale antibiotics in comparison to their small molecule counterparts.

UgpB, a periplasmic component of the UgpABCE ATP-binding cassette transporter, predominantly follows the Sec translocation pathway

Microorganisms secrete proteins to carry out different types of biological functions including biogenesis of flagella or pili, sequestration of nutrients, pathogenicity, etc. Translocation of these proteins across the plasma membrane to the periplasm or external environment takes place via two main pathways viz. Sec (secretory) and Tat (twin arginine translocation). Sec pathway translocates proteins in their unfolded state, whereas, Tat pathway favors the translocation of folded proteins. However, the selection of a particular translocation pathway must be accomplished during or immediately after the completion of protein biosynthesis. This suggests that the primary structure of proteins itself must have some specific signatures for Sec- and Tat-mediated translocation. To identify and further verify the signature motifs and properties of proteins translocated through Sec and Tat pathways, we took up a case study of periplasmic UgpB protein. UgpB is a periplasmic solute (or substrate) binding protein of UgpABCE ATP-binding cassette (ABC) transporter, which mediates the sequestration of either sn-glycerol-3-phosphate (G3P) or glycerophosphocholine (GPC) molecules in the periplasmic space. Recently, UgpB protein from Mycobacterium tuberculosis (MtUgpB) was reported to follow the Tat pathway for its translocation to the periplasm during its pathogenesis; hence it has been proposed as a promising candidate for drug target. Thus, in this study, we attempted to identify the translocation pathway for all UgpB proteins from different organisms available in protein databases. Our computational analysis of these proteins reveals that most of the UgpB proteins follow Sec pathway rather than Tat pathway, unlike MtUgpB. Moreover, this study reveals that the selection of pathway by UgpB proteins mainly depends on the characteristics of signal peptide. In addition, it suggests that the selection of the translocation pathway can also be influenced by the environment in which the organism thrives.