Uptake Of Peptides Research Articles

Rapid and Sensitive Quantification of Intracellular Glycyl-Sarcosine for Semi-High-Throughput Screening for Inhibitors of PEPT-1.

The peptide transporter PEPT-1 (SLC15A1) plays a major role in nutritional supply with amino acids by mediating the intestinal influx of dipeptides and tripeptides generated during food digestion. Its role in the uptake of small bioactive peptides and various therapeutics makes it an important target for the investigation of the systemic absorption of small peptide-like active compounds and prodrug strategies of poorly absorbed therapeutics. The dipeptide glycyl-sarcosine (Gly-Sar), which comprises an N-methylated peptide bond that increases stability against enzymatic degradation, is widely utilized for studying PEPT-1-mediated transport. To support experiments on PEPT-1 inhibitor screening to identify potential substrates, we developed a highly sensitive Gly-Sar quantification assay for Caco-2 cell lysates with a dynamic range of 0.1 to 1000 ng/mL (lower limit of quantification 0.68 nM) in 50 µL of cell lysate. The assay was validated following the applicable recommendations for bioanalytic method validation of the FDA and EMA. Sample preparation and quantification were established in 96-well cell culture plates that were also used for the cellular uptake studies, resulting in a rapid and robust screening assay for PEPT-1 inhibitors. This sample preparation principle, combined with the high sensitivity of the UPLC-MS/MS quantification, is suitable for screening assays for PEPT-1 inhibitors and substrates in high-throughput formats and holds the potential for automation. Applicability was demonstrated by IC50 determinations of the known PEPT-1 inhibitor losartan, the known substrates glycyl-proline (Gly-Pro), and valaciclovir, the prodrug of aciclovir, which itself is no substrate of PEPT-1 and consequently showed no inhibition in our assay.

Abstract 1310: Peptide delivery of siRNA in GBM

Abstract Glioblastoma Multiforme (GBM), a grade VI glioma, is an aggressive malignant tumorwith a poor prognosis. With an incidence rate of 3.19 per 100,000, GBM has a median survivalrate of 12-15 months and a five-year survival rate of less than 5%. Typical treatments involvesurgical removal, chemotherapy, and radiation; however, these treatments still result in a lowsurvival rate and can lead to potential harm to healthy cells. Therefore, a new therapeutic isneeded for GBM treatment to improve patient outcomes. RNAi, a naturally found process,utilizes siRNA to target specific mRNA for degradation to inhibit protein translation and hasshown clinical potential for targeting and silencing oncogenes. However, barriers to siRNAdelivery, such as cellular uptake, non-specificity, and lack of endosomal escape hinder clinicaltranslation of RNAi therapeutics. Peptides are a promising carrier for siRNAs and havedemonstrated the potential to overcome barriers to delivery. The purpose of this study is todesign a novel tandem peptide for siRNA delivery, evaluate ability to mediate knockdown of atarget oncogene, and compare the efficacy of the tandem peptide to the individual peptidesalone. The tandem peptide is a novel combination of a fusogenic peptide and a targetingpeptide. STAT3, the target gene, is an oncogene that plays a role in cell proliferation,differentiation, apoptosis, and angiogenesis in GBM. Initial results have confirmed basal expression of EGFR and STAT3 in all three GBM cell lines, LN18, U118, and T98G.Characterization of the fusogenic, targeting, and tandem peptides indicated formation of monodispere nanoparticles with diameters less than 150 nm when complexed with siRNA. Furthermore, gel shift assays were conducted and confirmed that the tandem peptide protectedthe siRNA from degradation in 50% FBS. MTS assays conducted in all three cell lines for eachpeptide confirmed that there was no significant cytotoxicity due to the vehicles alone. Finally, flow cytometry confirmed uptake of the tandem peptide in T98G cell line. Therefore, it ishypothesized that the tandem peptide composed of both fusogenic targeting peptide willenhance cell-specific uptake of STAT3 siRNA into glioblastoma cells for mediating endosomalescape, resulting in more effective gene silencing and associated downstream effects comparedto the fusogenic and targeting peptides alone. Future studies include immunofluorescence for uptake and endosomal escape, knockdown studies (MTS, Western Blot, and qPCR assays), and assays to examine the downstream effect of silencing STAT3, such as migration and invasion assays. Citation Format: Jessica Boulos, Angela Alexander-Bryant. Peptide delivery of siRNA in GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1310.

Antimicrobial Peptides: Delivery by Dendritic Mesoporous Silica Nanoparticles Enhances the Antimicrobial Activity of a Napsin‐Derived Peptide Against Intracellular Mycobacterium tuberculosis (Adv. Healthcare Mater. 14/2021)

In article number 2100453 by Steffen Stenger, Mika Lindén, and co-workers, the activity of the antimicrobial peptide NapFab against intracellular Mycobacterium tuberculosis (Mtb) is significantly increased by loading onto dendritic mesoporous silica nanoparticles (DMSN). This enhanced activity is based on a 30-fold increased, DMSN-mediated peptide uptake. Livecell fluorescence microscopy and electron microscopy reveal fast DMSN uptake in endosomal vesicles, intracellular degradation of the carrier, and a continuous peptide release.

Important Considerations Related to Permeability of Peptides.

This review on intracellular delivery and oral bioavailability of peptides reflects a number of principal investigations at Novartis. Our studies were aimed at either understanding features enabling peptides to interfere with intracellular protein-protein interactions, or to achieve a more patient-friendly delivery by the oral route. In the light of these objectives, we have also spent some effort on assay development to come up with alternative methods for monitoring cellular peptide uptake. This summary of our insights is intended to help in the assessment and development of peptide therapeutics requiring membrane transition.

The RagA and RagB proteins of Porphyromonas gingivalis.

RagA and RagB proteins are major components of the outer membrane of the oral pathogen Porphyromonas gingivalis and, while recently suggested to represent a novel peptide uptake system, their full function is still under investigation. Herein, we (a) discuss the evidence that the rag locus contributes to P. gingivalis virulence; (b) provide insight to Rag protein potential biological function in macromolecular transport and other aspects of bacterial physiology; (c) address the host response to Rag proteins which are immunodominant and immunomodulatory; and (d) review the potential of Rag-focused therapeutic strategies for the control of periodontal diseases.

Statin-boosted cellular uptake and endosomal escape of penetratin due to reduced membrane dipole potential.

Cell penetrating peptides are promising tools for delivery of cargo into cells, but factors limiting or facilitating their cellular uptake are largely unknown. We set out to study the effect of the biophysical properties of the cell membrane on the uptake of penetratin, a cell penetrating peptide. Using labelling with pH-insensitive and pH-sensitive dyes, the kinetics of cellular uptake and endo-lysosomal escape of penetratin were studied by flow cytometry. We report that escape of penetratin from acidic endo-lysosomal compartments is retarded compared with its total cellular uptake. The membrane dipole potential, known to alter transmembrane transport of charged molecules, is shown to be negatively correlated with the concentration of penetratin in the cytoplasmic compartment. Treatment of cells with therapeutically relevant concentrations of atorvastatin, an inhibitor of HMG-CoA reductase and cholesterol synthesis, significantly increased endosomal escape of penetratin in two different cell types. This effect of atorvastatin correlated with its ability to decrease the membrane dipole potential. These results highlight the importance of the dipole potential in regulating cellular uptake of cell penetrating peptides and suggest a clinically relevant way of boosting this process.

A novel self-assembled epitope peptide nanoemulsion vaccine targeting nasal mucosal epithelial cell for reinvigorating CD8+ T cell immune activity and inhibiting tumor progression

Epitope peptides are not suitable for nasal administration immunity due to their poor immunogenicity and low delivery efficiency. Here, we reported an intranasal self-assembled nanovaccine (I-OVA NE), which was loaded with the peptides IKVAV-OVA257–264 (I-OVA), a laminin peptide (Ile-Lys-Val-ala-Val, IKVAV) and OVA257–264 epitope conjugated peptide. This nanovaccine with I-OVA at a concentration of 4 mg/mL showed the average particle size of 30.37 ± 2.49 nm, zeta potential of −16.67 ± 1.76 mV, and encapsulation rate of 84.07 ± 7.59%. Moreover, the mucin did not alter its stability (size, PdI and zeta potential). And it also had no obvious acute pathological changes neither in the nasal mucosa nor lung tissues after nasal administration. Meanwhile, the antigen uptake of I-OVA NE was promoted, and the nasal residence time was also prolonged in vivo. Besides, the uptake rate of this nanovaccine was obviously higher than that of free I-OVA (P < 0.001) after blocking by the integrin antibody, suggesting that the binding of IKVAV to integrin is involved in the epitope peptide uptake. Importantly, this nanovaccine enhanced peptide-specific CD8+T cells exhibiting OVA257–264-specific CTL activity and Th1 immune response, leading to the induction of the protective immunity in E.G7-OVA tumor-bearing mice. Overall, these data indicate that I-OVA NE can be an applicable strategy of tumor vaccine development.

Influence of the Dabcyl group on the cellular uptake of cationic peptides: short oligoarginines as efficient cell-penetrating peptides

Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4–((4–(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity.

In Vitro and In Vivo Cell Uptake of a Cell-Penetrating Peptide Conjugated with Fluorescent Dyes Having Different Chemical Properties.

Simple SummaryIn fluorescence imaging employing a targeting strategy, fluorescent dyes conjugated with ligands may alter the pharmacokinetics of the conjugates. The aim of this study was to investigate whether in vitro and in vivo cell uptake are affected when fluorescent dyes with different chemical properties are conjugated with a ligand. The results show that attention should be paid to the chemical properties of fluorescent dyes in designing fluorescent imaging agents.In molecular imaging, a targeting strategy with ligands is widely used because specificity can be significantly improved. In fluorescence imaging based on a targeting strategy, the fluorescent dyes conjugated with ligands may affect the targeting efficiency depending on the chemical properties. Herein, we used a cell-penetrating peptide (CPP) as a ligand with a variety of fluorescent cyanine dye. We investigated in vitro and in vivo cell uptake of the dye-CPP conjugates when cyanine dyes with differing charge and hydrophilicity/lipophilicity were used. The results showed that the conjugates with positively charged and lipophilic cyanine dyes accumulated in cancer cells in vitro, but there was almost no accumulation in tumors in vivo. On the other hand, the conjugates with negatively charged and hydrophilic cyanine dyes did not accumulate in cancer cells in vitro, but fluorescence was observed in tumors in vivo. These results show that there are some cases in which the cell uptake of the dye-peptide conjugates may differ significantly between in vitro and in vivo experiments due to the chemical properties of the fluorescent dyes. This suggests that attention should be paid to the chemical properties of fluorescent dyes in fluorescence imaging based on a targeting strategy.

Generating An Exosome Packaged p38 Inhibitory Peptide To Block GPCR Induced Vascular Inflammation

A key mediator of angiogenesis and inflammatory response in chronic lung disease is the mitogen-activated protein kinase (MAPK), p38α. The typical activation pathway for p38α uses MAP kinase kinase 3 (MKK3), and MKK6 to phosphorylate and activate p38α. However, an atypical p38α signaling pathway activated by a family of pro-inflammatory G-protein coupled receptors (GPCRs) induces a direct interaction between transforming growth factor β activated kinase 1 binding protein-1 (TAB1) and p38α, bypassing MKK3/6-dependent p38α activation. The binding of TAB1 to p38α induces the autophosphorylation of p38α, triggering proinflammatory signaling in the vasculature. Recent studies have highlighted the use of cell-penetrating inhibitors that specifically block atypical p38α signaling, leaving MKK3/6-dependent signaling intact. Using the Xpack Lentiviral expression system, we have generated a stable cell line that expresses red fluorescent protein (RFP) labeled peptide inhibitors in endothelial cells. The Xpack system contains an exosomal packaging motif enabling RFP-linked inhibitor peptides to be targeted for exosomes. Using filter centrifugation and ultracentrifugation we have isolated RFP peptide loaded exosomes/extracellular vesicles. These inhibitor loaded extracellular vesicles can successfully deliver inhibitors to vascular endothelial cells in vitro. Our current studies focus on assessing whether cellular uptake of peptide loaded exosomes can efficiently attenuate GPCR-induced proinflammatory signaling. To investigate this we are using a combination of immunoblotting and live-cell Forster's Resonance Energy Transfer (FRET) assays. We predict that exosomally delivered peptides will inhibit p38 signaling by GPCR ligands in endothelial cells providing a tool to produce large quantities of bioavailable inhibitors that can be developed into therapeutics. These studies demonstrate the potential for exosomal nanocarriers to deliver peptide inhibitors to block GPCR inflammation.

Two ABC transport systems carry out peptide uptake in Enterococcus faecalis: Their roles in growth and in uptake of sex pheromones.

Enterococcal pheromone-inducible plasmids encode a predicted OppA-family secreted lipoprotein. In the case of plasmid pCF10, the protein is PrgZ, which enhances the mating response to cCF10 pheromone. OppA proteins generally function with associated OppBCDF ABC transporters to import peptides. In this study, we analyzed the potential interactions of PrgZ with two host-encoded Opp transporters using two pheromone-inducible fluorescent reporter constructs. Based on our results, we propose renaming these loci opp1 (OG1RF_10634-10639) and opp2 (OG1RF_12366-12370). We also examined the ability of the Opp1 and Opp2 systems to mediate import in the absence of PrgZ. Cells expressing PrgZ were able to import pheromone if either opp1 or opp2 was functional, but not if both opp loci were disrupted. In the absence of PrgZ, pheromone import was dependent on a functional opp2 system, including opp2A. Comparative structural analysis of the peptide-binding pockets of PrgZ, Opp1A, Opp2A, and the related Lactococcus lactis OppA protein, suggested that the robust pheromone-binding ability of PrgZ relates to a nearly optimal fit of the hydrophobic peptide, whereas binding ability of Opp2A likely results from a more open, promiscuous peptide-binding pocket similar to L. lactis OppA.

Circulating regulators of the wingless pathway in precapillary pulmonary hypertension.

Dysregulated Wnt signalling has been implicated in pulmonary hypertension (PH). We hypothesized that plasma levels of secreted Wnt proteins would be increased in patients with precapillary PH, correlate with indices of vascular resistance and cardiac function and give information on long-term prognosis. We measured the Wnt ligand Wnt5a and secreted Wnt antagonists Dickkopf (DKK) DKK1, DKK3, secreted frizzled-related protein 3 (sFRP3), Wnt inhibitory factor-1 (WIF1) and sclerostin (SOST) in 106 patients with precapillary PH and 40 healthy controls. A second sample was obtained after a median of 4months (n= 52). During a median of 90 months follow-up, 67 patients died. Our main findings were (i) Precapillary PH is characterized by enhanced systemic Wnt activity as reflected by elevated plasma levels of Wnt5a and secreted antagonists irrespective of diagnostic subgroups. (ii) WIF1 and in particular Wnt5a correlated with pulmonary vascular resistance and cardiac dysfunction. (iii) High levels of Wnt5a, sFRP3, DKK3 and WIF1 were associated with poor prognosis in age- and sex-adjusted analysis (hazard ratios per log/SD change ~1.4) and for DKK3 after further adjustment with right arterial pressure, pulmonary oxygen saturation, cardiac index, N-terminal pro B-type natriuretic peptide and peak oxygen uptake (VO2 ). Finally, an elevation of Wnt5a and DKK3 during follow-up was independently associated with poor prognosis. Our data indicate that Wnt signalling pathways could be implicated in the pathogenesis of precapillary PH, and that some of the Wnt-related molecules (i.e., Wnt5a and DKK3) should be further investigated in these patients.

Synthesis of six-membered carbocyclic ring α,α-disubstituted amino acids and arginine-rich peptides to investigate the effect of ring size on the properties of the peptide

Cell-penetrating peptides (CPPs) have been attracting attention as tools for intracellular delivery of membrane-impermeant functional molecules. Among the variety of CPPs that have been developed, many are composed of both natural and unnatural amino acids. We previously synthesized α,α-disubstituted α-amino acids (dAAs) containing a five-membered carbocyclic ring in its side chain and revealed the utility of dAAs for the development of novel CPPs. In the present study, we designed a six-membered carbocyclic ring dAA with an amino group on the ring and introduced it into arginine (Arg)-rich peptides to further investigate the value of dAAs for developing CPPs. We also assessed the effects of the size of the dAA carbocyclic ring on cellular uptake of dAA-containing peptides. The stability of the peptide’s secondary structure and its membrane permeability were both greater in dAA-containing peptides than in an Arg nonapeptide. However, the number of carbon atoms in the dAA side chain ring had little effect. Nevertheless, these results show the utility of cyclic dAAs in the design of novel CPPs containing unnatural amino acids.

Modeling Cell Selectivity of Antimicrobial Peptides: How Is the Selectivity Influenced by Intracellular Peptide Uptake and Cell Density.

Antimicrobial peptides (AMPs) are known to attack bacteria selectively over their host cells. Many attempts have been made to use them as a template for designing peptide antibiotics for fighting drug-resistant bacteria. A central concept in this endeavor is “peptide selectivity,” which measures the “quality” of peptides. However, the relevance of selectivity measurements has often been obscured by the cell-density dependence of the selectivity. For instance, the selectivity can be overestimated if the cell density is larger for the host cell. Furthermore, recent experimental studies suggest that peptide trapping in target bacteria magnifies the cell-density dependence of peptide activity. Here, we propose a biophysical model for peptide activity and selectivity, which assists with the correct interpretation of selectivity measurements. The resulting model shows how cell density and peptide trapping in cells influence peptide activity and selectivity: while these effects can alter the selectivity by more than an order of magnitude, peptide trapping works in favor of host cells at high host-cell densities. It can be used to correct selectivity overestimates.

Conformational Transition Pathway of GkPOT

GkPOT, a Proton-Coupled coupled Oligopeptide Transporter transporter (POTs) from Geobacillus Kaustophilus, is among the symporter members of the Major Facilitator Superfamily (MFS) of transporters. Functional and structural analysis of various POTs suggest that the uptake of small peptides and peptide- like molecules to the cell across the cell membrane, via the alternating access mechanism, utilizes an inward directed proton electrochemical gradient the source of energy for their active symport function. The transition between the inward-facing (IF) and outward-facing (OF) conformations is important in understanding the structural relationship between large-scale protein conformational changes and substrate translocation that has often times remained elusive due to the short time scales of molecular dynamics simulations (MD) limitations of computational and experimental effort techniques. Using an extensive set of enhanced sampling techniques, we have characterized the conformational transition pathway of GkPOT. We have aimed at describing the large-scale conformational changes between the IF and OF states while in the presence and absence of substrate (dialanine peptide). Nonequlibrium pulling simulations were initiated from the known inward facing IF conformation and steered the system toward the unknown outward facing OF conformation using orientation based collective variables defined on different transmembrane helices. The transition pathways are far from equilibrium and are smoothed with string method with swarms of trajectories (SMwST), thus generating microseconds of biased and unbiased simlulations. Free energy profiles of the conformational transition pathways were obtained from a novel maximum-likelihood estimation method. Our results indicate that the presence of ligand significantly changes the conformational landscape with respect to the apo system which contains a greater degree of conformational variability.

Interaction Between Antimicrobial Peptides and Non-Lipid Components in Bacterial Outer Membrane

Antimicrobial peptides (AMPs) offer advantages over conventional antibiotics; for example, bacteria develop less resistance to AMPs. The interaction of the AMPs with the lipopolysaccharide layer of the gram-negative bacteria cell envelope is not well understood. This first interaction is essential for the peptide uptake and specificity to the target organism. In this work, we simulated the AMPs interactions with the bacterial envelope. We build a coarse-grained system formed by a gram-negative bacterial outer membrane, including the LPS layer and the AMP Magainin 2 using CHARMM-GUI Martini Maker. We perform 20us of Molecular dynamics simulation using GROMAC on the free system. During this simulation, the AMP stays at 5nm in the Z-axis, corresponding to the negative charge groups in the LPS layer in the cell envelope. This result suggests interactions between the positive charge of the Magainin 2 and the LPS layer. Finally, we calculated the insertion Free Energy Profile (PMF) of the Magainin 2 in the membrane. For the PMF construction, the AMP was restrained to 200 different positions across the membrane, performing molecular dynamic simulation and to each window. The minimum energy was found around the 5nm, supporting the firth result and suggesting an interaction between the AMP and the negative charge groups of the LPS layer of the bacterial cell envelope.

Tentative Peptide‒Lipid Bilayer Models Elucidating Molecular Behaviors and Interactions Driving Passive Cellular Uptake of Collagen-Derived Small Peptides.

Collagen contains hydroxyproline (Hyp), which is a unique amino acid. Three collagen-derived small peptides (Gly-Pro-Hyp, Pro-Hyp, and Gly-Hyp) interacting across a lipid bilayer (POPC model membrane) for cellular uptakes of these collagen-derived small peptides were studied using accelerated molecular dynamics simulation. The ligands were investigated for their binding modes, hydrogen bonds in each coordinate frame, and mean square displacement (MSD) in the Z direction. The lipid bilayers were evaluated for mass and electron density profiles of the lipid molecules, surface area of the head groups, and root mean square deviation (RMSD). The simulation results show that hydrogen bonding between the small collagen peptides and plasma membrane plays a significant role in their internalization. The translocation of the small collagen peptides across the cell membranes was shown. Pro-Hyp laterally condensed the membrane, resulting in an increase in the bilayer thickness and rigidity. Perception regarding molecular behaviors of collagen-derived peptides within the cell membrane, including their interactions, provides the novel design of specific bioactive collagen peptides for their applications.

Host-specific functional compartmentalization within the oligopeptide transporter during the Borrelia burgdorferi enzootic cycle.

Borrelia burgdorferi must acquire all of its amino acids (AAs) from its arthropod vector and vertebrate host. Previously, we determined that peptide uptake via the oligopeptide (Opp) ABC transporter is essential for spirochete viability in vitro and during infection. Our prior study also suggested that B. burgdorferi employs temporal regulation in concert with structural variation of oligopeptide-binding proteins (OppAs) to meet its AA requirements in each biological niche. Herein, we evaluated the contributions to the B. burgdorferi enzootic cycle of three of the spirochete’s five OppAs (OppA1, OppA2, and OppA5). An oppA1 transposon (tn) mutant lysed in the hyperosmolar environment of the feeding tick, suggesting that OppA1 imports amino acids required for osmoprotection. The oppA2tn mutant displayed a profound defect in hematogenous dissemination in mice, yet persisted within skin while inducing only a minimal antibody response. These results, along with slightly decreased growth of the oppA2tn mutant within DMCs, suggest that OppA2 serves a minor nutritive role, while its dissemination defect points to an as yet uncharacterized signaling function. Previously, we identified a role for OppA5 in spirochete persistence within the mammalian host. We now show that the oppA5tn mutant displayed no defect during the tick phase of the cycle and could be tick-transmitted to naïve mice. Instead of working in tandem, however, OppA2 and OppA5 appear to function in a hierarchical manner; the ability of OppA5 to promote persistence relies upon the ability of OppA2 to facilitate dissemination. Structural homology models demonstrated variations within the binding pockets of OppA1, 2, and 5 indicative of different peptide repertoires. Rather than being redundant, B. burgdorferi’s multiplicity of Opp binding proteins enables host-specific functional compartmentalization during the spirochete lifecycle.

Mycobacterium tuberculosis PPE18 protein inhibits MHC class II antigen presentation and B cell response in mice.

PPE18 protein belongs to PE/PPE family of Mycobacterium tuberculosis. We reported earlier that PPE18 protein provides survival advantage to M. tuberculosis during infection. In the current study, we found that PPE18 inhibits MHC class II-mediated antigen presentation by macrophages in a dose-dependent manner without affecting the surface level of MHC class II or co-stimulatory molecules. PPE18 does not affect antigen uptake or presentation of preprocessed peptide by macrophages. Antigen degradation was found to be inhibited by PPE18 protein due to perturbation in phagolysosomal acidification. PPE18-mediated inhibition of MHC class II antigen presentation caused poorer activation of CD4 T cells. Mice infected with M. smegmatis expressing PPE18 exhibited reduced maturation and activation of B cells and had decreased Mycobacteria-specific antibody titers. Thus M. tuberculosis probably utilizes PPE18 to inhibit MHC class II antigen presentation causing poorer activation of adaptive immune responses. This study may be useful in understanding host-pathogen interaction and open up directions of designing novel therapeutics targeting PPE18 to tackle this nefarious pathogen.

Growth of Giant Peptide Vesicles Driven by Compartmentalized Transcription-Translation Activity.

Compartmentalization and spatial organization of biochemical reactions are essential for the establishment of complex metabolic pathways inside synthetic cells. Phospholipid and fatty acid membranes are the most natural candidates for this purpose, but also polymers have shown great potential as enclosures of artificial cell mimics. Herein, we report on the formation of giant vesicles in a size range of 1 μm–100 μm using amphiphilic elastin‐like polypeptides. The peptide vesicles can accommodate cell‐free gene expression reactions, which is demonstrated by the transcription of a fluorescent RNA aptamer and the production of a fluorescent protein. Importantly, gene expression inside the vesicles leads to a strong growth of their size—up to an order of magnitude in volume in several cases—which is driven by changes in osmotic pressure, resulting in fusion events and uptake of membrane peptides from the environment.