Structural basis for translation inhibition by the glycosylated drosocin peptide.

The proline-rich antimicrobial peptide (PrAMP) drosocin is produced by Drosophila species to combat bacterial infection. Unlike many PrAMPs, drosocin is O-glycosylated at threonine 11, a post-translation modification that enhances its antimicrobial activity. Here we demonstrate that the O-glycosylation not only influences cellular uptake of the peptide but also interacts with its intracellular target, the ribosome. Cryogenic electron microscopy structures of glycosylated drosocin on the ribosome at 2.0-2.8-Å resolution reveal that the peptide interferes with translation termination by binding within the polypeptide exit tunnel and trapping RF1 on the ribosome, reminiscent of that reported for the PrAMP apidaecin. The glycosylation of drosocin enables multiple interactions with U2609 of the 23S rRNA, leading to conformational changes that break the canonical base pair with A752. Collectively, our study reveals novel molecular insights into the interaction of O-glycosylated drosocin with the ribosome, which provide a structural basis for future development of this class of antimicrobials.

Selectivity among Anti-σ Factors by Mycobacterium tuberculosis ClpX Influences Intracellular Levels of Extracytoplasmic Function σ Factors.

Extracytoplasmic function σ factors that are stress inducible are often sequestered in an inactive complex with a membrane-associated anti-σ factor. Mycobacterium tuberculosis membrane-associated anti-σ factors have a small, stable RNA gene A (ssrA)-like degron for targeted proteolysis. Interaction between the unfoldase, ClpX, and a substrate with an accessible degron initiates energy-dependent proteolysis. Four anti-σ factors with a mutation in the degron provided a set of natural substrates to evaluate the influence of the degron on degradation strength in ClpX-substrate processivity. We note that a point mutation in the degron (X-Ala-Ala) leads to an order-of-magnitude difference in the dwell time of the substrate on ClpX. Differences in ClpX/anti-σ interactions were correlated with changes in unfoldase activities. Green fluorescent protein (GFP) chimeras or polypeptides with a length identical to that of the anti-σ factor degron also demonstrate degron-dependent variation in ClpX activities. We show that degron-dependent ClpX activity leads to differences in anti-σ degradation, thereby regulating the release of free σ from the σ/anti-σ complex. M. tuberculosis ClpX activity thus influences changes in gene expression by modulating the cellular abundance of ECF σ factors.IMPORTANCE The ability of Mycobacterium tuberculosis to quickly adapt to changing environmental stimuli occurs by maintaining protein homeostasis. Extracytoplasmic function (ECF) σ factors play a significant role in coordinating the transcription profile to changes in environmental conditions. Release of the σ factor from the anti-σ is governed by the ClpXP2P1 assembly. M. tuberculosis ECF anti-σ factors have an ssrA-like degron for targeted degradation. A point mutation in the degron leads to differences in ClpX-mediated proteolysis and affects the cellular abundance of ECF σ factors. ClpX activity thus synchronizes changes in gene expression with environmental stimuli affecting M. tuberculosis physiology.

Comparing naturally occurring glycosylated forms of proline rich antibacterial peptide, Drosocin.

Antimicrobial peptides (AMPs) are key players of innate immunity. Amongst various classes of AMPs, proline rich AMPs from insects enjoy special attention with few members of this class bearing O-glycosylation as post-translational modification. Drosocin, a 19 amino acid glycosylated AMP is a member of proline rich class, synthesized in the haemolymph of Drosophila melanogaster upon bacterial challenge. We report herein the chemical synthesis of drosocin carrying disaccharide (ß-Gal(1 â†’ 3)α-GalNAc) and comparison of its structural and functional properties with another naturally occurring monoglycosylated form of drosocin i.e. α-GalNAc-drosocin as well as with non-glycosylated drosocin. The disaccharide containing drosocin exhibited lower potency compared to monoglycosylated drosocin against all the tested Gram negative bacteria, suggesting the role of the distal sugar or increase in the sugar chain length on the activity. Circular dichroism studies failed to demonstrate the differential effect of sugars on the overall peptide conformation. Haemolytic and cytotoxic properties of drosocin were not altered due to an increase in the sugar chain length. In addition, we have also evaluated the effect of differentially glycosylated drosocins on two pro-inflammatory cytokines secreted by murine macrophages or LPS stimulated macrophages. All the drosocin forms tested, neither could stimulate the secretion of TNF-α and IL-6 nor could modulate LPS-induced levels of TNF-α and IL-6 in murine macrophages. This study provides insights about naturally occurring two different glycosylated forms of drosocin.

Effect of distal sugar and interglycosidic linkage of disaccharides on the activity of proline rich antimicrobial glycopeptides.

The effect of glycosylation on protein structure and function depends on a variety of intrinsic factors including glycan chain length. We have analyzed the effect of distal sugar and interglycosidic linkage of disaccharides on the properties of proline-rich antimicrobial glycopeptides, formaecin I and drosocin. Their glycosylated analogs-bearing lactose, maltose and cellobiose, as a glycan side chain on their conserved threonine residue, were synthesized where these disaccharides possess identical proximal sugar and vary in the nature of distal sugar and/or interglycosidic linkage. The structural and functional properties of these disaccharide-containing formaecin I and drosocin analogs were compared with their corresponding monoglycosylated forms, ß-D-glucosyl-formaecin I and ß-D-glucosyl-drosocin, respectively. We observed neither major secondary structural alterations studied by circular dichroism nor substantial differences in the toxicity with mammalian cells among all of these analogs. The comparative analyses of antibacterial activities of these analogs of formaecin I and drosocin displayed that ß-D-maltosyl-formaecin I and ß-D-maltosyl-drosocin were more potent than that of respective ß-D-Glc-analog, ß-D-cellobiosyl-analog and ß-D-lactosyl-analog. Despite the differences in their antibacterial activity, all the analogs exhibited comparable binding affinity to DnaK that has been reported as one of the targets for proline-rich class of antibacterial peptides. The comparative-quantitative internalization studies of differentially active analogs revealed the differences in their uptake into bacterial cells. Our results exhibit that the sugar chain length as well as interglycosidic linkage of disaccharide may influence the antibacterial activity of glycosylated analogs of proline-rich antimicrobial peptides and the magnitude of variation in antibacterial activity depends on the peptide sequence.

Understanding the importance of glycosylated threonine and stereospecific action of Drosocin, a Proline rich antimicrobial peptide.

Glycosylation is an essential post-translational modification for few antimicrobial peptides of Proline rich class. In the present study we have shown the importance of Thr glycosylation over Ser glycosylation in Drosocin. Difference of a methyl group makes glycosylated-Thr preferred over glycosylated-Ser and renders higher activity to the peptide, probably due to the rigid conformation provided by the glycosylated-Thr. The structural rigidity provided by glycosylated-Thr to Drosocin backbone was mimicked by substituting glycosylated-Thr11, Ser7 and Ser12 with Pro residues. The designed non-glycosylated analogue, P(7)P(11)P(12)-Drosocin, exhibited functional and structural properties similar to that of the native monoglycosylated peptide. The functional importance of stereospecificity of amino acids and sugar was further explored. Interestingly, (all D) p(7)p(11)p(12)-Drosocin failed to exhibit antimicrobial activity but had comparable binding affinity to DnaK, one of the proposed targets for Proline rich class of antibacterial peptides, as that of its L counterpart. However, Drosocin containing either L or D enantiomeric sugar, displayed antimicrobial activity and binding affinity to bacterial heat shock protein, DnaK. The flow cytometry (FACS) experiments revealed the internalization of Drosocins bearing enantiomeric sugars and P(7)P(11)P(12)-Drosocin but not of its d-enantiomer into bacteria suggesting the importance of stereospecificity of amino acids for membrane entry. Once internalized both enantiomeric peptides may behave similarly. This assumption was corroborated by in vitro activity of (all D) p(7)p(11)p(12)-Drosocin in cell free assay where it abrogated transcription/translation pathway similar to l-enantiomer but could not inhibit the same in whole cell assay. These research findings provide insights into the mode of action of Proline rich class of antibacterial peptides and guidelines for designing functionally equivalent non-glycosylated analogues of glycosylated antibacterial peptides.