Correction: Nonin-Lecomte et al. Bacterial Type I Toxins: Folding and Membrane Interactions. Toxins 2021, 13, 490.

The authors wish to make the following corrections to their paper [...].

Capsicumicine, a New Bioinspired Peptide from Red Peppers Prevents Staphylococcal Biofilm In Vitro and In Vivo via a Matrix Anti-Assembly Mechanism of Action.

Staphylococci are pathogenic biofilm-forming bacteria and a source of multidrug resistance and/or tolerance causing a broad spectrum of infections. These bacteria are enclosed in a matrix that allows them to colonize medical devices, such as catheters and tissues, and that protects against antibiotics and immune systems. Advances in antibiofilm strategies for targeting this matrix are therefore extremely relevant. Here, we describe the development of the Capsicum pepper bioinspired peptide "capsicumicine." By using microbiological, microscopic, and nuclear magnetic resonance (NMR) approaches, we demonstrate that capsicumicine strongly prevents methicillin-resistant Staphylococcus epidermidis biofilm via an extracellular "matrix anti-assembly" mechanism of action. The results were confirmed in vivo in a translational preclinical model that mimics medical device-related infection. Since capsicumicine is not cytotoxic, it is a promising candidate for complementary treatment of infectious diseases. IMPORTANCE Pathogenic biofilms are a global health care concern, as they can cause extensive antibiotic resistance, morbidity, mortality, and thereby substantial economic loss. So far, no effective treatments targeting the bacteria in biofilms have been developed. Plants are constantly attacked by a wide range of pathogens and have protective factors, such as peptides, to defend themselves. These peptides are common components in Capsicum baccatum (red pepper). Here, we provide insights into an antibiofilm strategy based on the development of capsicumicine, a natural peptide that strongly controls biofilm formation by Staphylococcus epidermidis, the most prevalent pathogen in device-related infections.

Acetonitrile allows indirect replacement of nondeuterated lipid detergents by deuterated lipid detergents for the nuclear magnetic resonance study of detergent-soluble proteins.

Detergent-soluble proteins (DSPs) are commonly dissolved in lipid buffers for NMR experiments, but the huge lipid proton signal prevents recording of high-quality spectra. The use of costly deuterated lipids is thus required to replace nondeuterated ones. With conventional methods, detergents like dodecylphosphocholine (DPC) cannot be fully exchanged due to their high binding affinity to hydrophobic proteins. We propose an original and simple protocol which combines the use of acetonitrile, dialysis and lyophilization to disrupt the binding of lipids to the protein and allow their indirect replacement by their deuterated equivalents, while maintaining the native structure of the protein. Moreover, by this protocol, the detergent-to-protein molar ratio can be controlled as it challenges the protein structure. This protocol was applied to solubilize the Vpx protein that was followed upon addition of DPC-d38 by 1 H-15 N SOFAST-HMQC spectra and the best detergent-to-DSPs molar ratio was obtained for structural studies.

Bacterial Type I Toxins: Folding and Membrane Interactions.

Bacterial type I toxin-antitoxin systems are two-component genetic modules that encode a stable toxic protein whose ectopic overexpression can lead to growth arrest or cell death, and an unstable RNA antitoxin that inhibits toxin translation during growth. These systems are widely spread among bacterial species. Type I antitoxins are cis- or trans-encoded antisense small RNAs that interact with toxin-encoding mRNAs by pairing, thereby inhibiting toxin mRNA translation and/or inducing its degradation. Under environmental stress conditions, the up-regulation of the toxin and/or the antitoxin degradation by specific RNases promote toxin translation. Most type I toxins are small hydrophobic peptides with a predicted α-helical transmembrane domain that induces membrane depolarization and/or permeabilization followed by a decrease of intracellular ATP, leading to plasmid maintenance, growth adaptation to environmental stresses, or persister cell formation. In this review, we describe the current state of the art on the folding and the membrane interactions of these membrane-associated type I toxins from either Gram-negative or Gram-positive bacteria and establish a chronology of their toxic effects on the bacterial cell. This review also includes novel structural results obtained by NMR concerning the sprG1-encoded membrane peptides that belong to the sprG1/SprF1 type I TA system expressed in Staphylococcus aureus and discusses the putative membrane interactions allowing the lysis of competing bacteria and host cells.

Pseudonajide peptide derived from snake venom alters cell envelope integrity interfering on biofilm formation in Staphylococcus epidermidis.

BACKGROUND: The increase in bacterial resistance phenotype cases is a global health problem. New strategies must be explored by the scientific community in order to create new treatment alternatives. Animal venoms are a good source for antimicrobial peptides (AMPs), which are excellent candidates for new antimicrobial drug development. Cathelicidin-related antimicrobial peptides (CRAMPs) from snake venoms have been studied as a model for the design of new antimicrobial pharmaceuticals against bacterial infections. RESULTS: In this study we present an 11 amino acid-long peptide, named pseudonajide, which is derived from a Pseudonaja textilis venom peptide and has antimicrobial and antibiofilm activity against Staphylococcus epidermidis. Pseudonajide was selected based on the sequence alignments of various snake venom peptides that displayed activity against bacteria. Antibiofilm activity assays with pseudonajide concentrations ranging from 3.12 to 100 µM showed that the lowest concentration to inhibit biofilm formation was 25 µM. Microscopy analysis demonstrated that pseudonajide interacts with the bacterial cell envelope, disrupting the cell walls and membranes, leading to morphological defects in prokaryotes. CONCLUSIONS: Our results suggest that pseudonajide's positives charges interact with negatively charged cell wall components of S. epidermidis, leading to cell damage and inhibiting biofilm formation.

The HIV-1 maturation inhibitor, EP39, interferes with the dynamic helix-coil equilibrium of the CA-SP1 junction of Gag.

During the maturation of HIV-1 particle, the Gag polyprotein is cleaved into several proteins by the HIV-1 protease. These proteins rearrange to form infectious virus particles. In this study, the solution structure and dynamics of a monomeric mutated domain encompassing the C-terminal of capsid, the spacer peptide SP1 and the nucleocapsid from Gag was characterized by Nuclear Magnetic Resonance in the presence of maturation inhibitor EP39, a more hydro-soluble derivative of BVM. We show that the binding of EP39 decreases the dynamics of CA-SP1 junction, especially the QVT motif in SP1, and perturbs the natural coil-helix equilibrium on both sides of the SP1 domain by stabilizing the transient alpha helical structure. Our results provide new insight into the structure and dynamics of the SP1 domain and how HIV-1 maturation inhibitors interfere with this domain. They offer additional clues for the development of new second generation inhibitors targeting HIV-1 maturation.

Reassembling green fluorescent protein for in vitro evaluation of trans-translation.

In order to discover new antibiotics with improved activity and selectivity, we created a reliable in vitro reporter system to detect trans-translation activity, the main mechanism for recycling ribosomes stalled on problematic messenger RNA (mRNA) in bacteria. This system is based on an engineered tmRNA variant that reassembles the green fluorescent protein (GFP) when trans-translation is active. Our system is adapted for high-throughput screening of chemical compounds by fluorescence.

In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution.

TmRNA is an abundant RNA in bacteria with tRNA and mRNA features. It is specialized in trans-translation, a translation rescuing system. We demonstrate that its partner protein SmpB binds the tRNA-like region (TLD) in vivo and chaperones the fold of the TLD-H2 region. We use an original approach combining the observation of tmRNA degradation pathways in a heterologous system, the analysis of the tmRNA digests by MS and NMR, and co-overproduction assays of tmRNA and SmpB. We study the conformation in solution of tmRNA alone or in complex with one SmpB before ribosome binding using SAXS. Our data show that Mg(2+) drives compaction of the RNA structure and that, in the absence of Mg(2+), SmpB has a similar effect albeit to a lesser extent. Our results show that tmRNA is intrinsically structured in solution with identical topology to that observed on complexes on ribosomes which should facilitate its subsequent recruitment by the 70S ribosome, free or preloaded with one SmpB molecule.

Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology.

RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA-protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA-His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

RNA-methyltransferase TrmA is a dual-specific enzyme responsible for C5-methylation of uridine in both tmRNA and tRNA.

In bacteria, trans-translation rescues stalled ribosomes by the combined action of tmRNA (transfer-mRNA) and its associated protein SmpB. The tmRNA 5' and 3' ends fold into a tRNA-like domain (TLD), which shares structural and functional similarities with tRNAs. As in tRNAs, the UUC sequence of the T-arm of the TLD is post-transcriptionally modified to m (5)UψC. In tRNAs of gram-negative bacteria, formation of m (5)U is catalyzed by the SAM-dependent methyltransferase TrmA, while formation of m (5)U at two different positions in rRNA is catalyzed by distinct site-specific methyltransferases RlmC and RlmD. Here, we show that m (5)U formation in tmRNAs is exclusively due to TrmA and should be considered as a dual-specific enzyme. The evidence comes from the lack of m (5)U in purified tmRNA or TLD variants recovered from an Escherichia coli mutant strain deleted of the trmA gene. Detection of m (5)U in RNA was performed by NMR analysis.

Functional and structural insights of a Staphylococcus aureus apoptotic-like membrane peptide from a toxin-antitoxin module.

We report a functional type I toxin-antitoxin (TA) module expressed by a human pathogen, Staphylococcus aureus. TA systems consist of stable toxins and labile antitoxins encoded within small genetic modules widespread in eubacteria and archaea. TA genes provide stress adaptation and protection against DNA loss or invasion. The genes encoding the SprA1 toxic peptide (PepA1) and the SprA1(AS) RNA antitoxin are within a pathogenicity island on opposite strands and possess a 3' overlap. To prevent peptide toxicity during S. aureus growth, PepA1 expression from stable (half-life > 3 h) SprA1 is repressed by elevated amounts of unstable (half-life = ∼10 mn) SprA1(AS). In vivo, PepA1 localizes at the bacterial membrane and triggers S. aureus death. Based on NMR and CD data, its solution structure was solved and is a long bent, interrupted helix. Molecular dynamics simulations indicate that PepA1 compaction and helical content fluctuate in accordance with its cytoplasm or membrane location. When inserted into the S. aureus membrane, the PepA1 conformation switches to a ∼7-nm-long continuous helix, presumably forming pores to alter membrane integrity. PepA1 expression is induced upon acidic and oxidative stresses by reducing SprA1(AS) levels. As an altruistic behavior during infection, some cells may induce the expression of that toxin that would facilitate departure from the host immune cells for spreading.

Selective RNase H cleavage of target RNAs from a tRNA scaffold.

In vivo overproduction of tRNA chimeras yields an RNA insert within a tRNA scaffold. For some applications, it may be necessary to discard the scaffold. Here we present a protocol for selective cleavage of the RNA of interest from the tRNA scaffold, using RNase H and two DNA oligonucleotides. After cleavage, we show that the RNA of interest can be isolated in a one-step purification. This method has, in particular, applications in structural investigations of RNA.

Crystal structure of an EAL domain in complex with reaction product 5'-pGpG.

FimX is a large multidomain protein containing an EAL domain and involved in twitching motility in Pseudomonas aeruginosa. We present here two crystallographic structures of the EAL domain of FimX (residues 438-686): one of the apo form and the other of a complex with 5'-pGpG, the reaction product of the hydrolysis of c-di-GMP. In both crystal forms, the EAL domains form a dimer delimiting a large cavity encompassing the catalytic pockets. The ligand is trapped in this cavity by its sugar phosphate moiety. We confirmed by NMR that the guanine bases are not involved in the interaction in solution. We solved here the first structure of an EAL domain bound to the reaction product 5'-pGpG. Though isolated FimX EAL domain has a very low catalytic activity, which would not be significant compared to other catalytic EAL domains, the structure with the product of the reaction can provides some hints in the mechanism of hydrolysis of the c-di-GMP by EAL domains.

A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold.

RNA production using in vivo transcription by Escherichia coli allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. We describe here a generic protocol for the overproduction and purification of recombinant RNA using liquid chromatography. The strategy utilizes a transfer RNA (tRNA) as a scaffold that can be removed from the RNA of interest by digestion of the fusion RNA at a designed site by RNase H. The tRNA scaffold serves to enhance the stability and to promote the proper expression of its fusion partners. This protocol describes how to construct a tRNA fusion RNA expression vector; to conduct a pilot experiment to assess the yield of the recombinant RNA both before and after processing of the fusion RNA by RNase H; and to purify the target RNA on a large scale for structural or functional studies. This protocol greatly facilitates production of RNA in a time frame of approximately 3 weeks from design to purification. As compared with in vitro methods (transcription, chemical synthesis), this approach is simple, cheap and well suited for large-scale expression and isotope labeling.

Ribosome hijacking: a role for small protein B during trans-translation.

Tight recognition of codon-anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the 'tmRNA-SmpB' system (transfer messenger RNA-small protein B). Remarkably, entry and accommodation of aminoacylated-tmRNA into stalled ribosomes occur without a codon-anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon-anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for DeltasmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon.

NMR structure of the Aquifex aeolicus tmRNA pseudoknot PK1: new insights into the recoding event of the ribosomal trans-translation.

The transfer-messenger RNA (tmRNA) pseudoknot PK1 is essential for bacterial trans-translation, a ribosomal rescue mechanism. We report the solution structure of PK1 from Aquifex aeolicus, which despite an unprecedented small number of nucleotides and thus an unprecented compact size, displays a very high thermal stability. Several unusual structural features account for these properties and indicate that PK1 belongs to the class of ribosomal frameshift pseudoknots. This suggests a similarity between the mechanism of programmed ribosomal frameshifting and trans-translation.

Self-organisation of an oligodeoxynucleotide containing the G- and C-rich stretches of the direct repeats of the human mitochondrial DNA.

Stretches of cytosines and guanosines have been shown in vitro to adopt non-canonical structures known as i-motifs and G-quartets, respectively. When combined, such sequences are expected to either retain their structure or form duplexes or triple helices. All these structures may occur in vivo whenever the sequence criteria are met. Such stretches are present in the circular genome of human mitochondria, as two 10 nucleotide-long perfect tandem direct repeats (DR1 and DR2). The DR1 and DR2 repeats are G-rich on the heavy strand and C-rich on the light strand. Previous results suggested that during replication, transient formation of a parallel GGC triple helix between the neo-synthesised G-rich DR1 and the double-stranded homologous DR2 could be involved in a rearrangement process leading to genome instability. In order to get structural insights into the interaction between the two repeats, we have studied by nuclear magnetic resonance (NMR) the assembly properties of a 24-mer oligodeoxyribonucleotide in which the C- and G-rich segments of the DRs are covalently tethered by a TTTT linker. We show here that this 24-mer self-associates into a triplex-containing symmetrical tetramer. The core of the structure is composed of anti-parallel Watson-Crick (WC) base pairs. Two additional strands are hydrogen-bonded to the Hoogsteen side of the Gs, thus forming CGC(+) triple helices, with G-rich ends folding into G-quartets. These results suggest that such structures could occur when the two DRs are put to close proximity in a biological context.

The tRNA-like domains of E coli and A.aeolicus transfer-messenger RNA: structural and functional studies.

Transfer-messenger RNA (tmRNA, 10Sa RNA or ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. The understanding at molecular level of this ubiquitous quality control system in eubacteria requires structural information. Here, we describe the purification and structural analysis of a functional fragment of both Aquifex aeolicus and Escherichia coli tmRNA, recapitulating their tRNA-like domain, which were expressed in vivo from synthetic genes. Both recombinant RNA are correctly processed at both 5' and 3' ends and are produced in quantities suitable for structural analysis by NMR and/or X-ray crystallography. The sequence and solution structure of the tRNA-like domains were analysed by various methods including structural mapping with chemical and enzymatic probes and 2D NMR spectroscopy. The minimalist RNAs contain two post-transcriptional base modifications, 5-methyluridine and pseudouridine, as the full-length tmRNA. Both RNAs fold into three stems, a D-analogue, a T-loop and a GAAA tetra-loop. 2D NMR analysis of the imino proton resonances of both RNAs allowed the assignment of the three stems and of a number of tertiary interactions. It shows the existence of interactions between the TPsiC-loop and the D-analogue, exhibiting a number of similarities and also differences with the canonical tRNA fold, indicating that RNA tertiary interactions can be modulated according to the sequence and secondary structure contexts. Furthermore, the E.coli minimalist RNA is aminoacylatable with alanine with a catalytic efficiency an order of magnitude higher than that for full-length tmRNA.