Peptidyl-tRNA hydrolase is the nascent chain release factor in bacterial ribosome-associated quality control.

Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis.

The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin-antitoxin and related phage defense systems.

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.

Structural basis for HflXr-mediated antibiotic resistance in Listeria monocytogenes.

HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.

Novel CRISPR-based detection of Leishmania species.

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10-2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

A low-cost and open-source protocol to produce key enzymes for molecular detection assays.

Here, we describe a detailed step-by-step protocol for the expression, purification, quantification, and activity determination of key enzymes for molecular detection of pathogens. Based on previous reports, we optimized the protocol for LbCas12a, Taq DNA polymerase, M-MLV reverse transcriptase, and TEV protease to make it compatible with minimal laboratory equipment, broadly available in low- and middle-income countries. The enzymes produced with this protocol have been successfully used for molecular detection applications. For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a, 2021b).

The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions.

During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both in vitro and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control. IMPORTANCE The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown. Our previous work revealed that (p)ppGpp can bind to and inhibit the enzymatic activity of four ribosome-associated GTPases (RA-GTPases), enzymes that facilitate maturation of the 50S and 30S ribosomal subunits. Here, we examine how this occurs mechanistically and demonstrate that this interaction prevents the accommodation of RA-GTPases on ribosomal subunits both in vitro and within bacterial cells, with the ppGpp-bound state structurally mimicking the inactive GDP-bound conformation of the enzyme. We additionally reveal that these GTPase enzymes have a greater affinity for OFF-state-inducing nucleotides, which is a mechanism likely to control ribosome assembly during growth. With this, we further our understanding of how ribosome function is controlled by (p)ppGpp, enabling bacterial survival during stress.

Unlocking SARS-CoV-2 detection in low- and middle-income countries.

Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch. The optimized one-step RT-PCR coupled to CRISPR-Cas12a-mediated detection showed a limit of detection of 102 ge/µL in a turnaround time of 2 h. The clinical validation indicated an overall sensitivity of 80%-88%, while for middle and high viral load samples (Cq ≤ 31) the sensitivity was 92%-100%. The specificity was 96%-100% regardless of viral load. Furthermore, we show that the toolkit can be used with the mobile laboratory Bento Lab, potentially enabling LMICs to implement detection services in unattended remote regions.

UnCovid: A versatile, low-cost, and open-source protocol for SARS-CoV-2 RNA detection.

Here, we describe a detailed step-by-step protocol to detect SARS-CoV-2 RNA using RT-PCR-mediated amplification and CRISPR/Cas-based visualization. The optimized assay uses basic molecular biology equipment such as conventional thermocyclers and transilluminators for qualitative detection. Alternatively, a fluorescence plate reader can be used for quantitative measurements. The protocol detects two regions of the SARS-CoV-2 genome in addition to the human RNaseP sample control. Aiming to reach remote regions, this work was developed to use the portable molecular workstation from BentoLab. For complete details on the use and execution of this protocol, please refer to Alcántara et al., 2021.

The dynamic cycle of bacterial translation initiation factor IF3.

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.

DNA aptamers for the recognition of HMGB1 from Plasmodium falciparum.

Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable, and present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in a few seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a candidate biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection.