CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis.

The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T. vaginalis cell. We then created DNA constructs required to express several components essential to drive CRISPR/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the T. vaginalis genome. This first report of the use of the CRISPR/Cas9 system in T. vaginalis greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.

YoeB toxin is activated during thermal stress.

Type II toxin-antitoxin (TA) modules are thought to mediate stress-responses by temporarily suppressing protein synthesis while cells redirect transcription to adapt to environmental change. Here, we show that YoeB, a ribosome-dependent mRNase toxin, is activated in Escherichia coli cells grown at elevated temperatures. YoeB activation is dependent on Lon protease, suggesting that thermal stress promotes increased degradation of the YefM antitoxin. Though YefM is efficiently degraded in response to Lon overproduction, we find that Lon antigen levels do not increase during heat shock, indicating that another mechanism accounts for temperature-induced YefM proteolysis. These observations suggest that YefM/YoeB functions in adaptation to temperature stress. However, this response is distinct from previously described models of TA function. First, YoeB mRNase activity is maintained over several hours of culture at 42°C, indicating that thermal activation is not transient. Moreover, heat-activated YoeB does not induce growth arrest nor does it suppress global protein synthesis. In fact, E. coli cells proliferate more rapidly at elevated temperatures and instantaneously accelerate their growth rate in response to acute heat shock. We propose that heat-activated YoeB may serve a quality control function, facilitating the recycling of stalled translation complexes through ribosome rescue pathways.

Trichomonas vaginalis flavin reductase 1 and its role in metronidazole resistance.

The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defence in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis.

A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging.

In Escherichia coli, prolonged translational arrest allows mRNA degradation into the A site of stalled ribosomes. The enzyme that cleaves the A-site codon is not known, but its activity requires RNase II to degrade mRNA downstream of the ribosome. This A-site mRNA cleavage process is thought to function in translation quality control because stalled ribosomes are recycled from A-site truncated transcripts by the tmRNA-SmpB "ribosome rescue" system. During rescue, the tmRNA-encoded ssrA peptide is added to the nascent chain, thereby targeting the tagged protein for degradation after release from the ribosome. Here, we examine the influence of A-site mRNA cleavage upon tmRNA-SmpB activity. Using a model transcript that undergoes stop-codon cleavage in response to inefficient translation termination, we quantify ssrA-peptide tagging of the encoded protein in cells that contain (rnb(+)) or lack (Δrnb) RNase II. A-site mRNA cleavage is reduced approximately three-fold in Δrnb backgrounds, but the efficiency of ssrA-tagging is identical to that of rnb(+) cells. Additionally, pulse-chase analysis demonstrates that paused ribosomes recycle from the test transcripts at similar rates in rnb(+) and Δrnb cells. Together, these results indicate that A-site truncated transcripts are not required for tmRNA-SmpB-mediated ribosome rescue and suggest that A-site mRNA cleavage process may play a role in other recycling pathways.

Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis.

During protein synthesis, ribosomes translate the genetic information encoded within messenger RNAs into defined amino acid sequences. Transfer RNAs (tRNAs) are crucial adaptor molecules in this process, delivering amino acid residues to the ribosome and holding the nascent peptide chain as it is assembled. Here, we present methods for the analysis of aminoacyl- and peptidyl-tRNA species isolated from Escherichia coli. These approaches utilize denaturing gel electrophoresis at acidic pH to preserve the labile ester bonds that link amino acids to tRNA. Specific aminoacyl- and peptidyl-tRNAs are detected by Northern blot hybridization using probes for tRNA isoacceptors. Small peptidyl-tRNAs can be differentiated from aminoacyl-tRNA through selective deacylation of the latter with copper sulfate. Additionally, peptidyl-tRNAs can be detected through metabolic labeling of the nascent peptide. This approach is amenable to pulse-chase analysis to examine peptidyl-tRNA turnover in vivo. We have applied these methods to study programmed translational arrests and the kinetics of paused ribosome turnover.

The tmRNA ribosome-rescue system.

The bacterial tmRNA quality control system monitors protein synthesis and recycles stalled translation complexes in a process termed "ribosome rescue." During rescue, tmRNA acts first as a transfer RNA to bind stalled ribosomes, then as a messenger RNA to add the ssrA peptide tag to the C-terminus of the nascent polypeptide chain. The ssrA peptide targets tagged peptides for proteolysis, ensuring rapid degradation of potentially deleterious truncated polypeptides. Ribosome rescue also facilitates turnover of the damaged messages responsible for translational arrest. Thus, tmRNA increases the fidelity of gene expression by promoting the synthesis of full-length proteins. In addition to serving as a global quality control system, tmRNA also plays important roles in bacterial development, pathogenesis, and environmental stress responses. This review focuses on the mechanism of tmRNA-mediated ribosome rescue and the role of tmRNA in bacterial physiology.

Alternative fates of paused ribosomes during translation termination.

The bacterial tmRNA·SmpB system facilitates recycling of stalled translational complexes in a process termed "ribosome rescue." During ribosome rescue, the nascent chain is tagged with the tmRNA-encoded ssrA peptide, which targets the tagged polypeptide for degradation. Translational pausing also induces a variety of recoding events such as frameshifts, ribosome hops, and stop codon readthrough. To examine the interplay between recoding and ribosome rescue, we determined the various fates of ribosomes that pause during translation termination. We expressed a model protein containing the C-terminal Asp-Pro nascent peptide motif (which interferes with translation termination) and quantified the protein chains produced by recoding and ssrA-peptide tagging. The nature and extent of translational recoding depended upon the codon for the C-terminal Pro residue, with CCU and CCC promoting efficient +1 frameshifting. In contrast, ssrA-peptide tagging was unaffected by C-terminal Pro coding. Moreover, +1 frameshifting was not suppressed by tmRNA·SmpB activity, suggesting that recoding and ribosome rescue are not competing events. However, cells lacking ribosomal protein L9 (ΔL9) exhibited a significant increase in recoding and a concomitant decrease in ssrA-peptide tagging. Pulse-chase analysis revealed that pre-termination ribosomes turn over more rapidly in ΔL9 cells, suggesting that increased recoding alleviates the translational arrest. Together, these results indicate that tmRNA·SmpB does not suppress transient ribosome pauses, but responds to prolonged translational arrest.

tmRNA regulates synthesis of the ArfA ribosome rescue factor.

Translation of mRNA lacking an in-frame stop codon leads to ribosome arrest at the 3' end of the transcript. In bacteria, the tmRNA quality control system recycles these stalled ribosomes and tags the incomplete nascent chains for degradation. Although ubiquitous in eubacteria, the ssrA gene encoding tmRNA is not essential for the viability of Escherichia coli and other model bacterial species. ArfA (YhdL) is a mediator of tmRNA-independent ribosome rescue that is essential for the viability of E. coliΔssrA mutants. Here, we demonstrate that ArfA is synthesized from truncated mRNA and therefore regulated by tmRNA tagging activity. RNase III cleaves a hairpin structure within the arfA-coding sequence to produce transcripts that lack stop codons. In the absence of tmRNA tagging, truncated ArfA chains are released from the ribosome. The truncated ArfAΔ18 protein (which lacks 18 C-terminal residues) is functional in ribosome rescue and supports ΔssrA cell viability when expressed from the arfA locus. Other proteobacterial arfA genes also encode hairpins, and transcripts from Dickeya dadantii and Salmonella typhimurium are cleaved by RNase III when expressed in E. coli. Thus, synthesis of ArfA from truncated mRNA appears to be a general mechanism to regulate alternative ribosome rescue activity.

Translation factor LepA contributes to tellurite resistance in Escherichia coli but plays no apparent role in the fidelity of protein synthesis.

LepA is a translational GTPase highly conserved in bacterial lineages. While it has been shown that LepA can catalyze reverse ribosomal translocation in vitro, the role of LepA in the cell remains unclear. Here, we show that deletion of the lepA gene (DeltalepA) in Escherichia coli causes hypersensitivity to potassium tellurite and penicillin G, but has no appreciable effect on growth under many other conditions. DeltalepA does not increase miscoding or frameshifting errors under normal or stress conditions, indicating that LepA does not contribute to the fidelity of translation. Overexpression of LepA interferes with tmRNA-mediated peptide tagging and A-site mRNA cleavage, suggesting that LepA is a bona fide translation factor that can act on stalled ribosomes with a vacant A site in vivo. Together these results lead us to hypothesize that LepA is involved in co-translational folding of proteins that are otherwise vulnerable to tellurite oxidation.

Kinetics of paused ribosome recycling in Escherichia coli.

The bacterial tmRNA.SmpB system recycles stalled translation complexes in a process termed 'ribosome rescue.' tmRNA.SmpB specifically recognizes ribosomes that are paused at or near the 3' end of truncated mRNA; therefore, nucleolytic mRNA processing is required before paused ribosomes can be rescued from full-length transcripts. Here, we examine the recycling of ribosomes paused on both full-length and truncated mRNAs. Peptidyl-tRNAs corresponding to each paused translation complex were identified, and their turnover kinetics was used to estimate the half-lives of paused ribosomes in vivo. Ribosomes were paused at stop codons on full-length mRNA using a nascent peptide motif that interferes with translation termination and elicits tmRNA.SmpB activity. Peptidyl-tRNA turnover from these termination-paused ribosomes was slightly more rapid in tmRNA(+) cells (T(1/2)=22+/-2.2 s) than in DeltatmRNA cells (T(1/2)=32+/-1.6 s). Overexpression of release factor (RF) 1 greatly accelerated peptidyl-tRNA turnover from termination-paused ribosomes in both tmRNA(+) and DeltatmRNA cells, whereas other termination factors had little or no effect on recycling. In contrast to inefficient translation termination, ribosome recycling from truncated transcripts lacking in-frame stop codons was dramatically accelerated by tmRNA.SmpB. However, peptidyl-tRNA still turned over from nonstop-paused ribosomes at a significant rate (t(1/2)=61+/-7.3 s) in DeltatmRNA cells. Overexpression of RF-1, RF-3, and ribosome recycling factor in DeltatmRNA cells failed to accelerate ribosome recycling from nonstop mRNA. These results indicate that tmRNA.SmpB activity is rate limited by mRNA cleavage, and that RF-3 and ribosome recycling factor do not constitute a tmRNA-independent rescue pathway, as previously suggested. Peptidyl-tRNA turnover from nonstop-paused ribosomes in DeltatmRNA cells suggests the existence of another uncharacterized ribosome rescue pathway.

Prolyl-tRNA(Pro) in the A-site of SecM-arrested ribosomes inhibits the recruitment of transfer-messenger RNA.

Translational pausing can lead to cleavage of the A-site codon and facilitate recruitment of the transfer-messenger RNA (tmRNA) (SsrA) quality control system to distressed ribosomes. We asked whether aminoacyl-tRNA binding site (A-site) mRNA cleavage occurs during regulatory translational pausing using the Escherichia coli SecM-mediated ribosome arrest as a model. We find that SecM ribosome arrest does not elicit efficient A-site cleavage, but instead allows degradation of downstream mRNA to the 3'-edge of the arrested ribosome. Characterization of SecM-arrested ribosomes shows the nascent peptide is covalently linked via glycine 165 to tRNA(3Gly) in the peptidyl-tRNA binding site, and prolyl-tRNA(2Pro) is bound to the A-site. Although A-site-cleaved mRNAs were not detected, tmRNA-mediated ssrA tagging after SecM glycine 165 was observed. This tmRNA activity results from sequestration of prolyl-tRNA(2Pro) on overexpressed SecM-arrested ribosomes, which produces a second population of stalled ribosomes with unoccupied A-sites. Indeed, compensatory overexpression of tRNA(2Pro) readily inhibits ssrA tagging after glycine 165, but has no effect on the duration of SecM ribosome arrest. We conclude that, under physiological conditions, the architecture of SecM-arrested ribosomes allows regulated translational pausing without interference from A-site cleavage or tmRNA activities. Moreover, it seems likely that A-site mRNA cleavage is generally avoided or inhibited during regulated ribosome pauses.