Inactivation of the ribosome assembly factor RimP causes streptomycin resistance and impairs motility in Salmonella.

The ribosome is the central hub for protein synthesis and the target of many antibiotics. Although the majority of ribosome-targeting antibiotics inhibit protein synthesis and are bacteriostatic, aminoglycosides promote protein mistranslation and are bactericidal. Understanding the resistance mechanisms of bacteria against aminoglycosides is not only vital for improving the efficacy of this critically important group of antibiotics but also crucial for studying the molecular basis of translational fidelity. In this work, we analyzed Salmonella mutants evolved in the presence of the aminoglycoside streptomycin (Str) and identified a novel gene rimP to be involved in Str resistance. RimP is a ribosome assembly factor critical for the maturation of the 30S small subunit that binds Str. Deficiency in RimP increases resistance against Str and facilitates the development of even higher resistance. Deleting rimP decreases mistranslation and cellular uptake of Str and further impairs flagellar motility. Our work thus highlights a previously unknown mechanism of aminoglycoside resistance via defective ribosome assembly.

Salmonella Typhimurium infection inhibits macrophage IFNß signaling in a TLR4-dependent manner.

Type I Interferons (IFNs) generally have a protective role during viral infections, but their function during bacterial infections is dependent on the bacterial species. Legionella pneumophila, Shigella sonnei and Mycobacterium tuberculosis can inhibit type I IFN signaling. Here we examined the role of type I IFN, specifically IFNß, in the context of Salmonella enterica serovar Typhimurium (STm) macrophage infections and the capacity of STm to inhibit type I IFN signaling. We demonstrate that IFNß has no effect on the intracellular growth of STm in infected bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice. STm infection inhibits IFNß signaling but not IFNγ signaling in a murine macrophage cell line. We show that this inhibition is independent of the type III and type VI secretion systems expressed by STm and is also independent of bacterial phagocytosis. The inhibition is Toll-like receptor 4 (TLR4)-dependent as the TLR4 ligand, lipopolysaccharide (LPS), alone is sufficient to inhibit IFNß-mediated signaling and STm-infected, TLR4-deficient BMDMs do not exhibit inhibited IFNß signaling. In summary, we show that macrophages exposed to STm have reduced IFNß signaling via crosstalk with TLR4 signaling, and that IFNß signaling does not affect cell autonomous host defense against STm.

Serine mistranslation induces the integrated stress response without accumulation of uncharged tRNAs.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that support robust and accurate protein synthesis. A rapidly expanding number of studies show that mutations in aaRSs lead to multiple human diseases, including neurological disorders and cancer. Much remains unknown about how aaRS mutations impact human health. In particular, how aminoacylation errors affect stress responses and fitness in eukaryotic cells remains poorly understood. The integrated stress response (ISR) is an adaptive mechanism in response to multiple stresses. However, chronic activation of the ISR contributes to the development of multiple diseases (e.g., neuropathies). Here we show that Ser misincorporation into Ala and Thr codons, resulting from aaRS editing defects or mutations in tRNAs, constitutively active the ISR. Such activation does not appear to depend on the accumulation of uncharged tRNAs, implicating that Ser mistranslation may lead to ribosome stalling and collision.

Deficiency in ribosome biogenesis causes streptomycin resistance and impairs motility in Salmonella.

The ribosome is the central hub for protein synthesis and the target of many antibiotics. Whereas the majority of ribosome-targeting antibiotics inhibit protein synthesis and are bacteriostatic, aminoglycosides promote protein mistranslation and are bactericidal. Understanding the resistance mechanisms of bacteria against aminoglycosides is not only vital for improving the efficacy of this critically important group of antibiotics but also crucial for studying the molecular basis of translational fidelity. In this work, we analyzed Salmonella mutants evolved in the presence of the aminoglycoside streptomycin (Str) and identified a novel gene rimP to be involved in Str resistance. RimP is a ribosome assembly factor critical for the maturation of the 30S small subunit that binds Str. Deficiency in RimP increases resistance against Str and facilitates the development of even higher resistance. Deleting rimP decreases mistranslation and cellular uptake of Str, and further impairs flagellar motility. Our work thus highlights a previously unknown mechanism of aminoglycoside resistance via defective ribosome assembly.

BBQ methods: streamlined workflows for bacterial burden quantification in infected cells by confocal microscopy.

Accurate quantification of bacterial burden within macrophages, termed bacterial burden quantification (BBQ), is crucial for understanding host-pathogen interactions. Various methods have been employed, each with strengths and weaknesses. This article addresses limitations in existing techniques and introduces two novel, automated methods for BBQ within macrophages based on confocal microscopy data analysis. The first method refines total fluorescence quantification by incorporating filtering steps to exclude uninfected cells, while the second method calculates total bacterial volume per cell to mitigate potential biases in fluorescence-based readouts. These workflows utilize PyImageJ and Cellpose software, providing reliable, unbiased, and rapid quantification of bacterial load. The proposed workflows were validated using Salmonella enterica serovar Typhimurium and Mycobacterium tuberculosis models, demonstrating their effectiveness in accurately assessing bacterial burden. These automated workflows offer valuable tools for studying bacterial interactions within host cells and provide insights for various research applications.

BBQ methods: Streamlined workflows for Bacterial Burden Quantification in infected cells by confocal microscopy.

Accurate quantification of bacterial burden within macrophages, termed Bacterial Burden Quantification (BBQ), is crucial for understanding host-pathogen interactions. Various methods have been employed, each with strengths and weaknesses. This article addresses limitations in existing techniques and introduces two novel automated methods for BBQ within macrophages based on confocal microscopy data analysis. The first method refines total fluorescence quantification by incorporating filtering steps to exclude uninfected cells, while the second method calculates total bacterial volume per cell to mitigate potential biases in fluorescence-based readouts. These workflows utilize PyImageJ and Cellpose software, providing reliable, unbiased, and rapid quantification of bacterial load. The proposed workflows were validated using Salmonella enterica serovar Typhimurium and Mycobacterium tuberculosis models, demonstrating their effectiveness in accurately assessing bacterial burden. These automated workflows offer valuable tools for studying bacterial interactions within host cells and provide insights for various research applications.

Coordination between aminoacylation and editing to protect against proteotoxicity.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ligate amino acids to tRNAs, and often require editing to ensure accurate protein synthesis. Recessive mutations in aaRSs cause various neurological disorders in humans, yet the underlying mechanism remains poorly understood. Pathogenic aaRS mutations frequently cause protein destabilization and aminoacylation deficiency. In this study, we report that combined aminoacylation and editing defects cause severe proteotoxicity. We show that the ths1-C268A mutation in yeast threonyl-tRNA synthetase (ThrRS) abolishes editing and causes heat sensitivity. Surprisingly, experimental evolution of the mutant results in intragenic mutations that restore heat resistance but not editing. ths1-C268A destabilizes ThrRS and decreases overall Thr-tRNAThr synthesis, while the suppressor mutations in the evolved strains improve aminoacylation. We further show that deficiency in either ThrRS aminoacylation or editing is insufficient to cause heat sensitivity, and that ths1-C268A impairs ribosome-associated quality control. Our results suggest that aminoacylation deficiency predisposes cells to proteotoxic stress.

Genome-wide screening reveals metabolic regulation of stop-codon readthrough by cyclic AMP.

Translational fidelity is critical for microbial fitness, survival and stress responses. Much remains unknown about the genetic and environmental control of translational fidelity and its single-cell heterogeneity. In this study, we used a high-throughput fluorescence-based assay to screen a knock-out library of Escherichia coli and identified over 20 genes critical for stop-codon readthrough. Most of these identified genes were not previously known to affect translational fidelity. Intriguingly, we show that several genes controlling metabolism, including cyaA and crp, enhance stop-codon readthrough. CyaA catalyzes the synthesis of cyclic adenosine monophosphate (cAMP). Combining RNA sequencing, metabolomics and biochemical analyses, we show that deleting cyaA impairs amino acid catabolism and production of ATP, thus repressing the transcription of rRNAs and tRNAs to decrease readthrough. Single-cell analyses further show that cAMP is a major driver of heterogeneity in stop-codon readthrough and rRNA expression. Our results highlight that carbon metabolism is tightly coupled with stop-codon readthrough.

Translational Fidelity during Bacterial Stresses and Host Interactions.

Translational fidelity refers to accuracy during protein synthesis and is maintained in all three domains of life. Translational errors occur at base levels during normal conditions and may rise due to mutations or stress conditions. In this article, we review our current understanding of how translational fidelity is perturbed by various environmental stresses that bacterial pathogens encounter during host interactions. We discuss how oxidative stress, metabolic stresses, and antibiotics affect various types of translational errors and the resulting effects on stress adaption and fitness. We also discuss the roles of translational fidelity during pathogen-host interactions and the underlying mechanisms. Many of the studies covered in this review will be based on work with Salmonella enterica and Escherichia coli, but other bacterial pathogens will also be discussed.

Increase in Ribosomal Fidelity Benefits Salmonella upon Bile Salt Exposure.

Translational fidelity is maintained by multiple quality control steps in all three domains of life. Increased translational errors (mistranslation) occur due to genetic mutations and external stresses. Severe mistranslation is generally harmful, but moderate levels of mistranslation may be favored under certain conditions. To date, little is known about the link between translational fidelity and host-pathogen interactions. Salmonella enterica can survive in the gall bladder during systemic or chronic infections due to bile resistance. Here we show that increased translational fidelity contributes to the fitness of Salmonella upon bile salt exposure, and the improved fitness depends on an increased level of intracellular adenosine triphosphate (ATP). Our work thus reveals a previously unknown linkage between translational fidelity and bacterial fitness under bile stress.

Impact of alanyl-tRNA synthetase editing deficiency in yeast.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that provide the ribosome with aminoacyl-tRNA substrates for protein synthesis. Mutations in aaRSs lead to various neurological disorders in humans. Many aaRSs utilize editing to prevent error propagation during translation. Editing defects in alanyl-tRNA synthetase (AlaRS) cause neurodegeneration and cardioproteinopathy in mice and are associated with microcephaly in human patients. The cellular impact of AlaRS editing deficiency in eukaryotes remains unclear. Here we use yeast as a model organism to systematically investigate the physiological role of AlaRS editing. Our RNA sequencing and quantitative proteomics results reveal that AlaRS editing defects surprisingly activate the general amino acid control pathway and attenuate the heatshock response. We have confirmed these results with reporter and growth assays. In addition, AlaRS editing defects downregulate carbon metabolism and attenuate protein synthesis. Supplying yeast cells with extra carbon source partially rescues the heat sensitivity caused by AlaRS editing deficiency. These findings are in stark contrast with the cellular effects caused by editing deficiency in other aaRSs. Our study therefore highlights the idiosyncratic role of AlaRS editing compared with other aaRSs and provides a model for the physiological impact caused by the lack of AlaRS editing.

Heterogeneous Flagellar Expression in Single Salmonella Cells Promotes Diversity in Antibiotic Tolerance.

Phenotypic heterogeneity among single cells in a genetically identical population leads to diverse environmental adaptation. The human and animal pathogen Salmonella enterica serovar Typhimurium exhibits heterogeneous expression of virulence genes, including flagellar and Salmonella pathogenicity island (SPI) genes. Little is known about how the differential expression of flagellar genes among single cells affects bacterial adaptation to stresses. Here, we have developed a triple-fluorescence reporter to simultaneously monitor the expression of flagellar and SPI-1 pathways. We show that the two pathways cross talk at the single-cell level. Intriguingly, cells expressing flagella (fliC-ON) exhibit decreased tolerance to antibiotics compared to fliC-OFF cells. Such variation depends on TolC-dependent efflux pumps. We further show that fliC-ON cells contain higher intracellular proton concentrations. This suggests that the assembly and rotation of flagella consume the proton motive force and decrease the efflux activity, resulting in antibiotic sensitivity. Such a trade-off between motility and efflux highlights a novel mechanism of antibiotic tolerance. IMPORTANCE Antibiotic resistance and tolerance pose a severe threat to human health. How bacterial pathogens acquire antibiotic tolerance is not clear. Here, we show that the human and animal pathogen Salmonella divides its population into subgroups that are different in their abilities to tolerate antibiotic treatments. In a Salmonella population that is genetically identical, some cells express flagella to move toward nutrients, while other cells do not express flagella. Interestingly, we show that Salmonella cells that do not express flagella are more tolerant to antibiotics. We have further determined the mechanism underlying such diverse responses to antibiotics. Flagellar motility uses cellular energy stored in the form of proton motive force and makes cells less efficient in pumping out toxic molecules such as antibiotics. The overall bacterial population therefore gains benefits from such diversity to quickly adapt to different environmental conditions.

Metabolic stress promotes stop-codon readthrough and phenotypic heterogeneity.

Accurate protein synthesis is a tightly controlled biological process with multiple quality control steps safeguarded by aminoacyl-transfer RNA (tRNA) synthetases and the ribosome. Reduced translational accuracy leads to various physiological changes in both prokaryotes and eukaryotes. Termination of translation is signaled by stop codons and catalyzed by release factors. Occasionally, stop codons can be suppressed by near-cognate aminoacyl-tRNAs, resulting in protein variants with extended C termini. We have recently shown that stop-codon readthrough is heterogeneous among single bacterial cells. However, little is known about how environmental factors affect the level and heterogeneity of stop-codon readthrough. In this study, we have combined dual-fluorescence reporters, mass spectrometry, mathematical modeling, and single-cell approaches to demonstrate that a metabolic stress caused by excess carbon substantially increases both the level and heterogeneity of stop-codon readthrough. Excess carbon leads to accumulation of acid metabolites, which lower the pH and the activity of release factors to promote readthrough. Furthermore, our time-lapse microscopy experiments show that single cells with high readthrough levels are more adapted to severe acid stress conditions and are more sensitive to an aminoglycoside antibiotic. Our work thus reveals a metabolic stress that promotes translational heterogeneity and phenotypic diversity.

Trade-Offs between Speed, Accuracy, and Dissipation in tRNAIle Aminoacylation.

Living systems maintain a high fidelity in information processing through kinetic proofreading, a mechanism for preferentially removing incorrect substrates at the cost of energy dissipation and slower speed. Proofreading mechanisms must balance their demand for higher speed, fewer errors, and lower dissipation, but it is unclear how rates of individual reaction steps are evolutionarily tuned to balance these needs, especially when multiple proofreading mechanisms are present. Here, using a discrete-state stochastic model, we analyze the optimization strategies in Escherichia coli isoleucyl-tRNA synthetase. Surprisingly, this enzyme adopts an economic proofreading strategy and improves speed and dissipation as long as the error is tolerable. Through global parameter sampling, we reveal a fundamental dissipation-error relation that bounds the enzyme's optimal performance and explains the importance of the post-transfer editing mechanism. The proximity of native system parameters to this bound demonstrates the importance of energy dissipation as an evolutionary force affecting fitness.

Increased mistranslation protects E. coli from protein misfolding stress due to activation of a RpoS-dependent heat shock response.

The misincorporation of an incorrect amino acid into a polypeptide during protein synthesis is considered a detrimental phenomenon. A mistranslated protein is often misfolded and degraded or nonfunctional and results in an increased cost to quality control machinery. Despite these costs, errors during protein synthesis are common in bacteria. Here, we report that mistranslation in Escherichia coli increase the protein level of the heat shock sigma factor RpoH and protect cells against heat stress. Surprisingly, this increase in RpoH due to mistranslation is dependent on the presence of the general stress response sigma factor RpoS. This report provides evidence for a protective function of mistranslation and suggests a novel regulatory role of RpoS in the heat shock response.

Optimal translational fidelity is critical for Salmonella virulence and host interactions.

Translational fidelity is required for accurate flow of genetic information, but is frequently altered by genetic changes and environmental stresses. To date, little is known about how translational fidelity affects the virulence and host interactions of bacterial pathogens. Here we show that surprisingly, either decreasing or increasing translational fidelity impairs the interactions of the enteric pathogen Salmonella Typhimurium with host cells and its fitness in zebrafish. Host interactions are mediated by Salmonella pathogenicity island 1 (SPI-1). Our RNA sequencing and quantitative RT-PCR results demonstrate that SPI-1 genes are among the most down-regulated when translational fidelity is either increased or decreased. Further, this down-regulation of SPI-1 genes depends on the master regulator HilD, and altering translational fidelity destabilizes HilD protein via enhanced degradation by Lon protease. Our work thus reveals that optimal translational fidelity is pivotal for adaptation of Salmonella to the host environment, and provides important mechanistic insights into this process.

Global analysis of tRNA and translation factor expression reveals a dynamic landscape of translational regulation in human cancers.

The protein translational system, including transfer RNAs (tRNAs) and several categories of enzymes, plays a key role in regulating cell proliferation. Translation dysregulation also contributes to cancer development, though relatively little is known about the changes that occur to the translational system in cancer. Here, we present global analyses of tRNAs and three categories of enzymes involved in translational regulation in ~10,000 cancer patients across 31 cancer types from The Cancer Genome Atlas. By analyzing the expression levels of tRNAs at the gene, codon, and amino acid levels, we identified unequal alterations in tRNA expression, likely due to the uneven distribution of tRNAs decoding different codons. We find that overexpression of tRNAs recognizing codons with a low observed-over-expected ratio may overcome the translational bottleneck in tumorigenesis. We further observed overall overexpression and amplification of tRNA modification enzymes, aminoacyl-tRNA synthetases, and translation factors, which may play synergistic roles with overexpression of tRNAs to activate the translational systems across multiple cancer types.

Errors during Gene Expression: Single-Cell Heterogeneity, Stress Resistance, and Microbe-Host Interactions.

Gene expression has been considered a highly accurate process, and deviation from such fidelity has been shown previously to be detrimental for the cell. More recently, increasing evidence has supported the notion that the accuracy of gene expression is indeed flexibly variable. The levels of errors during gene expression differ from condition to condition and even from cell to cell within genetically identical populations grown under the same conditions. The different levels of errors resulting from inaccurate gene expression are now known to play key roles in regulating microbial stress responses and host interactions. This minireview summarizes the recent development in understanding the level, regulation, and physiological impact of errors during gene expression.