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.

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.

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.

Broadband Terahertz Detection by Laser Plasma with Balanced Optical Bias.

Using a controlled optical bias and balanced geometry, we propose a new scheme for broadband terahertz detection by laser-gas interaction without high-voltage manipulation. Compared to the conventional optical bias scheme, the common noise is reduced and the dynamic range as well as the signal-to-noise ratio are doubled. It provides a simple alternative for coherent broadband terahertz detection. The influence of optical bias on terahertz waveform is also investigated, and the evolution of the terahertz-induced second harmonic with probe delay is further revealed. This new detection scheme for broadband terahertz will boost the application of terahertz time-domain spectroscopy for its miniaturization and integrability.

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.

Selection and Identification of Novel Antibacterial Agents against Planktonic Growth and Biofilm Formation of Enterococcus faecalis.

YycFG, one of the two-component systems involved in the regulation of biofilm formation, has attracted increasing interest as a potential target of antibacterial and antibiofilm agents. YycG inhibitors for Staphylococcus aureus and Staphylococcus epidermidis have been developed, but Enterococcus faecalis remains underexplored. Herein, we selected and identified novel candidate molecules against E. faecalis targeting histidine kinase YycG using high-throughput virtual screening; six molecules (compound-16, -30, -42, -46, -59, and -62) with low cytotoxicity toward mammalian cells were verified as potential YycG inhibitors through an autophosphorylation test and binding kinetics. Compound-16 inhibited planktonic cells of E. faecalis, including the vancomycin- or linezolid-resistant strains. In contrast, compound-62 did not affect planktonic growth but significantly inhibited biofilm formation in static and dynamic conditions. Compound-62 combined with ampicillin could synergistically eradicate the biofilm-embedded viable bacteria. The study demonstrates that YycG inhibitors may be valuable approaches for the development of novel antimicrobial agents for difficult-to-treat bacterial infections.

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.

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.

Monoclonal Antibodies Specific to the Extracellular Domain of Histidine Kinase YycG of Staphylococcus epidermidis Inhibit Biofilm Formation.

Staphylococcus epidermidis is frequently associated with biofilm-related infections. Biofilms drastically reduce the efficacy of conventional antibiotics and the host immune system. In S. epidermidis biofilm formation, a major role is played by the YycG/YycF two-component system, and previous findings have indicated that inhibitors targeting the cytoplasmic HATPase_c domain of YycG kinase in S. epidermidis exhibit bactericidal and biofilm-killing activities. Therefore, we hypothesized that monoclonal antibodies (mAbs) against YycG extracellular (YycGex) domain would block the signal transduction and influence the biofilm formation of S. epidermidis. In this study, we screened out two YycGex-specific mAbs showing the highest affinity for the target, mAbs 2F3 and 1H1. These mAbs inhibited S. epidermidis biofilm formation in a dose-dependent manner, and at a concentration of 160 µg/mL, mAbs 2F3 and 1H1 caused 78.3 and 93.1% biofilm reduction, respectively, relative to normal mouse IgG control. When co-cultivated with YycGex mAbs, S. epidermidis cells showed diminished initial-adherence capacity, and the antibody treatment further led to a marked decrease in the synthesis of polysaccharide intercellular adhesin and in the transcriptional level of genes encoding proteins involved in biofilm formation. Lastly, we determined that the epitopes recognized by the two YycGex mAbs are located within aa 59-70 of the YycGex domain. It indicates that the YycGex domain may be a potential candidate as a vaccine for the prevention of S. epidermidis biofilm infections.

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.

Staphylococcus aureus PhoU Homologs Regulate Persister Formation and Virulence.

PhoU homologs are one of the determinant factors in the regulation of persister formation and phosphate metabolism in many bacterial species; however, the functions of PhoU homologs exhibit species-specific characteristics. The pathogenesis of Staphylococcus aureus is closely correlated with persister formation and virulence factors. The functions of two PhoU homologs, PhoU1 and PhoU2, in S. aureus are unclear yet. In this study, single- and double-deletion mutants of phoU1 and phoU2 were generated in strain USA500 2395. The ΔphoU1 or ΔphoU2 mutants displayed a change in persister formation and virulence compared to the parent strain; the persisters to vancomycin and levofloxacin were decreased at least 1,000-fold, and the number of intracellular bacteria surviving in the A549 cells for 24 h decreased to 82 or 85%. The α-hemolysin expression and activity were increased in the ΔphoU2 mutants. Transcriptome analysis revealed that 573 or 285 genes were differentially expressed by at least 2.0-fold in the ΔphoU1 or ΔphoU2 mutant vs. the wild type. Genes involved in carbon and pyruvate metabolism were up-regulated, and virulence genes and virulence regulatory genes were down-regulated, including type VII secretion system, serine protease, leukocidin, global regulator (sarA, rot), and the two-component signal transduction system (saeS). Correspondingly, the deletion of the phoU1 or phoU2 resulted in increased levels of intracellular pyruvate and ATP. Deletion of the phoU2, but not the phoU1, resulted in the up-regulation of inorganic phosphate transport genes and increased levels of intracellular inorganic polyphosphate. In conclusion, both PhoU1 and PhoU2 in S. aureus regulate virulence by the down-regulation of multiple virulence factors (type VII secretion system, serine protease, and leucocidin) and the persister generation by hyperactive carbon metabolism accompanied by increasing intracellular ATP. The results in S. aureus are different from what we have previously found in Staphylococcus epidermis, where only PhoU2 regulates biofilm and persister formation. The different functions of PhoU homologs between the two species of Staphylococcus warrant further investigation.

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.