Targeting intracellular nontuberculous mycobacteria and M. tuberculosis with a bactericidal enzymatic cocktail.

To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This cocktail is delivered to macrophages, through a targeted liposome presented here as ENTX_001. Endolytix Cocktail 1 (EC1) leverages mycobacteriophage lysin enzymes LysA and LysB, while also including α-amylase and isoamylase for degradation of the mycobacterial envelope from outside of the cell. The LysA family of proteins from mycobacteriophages has been shown to cleave the peptidoglycan layer, whereas LysB is an esterase that hydrolyzes the linkage between arabinogalactan and mycolic acids of the mycomembrane. The challenge of gaining access to the substrates of LysA and LysB provided exogenously was addressed by adding amylase enzymes that degrade the extracellular capsule shown to be present in Mycobacterium tuberculosis. This enzybiotic approach avoids antimicrobial resistance, specific receptor-mediated binding, and intracellular DNA surveillance pathways that limit many bacteriophage applications. We show this cocktail of enzymes is bactericidal in vitro against both rapid- and slow-growing nontuberculous mycobacteria (NTM) as well as M. tuberculosis strains. The EC1 cocktail shows superior killing activity when compared to previously characterized LysB alone. EC1 is also powerfully synergistic with standard-of-care antibiotics. In addition to in vitro killing of NTM, ENTX_001 demonstrates the rescue of infected macrophages from necrotic death by Mycobacteroides abscessus and Mycobacterium avium. Here, we demonstrate shredding of mycobacterial cells by EC1 into cellular debris as a mechanism of bactericide.IMPORTANCEThe world needs entirely new forms of antibiotics as resistance to chemical antibiotics is a critical problem facing society. We addressed this need by developing a targeted enzyme therapy for a broad range of species and strains within mycobacteria and highly related genera including nontuberculous mycobacteria such as Mycobacteroides abscessus, Mycobacterium avium, Mycobacterium intracellulare, as well as Mycobacterium tuberculosis. One advantage of this approach is the ability to drive our lytic enzymes through encapsulation into macrophage-targeted liposomes resulting in attack of mycobacteria in the cells that harbor them where they hide from the adaptive immune system and grow. Furthermore, this approach shreds mycobacteria independent of cell physiology as the drug targets the mycobacterial envelope while sidestepping the host range limitations observed with phage therapy and resistance to chemical antibiotics.

Spatial heterogeneity in biofilm metabolism elicited by local control of phenazine methylation.

Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacterium Pseudomonas aeruginosa produces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. In this study, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone of P. aeruginosa biofilms. Deletion of rpoS or crc therefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity-an apparent beneficial effect-in hypoxic/anoxic subpopulations within biofilms. However, we also find that under specific conditions, biofilms lacking RpoS and/or Crc show increased sensitivity to phenazines indicating that the increased metabolic activity in these mutants comes at a cost. Together, these results suggest that complex regulation of PhzM allows P. aeruginosa to simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.

Spatial heterogeneity in biofilm metabolism elicited by local control of phenazine methylation.

Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacterium Pseudomonas aeruginosa produces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. Here, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone of P. aeruginosa biofilms. Deletion of rpoS or crc therefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity-an apparent beneficial effect-in hypoxic/anoxic subpopulations within biofilms. However, we also find that biofilms lacking Crc show increased sensitivity to an exogenously added methylated phenazine, indicating that the increased metabolic activity in this mutant comes at a cost. Together, these results suggest that complex regulation of PhzM allows P. aeruginosa to simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.

The survivorship of revision total hip replacement with severe proximal bone deficiency using a modular taper fluted prosthesis.

Contemporary uncemented femoral revision hip systems have become commonly used over the past decade and have enabled the reconstruction of leg length, offset and anteversion as independent variables through the use of modular junctions. Modular junction failures between the proximal body and distal stem have been described with revision systems, although this is rare. We sought to identify the survivorship of one revision system in a salvage arthroplasty scenario where no host bone support of the modular junction was present. From a series of 136 patients, 15 patients (16 hips) were identified without host bone support of the modular junction with a mean radiological follow up of over 6 years (76 months +/- 35 months). There have been no cases of prosthetic fracture over the follow-up duration, with two revisions performed for reasons of aseptic loosening and infection. The mean BMI of the study group was 30.2 with 78% of the cohort classified as overweight or obese. It is well recognised that, host bone support of the modular junction is preferable, however the satisfactory outcomes over the midterm in these complex patients suggests that modular revision systems remain an option.

Anthropometric method for estimating component sizes in total hip arthroplasty.

BACKGROUND: Preoperative templating is essential in total hip arthroplasty (THA) as it not only helps to facilitate the correct implant type and size but also determines the post-operative biomechanics. Templating is also increasingly important from a medico-legal perspective and recommended in the British Orthopaedic Association Guide to Good Practice. Although templating has become increasingly digitised, there are no simple anthropometric models to predict implant sizes in the absence of digital methods. AIM: To assess the accuracy of using an easily obtainable measurement (shoe size) to predict component sizes in THA compared with digital templating. METHODS: Digital radiographs from a cohort of 102 patients (40 male, 62 female) who had undergone uncemented or hybrid THA at a single centre were retrospectively templated to desired cup and stem sizes using TraumaCad ® . We compared the templated size to the actual size of the implant and assessed if there was any correlation with the patient's shoe size. RESULTS: Statistically significant positive correlations were observed between: shoe size and templated cup size (ρ = 0.92, P < 0.001); shoe size with implanted cup size (ρ = 0.71, P < 0.001); shoe size and templated stem size (ρ = 0.87, P < 0.001); and shoe size with implanted stem size (ρ = 0.57, P < 0.001). Templated and implanted acetabular cup sizes were positively correlated (ρ = 0.76, P < 0.001) and were exact in 43.1% cases; 80.4% of implanted cup sizes were within 1 size (+/- 2 mm) of the template and 100% within 2 sizes (+/- 4 mm). Positive correlation was also demonstrated between templated and implanted femoral stem sizes (ρ = 0.69, P < 0.001) and were exact in 52.6% cases; 92.6% were within 1 size of the template and 98% within 2 sizes. CONCLUSION: This study has shown there to be a significant positive correlation between shoe size and templated size. Anthropometric measurements are easily obtainable and can be used to predict uncemented component sizes in the absence of digital methods.

Loss of the ClpXP Protease Leads to Decreased Resistance to Cell-Envelope Targeting Antimicrobials in Bacillus anthracis Sterne.

The ClpX ATPase is critical for resistance to cell envelope targeting antibiotics in Bacillus anthracis, however, it is unclear whether this is due to its function as an independent chaperone or as part of the ClpXP protease. In this study, we demonstrate that antibiotic resistance is due to formation of the ClpXP protease through construction of a ClpX complementation plasmid that is unable to interact with ClpP. Additionally, we genetically disrupted both clpP genes, clpP1 and clpP2, found in B. anthracis Sterne and find that the loss of either increases susceptibility to cell envelope targeting antimicrobials, although neither has as strong of a phenotype as loss of clpX and neither clpP gene is essential for virulence in a G. mellonella model of infection. Lastly, we looked at changes to cell envelope morphology that could contribute to increased antibiotic sensitivity. We find no difference in cell charge or cell lysis, although we do see increased hydrophobicity in the ΔclpX strain, decreased cellular density and slightly thinner cells walls. We also see significant cell division defects in ΔclpX, although only when cells are grown in the mammalian cell culture medium, RPMI. We conclude that the intrinsic resistance of B. anthracis to cell wall active antimicrobials is dependent on formation of the ClpXP protease and that this could be due, at least in part, to the role of ClpX in regulating cell envelope morphology.

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.

Metabolic Heterogeneity and Cross-Feeding in Bacterial Multicellular Systems.

Cells in assemblages differentiate and perform distinct roles. Though many pathways of differentiation are understood at the molecular level in multicellular eukaryotes, the elucidation of similar processes in bacterial assemblages is recent and ongoing. Here, we discuss examples of bacterial differentiation, focusing on cases in which distinct metabolisms coexist and those that exhibit cross-feeding, with one subpopulation producing substrates that are metabolized by a second subpopulation. We describe several studies of single-species systems, then segue to studies of multispecies metabolic heterogeneity and cross-feeding in the clinical setting. Many of the studies described exemplify the application of new techniques and modeling approaches that provide insights into metabolic interactions relevant for bacterial growth outside the laboratory.

Mid-infrared metabolic imaging with vibrational probes.

Understanding metabolism is indispensable in unraveling the mechanistic basis of many physiological and pathological processes. However, in situ metabolic imaging tools are still lacking. Here we introduce a framework for mid-infrared (MIR) metabolic imaging by coupling the emerging high-information-throughput MIR microscopy with specifically designed IR-active vibrational probes. We present three categories of small vibrational tags including azide bond, 13C-edited carbonyl bond and deuterium-labeled probes to interrogate various metabolic activities in cells, small organisms and mice. Two MIR imaging platforms are implemented including broadband Fourier transform infrared microscopy and discrete frequency infrared microscopy with a newly incorporated spectral region (2,000-2,300 cm-1). Our technique is uniquely suited to metabolic imaging with high information throughput. In particular, we performed single-cell metabolic profiling including heterogeneity characterization, and large-area metabolic imaging at tissue or organ level with rich spectral information.

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.

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.

Transcriptional profiling of the clpX mutant in Bacillus anthracis reveals regulatory connection with the lrgAB operon.

ClpX functions as either an independent chaperone or a component of the ClpXP protease, a conserved intracellular protease that acts as a global regulator in the bacterial cell by degrading regulatory proteins, stress response proteins and rate-limiting enzymes. Previously, we found that loss of clpX in Bacillus anthracis Sterne leads to increased susceptibility to antimicrobial agents that target the cell envelope. The aim of this study was to identify genes within the regulatory network of clpX that contribute to antimicrobial resistance. Using microarray analysis, we found 119 genes that are highly differentially expressed in the ∆clpX mutant, with the majority involved in metabolic, transport or regulatory functions. Several of these differentially expressed genes, including glpF, sigM, mrsA, lrgA and lrgB, are associated with cell wall-active antibiotics in other bacterial species. We focused on lrgA and lrgB, which form the lrgAB operon and are downregulated in ∆clpX, because loss of lrgAB increases autolytic activity and penicillin susceptibility in Staphylococcus aureus. While we observed no changes in autolytic activity in either ∆clpX or ∆lrgAB B. anthracis Sterne, we find that both mutants have increased susceptibility to the antimicrobial peptide LL-37 and daptomycin. However, phenotypes between ∆clpX and ∆lrgAB are not identical as ∆clpX also displays increased susceptibility to penicillin and nisin but ∆lrgAB does not. Therefore, while decreased expression of lrgAB may be partially responsible for the increased antimicrobial susceptibility seen in the ∆clpX mutant, disruption of other pathways must also contribute to this phenotype.

Visualizing translational errors: one cell at a time.

Physiological heterogeneity among single cells with identical genetic information has been observed in a large number of bacterial phenotypes, including growth, stress responses, cell size, and antibiotic tolerance. Despite the widespread observation of this phenomenon in bacterial populations, not much is known about the molecular mechanisms behind phenotypic heterogeneity. Currently, our understanding is primarily limited to transcriptional profile of single cells using fluorescence reporters. Although the development of these tools has been extremely informative, it cannot fully explain the heterogeneity seen in populations. In a recent publication, Fan et al. have developed a dual-fluorescent reporter system that is capable of quantitatively measuring translational fidelity in single cells. It is shown that translational fidelity is heterogeneous and affects the growth characteristics of single cells. The development of tools for analysis of molecular heterogeneity downstream of transcription may play an important role in advancing our understanding of the physiology of bacterial populations.

Heterogeneity of Stop Codon Readthrough in Single Bacterial Cells and Implications for Population Fitness.

Gene expression noise (heterogeneity) leads to phenotypic diversity among isogenic individual cells. Our current understanding of gene expression noise is mostly limited to transcription, as separating translational noise from transcriptional noise has been challenging. It also remains unclear how translational heterogeneity originates. Using a transcription-normalized reporter system, we discovered that stop codon readthrough is heterogeneous among single cells, and individual cells with higher UGA readthrough grow faster from stationary phase. Our work also revealed that individual cells with lower protein synthesis levels exhibited higher UGA readthrough, which was confirmed with ribosome-targeting antibiotics (e.g., chloramphenicol). Further experiments and mathematical modeling suggest that varied competition between ternary complexes and release factors perturbs the UGA readthrough level. Our results indicate that fluctuations in the concentrations of translational components lead to UGA readthrough heterogeneity among single cells, which enhances phenotypic diversity of the genetically identical population and facilitates its adaptation to changing environments.

Rewiring protein synthesis: From natural to synthetic amino acids.

BACKGROUND: The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. SCOPE OF REVIEW: This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. MAJOR CONCLUSIONS: The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. GENERAL SIGNIFICANCE: Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Reduced Protein Synthesis Fidelity Inhibits Flagellar Biosynthesis and Motility.

Accurate translation of the genetic information from DNA to protein is maintained by multiple quality control steps from bacteria to mammals. Genetic and environmental alterations have been shown to compromise translational quality control and reduce fidelity during protein synthesis. The physiological impact of increased translational errors is not fully understood. While generally considered harmful, translational errors have recently been shown to benefit cells under certain stress conditions. In this work, we describe a novel regulatory pathway in which reduced translational fidelity downregulates expression of flagellar genes and suppresses bacterial motility. Electron microscopy imaging shows that the error-prone Escherichia coli strain lacks mature flagella. Further genetic analyses reveal that translational errors upregulate expression of a small RNA DsrA through enhancing its transcription, and deleting DsrA from the error-prone strain restores motility. DsrA regulates expression of H-NS and RpoS, both of which regulate flagellar genes. We demonstrate that an increased level of DsrA in the error-prone strain suppresses motility through the H-NS pathway. Our work suggests that bacteria are capable of switching on and off the flagellar system by altering translational fidelity, which may serve as a previously unknown mechanism to improve fitness in response to environmental cues.