Covalent Modifiers of Botulinum Neurotoxin Counteract Toxin Persistence
ACS CHEMICAL BIOLOGY
2019; 14 (1): 76–87
The dormancy-specific regulator, SutA, is intrinsically disordered and modulates transcription initiation in Pseudomonas aeruginosa.
Botulinum neurotoxins (BoNTs) are the most potent toxin known to man and a significant threat as a weapon of bioterrorism. BoNTs contain a metalloprotease domain that blocks neurotransmitter release in nerve terminals, resulting in a descending, flaccid paralysis with a 5-10% mortality rate. Existing treatment options cannot access or neutralize toxin following its endocytosis, so there is a clear need to develop novel therapies. Numerous substrate-based and zinc-chelating small-molecule inhibitors have been reported, however none have progressed to the clinic. This is likely due to the difficulty of reversible inhibitors to achieve sustained inhibition of the toxin, which has a months-long half-life in vivo. An alternative strategy to mitigate BoNT persistence is through covalent, irreversible inhibition of toxin function. However, few examples of covalent BoNT inhibitors have been reported. Here, we describe a competition-based screen to identify covalent modifiers of the conserved active-site adjacent cysteine C165 in the BoNT/A serotype. We found that compounds containing cysteine-reactive electrophiles designed to target cysteine proteases failed to bind C165 while selenide compounds were efficient covalent binders of this cysteine. Importantly, covalent modification at C165 resulted in sustained, irreversible inhibition of BoNT/A protease activity. Covalent selenide inhibitors were non-toxic and protective in a neuronal assay of intoxication making them promising new scaffolds for the study of the BoNT/A toxin as well as for the design of novel therapy agents.
View details for PubMedID 30571080
Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms.
2017; 8 (5)
Though most bacteria in nature are nutritionally limited and grow slowly, our understanding of core processes like transcription comes largely from studies in model organisms doubling rapidly. We previously identified a small protein of unknown function, SutA, in a screen of proteins synthesized in Pseudomonas aeruginosa during dormancy. SutA binds RNA polymerase (RNAP), causing widespread changes in gene expression, including upregulation of the ribosomal RNA genes. Here, using biochemical and structural methods, we examine how SutA interacts with RNAP and the functional consequences of these interactions. We show that SutA comprises a central α-helix with unstructured N- and C-terminal tails, and binds to the β1 domain of RNAP. It activates transcription from the rrn promoter by both the housekeeping sigma factor holoenzyme (Eσ70 ) and the stress sigma factor holoenzyme (EσS ) in vitro, but has a greater impact on EσS . In both cases, SutA appears to affect intermediates in open complex formation, and its N-terminal tail is required for activation. The small magnitudes of in vitro effects are consistent with a role in maintaining activity required for homeostasis during dormancy. Our results add SutA to a growing list of transcription regulators that use their intrinsically disordered regions to remodel transcription complexes. This article is protected by copyright. All rights reserved.
View details for DOI 10.1111/mmi.14337
View details for PubMedID 31254296
SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (5): E597-E605
Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance.IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.
View details for PubMedID 29066549
View details for PubMedCentralID PMC5654934
In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry
2014; 16 (8): 2568-2590
Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteome-labeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally up-regulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A).
View details for DOI 10.1073/pnas.1514412113
View details for Web of Science ID 000369085100016
View details for PubMedID 26787849
View details for PubMedCentralID PMC4747698
State-selective Metabolic Labeling of Cellular Proteins
ACS CHEMICAL BIOLOGY
2012; 7 (8): 1326-1330
Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for l-methionine, followed by fluorescent labelling of AHA-containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano-scale secondary ion mass spectrometry ((15)NH(3) assimilation) for individual cells within a sediment-sourced enrichment culture showed concordance between AHA-positive cells and (15)N enrichment. BONCAT-FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single-cell level.
View details for DOI 10.1111/1462-2920.12436
View details for Web of Science ID 000340522500017
View details for PubMedID 24571640
View details for PubMedCentralID PMC4122687
Noninvasive characterization of in situ forming implants using diagnostic ultrasound
JOURNAL OF CONTROLLED RELEASE
2010; 143 (2): 183-190
Transcriptional activity from a specified promoter can provide a useful marker for the physiological state of a cell. Here we introduce a method for selective tagging of proteins made in cells in which specified promoters are active. Tagged proteins can be modified with affinity reagents for enrichment or with fluorescent dyes for visualization. The method allows state-selective analysis of the proteome, whereby proteins synthesized in predetermined physiological states can be identified. The approach is demonstrated by proteome-wide labeling of bacterial proteins upon activation of the P(BAD) promoter and the SoxRS regulon and provides a basis for analysis of more complex systems including spatially heterogeneous microbial cultures and biofilms.
View details for DOI 10.1021/cb300238w
View details for Web of Science ID 000307526600003
View details for PubMedID 22692961
View details for PubMedCentralID PMC3423470
A multipurpose microfluidic device designed to mimic microenvironment gradients and develop targeted cancer therapeutics
LAB ON A CHIP
2009; 9 (4): 545-554
In situ forming drug delivery systems provide a means by which a controlled release depot can be physically inserted into a target site without the use of surgery. The release rate of drugs from these systems is often related to the rate of implant formation. Currently, only a limited number of techniques are available to monitor phase inversion, and none of these methods can be used to visualize the process directly and noninvasively. In this study, diagnostic ultrasound was used to visualize and quantify the process of implant formation in a phase inversion based system both in vitro and in vivo. Concurrently, sodium fluorescein was used as a mock drug to evaluate the drug release profiles and correlate drug release and implant formation processes. Implants comprised of three different molecular weight poly(lactic-co-glycolic acid) (PLGA) polymers dissolved in 1-methyl-2-pyrrolidinone (NMP) were studied in vitro and a 29 kDa PLGA solution was evaluated in vivo. The implants were encapsulated in a 1% agarose tissue phantom for five days, or injected into a rat subcutaneously and evaluated for 48 h. Quantitative measurements of the gray-scale value (corresponding to the rate of implant formation), swelling, and precipitation were evaluated using image analysis techniques, showing that polymer molecular weight has a considerable effect on the swelling and formation of the in situ drug delivery depots. A linear correlation was also seen between the in vivo release and depot formation (R(2)=0.93). This study demonstrates, for the first time, that ultrasound can be used to noninvasively and nondestructively monitor and evaluate the phase inversion process of in situ forming drug delivery implants, and that the formation process can be directly related to the initial phase of drug release dependent on this formation.
View details for DOI 10.1016/j.jconrel.2010.01.001
View details for Web of Science ID 000277219200004
View details for PubMedID 20060859
View details for PubMedCentralID PMC2847659
The heterogeneity of cellular microenvironments in tumors severely limits the efficacy of most cancer therapies. We have designed a microfluidic device that mimics the microenvironment gradients present in tumors that will enable the development of more effective cancer therapies. Tumor cell masses were formed within micron-scale chambers exposed to medium perfusion on one side to create linear nutrient gradients. The optical accessibility of the PDMS and glass device enables quantitative transmitted and fluorescence microscopy of all regions of the cell masses. Time-lapse microscopy was used to measure the growth rate and show that the device can be used for long-term efficacy studies. Fluorescence microscopy was used to demonstrate that the cell mass contained viable, apoptotic, and acidic regions similar to in vivo tumors. The diffusion coefficient of doxorubicin was accurately measured, and the accumulation of therapeutic bacteria was quantified. The device is simple to construct, and it can easily be reproduced to create an array of in vitro tumors. Because microenvironment gradients and penetration play critical roles controlling drug efficacy, we believe that this microfluidic device will be vital for understanding the behavior of common cancer drugs in solid tumors and designing novel intratumorally targeted therapeutics.
View details for DOI 10.1039/b810571e
View details for Web of Science ID 000263465900007
View details for PubMedID 19190790
View details for PubMedCentralID PMC2855303