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Microbiology Spectrum Jul 2019Diphtheria is one of the most well studied of all the bacterial infectious diseases. These milestone studies of toxigenic along with its primary virulence determinant,... (Review)
Review
Diphtheria is one of the most well studied of all the bacterial infectious diseases. These milestone studies of toxigenic along with its primary virulence determinant, diphtheria toxin, have established the paradigm for the study of other related bacterial protein toxins. This review highlights those studies that have contributed to our current understanding of the structure-function relationships of diphtheria toxin, the molecular mechanism of its entry into the eukaryotic cell cytosol, the regulation of diphtheria expression by holo-DtxR, and the molecular basis of transition metal ion activation of apo-DtxR itself. These seminal studies have laid the foundation for the protein engineering of diphtheria toxin and the development of highly potent eukaryotic cell-surface receptor-targeted fusion protein toxins for the treatment of human diseases that range from T cell malignancies to steroid-resistant graft-versus-host disease to metastatic melanoma. This deeper scientific understanding of diphtheria toxin and the regulation of its expression have metamorphosed the third-most-potent bacterial toxin known into a life-saving targeted protein therapeutic, thereby at least partially fulfilling Paul Erlich's concept of a magic bullet-"a chemical that binds to and specifically kills microbes or tumor cells."
Topics: Animals; Bacterial Proteins; Corynebacterium diphtheriae; DNA-Binding Proteins; Diphtheria; Diphtheria Toxin; Gene Expression Regulation, Bacterial; Humans; Iron; Operon
PubMed: 31267892
DOI: 10.1128/microbiolspec.GPP3-0063-2019 -
Molecules (Basel, Switzerland) Mar 2011Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography... (Review)
Review
Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA) which combines with lethal factor (LF) and edema factor (EF), forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.
Topics: Amino Acid Sequence; Bacillus anthracis; Bacterial Proteins; Limit of Detection; Molecular Sequence Data; Sequence Homology, Amino Acid; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry
PubMed: 21403598
DOI: 10.3390/molecules16032391 -
Microbiology and Molecular Biology... Dec 2004Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have... (Review)
Review
Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function.
Topics: Amino Acid Sequence; Bacterial Proteins; Gram-Negative Bacteria; Molecular Sequence Data; Phylogeny; Protein Transport
PubMed: 15590781
DOI: 10.1128/MMBR.68.4.692-744.2004 -
PloS One 2012The processing and MHC class I-restricted presentation of antigenic peptides derived from the p60 protein of the facultative intracellular bacterium Listeria...
The processing and MHC class I-restricted presentation of antigenic peptides derived from the p60 protein of the facultative intracellular bacterium Listeria monocytogenes is tightly linked to bacterial protein synthesis. We used non-linear regression analysis to fit a mathematical model of bacterial antigen processing to a published experimental data set showing the accumulation and decay of p60-derived antigenic peptides in L. monocytogenes-infected cells. Two alternative models equally describe the experimental data. The simulation accounting for a stable and a hypothetical rapidly degraded form of antigen predicts that the antigenic peptides p60 217-225 and p60 449-457 are derived from a putative instable form of p60 with an average intracellular half-life of approximately 3 minutes accounting for approximately 31% of all p60 molecules synthesized. The alternative model predicts that both antigenic peptides are processed from p60 degraded intracellularly with a half-life of 109 min and that antigen processing only occurs as long as bacterial protein synthesis is not inhibited. In order to decide between both models the intracellular accumulation of p60 in infected cells was studied experimentally and compared with model predictions. Inhibition of p60 degradation by the proteasome inhibitor epoxomicin revealed that during the first 3 h post infection approximately 30% of synthesized p60 molecules were degraded. This value is significantly lower than the approximately 50% degradation of p60 that would be expected in the presence of the predicted putative short-lived state of p60 and also fits precisely with the predictions of the alternative model, indicating that the tight connection of bacterial protein biosynthesis and antigen processing and presentation of L. monocyctogenes-derived antigenic peptides is not caused by the presence of a highly instable antigenic substrate.
Topics: Antigen Presentation; Antigens, Bacterial; Bacterial Proteins; Blotting, Western; Genes, MHC Class I; Listeria monocytogenes; Models, Biological; Oligopeptides; Protein Biosynthesis; Proteolysis; Regression Analysis
PubMed: 22428021
DOI: 10.1371/journal.pone.0033335 -
Annual Review of Microbiology 2014One of the most exciting developments in the field of bacterial pathogenesis in recent years is the discovery that many pathogens utilize complex nanomachines to deliver... (Review)
Review
One of the most exciting developments in the field of bacterial pathogenesis in recent years is the discovery that many pathogens utilize complex nanomachines to deliver bacterially encoded effector proteins into target eukaryotic cells. These effector proteins modulate a variety of cellular functions for the pathogen's benefit. One of these protein-delivery machines is the type III secretion system (T3SS). T3SSs are widespread in nature and are encoded not only by bacteria pathogenic to vertebrates or plants but also by bacteria that are symbiotic to plants or insects. A central component of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins across the bacterial envelope. Working in conjunction with several cytoplasmic components, the needle complex engages specific substrates in sequential order, moves them across the bacterial envelope, and ultimately delivers them into eukaryotic cells. The central role of T3SSs in pathogenesis makes them great targets for novel antimicrobial strategies.
Topics: Animals; Bacteria; Bacterial Infections; Bacterial Proteins; Bacterial Secretion Systems; Humans; Protein Transport
PubMed: 25002086
DOI: 10.1146/annurev-micro-092412-155725 -
Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells.MBio Apr 2020RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its...
RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent of mitochondrial impairment in human cells affected by neurodegeneration. To fulfill all the criteria to qualify as a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1 wild type [RepA-WH1(WT)] and assayed its response to exposure to -assembled RepA-WH1(A31V) amyloid fibers. In parallel, murine cells releasing RepA-WH1(A31V) aggregates were cocultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibers and donor-derived RepA-WH1(A31V) aggregates induced, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells pointed to alterations in mitochondria, protein quality triage, signaling, and intracellular traffic. Thus, a synthetic prion-like protein can be propagated to, and become cytotoxic to, cells of organisms placed at such distant branches of the tree of life as bacteria and mammalia, suggesting that mechanisms of protein aggregate spreading and toxicity follow default pathways. Proteotoxic amyloid seeds can be transmitted between mammalian cells, arguing that the intercellular exchange of prion-like protein aggregates can be a common phenomenon. RepA-WH1 is derived from a bacterial intracellular functional amyloid protein, engineered to become cytotoxic in Here, we have studied if such bacterial aggregates can also be transmitted to, and become cytotoxic to, mammalian cells. We demonstrate that RepA-WH1 is capable of entering naive cells, thereby inducing the cytotoxic aggregation of a soluble RepA-WH1 variant expressed in the cytosol, following the same trend that had been described in bacteria. These findings highlight the universality of one of the central principles underlying prion biology: No matter the biological origin of a given prion-like protein, it can be transmitted to a phylogenetically unrelated recipient cell, provided that the latter expresses a soluble protein onto which the incoming protein can readily template its amyloid conformation.
Topics: Animals; Bacterial Proteins; Cell Line, Tumor; Coculture Techniques; HeLa Cells; Humans; Intercellular Junctions; Membrane Fusion; Mice; Neuroblastoma; Prions
PubMed: 32291306
DOI: 10.1128/mBio.02937-19 -
Science Progress 2020Protein secretion is almost universally employed by bacteria. Some proteins are retained on the cell surface, whereas others are released into the extracellular milieu,... (Review)
Review
Protein secretion is almost universally employed by bacteria. Some proteins are retained on the cell surface, whereas others are released into the extracellular milieu, often playing a key role in virulence. In this review, we discuss the diverse types and potential functions of post-translational modifications (PTMs) occurring to extracellular bacterial proteins.
Topics: Bacteria; Bacterial Proteins; Protein Processing, Post-Translational; Proteomics; Virulence
PubMed: 33148128
DOI: 10.1177/0036850420964317 -
ELife Jul 2021Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no...
Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to and , we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently oriented genes, consistent with the 'twin-domain' model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin-binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.
Topics: Bacterial Proteins; Binding Sites; Cell Cycle Proteins; Chromatin Immunoprecipitation; Chromosomal Proteins, Non-Histone; Chromosome Structures; Chromosomes; Chromosomes, Bacterial; DNA; DNA Replication; DNA, Bacterial; Escherichia coli; Protein Binding; Saccharomyces cerevisiae; Transcription, Genetic; Cohesins
PubMed: 34279217
DOI: 10.7554/eLife.67236 -
Journal of Clinical Microbiology Jun 2021diagnostic imaging of bacterial infections is currently reliant on targeting their metabolic pathways, an ineffective method to identify microbial species with low...
diagnostic imaging of bacterial infections is currently reliant on targeting their metabolic pathways, an ineffective method to identify microbial species with low metabolic activity. Here, we establish HS-198 as a small-molecule fluorescent conjugate that selectively targets the highly conserved bacterial protein HtpG (high-temperature protein G), within Borrelia burgdorferi, the bacterium responsible for Lyme disease. We describe the use of HS-198 to target morphologic forms of B. burgdorferi in both the logarithmic growth phase and the metabolically dormant stationary phase as well as in inactivated spirochetes. Furthermore, in a murine infection model, systemically injected HS-198 identified B. burgdorferi as revealed by imaging in postnecropsy tissue sections. These findings demonstrate how small-molecule probes directed at conserved bacterial protein targets can function to identify the microbe using noninvasive imaging and potentially as scaffolds to deliver antimicrobial agents to the pathogen.
Topics: Animals; Bacterial Proteins; Borrelia burgdorferi; Diagnostic Imaging; Humans; Lyme Disease; Mice
PubMed: 33910962
DOI: 10.1128/JCM.02313-20 -
FEMS Immunology and Medical Microbiology Apr 2005Various bacterial components (e.g., endotoxin, teichoic and lipoteichoic acids, peptidoglycans, DNA) induce or enhance inflammation by stimulating the innate immune... (Review)
Review
Various bacterial components (e.g., endotoxin, teichoic and lipoteichoic acids, peptidoglycans, DNA) induce or enhance inflammation by stimulating the innate immune system and/or are directly toxic in eukariotic cells (e.g., hemolysins). When antibiotics which inhibit bacterial protein synthesis kill bacteria, smaller quantities of proinflammatory or toxic compounds are released in vitro and in vivo than during killing of bacteria by beta-lactams and other cell-wall active drugs. In general, high antibiotic concentrations liberate lower quantities of bacterial proinflammatory or toxic compounds than concentrations close to the minimum inhibitory concentration. In animal models of Escherichia coli Pseudomonas aeruginosa and Staphylococcus aureus peritonitis/sepsis and of Streptococcus pneumoniae meningitis, a lower release of proinflammatory bacterial compounds was associated with a reduced mortality or neuronal injury. Pre-treatment with a bacterial protein synthesis inhibitor reduced the strong release of bacterial products usually observed during treatment with a beta-lactam antibiotic. Data available strongly encourage clinical trials comparing antibiotic regimens with different release of proinflammatory/toxic bacterial products. The benefit of the approach to reduce the liberation of bacterial products should be greatest in patients with a high bacterial load.
Topics: Animals; Anti-Bacterial Agents; Bacterial Infections; Bacterial Proteins; Bacterial Toxins; DNA, Bacterial; Humans; Immunity, Innate; Immunologic Factors; In Vitro Techniques; Inflammation Mediators; Lipopolysaccharides; Protein Synthesis Inhibitors; Teichoic Acids
PubMed: 15780573
DOI: 10.1016/j.femsim.2005.01.001