Target For Development Of Antibiotics Research Articles

Metabolic Profiling Reveals the Importance of Arginine Metabolism via the Arginine Deiminase Pathway in Vancomycin-intermediate S. aureus

Gene mutations located in VraSR and GraSR, two-component regulatory systems have been reported to cause vancomycin resistance in vancomycin-intermediate Staphylococcus aureus (VISA). Subsequent comparative proteomic profiling between vancomycin-susceptible S. aureus (VSSA) and VISAs of the Mu50 genetic lineage revealed up-regulated level of catabolic ornithine carbamoyltransferase (ArcB) in the latter, suggesting a role of arginine catabolism in VISA development. This study aimed to further investigate metabolic pathways associated with VISA development using liquid chromatography mass spectrometry (LCMS)-based untargeted metabolomic profiling. Metabolite profiles were compared among 3 isogenic strains of the Mu50 lineage: Mu50Ω (VSSA), Mu50Ω-vraSm (VISA) and Mu50Ω-vraSm-graRm (VISA). Intracellular levels of α-hydroxyglutaric acid, choline, lysine, malic acid, N-acetylornithine, nicotinamide, and cystathionine were found to be significantly different between VISAs compared to VSSA. VISA cells were found to have lower levels of intracellular citrulline and extracellular arginine, with higher extracellular level of ornithine compared to VSSA. These differences in metabolite levels between VISA and VSSA suggested the importance of arginine metabolism. Activation of the arginine deiminase pathway (ADI) in VISAs of the Mu50 lineage could be further explored as a target for antibiotic development.

Bacterial Signal Peptides- Navigating the Journey of Proteins.

In 1971, Blobel proposed the first statement of the Signal Hypothesis which suggested that proteins have amino-terminal sequences that dictate their export and localization in the cell. A cytosolic binding factor was predicted, and later the protein conducting channel was discovered that was proposed in 1975 to align with the large ribosomal tunnel. The 1975 Signal Hypothesis also predicted that proteins targeted to different intracellular membranes would possess distinct signals and integral membrane proteins contained uncleaved signal sequences which initiate translocation of the polypeptide chain. This review summarizes the central role that the signal peptides play as address codes for proteins, their decisive role as targeting factors for delivery to the membrane and their function to activate the translocation machinery for export and membrane protein insertion. After shedding light on the navigation of proteins, the importance of removal of signal peptide and their degradation are addressed. Furthermore, the emerging work on signal peptidases as novel targets for antibiotic development is described.

Toward Understanding the Role of an Active Site Network in LThDP Stabilization on DXPS

Microbial metabolic pathways may provide novel, bacteria‐specific targets for antibiotic development, thus providing new strategies to combat the evolving conflict of antibiotic resistance. The bacterial metabolic enzyme 1‐deoxy‐D‐xylulose 5‐phosphate synthase (DXPS), which is absent in humans, catalyzes the thiamine diphosphate (ThDP)‐dependent formation of DXP from pyruvate and glyceraldehyde‐3‐phosphate (GAP). DXPS has emerged as an appealing target due to its essentiality in bacterial pathogens, as DXP feeds into the biosynthesis of isoprenoids, ThDP, and pyridoxal phosphate (PLP), positioning DXPS as a central player in bacterial metabolism. DXPS has a unique mechanism in ThDP enzymology, in which binding of a small molecule “trigger” is required to induce a conformational change from the closed to an open conformation, essential for decarboxylation of the first enzyme bound intermediate, C2α−lactyl‐ThDP (LThDP). GAP is one such trigger molecule, likely binding at a site that is distinct from its acceptor binding site. However, how DXPS stabilizes LThDP prior to GAP‐induced decarboxylation remains unknown. In this study, we propose an active site network connects key active site histidine residues to induce the closed conformation upon reaction with pyruvate, stabilizing the LThDP intermediate. E. coli Arginine 99 may be an essential component of this network. We found that artificial disruption of this network through substitution of R99 (R99A) 1) increases KmGAP three‐fold, 2) destabilizes LThDP (detected by Circular Dichroism), and 3) shifts the conformation to the open state believed to induce decarboxylation (detected by HDX‐MS and limited proteolysis). Overall, the results support the hypothesis that R99 serves to maintain the closed conformation by connecting the key histidine residues, providing preliminary evidence for the existence of an active site network that links catalysis to conformational dynamics. Targeting the unique gated mechanism of DXPS could prove to be an effective strategy for developing a selective antibiotic that has far reaching impacts on bacterial metabolism in a pathogenic state.

Characterization of ribonucleotide reductases of emerging pathogens Elizabethkingia anophelis and Elizabethkingia meningoseptica and streptonigrin as their inhibitor: a computational study

Antibiotic resistance is a global concern. Two members of the bacterial genus Elizabethkingia, namely, E. anophelis and E. meningoseptica have raised much concern in recent years because of their resistance to multiple commonly used antibiotics. Identification of multidrug resistant and pan-drug resistant bacteria has propelled the search for new antibiotics that can act on unconventional targets. Researches are going on to find out the possibility of using bacterial ribonucleotide reductases as a novel target for antibiotic development. Through in silico evaluations, this study aims for characterization and functional annotation of ribonucleotide reductase enzymes of E. anophelis and E. meningoseptica. Binding affinities with these enzymes of the compounds that have shown promising results in inhibiting Pseudomonas aeruginosa growth by acting on its ribonucleotide reductase were also assessed by molecular docking and dynamics simulations. Insights from this study will help in battling these infections in the near future. Communicated by Ramaswamy H. Sarma

Natural Product Inhibition and Enzyme Kinetics Related to Phylogenetic Characterization for Bacterial Peptidyl-tRNA Hydrolase 1.

With the relentless development of drug resistance and re-emergence of many pathogenic bacteria, the need for new antibiotics and new antibiotic targets is urgent and growing. Bacterial peptidyl-tRNA hydrolase, Pth1, is emerging as a promising new target for antibiotic development. From the conserved core and high degree of structural similarity, broad-spectrum inhibition is postulated. However, Pth1 small-molecule inhibition is still in the earliest stages. Focusing on pathogenic bacteria, herein we report the phylogenetic classification of Pth1 and natural product inhibition spanning phylogenetic space. While broad-spectrum inhibition is found, narrow-spectrum and even potentially clade-specific inhibition is more frequently observed. Additionally reported are enzyme kinetics and general in vitro Pth1 solubility that follow phylogenetic boundaries along with identification of key residues in the gate loop region that appear to govern both. The studies presented here demonstrate the sizeable potential for small-molecule inhibition of Pth1, improve understanding of Pth enzymes, and advance Pth1 as a much-needed novel antibiotic target.

Discovery of Prenyltransferase Inhibitors with In Vitro and In Vivo Antibacterial Activity.

Cis-prenyltransferases such as undecaprenyl diphosphate synthase (UPPS) and decaprenyl diphosphate synthase (DPPS) are essential enzymes in bacteria and are involved in cell wall biosynthesis. UPPS and DPPS are absent in the human genome, so they are of interest as targets for antibiotic development. Here, we screened a library of 750 compounds from National Cancer Institute Diversity Set V for the inhibition of Mycobacterium tuberculosis DPPS and found 17 hits, and then IC50s were determined using dose-response curves. Compounds were tested for growth inhibition against a panel of bacteria, for in vivo activity in a Staphylococcus aureus/Caenorhabditis elegans model, and for mammalian cell toxicity. The most active DPPS inhibitor was the dicarboxylic acid redoxal (compound 10), which also inhibited undecaprenyl diphosphate synthase (UPPS) as well as farnesyl diphosphate synthase. 10 was active against S. aureus, Clostridiodes difficile, Bacillus anthracis Sterne, and Bacillus subtilis, and there was a 3.4-fold increase in IC50 on addition of a rescue agent, undecaprenyl monophosphate. We found that 10 was also a weak protonophore uncoupler, leading to the idea that it targets both isoprenoid biosynthesis and the proton motive force. In an S. aureus/C. elegans in vivo model, 10 reduced the S. aureus burden 3 times more effectively than did ampicillin.

Endophyte-produced antimicrobials: a review of potential lead compounds with a focus on quorum-sensing disruptors

Endophytes are defined as microorganisms which live in plant tissues without causing any apparent negative effects to the host plant. Endophytes have been considered rich sources for natural products since they produce an array of chemically diverse bioactive secondary metabolites. Consistent with the hypothesis that endophytes help the survival and growth of host plants by producing secondary metabolites, many compounds derived from endophytes exhibit potent antimicrobial activity. Interestingly, some endophytes have been reported to produce the same metabolites as the host plants, probably due to horizontal gene transfer. In this review, metabolites produced by endophytes as well as their host plants are briefly introduced, with the emphasis on their value as pharmaceutical lead compounds. We have defined lead compounds as secondary metabolites with antimicrobial activities with Minimal Inhibitory Concentration values less than 20 μM (5 μg/mL). The endophytic metabolites with antimicrobial activities are classified into two groups according to the mode of inhibition: (1) antibiotic metabolites that kill or inhibit growth of microorganisms (2) endophyte-derived compounds with inhibitory activity against quorum sensing. Disruption of quorum sensing, which is a microbial communication system, has recently emerged as a novel target for antibiotic development since it affords a new modality to reduce microbial infection. Although currently there are no commercially-available antibiotics derived from endophytes, it is expected that new antibiotics based on several modes of action will be developed and marketed due to increasing research emphasis in this field.

Posttranslational modification of Elongation Factor P from Staphylococcusaureus.

Antibiotic‐resistant Staphylococcus aureus is becoming a major burden on health care systems in many countries, necessitating the identification of new targets for antibiotic development. Elongation Factor P (EF‐P) is a highly conserved elongation protein factor that plays an important role in protein synthesis and bacteria virulence. EF‐P undergoes unique posttranslational modifications in a stepwise manner to function correctly, but experimental information on EF‐P posttranslational modifications is currently lacking for S. aureus. Here, we expressed EF‐P in S. aureus to analyze its posttranslational modifications by mass spectrometry and report experimental proof of 5‐aminopentanol modification of S. aureus EF‐P.

GammaBOriS: Identification and Taxonomic Classification of Origins of Replication in Gammaproteobacteria using Motif-based Machine Learning

The biology of bacterial cells is, in general, based on information encoded on circular chromosomes. Regulation of chromosome replication is an essential process that mostly takes place at the origin of replication (oriC), a locus unique per chromosome. Identification of high numbers of oriC is a prerequisite for systematic studies that could lead to insights into oriC functioning as well as the identification of novel drug targets for antibiotic development. Current methods for identifying oriC sequences rely on chromosome-wide nucleotide disparities and are therefore limited to fully sequenced genomes, leaving a large number of genomic fragments unstudied. Here, we present gammaBOriS (Gammaproteobacterial oriCSearcher), which identifies oriC sequences on gammaproteobacterial chromosomal fragments. It does so by employing motif-based machine learning methods. Using gammaBOriS, we created BOriS DB, which currently contains 25,827 gammaproteobacterial oriC sequences from 1,217 species, thus making it the largest available database for oriC sequences to date. Furthermore, we present gammaBOriTax, a machine-learning based approach for taxonomic classification of oriC sequences, which was trained on the sequences in BOriS DB. Finally, we extracted the motifs relevant for identification and classification decisions of the models. Our results suggest that machine learning sequence classification approaches can offer great support in functional motif identification.

Crystal structure and catalytic mechanism of the essential m1G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa

The tRNA (m1G37) methyltransferase TrmD catalyzes m1G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-l-methionine (SAM) and tRNALeu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (Ki = 0.41 ± 0.07 µM) and uncompetitive for tRNA (Ki = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.

Molecular Basis of Bacillus subtilis ATCC 6633 Self-Resistance to the Phosphono-oligopeptide Antibiotic Rhizocticin.

Rhizocticins are phosphono-oligopeptide antibiotics that contain a toxic C-terminal ( Z) -l -2-amino-5-phosphono-3-pentenoic acid (APPA) moiety. APPA is an irreversible inhibitor of threonine synthase (ThrC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-phospho-l-homoserine to l-threonine. ThrCs are essential for the viability of bacteria, plants, and fungi and are a target for antibiotic development, as de novo threonine biosynthetic pathway is not found in humans. Given the ability of APPA to interfere in threonine metabolism, it is unclear how the producing strain B. subtilis ATCC 6633 circumvents APPA toxicity. Notably, in addition to the housekeeping APPA-sensitive ThrC ( BsThrC), B. subtilis encodes a second threonine synthase (RhiB) encoded within the rhizocticin biosynthetic gene cluster. Kinetic and spectroscopic analyses show that PLP-dependent RhiB is an authentic threonine synthase, converting O-phospho-l-homoserine to threonine with a catalytic efficiency comparable to BsThrC. To understand the structural basis of inhibition, we determined the crystal structure of APPA bound to the housekeeping BsThrC, revealing a covalent complex between the inhibitor and PLP. Structure-based sequence analyses reveal structural determinants within the RhiB active site that contribute to rendering this ThrC homologue resistant to APPA. Together, this work establishes the self-resistance mechanism utilized by B. subtilis ATCC 6633 against APPA exemplifying one of many ways by which bacteria can overcome phosphonate toxicity.

Functional annotation and distribution overview of RNA families in 27\xa0Streptococcus agalactiae genomes

BackgroundStreptococcus agalactiae, also known as Group B Streptococcus (GBS), is a Gram-positive bacterium that colonizes the gastrointestinal and genitourinary tract of humans. This bacterium has also been isolated from various animals, such as fish and cattle. Non-coding RNAs (ncRNAs) can act as regulators of gene expression in bacteria, such as Streptococcus pneumoniae and Streptococcus pyogenes. However, little is known about the genomic distribution of ncRNAs and RNA families in S. agalactiae.ResultsComparative genome analysis of 27 S. agalactiae strains showed more than 5 thousand genomic regions identified and classified as Core, Exclusive, and Shared genome sequences. We identified 27 to 89 RNA families per genome distributed over these regions, from these, 25 were in Core regions while Shared and Exclusive regions showed variations amongst strains. We propose that the amount and type of ncRNA present in each genome can provide a pattern to contribute in the identification of the clonal types.ConclusionsThe identification of RNA families provides an insight over ncRNAs, sRNAs and ribozymes function, that can be further explored as targets for antibiotic development or studied in gene regulation of cellular processes. RNA families could be considered as markers to determine infection capabilities of different strains. Lastly, pan-genome analysis of GBS including the full range of functional transcripts provides a broader approach in the understanding of this pathogen.

Biophysical characterization and stabilization of detergent-solubilized lipoprotein N-acyl transferase from P. aeruginosa and E. coli

Lipoproteins are important for bacterial growth and virulence and interest in them as targets for antibiotic development is growing. Lipoprotein N-acyl transferase (Lnt) catalyzes the final step in the lipoprotein posttranslational processing pathway. The mature lipoprotein can remain in the inner membrane or be trafficked to the outer membrane in the case of diderm prokaryotes. With a view to obtaining high-resolution crystal structures of membrane integral Lnt for use in drug discovery a program was undertaken to generate milligram quantities of stable, homogenous and functional protein. This involved screening across bacterial species for suitable orthologues and optimization at the level of protein expression, solubilization and stability. Combining biophysical and functional characterization, orthologous Lnt from Escherichia coli and the opportunistic human pathogen Pseudomonas aeruginosa was identified as suitable for the proposed structure determination campaign that ultimately yielded crystal structures. The rational approaches taken that eventually provided structure-quality protein are presented in this report.

The Mechanism of Regulation of Pantothenate Biosynthesis by the PanD-PanZ·AcCoA Complex Reveals an Additional Mode of Action for the Antimetabolite N-Pentyl Pantothenamide (N5-Pan).

The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of β-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked β-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.

Microbiology: The bacterial cell wall takes centre stage.

An unexpected function has been assigned to part of the molecular machinery that synthesizes the bacterial cell wall — a dramatic shift in our understanding that may have major implications for antibiotic development. See Article p.634 It has been generally accepted that the cell wall peptidoglycans of the bacterial exoskeleton are synthesized by penicillin binding proteins (PBPs) known as class A PBPs. Now, using genetic manipulation, phylogenetic analysis and functional experiments in Bacillus subtilis, David Rudner and colleagues have identified SEDS family proteins as the main peptidoglycan polymerases more broadly conserved than class A PBPs. Specifically in B. subtilis, they show that the SEDS protein RodA, a widely conserved component of the Rod complex involved in elongation of rod-shaped bacteria, acts with class B PBPs as the core cell wall synthase of the cell elongation and division machinery. The authors conclude that B. subtilis and probably most bacteria use two distinct classes of polymerases to synthesize their exoskeleton. This work also suggests that SEDS family proteins should be attractive targets for antibiotic development.

The Power of Asymmetry: Architecture and Assembly of the Gram-Negative Outer Membrane Lipid Bilayer

Determining the chemical composition of biological materials is paramount to the study of natural phenomena. Here, we describe the composition of model gram-negative outer membranes, focusing on the predominant assembly, an asymmetrical bilayer of lipid molecules. We also give an overview of lipid biosynthetic pathways and molecular mechanisms that organize this material into the outer membrane bilayer. An emphasis is placed on the potential of these pathways as targets for antibiotic development. We discuss deviations in composition, through bacterial cell surface remodeling, and alternative modalities to the asymmetric lipid bilayer. Outer membrane lipid alterations of current microbiological interest, such as lipid structures found in commensal bacteria, are emphasized. Additionally, outer membrane components could potentially be engineered to develop vaccine platforms. Observations related to composition and assembly of gram-negative outer membranes will continue to generate novel discoveries, broaden biotechnologies, and reveal profound mysteries to compel future research.

Expression, purification, and buffer solubility optimization of the putative human peptidyl-tRNA hydrolase PTRHD1

Performing the essential function of recycling peptidyl-tRNAs, peptidyl-tRNA hydrolases are ubiquitous in all domains of life. The multicomponent eukaryotic Pth system differs greatly from the bacterial system composed predominantly of a single Pth1 enzyme. While bacterial Pth1s are structurally well characterized and promising new targets for antibiotic development, eukaryotic Pths are largely understudied. From amino acid sequence alignment and secondary structure predictions, the human gene product PTRHD1 was classified as a eukaryotic Pth. Herein, we report cloning, recombinant bacterial expression, and weak binding to peptidyl-tRNA for PTRHD1. Additionally, we report binding to tRNA but absence of peptidyl-tRNA hydrolase activity. Thus, PTRHD1 is not a Pth and the functional consequence of nucleotide binding remains undefined.

Trans‐Translation As A Target For Novel Antibiotics

The trans‐translation pathway rescues ribosomes stalled at the end of mRNAs in bacteria. This pathway is required for viability or virulence in many species, but is not targeted by existing antibiotics. Using a high‐throughput screen, we identified compounds that inhibit trans‐translation but not protein synthesis. These compounds are not toxic to eukaryotic cells, but inhibit growth of a broad spectrum of bacterial species, including Staphylococcus aureus, Bacillus anthracis, Shigella flexneri, Mycobacterium tuberculosis, and Francisella tularensis, both in vitro and during ex vivo infection. Chemical cross‐linking was used to determine the molecular targets of these compounds in bacteria. One family of inhibitors was found to bind to the base of helix 89 in 23S rRNA, a site that is not bound by other antibiotics. These data validate trans‐translation as a target for antibiotic development and provide new insights into the early steps of ribosome rescue pathways.Support or Funding InformationThis work was supported by grants GM068720 and AI111692 from the National Institutes of Health.

Comparison of the sulfonamide inhibition profiles of the α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae

Carbonic anhydrases (CA, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyze the conversion of carbon dioxide (CO2) to bicarbonate (HCO3−) and protons (H+). In prokaryotes, the existence of genes encoding for α-, β- and γ-classes suggests that these enzymes play an important role in the prokaryotic physiology. It has been demonstrated, in fact, that their inhibition in vivo leads to growth impairment or growth defects of the microorganism. Ultimately, we started to investigate the biochemical properties and the inhibitory profiles of the α- and β-CAs identified in the genome of Vibrio cholerae, which is the causative agent of cholera. The genome of this pathogen encodes for CAs belonging to α, β and γ classes. Here, we report a sulfonamide inhibition study of the γ-CA (named VchCAγ) comparing it with data obtained for the α- and β-CA enzymes. VchCAγ activity (kcat=7.39×105s−1) was significantly higher than the other γ-CAs. The inhibition study with a panel of sulfonamides and one sulfamate led to the detection of a large number of nanomolar VchCAγ inhibitors, including simple aromatic/heterocyclic sulfonamides (compounds 2–9, 11, 13–15, 24) as well as EZA, DZA, BRZ, BZA, TPM, ZNS, SLP, IND (KIs in the range of 66.2–95.3nM). As it was proven that bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit this virulence in vivo, we propose that VchCA, VchCAβ and VchCAγ may be a target for antibiotic development, exploiting a mechanism of action rarely considered up until now, i.e., interference with bicarbonate supply as a virulence factor.

On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer

Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3(-) complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3 on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.