scholarly journals Several New Putative Bacterial ADP-Ribosyltransferase Toxins Are Revealed from In Silico Data Mining, Including the Novel Toxin Vorin, Encoded by the Fire Blight Pathogen Erwinia amylovora

Toxins ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 792
Author(s):  
Olivier Tremblay ◽  
Zachary Thow ◽  
A. Rod Merrill
Keyword(s):  
Host Cell ◽  
In Silico ◽  
Pathogenic Bacteria ◽  
Erwinia Amylovora ◽  
Genomic Sequence ◽  
Whooping Cough ◽  
Sequence Data ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Adp Ribosyltransferase

Mono-ADP-ribosyltransferase (mART) toxins are secreted by several pathogenic bacteria that disrupt vital host cell processes in deadly diseases like cholera and whooping cough. In the last two decades, the discovery of mART toxins has helped uncover the mechanisms of disease employed by pathogens impacting agriculture, aquaculture, and human health. Due to the current abundance of mARTs in bacterial genomes, and an unprecedented availability of genomic sequence data, mART toxins are amenable to discovery using an in silico strategy involving a series of sequence pattern filters and structural predictions. In this work, a bioinformatics approach was used to discover six bacterial mART sequences, one of which was a functional mART toxin encoded by the plant pathogen, Erwinia amylovora, called Vorin. Using a yeast growth-deficiency assay, we show that wild-type Vorin inhibited yeast cell growth, while catalytic variants reversed the growth-defective phenotype. Quantitative mass spectrometry analysis revealed that Vorin may cause eukaryotic host cell death by suppressing the initiation of autophagic processes. The genomic neighbourhood of Vorin indicated that it is a Type-VI-secreted effector, and co-expression experiments showed that Vorin is neutralized by binding of a cognate immunity protein, VorinI. We demonstrate that Vorin may also act as an antibacterial effector, since bacterial expression of Vorin was not achieved in the absence of VorinI. Vorin is the newest member of the mART family; further characterization of the Vorin/VorinI complex may help refine inhibitor design for mART toxins from other deadly pathogens.

scholarly journals Biochemical Analysis of Lpt3, a Protein Responsible forPhosphoethanolamine Addition to Lipooligosaccharide ofPathogenic Neisseria

2006 ◽  
Vol 188 (3) ◽  
pp. 1039-1048 ◽  
Author(s):  
Ellen T. O'Connor ◽  
Andrzej Piekarowicz ◽  
Karen V. Swanson ◽  
J. McLeod Griffiss ◽  
Daniel C. Stein
Keyword(s):  
Genomic Dna ◽  
Biochemical Analysis ◽  
Expression Vector ◽  
Inner Core ◽  
Genomic Sequence ◽  
Mass Spectrometry Analysis ◽  
Dna Encoding ◽  
Spectrometry Analysis ◽  
Transposon Insertion ◽  
Genomic Dna Sequence

ABSTRACT The inner core of neisserial lipooligosaccharide (LOS) contains heptose residues that can be decorated by phosphoethanolamine (PEA). PEA modification of heptose II (HepII) can occur at the 3, 6, or 7 position(s). We used a genomic DNA sequence of lpt3, derived from Neisseria meningitidis MC58, to search the genomic sequence of N. gonorrhoeae FA1090 and identified a homolog of lpt3 in N. gonorrhoeae. A PCR amplicon containing lpt3 was amplified from F62ΔLgtA, cloned, mutagenized, and inserted into the chromosome of N. gonorrhoeae strain F62ΔLgtA, producing strain F62ΔLgtAlpt3::Tn5. LOS isolated from this strain lost the ability to bind monoclonal antibody (MAb) 2-1-L8. Complementation of this mutation by genetic removal of the transposon insertion restored MAb 2-1-L8 binding. Mass spectrometry analysis of LOS isolated from the F62ΔLgtA indicated that this strain contained two PEA modifications on its LOS. F62ΔLgtAlpt3::Tn5 lacked a PEA modification on its LOS, a finding consistent with the hypothesis that lpt3 encodes a protein mediating PEA addition onto gonococcal LOS. The DNA encoding lpt3 was cloned into an expression vector and Lpt3 was purified. Purified Lpt3 was able to mediate the addition of PEA to LOS isolated from F62ΔLgtAlpt3::Tn5.

scholarly journals Description of Klebsiella spallanzanii sp. nov. and of Klebsiella pasteurii sp. nov

2019 ◽  
Author(s):  
Cristina Merla ◽  
Carla Rodrigues ◽  
Virginie Passet ◽  
Marta Corbella ◽  
Harry A. Thorpe ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Type Strain ◽  
Genomic Sequence ◽  
Phylogenetic Analyses ◽  
Klebsiella Oxytoca ◽  
Mass Spectrometry Analysis ◽  
Nucleotide Identity ◽  
Average Nucleotide Identity ◽  
Single Strain ◽  
Spectrometry Analysis

AbstractKlebsiella oxytoca causes opportunistic human infections and post-antibiotic haemorrhagic diarrhoea. This Enterobacteriaceae species is genetically heterogeneous and is currently subdivided into seven phylogroups (Ko1 to Ko4, Ko6 to Ko8). Here we investigated the taxonomic status of phylogroups Ko3 and Ko4. Genomic sequence-based phylogenetic analyses demonstrate that Ko3 and Ko4 formed well-defined sequence clusters related to, but distinct from, Klebsiella michiganensis (Ko1), Klebsiella oxytoca (Ko2), K. huaxiensis (Ko8) and K. grimontii (Ko6). The average nucleotide identity of Ko3 and Ko4 were 90.7% with K. huaxiensis and 95.5% with K. grimontii, respectively. In addition, three strains of K. huaxiensis, a species so far described based on a single strain from a urinary tract infection patient in China, were isolated from cattle and human faeces. Biochemical and MALDI-ToF mass spectrometry analysis allowed differentiating Ko3, Ko4 and Ko8 from the other K. oxytoca species. Based on these results, we propose the names Klebsiella spallanzanii for the Ko3 phylogroup, with SPARK_775_C1T (CIP 111695T, DSM 109531T) as type strain, and Klebsiella pasteurii for Ko4, with SPARK_836_C1T (CIP 111696T, DSM 109530T) as type strain. Strains of K. spallanzanii were isolated from human urine, cow faeces and farm surfaces, while strains of K. pasteurii were found in faecal carriage from humans, cows and turtles.Accession numbersThe nucleotide sequences generated in this study were deposited in ENA and are available through the INSDC databases under accession numbers MN091365 (SB6411T = SPARK775C1T), MN091366 (SB6412 T = SPARK836C1T) and MN104661 to MN104677 (16S rRNA), MN076606 to MN076643 (gyrA and rpoB), and MN030558 to MN030567 (blaOXY). Complete genomic sequences were submitted to European Nucleotide Archive under the BioProject number PRJEB15325.AbbreviationsANI, average nucleotide identity; HCCA, a-cyano-4-hydroxycinnamic acid; isDDH, in silico DNA-DNA hybridization; SCAI, Simmons citrate agar with inositol; MALDI57 ToF MS: Matrix-assisted laser desorption/ionization time of flight mass spectrometry

scholarly journals Mass spectrometry analysis reveals differences in the host cell protein species found in pseudotyped lentiviral vectors

Biologicals ◽  
2018 ◽  
Vol 52 ◽  
pp. 59-66 ◽  
Author(s):  
Sabine Johnson ◽  
Jun X. Wheeler ◽  
Robin Thorpe ◽  
Mary Collins ◽  
Yasuhiro Takeuchi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Host Cell ◽  
Lentiviral Vectors ◽  
Mass Spectrometry Analysis ◽  
Cell Protein ◽  
Protein Species ◽  
Host Cell Protein ◽  
Spectrometry Analysis

scholarly journals Chemical characterization and mode of action of Ligustrum lucidum flower essential oil against food-borne pathogenic bacteria

2016 ◽  
Vol 11 (1) ◽  
pp. 269 ◽  
Author(s):  
Vivek K. Bajpai ◽  
Siddhartha Singh ◽  
Archana Mehta
Keyword(s):  
Essential Oil ◽  
Mode Of Action ◽  
Pathogenic Bacteria ◽  
Membrane Integrity ◽  
Chemical Characterization ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Ligustrum Lucidum ◽  
Food Borne

<p class="Abstract">Current research analyzes the chemical composition of <em>Ligustrum lucidum</em> flower essential oil obtained by the hydrodistillation, and examines its anti-microbial mode of action against food-borne pathogenic bacteria. Gas chromatography-mass spectrometry analysis of the oil resulted in the determination of 44 different compounds, representing 85.2% of the total oil. The oil (1 mg/disc) showed significant antibacterial effect as diameters of inhibition zones (14.6 ± 0.2 – 19.7 ± 0.3 mm), as well as minimum inhibitory and minimum bactericidal concentrations values (250–1000 and 250–2000 µg/mL), respectively. Based on the susceptibility, <em>L. lucidum</em> flower oil revealed its mode of action on membrane integrity as confirmed by increased release of extracellular ATP (2.5 and 2.2 pg/mL), leakage of potassium ions (950 and 900 mM/L), loss of 260-nm absorbing materials (4.2 and 3.9 optical density), and increase in relative electrical conductivity (10.6 and 9.8%) against <em>Staphylococcus aureus</em> KCTC-1621 (Gram-positive) and <em>Salmonella enterica</em> ATCC-4731 (Gram-negative), respectively.</p><p> </p>

scholarly journals Regulation of Insulin Secretion by SIRT4, a Mitochondrial ADP-ribosyltransferase

2007 ◽  
Vol 282 (46) ◽  
pp. 33583-33592 ◽  
Author(s):  
Nidhi Ahuja ◽  
Bjoern Schwer ◽  
Stefania Carobbio ◽  
David Waltregny ◽  
Brian J. North ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Insulin Secretion ◽  
Amino Acid ◽  
Bovine Serum Albumin ◽  
Matrix Protein ◽  
Mass Spectrometry Analysis ◽  
Carrier Proteins ◽  
Β Cells ◽  
Spectrometry Analysis ◽  
Adp Ribosyltransferase

Sirtuins are homologues of the yeast transcriptional repressor Sir2p and are conserved from bacteria to humans. We report that human SIRT4 is localized to the mitochondria. SIRT4 is a matrix protein and becomes cleaved at amino acid 28 after import into mitochondria. Mass spectrometry analysis of proteins that coimmunoprecipitate with SIRT4 identified insulindegrading enzyme and the ADP/ATP carrier proteins, ANT2 and ANT3. SIRT4 exhibits no histone deacetylase activity but functions as an efficient ADP-ribosyltransferase on histones and bovine serum albumin. SIRT4 is expressed in islets of Langerhans and colocalizes with insulin-expressing β cells. Depletion of SIRT4 from insulin-producing INS-1E cells results in increased insulin secretion in response to glucose. These observations define a new role for mitochondrial SIRT4 in the regulation of insulin secretion.

scholarly journals Identification of Extracellular N-Acylhomoserine Lactone Acylase from a Streptomyces sp. and Its Application to Quorum Quenching

2005 ◽  
Vol 71 (5) ◽  
pp. 2632-2641 ◽  
Author(s):  
Sun-Yang Park ◽  
Hye-Ok Kang ◽  
Hak-Sun Jang ◽  
Jung-Kee Lee ◽  
Bon-Tag Koo ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Pathogenic Bacteria ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Amino Acid Sequences ◽  
Amide Bond ◽  
Mass Spectrometry Analysis ◽  
Penicillin G ◽  
Spectrometry Analysis ◽  
Acyl Chains

ABSTRACT N-Acylhomoserine lactones (AHLs) play an important role in regulating virulence factors in pathogenic bacteria. Recently, the enzymatic inactivation of AHLs, which can be used as antibacterial targets, has been identified in several soil bacteria. In this study, strain M664, identified as a Streptomyces sp., was found to secrete an AHL-degrading enzyme into a culture medium. The ahlM gene for AHL degradation from Streptomyces sp. strain M664 was cloned, expressed heterologously in Streptomyces lividans, and purified. The enzyme was found to be a heterodimeric protein with subunits of approximately 60 kDa and 23 kDa. A comparison of AhlM with known AHL-acylases, Ralstonia strain XJ12B AiiD and Pseudomonas aeruginosa PAO1 PvdQ, revealed 35% and 32% identities in the deduced amino acid sequences, respectively. However, AhlM was most similar to the cyclic lipopeptide acylase from Streptomyces sp. strain FERM BP-5809, exhibiting 93% identity. A mass spectrometry analysis demonstrated that AhlM hydrolyzed the amide bond of AHL, releasing homoserine lactone. AhlM exhibited a higher deacylation activity toward AHLs with long acyl chains rather than short acyl chains. Interestingly, AhlM was also found to be capable of degrading penicillin G by deacylation, showing that AhlM has a broad substrate specificity. The addition of AhlM to the growth medium reduced the accumulation of AHLs and decreased the production of virulence factors, including elastase, total protease, and LasA, in P. aeruginosa. Accordingly, these results suggest that AHL-acylase, AhlM could be effectively applied to the control of AHL-mediated pathogenicity.

scholarly journals Holomycin, an antibiotic secondary metabolite, is required for biofilm formation of the native producer Photobacterium galatheae S2753

2021 ◽  
Author(s):  
Sheng-Da Zhang ◽  
Thomas Isbrandt ◽  
Laura Louise Lindqvist ◽  
Thomas Ostenfeld Larsen ◽  
Lone Gram
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Genome Mining ◽  
Core Gene ◽  
Mass Spectrometry Analysis ◽  
Distinct Advantage ◽  
Spectrometry Analysis ◽  
Cell Extracts ◽  
Antibiotic Compounds ◽  
Ecological Importance

Whilst the effects of antibiotics on microorganisms are widely studied, it remains less well-understood how antibiotics affect the physiology of the native producing organisms. Here, using a marine bacterium Photobacterium galatheae S2753 that produces the antibiotic holomycin, we generated a holomycin deficient strain by in-frame deletion of hlmE, the core gene responsible for holomycin production. Mass spectrometry analysis of cell extracts confirmed that ΔhlmE did not produce holomycin and that the mutant was devoid of antibacterial activity. Biofilm formation of ΔhlmE was significantly reduced compared to that of the wild-type S2753 and was restored in an hlmE complementary mutant. Consistently, exogenous holomycin, but not its dimethylated and less antibacterial derivative, S,S’-dimethyl holomycin, restored the biofilm formation of ΔhlmE. Furthermore, zinc starvation was found essential for both holomycin production and biofilm formation of S2753, although the molecular mechanism remains elusive. Collectively, these data suggest that holomycin promotes biofilm formation of S2753 via its ene-disulfide group. Lastly, the addition of holomycin in sub-inhibitory concentrations also enhanced the biofilm of four other Vibrionaceae strains. P. galatheae likely gains an ecological advantage from producing holomycin as both an antibiotic and a biofilm stimulator, which facilitates the nutrition acquisition and protects P. galatheae from environmental stresses. Studying the function of antibiotic compounds in the native producer will shed light on their role in nature and could potentially point to novel bioprospecting strategies. Importance Despite the societal impact of antibiotics, their ecological functions remain elusive and have mostly been studied by exposing non-producing bacteria to sub-inhibitory concentrations. Here, we studied the effects of the antibiotic holomycin on its native producer, Photobacterium galatheae S2753, a Vibrionaceae bacterium. Holomycin provides a distinct advantage to S2753 both as an antibiotic and by enhancing biofilm formation in the producer. Vibrionaceae species successfully thrive in global marine ecosystems, where they play critical ecological roles as free-living, symbiotic, or pathogenic bacteria. Genome mining has demonstrated that many have the potential to produce several bioactive compounds, including P. galaltheae. To unravel the contribution of the microbial metabolites to the development of marine microbial ecosystems, better insight into the function of these compounds in the producing organisms is needed. Our finding provides a model to pursue this and highlights the ecological importance of antibiotics to the fitness of the producing organisms.

scholarly journals Investigation of the In-Vivo Cytotoxicity and the In Silico-Prediction of MDM2-p53 Inhibitor Potential of Euphorbia peplus Methanolic Extract in Rats

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 642 ◽  
Author(s):  
Yasmina M. Abd-Elhakim ◽  
Mohamed Abdo Nassan ◽  
Gamal A. Salem ◽  
Abdelkarim Sasi ◽  
Adil Aldhahrani ◽  
...  
Keyword(s):  
In Silico ◽  
Methanolic Extract ◽  
P53 Protein ◽  
Underlying Mechanism ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
P53 Expression ◽  
Spectrometry Analysis ◽  
Euphorbia Peplus

This study explored the probable in vivo cardiac and renal toxicities together with in silico approaches for predicting the apoptogenic potential of Euphorbia peplus methanolic extract (EPME) in rats. Cardiac and renal injury biomarkers were estimated with histopathological and immunohistochemical evaluations of both kidney and heart. The probable underlying mechanism of E. peplus compounds to potentiate p53 activity is examined using Molecular Operating Environment (MOE) docking software and validated experimentally by immunohistochemical localization of p53 protein in the kidney and heart tissues. The gas chromatography/mass spectrometry analysis of E. peplus revealed the presence of nine different compounds dominated by di-(2-ethylhexyl) phthalate (DEHP). Significant elevations of troponin, creatine phosphokinase, creatine kinase–myocardium bound, lactate dehydrogenase, aspartate transaminase, alkaline phosphatase, urea, creatinine, and uric acid were evident in the EPME treated rats. The EPME treated rats showed strong renal and cardiac p53 expression and moderate cardiac TNF-α expression. Further, our in silico results predicted the higher affinity and good inhibition of DEHP, glyceryl linolenate, and lucenin 2 to the MDM2-p53 interface compared to the standard reference 15 a compound. Conclusively, EPME long-term exposure could adversely affect the cardiac and renal tissues probably due to their inflammatory and apoptotic activity. Moreover, the in silico study hypothesizes that EPME inhibits MDM2-mediated degradation of p53 suggesting possible anticancer potentials which confirmed experimental by strong p53 expression in renal and cardiac tissues.

scholarly journals Mycobacterium smegmatis Erm(38) Is a Reluctant Dimethyltransferase

2005 ◽  
Vol 49 (9) ◽  
pp. 3803-3809 ◽  
Author(s):  
Christian Toft Madsen ◽  
Lene Jakobsen ◽  
Stephen Douthwaite
Keyword(s):  
Mycobacterium Smegmatis ◽  
Cell Walls ◽  
Pathogenic Bacteria ◽  
Specific Resistance ◽  
Methylation Pattern ◽  
Mass Spectrometry Analysis ◽  
23S Rrna ◽  
Resistance Pattern ◽  
Type I ◽  
Spectrometry Analysis

ABSTRACT The waxy cell walls of mycobacteria provide intrinsic tolerance to a broad range of antibiotics, and this effect is augmented by specific resistance determinants. The inducible determinant erm(38) in the nontuberculous species Mycobacterium smegmatis confers high resistance to lincosamides and some macrolides, without increasing resistance to streptogramin B antibiotics. This is an uncharacteristic resistance pattern falling between the type I and type II macrolide, lincosamide, and streptogramin B (MLSB) phenotypes that are conferred, respectively, by Erm monomethyltransferases and dimethyltransferases. Erm dimethyltransferases are typically found in pathogenic bacteria and confer resistance to all MLSB drugs by addition of two methyl groups to nucleotide A2058 in 23S rRNA. We show here by mass spectrometry analysis of the mycobacterial rRNA that Erm(38) is indeed an A2058-specific dimethyltransferase. The activity of Erm(38) is lethargic, however, and only a meager proportion of the rRNA molecules become dimethylated in M. smegmatis, while most of the rRNAs are either monomethylated or remain unmethylated. The methylation pattern produced by Erm(38) clarifies the phenotype of M. smegmatis, as it is adequate to confer resistance to lincosamides and 14-member ring macrolides such as erythromycin, but it is insufficient to raise the level of resistance to streptogramin B drugs above the already high intrinsic tolerance displayed by this species.

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