scholarly journals Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli

2020 ◽  
Author(s):  
Jenelle A. Patterson ◽  
Hai He ◽  
Jacob S. Folz ◽  
Qiang Li ◽  
Mark A. Wilson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Damage Control ◽  
Genomic Analysis ◽  
Relevant Information ◽  
Comparative Genomic ◽  
Control Mechanisms ◽  
One Carbon Metabolism ◽  
Formaldehyde Toxicity

ABSTRACTFormaldehyde (HCHO) is a reactive carbonyl compound that formylates and cross-links proteins, DNA, and small molecules. It is of specific concern as a toxic intermediate in the design of engineered pathways involving methanol oxidation or formate reduction. The high interest in engineering these pathways is not, however, matched by engineering-relevant information on precisely why HCHO is toxic or on what damage-control mechanisms cells deploy to manage HCHO toxicity. The only well-defined mechanism for managing HCHO toxicity is formaldehyde dehydrogenase-mediated oxidation to formate, which is counterproductive if HCHO is a desired pathway intermediate. We therefore sought alternative HCHO damage-control mechanisms via comparative genomic analysis. This analysis associated homologs of the Escherichia coli pepP gene with HCHO-related one-carbon metabolism. Furthermore, deleting pepP increased the sensitivity of E. coli to supplied HCHO but not other carbonyl compounds. PepP is a proline aminopeptidase that cleaves peptides of the general formula X-Pro-Y, yielding X + Pro-Y. HCHO is known to react spontaneously with cysteine to form the close proline analog thioproline (thiazolidine-4-carboxylate), which is incorporated into proteins and hence into proteolytic peptides. We therefore hypothesized that thioproline-containing peptides are toxic and that PepP cleaves these aberrant peptides. Supporting this hypothesis, PepP cleaved the model peptide Ala-thioproline-Ala as efficiently as Ala-Pro-Ala in vitro and in vivo, and deleting pepP increased sensitivity to supplied thioproline. Our data thus (i) provide biochemical genetic evidence that thioproline formation contributes substantially to HCHO toxicity and (ii) make PepP a candidate damage-control enzyme for engineered pathways having HCHO as an intermediate.

scholarly journals Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli

2020 ◽  
Vol 477 (9) ◽  
pp. 1745-1757 ◽  
Author(s):  
Jenelle A. Patterson ◽  
Hai He ◽  
Jacob S. Folz ◽  
Qiang Li ◽  
Mark A. Wilson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Damage Control ◽  
Genomic Analysis ◽  
Relevant Information ◽  
Comparative Genomic ◽  
Control Mechanisms ◽  
One Carbon Metabolism ◽  
Formaldehyde Toxicity

Formaldehyde (HCHO) is a reactive carbonyl compound that formylates and cross-links proteins, DNA, and small molecules. It is of specific concern as a toxic intermediate in the design of engineered pathways involving methanol oxidation or formate reduction. The interest in engineering these pathways is not, however, matched by engineering-relevant information on precisely why HCHO is toxic or on what damage-control mechanisms cells deploy to manage HCHO toxicity. The only well-defined mechanism for managing HCHO toxicity is formaldehyde dehydrogenase-mediated oxidation to formate, which is counterproductive if HCHO is a desired pathway intermediate. We therefore sought alternative HCHO damage-control mechanisms via comparative genomic analysis. This analysis associated homologs of the Escherichia coli pepP gene with HCHO-related one-carbon metabolism. Furthermore, deleting pepP increased the sensitivity of E. coli to supplied HCHO but not other carbonyl compounds. PepP is a proline aminopeptidase that cleaves peptides of the general formula X-Pro-Y, yielding X + Pro-Y. HCHO is known to react spontaneously with cysteine to form the close proline analog thioproline (thiazolidine-4-carboxylate), which is incorporated into proteins and hence into proteolytic peptides. We therefore hypothesized that certain thioproline-containing peptides are toxic and that PepP cleaves these aberrant peptides. Supporting this hypothesis, PepP cleaved the model peptide Ala-thioproline-Ala as efficiently as Ala-Pro-Ala in vitro and in vivo, and deleting pepP increased sensitivity to supplied thioproline. Our data thus (i) provide biochemical genetic evidence that thioproline formation contributes substantially to HCHO toxicity and (ii) make PepP a candidate damage-control enzyme for engineered pathways having HCHO as an intermediate.

Extended-spectrum resistance to β-lactams/β-lactamase inhibitors (ESRI) evolved from low-level resistant Escherichia coli

2019 ◽  
Author(s):  
Ángel Rodríguez-Villodres ◽  
María Luisa Gil-Marqués ◽  
Rocío Álvarez-Marín ◽  
Rémy A Bonnin ◽  
María Eugenia Pachón-Ibáñez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clavulanic Acid ◽  
Genomic Analysis ◽  
E Coli ◽  
Extended Spectrum ◽  
Copy Numbers ◽  
Resistance Patterns ◽  
Amoxicillin Clavulanic Acid

Abstract Objectives Escherichia coli is characterized by three resistance patterns to β-lactams/β-lactamase inhibitors (BLs/BLIs): (i) resistance to ampicillin/sulbactam and susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (RSS); (ii) resistance to ampicillin/sulbactam and amoxicillin/clavulanic acid, and susceptibility to piperacillin/tazobactam (RRS); and (iii) resistance to ampicillin/sulbactam, amoxicillin/clavulanic acid and piperacillin/tazobactam (RRR). These resistance patterns are acquired consecutively, indicating a potential risk of developing resistance to piperacillin/tazobactam, but the precise mechanism of this process is not completely understood. Methods Clinical isolates incrementally pressured by piperacillin/tazobactam selection in vitro and in vivo were used. We determined the MIC of piperacillin/tazobactam in the presence and absence of piperacillin/tazobactam pressure. We deciphered the role of the blaTEM genes in the new concept of extended-spectrum resistance to BLs/BLIs (ESRI) using genomic analysis. The activity of β-lactamase was quantified in these isolates. Results We show that piperacillin/tazobactam resistance is induced in E. coli carrying blaTEM genes. This resistance is due to the increase in copy numbers and transcription levels of the blaTEM gene, thus increasing β-lactamase activity and consequently increasing piperacillin/tazobactam MICs. Genome sequencing of two blaTEM-carrying representative isolates showed that piperacillin/tazobactam treatment produced two types of duplications of blaTEM (8 and 60 copies, respectively). In the clinical setting, piperacillin/tazobactam treatment of patients infected by E. coli carrying blaTEM is associated with a risk of therapeutic failure. Conclusions This study describes for the first time the ESRI in E. coli. This new concept is very important in the understanding of the mechanism involved in the acquisition of resistance to BLs/BLIs.

scholarly journals Characterization and Comparative Genomic Analysis of a Novel Bacteriophage, SFP10, Simultaneously Inhibiting both Salmonella enterica and Escherichia coli O157:H7

2011 ◽  
Vol 78 (1) ◽  
pp. 58-69 ◽  
Author(s):  
Minjung Park ◽  
Ju-Hoon Lee ◽  
Hakdong Shin ◽  
Minsik Kim ◽  
Jeongjoon Choi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Burst Size ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Tail Fiber ◽  
Content Type ◽  
E Coli ◽  
Development Of Resistance

ABSTRACTSalmonella entericaandEscherichia coliO157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of bothS. entericaandE. coliO157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like familyMyoviridae.In vitroadsorption assays showed that the adsorption constant rates to bothSalmonella entericaserovar Typhimurium andE. coliO157:H7 were 2.50 × 10−8ml/min and 1.91 × 10−8ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinaryMyoviridaephages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 inS.Typhimurium andE. coliO157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 × 10−2CFU/ml forS.Typhimurium and 4.58 × 10−5CFU/ml forE. coliO157:H7 were found, indicating that SFP10 should be active and stable for control ofE. coliO157:H7 with minimal emergence of SFP10-resistant pathogens but may not be forS.Typhimurium. Specific mutation ofrfaLinS.Typhimurium andE. coliO157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologousSalmonellaVi01 andShigellaphiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition ofSalmonellaTyphimurium andE. coliO157:H7 by a single bacteriophage.

scholarly journals Antibiofilm agents with therapeutic potential against enteroaggregative Escherichia coli

2021 ◽  
Author(s):  
David A Kwasi ◽  
Chinedum Peace Babalola ◽  
Olujide O Olubiyi ◽  
Jennifer Hoffmann ◽  
Ikemefuna Uzochukwu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mechanism Of Action ◽  
Time Course ◽  
Therapeutic Potential ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Antibiofilm Activity ◽  
Docking Simulations ◽  
New Therapeutic Approaches

Abstract Background Enteroaggregative Escherichia coli (EAEC) is a predominant but neglected enteric pathogen implicated in infantile diarrhoea and nutrient malabsorption. There are no non-antibiotic approaches to dealing with persistent infection by these exceptional colonizers, which form copious biofilms. We screened the Medicines for Malaria Venture Pathogen Box for chemical entities that inhibit EAEC biofilm formation. Methodology We used two EAEC strains, 042 and MND005E, in a medium-throughput crystal violet-based antibiofilm screen. Hits were confirmed in concentration-dependence, growth kinetic and time course assays and activity spectra were determined against a panel of genome-sequenced EAEC. Antibiofilm activity against isogenic EAEC mutants, molecular docking simulations and comparative genomic analysis were used to identify the mechanism of action of one hit. Principal findings In all, five compounds (1.25%) reproducibly inhibited biofilm accumulation by at least one strain by 30-85% while inhibiting growth by under 10%. Hits exhibited at least 10-fold greater antibiofilm activity than nitazoxanide, the only known EAEC biofilm inhibitor. Reflective of known EAEC heterogeneity, only one hit was active against both screen isolates, but three hits showed broad antibiofilm activity against a larger panel of strains. Mechanism of action studies point to the EAEC anti-aggregation protein (Aap), dispersin, as the target of compound MMV687800. Conclusions This study identified five compounds not previously described as anti-adhesins or Gram-negative antibacterials with significant and specific EAEC antibiofilm activity. One molecule, MMV687800, targets the EAEC Aap. In vitro small-molecule inhibition of EAEC colonization opens a way to new therapeutic approaches to preventing and treating EAEC infection.

scholarly journals Phage Selective Pressure Reduces Virulence of Hypervirulent Klebsiella pneumoniae Through Mutation of the wzc Gene

2021 ◽  
Vol 12 ◽  
Author(s):  
Lingjie Song ◽  
Xianggui Yang ◽  
Jinwei Huang ◽  
Xiaokui Zhu ◽  
Guohui Han ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Genomic Analysis ◽  
Resistant Bacteria ◽  
Comparative Genomic ◽  
Wild Type ◽  
Bacteriophage Therapy ◽  
Deletion Mutations ◽  
Invasive Infections

Hypervirulent Klebsiella pneumoniae (hvKp), one of the major community-acquired pathogens, can cause invasive infections such as liver abscess. In recent years, bacteriophages have been used in the treatment of K. pneumoniae, but the characteristics of the phage-resistant bacteria produced in the process of phage therapy need to be evaluated. In this study, two Podoviridae phages, hvKpP1 and hvKpP2, were isolated and characterized. In vitro and in vivo experiments demonstrated that the virulence of the resistant bacteria was significantly reduced compared with that of the wild type. Comparative genomic analysis of monoclonal sequencing showed that nucleotide deletion mutations of wzc and wcaJ genes led to phage resistance, and the electron microscopy and mucoviscosity results showed that mutations led to the loss of the capsule. Meanwhile, animal assay indicated that loss of capsule reduced the virulence of hvKp. These findings contribute to a better understanding of bacteriophage therapy, which not only can kill bacteria directly but also can reduce the virulence of bacteria by phage screening.

scholarly journals Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte–specific expression in vivo

2003 ◽  
Vol 162 (5) ◽  
pp. 833-842 ◽  
Author(s):  
Qiping Zheng ◽  
Guang Zhou ◽  
Roy Morello ◽  
Yuqing Chen ◽  
Xavier Garcia-Rojas ◽  
...  
Keyword(s):  
Binding Sites ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Collagen Gene ◽  
Runt Domain ◽  
Specific Expression ◽  
Hypertrophic Chondrocyte ◽  
In Vitro Transfection

The α1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte–specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive β-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2+/− background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2-null mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

scholarly journals N-methyl-bacillithiol, a new metabolite discovered in theChlorobiaceae, indicates that bacillithiol and derivatives are widely phylogenetically distributed

2017 ◽  
Author(s):  
Jennifer Hiras ◽  
Sunil V. Sharma ◽  
Vidhyavathi Raman ◽  
Ryan A. J. Tinson ◽  
Miriam Arbach ◽  
...  
Keyword(s):  
Redox Homeostasis ◽  
Genomic Analysis ◽  
Sulfur Oxidation ◽  
Comparative Genomic ◽  
Molecular Formula ◽  
Gram Positive Bacteria ◽  
Redox Responsive ◽  
Functional Consequences

AbstractLow-molecular weight (LMW) thiols are metabolites that mediate redox homeostasis and the detoxification of chemical stressors in cells. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including theChlorobiaceae. Fluorescent thiol labeling of metabolite extracts coupled with HPLC showed thatChlorobaculum tepidumcontained a novel LMW thiol with a mass of 412 ± 1 Da corresponding to a molecular formula of C14H24N2O10S. These data suggested the new thiol is closely related to bacillithiol (BSH), the major LMW thiol from low G+C% Gram-positive bacteria. By comparing the as-isolated bimane adduct with chemically synthesized candidate structures, theCba. tepidumthiol structure was identified as N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, a rarely observed modification in metabolism. Orthologs of bacillithiol biosynthetic genes in theCba. tepidumgenome were required for the biosynthesis of N-Me-BSH. Furthermore, the CT1040 gene product was genetically identified as the BSH N-methyltransferase. N-Me-BSH was found in allChlorobiexamined as well asPolaribactersp. strain MED152, a member of theBacteroidetes.A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of theChlorobi,Bacteroidetes,Deinococcus-Thermus, andFirmicutes, but also byAcidobacteria,Chlamydiae,Gemmatimonadetes, andProteobacteria.Significance StatementHere, N-Me-BSH is shown to be a redox-responsive LMW thiol cofactor inCba. tepidumand the gene,nmbA, encoding the BSH N-methyltransferase responsible for its synthesis is identified. The co-occurrence of orthologs to BSH biosynthesis genes and bacillithiol N-methyltransferase was confirmed to correctly predict LMW thiol biosynthesis in phylogenetically distant genomes. The analysis indicates that BSH/N-Me-BSH are likely the most widely distributed class of LMW thiols in biology. This finding sheds light on the evolution of LMW thiol metabolism, which is central to redox homeostasis, regulation and stress resistance in all cellular life. It also sheds light on a rare chemical modification. N-Me-BSH is the fourth instance of cysteinyl nitrogen methylation in metabolism. Identification of the BSH N-methyltransferase reported here will enable detailedin vivoandin vitrodissection of the functional consequences of this modification. As a standalone N-methyltransferase, NmbA may be useful as a component of constructed biosynthetic pathways for novel product (bio)synthesis.

scholarly journals Inactivation of thymidine kinase as a cause of resistance to zidovudine in clinical isolates of Escherichia coli: a phenotypic and genomic study

2020 ◽  
Vol 75 (6) ◽  
pp. 1410-1414
Author(s):  
Lucie Peyclit ◽  
Maryem Ben Khedher ◽  
Lotfi Zerrouki ◽  
Seydina M Diene ◽  
Sophie Alexandra Baron ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Thymidine Kinase ◽  
Gene Sequence ◽  
Stop Codon ◽  
Genomic Analysis ◽  
Clinical Isolates ◽  
Comparative Genomic ◽  
E Coli ◽  
Genomic Study

Abstract Objectives The antiviral zidovudine has been recently identified as an active drug against resistant Enterobacteriaceae, but prevalence of resistance to this compound remains unknown. The aim was to estimate the prevalence of clinical Escherichia coli isolates resistant to zidovudine and to decipher the mechanism of zidovudine resistance. Methods We screened 537 isolates on zidovudine-containing agar plates and studied their thymidine kinase (tdk) gene sequences, the putative target involved in zidovudine resistance. Moreover, sequence analysis of 633 complete genomes of E. coli was performed to investigate mutation in the tdk gene. A comparative genomic analysis was done on an in vitro zidovudine-resistant mutant. Results After screening on our medium containing 2.7 mg/L (10 μM) zidovudine, nine strains had a zidovudine MIC >26.7 mg/L. The gene was absent in three isolates, inactivated by an IS (IS1X2 and ISApl1) in two isolates and mutated in four isolates. A genomic analysis of 633 E. coli genomes showed heterogeneity of the tdk gene sequence, with 27 different sequences. Among them, three genomes showed an inactivation of the gene (IS, stop codon and no tdk gene sequence). The in vitro mutant E. coli had 27 SNPs in eight genes of the core genome compared with the initial strain. Conclusions Our study reports zidovudine-resistant clinical isolates of E. coli, presumably related to tdk inactivation. Diversity of Tdk in bacterial genomes can be large. Other mechanisms need to be considered in zidovudine resistance. The use of zidovudine in antibiotic-resistant infections needs to be in combination and should be tested before clinical administration.

Безопасность соединений из группы терпенов ментанового ряда in vitro и in vivo

2020 ◽  
Vol 83 (4) ◽  
pp. 24-30
Author(s):  
Ирина Владимировна Акулина ◽  
Светлана Ивановна Павлова ◽  
Ирина Семеновна Степаненко ◽  
Назира Сунагатовна Карамова ◽  
Александр Владиславович Сергеев ◽  
...  
Keyword(s):  
Escherichia Coli

Проведено токсикологическое исследование соединений с антибактериальными свойствами из группы терпенов ментанового ряда в условиях in vitro и in vivo: лимонена (B34), его производного (+)-1,2-оксида лимонена (B60) и серосодержащего монотерпенового соединения 2-(1’-гидрокси-4’-изопренил-1’-метилциклогексил-2’-тио)метилэтаноата (B65). В условиях in vitro (культура опухолевых клеток HeLa) изучаемые монотерпены в диапазоне концентраций 2 – 200 мкг/мл обладали цитотоксичностью. Ингибирующая концентрация (ИК50) для B34 составила 231 (167 – 295) мкг/мл, для B60 – 181 (105 – 257) мкг/мл, ИК50 B65 – 229 (150 – 308) мкг/мл. Исследование генотоксичности показало, что B34 и B65 в диапазоне концентраций 50 – 1000 мкг/мл не индуцируют SOS мутагенез в клетках Escherichia coli PQ37, тогда как B60 в концентрациях 500 и 1000 мкг/мл проявляет генотоксичность. In vivo в остром эксперименте на беспородных мышах установлена низкая токсичность B34 и его производных при различных путях введения. Наименьший показатель острой токсичности имеет B65, в связи с чем дополнительно на крысах проведено изучение его хронической токсичности. Ежедневное внутрижелудочное введение B65 в разовых дозах, составляющих 1/10 и 1/20 ЛД50 (1000 мг/кг и 500 мг/кг), в течение 1 мес не вызывало гибели животных, значимых нарушений общего состояния, изменения динамики массы тела, морфопатологических изменений. Внутрижелудочное введение B65 крысам в высокой токсической дозе 2000 мг/кг (1/5 ЛД50) в течение месяца вызывает патоморфологические изменения структуры печени.

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