Pathways of Pyrimidine Salvage in Pseudomonas and Former Pseudomonas: Detection of Recycling Enzymes Using High-Performance Liquid Chromatography

Beck, Debrah A.; O’Donovan, Gerard A.

doi:10.1007/s00284-007-9050-3

Cited by 17 publications

(13 citation statements)

References 26 publications

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“…Very recently it has been shown that P. aeruginosa mutants resistant to 5-FC spontaneously arise after long-term treatment with 5-FC in human serum, and whole genome sequencing revealed that these mutants invariably carried mutations in the upp gene (Rezzoagli et al, 2018), which encodes the uracil phosphoribosyltransferase responsible for conversion of uracil into the nucleotide precursor UMP in the pyrimidine salvage pathway (Beck and O'Donovan, 2008). To verify whether upp mutations were also present in our spontaneous 5-FU resistant mutants, we sequenced the entire upp gene, including its promoter.…”

Section: Resultsmentioning

confidence: 99%

“…To investigate this aspect, in this work we analyzed two highly-related antivirulence drugs endowed with significant antivirulence activity and different impact on P. aeruginosa growth, namely the fluorinated pyrimidine analogs 5-FU and 5-FC (Figure 1, Supplementary Figure 1; Kirienko et al, 2016). The growth inhibitory activity of 5-FU allowed us to easily select spontaneous 5-FU-resistant mutants, which were characterized by loss-of-function mutations in the upp gene, responsible for conversion of uracil into the nucleotide precursor UMP in the pyrimidine salvage pathway (Figures 3, 4, Supplementary Table 1; Beck and O'Donovan, 2008). This implies that the growth inhibitory activity of 5-FU is mainly related to inhibition of nucleic acid synthesis, as it has been proposed to occur in yeasts and cancer cells (Vermes et al, 2000; Longley et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

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Activity and Impact on Resistance Development of Two Antivirulence Fluoropyrimidine Drugs in Pseudomonas aeruginosa

Imperi

Fiscarelli

Visaggio

et al. 2019

Front. Cell. Infect. Microbiol.

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The rise in antibiotic resistance among bacterial pathogens has prompted the exploitation of alternative antibacterial strategies, such as antivirulence therapy. By inhibiting virulence traits, antivirulence drugs are expected to lessen pathogenicity without affecting bacterial growth, therefore avoiding the spread of resistance. However, some studies argued against this assumption, and the lack of antivirulence drugs in clinical use hampers the empirical assessment of this concept. Here we compared the mode of action and range of activity of two drugs which have been proposed for repurposing as quorum sensing and pyoverdine inhibitors in the human pathogen Pseudomonas aeruginosa: the anticancer drug 5-fluorouracil (5-FU) and the antimycotic drug 5-fluorocytosine (5-FC), respectively. The effect on bacterial growth, emergence and spread of resistance, and activity against clinical isolates were assessed. Our results confirm that 5-FU has growth inhibitory activity on reference strains and can rapidly select for spontaneous resistant mutants with loss-of-function mutations in the upp gene, responsible for uracil conversion into UMP. These mutants were also insensitive to the anti-pyoverdine effect of 5-FC. Conversely, 5-FC did not cause relevant growth inhibition, likely because of poor enzymatic conversion into 5-FU by cytosine deaminase. However, coculturing experiments showed that 5-FU resistant mutants can outcompete sensitive cells in mixed populations, in the presence of not only 5-FU but also 5-FC. Moreover, we observed that serial passages of wild-type cells in 5-FC-containing medium leads to the appearance and spread of 5-FC insensitive sub-populations of 5-FU resistant cells. The different effect on growth of 5-FU and 5-FC was overall conserved in a large collection of cystic fibrosis (CF) isolates, corresponding to different infection stages and antibiotic resistance profiles, although high variability was observed among strains. Notably, this analysis also revealed a significant number of pyoverdine-deficient isolates, whose proportion apparently increases over the course of the CF infection. This study demonstrates that the efficacy of an antivirulence drug with no apparent effect on growth can be significantly influenced by the emergence of insensitive mutants, and highlights the importance of the assessment of resistance-associated fitness cost and activity on clinical isolates for the development of “resistance-proof” antivirulence drugs.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Activity and Impact on Resistance Development of Two Antivirulence Fluoropyrimidine Drugs in Pseudomonas aeruginosa

Imperi

Fiscarelli

Visaggio

et al. 2019

Front. Cell. Infect. Microbiol.

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“…Also, the overexpression of CTP synthase (citidine triphosphate synthase, pyr G gene, Q8CNI2) supports the de novo biosynthesis of the nucleotide cytosine by catalyzing the ATP-dependent amination of the UTP pyrimidine ring at 4-position to obtain CTP using either L-glutamine or ammonia as a nitrogen source (Endrizzi et al, 2004). Other proteins such as uridine kinase (udk gene, Q8CSB2) highlight the effort bacteria mount in pyrimidine biosynthesis by activating the salvage pathway, resulting in pyrimidine biosynthesis in a more cost-effective way (Beck and O’Donovan, 2008).…”

Section: Discussionmentioning

confidence: 99%

Proteomic Analysis Reveals a Biofilm-Like Behavior of Planktonic Aggregates of Staphylococcus epidermidis Grown Under Environmental Pressure/Stress

et al. 2019

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Prosthetic joint replacement failure has a huge impact on quality of life and hospitalization costs. A leading cause of prosthetic joint infection is bacteria-forming biofilm on the surface of orthopedic devices. Staphylococcus epidermidis is an emergent, lowvirulence pathogen implicated in chronic infections, barely indistinguishable from aseptic loosening when embedded in a mature matrix. The literature on the behavior of quiescent S. epidermidis in mature biofilms is scarce. To fill this gap, we performed comparative analysis of the whole proteomic profiles of two methicillin-resistant S. epidermidis strains growing in planktonic and in sessile form to investigate the molecular mechanisms underlying biofilm stability. After 72-h culture of biofilmforming S. epidermidis, overexpression of proteins involved in the synthesis of nucleoside triphosphate and polysaccharides was observed, whereas planktonic bacteria expressed proteins linked to stress and anaerobic growth. Cytological analysis was performed to determine why planktonic bacteria unexpectedly expressed proteins typical of sessile culture. Images evidenced that prolonged culture under vigorous agitation can create a stressful growing environment that triggers microorganism aggregation in a biofilm-like matrix as a mechanism to survive harsh conditions. The choice of a unique late time point provided an important clue for future investigations into the biofilm-like behavior of planktonic cells. Our preliminary results may inform comparative proteomic strategies in the study of mature bacterial biofilm. Finally, there is an increasing number of studies on the aggregation of free-floating bacteria embedded in an extracellular matrix, prompting the need to gain further insight into this mode of bacterial growth.

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“…There were 4 different types of mutations, including two different nonsynonymous SNPs, a 15-bp deletion and a 1-bp insertion ( Supplementary Table S3 ). The upp gene encodes for a uracil phosphoribosyl-transferase, an enzyme required for the intra-cellular activation of flucytosine [ 56 , 57 ].…”

Section: Resultsmentioning

confidence: 99%

Probing the evolutionary robustness of two repurposed drugs targeting iron uptake in Pseudomonas aeruginosa

Rezzoagli

Wilson

Weigert

et al. 2018

Evolution, Medicine, and Public Health

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Lay SummaryWe probed the evolutionary robustness of two antivirulence drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine in the opportunistic pathogen Pseudomonas aeruginosa. Using an experimental evolution approach in human serum, we showed that antivirulence treatments are not evolutionarily robust per se, but vary in their propensity to select for resistance.Background and objectivesTreatments that inhibit the expression or functioning of bacterial virulence factors hold great promise to be both effective and exert weaker selection for resistance than conventional antibiotics. However, the evolutionary robustness argument, based on the idea that antivirulence treatments disarm rather than kill pathogens, is controversial. Here, we probe the evolutionary robustness of two repurposed drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine of the opportunistic human pathogen Pseudomonas aeruginosa.MethodologyWe subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an ex vivo infection model. We screened evolved populations and clones for resistance phenotypes, including the restoration of growth and pyoverdine production, and the evolution of iron uptake by-passing mechanisms. We whole-genome sequenced evolved clones to identify the genetic basis of resistance.ResultsWe found that mutants resistant against antivirulence treatments readily arose, but their selective spreading varied between treatments. Flucytosine resistance quickly spread in all populations due to disruptive mutations in upp, a gene encoding an enzyme required for flucytosine activation. Conversely, resistance against gallium arose only sporadically, and was based on mutations in transcriptional regulators, upregulating pyocyanin production, a redox-active molecule promoting siderophore-independent iron acquisition. The spread of gallium resistance was presumably hampered because pyocyanin-mediated iron delivery benefits resistant and susceptible cells alike.Conclusions and implicationsOur work highlights that antivirulence treatments are not evolutionarily robust per se. Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.

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Pathways of Pyrimidine Salvage in Pseudomonas and Former Pseudomonas: Detection of Recycling Enzymes Using High-Performance Liquid Chromatography

Cited by 17 publications

References 26 publications

Activity and Impact on Resistance Development of Two Antivirulence Fluoropyrimidine Drugs in Pseudomonas aeruginosa

Activity and Impact on Resistance Development of Two Antivirulence Fluoropyrimidine Drugs in Pseudomonas aeruginosa

Proteomic Analysis Reveals a Biofilm-Like Behavior of Planktonic Aggregates of Staphylococcus epidermidis Grown Under Environmental Pressure/Stress

Probing the evolutionary robustness of two repurposed drugs targeting iron uptake in Pseudomonas aeruginosa

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