Novel pathways for ameliorating the fitness cost of gentamicin resistant small colony variants
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB15409 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Other | ||||
| ERR1633560_1.fastq.gz | Fastqsanger.gz | |||
| ERR1633560_2.fastq.gz | Fastqsanger.gz | |||
| ERR1633570_1.fastq.gz | Fastqsanger.gz | |||
| ERR1633570_2.fastq.gz | Fastqsanger.gz |
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Project description:Enterobacter cloacae is a Gram-negative nosocomial pathogen of the ESKAPE priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However, E. cloacae can also display phenotypic antimicrobial resistance, such as heteroresistance or persistence. Here we report that E. cloacae ATCC 13047 and six strains isolated from patients with blood infections display heteroresistance or persistence to aminoglycosides. E. cloacae heteroresistance is transient, accompanied with formation of "petite" colonies and increased MIC against gentamicin and other aminoglycosides used in the clinic, but not other antibiotic classes. To explore the underlying mechanisms, we performed RNA sequencing of heteroresistant bacteria, which revealed global gene-expression changes and a signature of the CpxRA cell envelope stress response. Deletion of the cpxRA two-component system abrogated aminoglycoside heteroresistance and petite colony formation, pointing to its indispensable role in phenotypic resistance. The introduction of a constitutively active allele of cpxA led to high aminoglycoside MICs, consistent with cell envelope stress driving these behaviours in E. cloacae. Cell envelope stress can be caused by environmental cues, including heavy metals. Indeed, bacterial exposure to copper increased gentamicin MIC in the wild type, but not the ΔcpxRA mutant. Moreover, copper exposure also elevated the gentamicin MICs of bloodstream isolates, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved in E. cloacae strains. Altogether, we establish that E. cloacae relies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic.
2023-10-18 | GSE236124 | GEO
Project description:Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10-5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAOSCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexABoprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system. We used microarrays to study changes in gene expression during early and late stationary phase of SCV and WT strains.
2011-12-06 | GSE34141 | GEO
Project description:Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10-5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAOSCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexABoprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system. We used microarrays to study changes in gene expression during early and late stationary phase of SCV and WT strains. SCV and WT cultures were grown in triplicate until early (24h) and late (48h) stationary phase. RNA was extracted, labelled and hybridised on Affymetrix P. aeruginosa expression microarrays.
2011-12-06 | E-GEOD-34141 | biostudies-arrayexpress
Project description:For this study, we created three highly representational different sized genomic libraries of P. aeruginosa PAO1 within the vector pBTB-1. Vector pBTB-1 is a low copy number broad host range plasmid containing a Ã-lactamase resistance marker, a pBAD promoter upstream of the cloning site, as well as transcriptional terminators flanking the cloning site to aid in insert stability. These libraries were transformed into the recombination-deficient P. aeruginosa PAO1 mutant, PAO2003, pooled, and parallel selections performed to identify via traditional sequencing or SCALEs the genomic regions capable of conferring increased tolerance to amikacin, gentamicin, or tobramycin. These antibiotics are all structurally related but have differing substitution patterns on their aminoglycoside backbones. These differences in structure and substitution patterns impact the activity of each antibiotic. We chose to study three different aminoglycosides in attempts to identify genomic regions capable of conferring resistance to not only a specific aminoglycoside, but also to the more general class of aminoglycosides.
2009-12-19 | E-MEXP-2396 | biostudies-arrayexpress
Project description:End-to-end chromosome fusions that occur in the context of telomerase deficiency can trigger genomic duplications. These duplications are suggested to arise via Breakage-Fusion-Bridge cycles. To test this hypothesis, we examined end-to-end fusions isolated from C. elegans telomere replication mutants. Genome level rearrangements revealed fused chromosome ends possessing interrupted terminal duplications accompanied by template switching events. These features are very similar to disease-associated duplications of interstitial segments of the human genome. A model termed Fork Stalling and Template Switching has been proposed previously to explain such duplications, where promiscuous replication of large, non-contiguous segments of the genome occurs. Thus, a DNA synthesis-based process can create duplications that seal end-to-end fusions, in the absence of Breakage-Fusion-Bridge cycles. Numerous C. elegans mutant samples were studied with comparative genomic hybridization. There were no replicates or dye-swap hybridizations.
2011-01-21 | E-GEOD-26691 | biostudies-arrayexpress
Project description:Aminoglycoside antibiotics display broad-spectrum activity against Gram-negative and Gram-positive bacteria by targeting their ribosomes. Herein, we have demonstrated that energy metabolism plays a crucial role in aminoglycoside tolerance, as knockout strains associated with the tricarboxylic acid cycle (TCA) and the electron transport chain (ETC) exhibited increased tolerance to aminoglycosides in the mid-exponential growth phase of Escherichia coli cells. Given that aminoglycoside uptake relies on the energy-driven electrochemical potential across the cytoplasmic membrane, our initial expectation was that these genetic perturbations would decrease the proton motive force (PMF), subsequently affecting the uptake of aminoglycosides. However, our results did not corroborate this assumption. We found no consistent metabolic changes, ATP levels, cytoplasmic pH variations, or membrane potential differences in the mutant strains compared to the wild type. Additionally, intracellular concentrations of fluorophore-labeled gentamicin remained similar across all strains. To uncover the mechanism responsible for the observed tolerance in mutant strains, we employed untargeted mass spectrometry to quantify the proteins within these mutants and subsequently compared them to their wild-type counterparts. Our comprehensive analysis, which encompassed protein-protein association networks and functional enrichment, unveiled a noteworthy upregulation of proteins linked to the TCA cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research has the potential to uncover mechanisms behind aminoglycoside tolerance, paving the way for novel strategies to combat such cells.
2024-06-21 | PXD053082 | Pride
Project description:Elucidating antibiotic resistance mechanisms is necessary for developing novel therapeutic strategies. The increasing incidence of antibiotic-resistant Vibrio alginolyticus infection threatens both human health and aquaculture, but the mechanism has not been fully elucidated. Here, an isobaric tags for relative and absolute quantification (iTRAQ) functional proteomics analysis was performed on gentamicin-resistant V. alginolyticus (VA-RGEN) and a gentamicin-sensitive strain in order to characterize the global protein expression changes upon gentamicin resistance. Proteomics analysis demonstrated a global metabolic downshift in VA-RGEN, where the pyruvate cycle (the P cycle) was severely compromised. Exogenous pyruvate restored the P cycle activity, disrupting the redox state and increasing the membrane potential. It thereby potentiated gentamicin-mediated killing by approximately 3000- and 150-fold in vitro and in vivo, respectively. More importantly, bacterial gentamicin pharmacokinetics indicated that pyruvate enhanced gentamicin influx to a degree that exceeded the gentamicin expelled by the bacteria, increasing the intracellular gentamicin. Thus, our study suggests a metabolism-based approach to combating gentamicin-resistant V. algonolyticus, which paves the way for combating other types of antibiotic-resistant bacterial pathogens.
2023-09-29 | PXD045938 | iProX
Project description:End-to-end chromosome fusions that occur in the context of telomerase deficiency can trigger genomic duplications. These duplications are suggested to arise via Breakage-Fusion-Bridge cycles. To test this hypothesis, we examined end-to-end fusions isolated from C. elegans telomere replication mutants. Genome level rearrangements revealed fused chromosome ends possessing interrupted terminal duplications accompanied by template switching events. These features are very similar to disease-associated duplications of interstitial segments of the human genome. A model termed Fork Stalling and Template Switching has been proposed previously to explain such duplications, where promiscuous replication of large, non-contiguous segments of the genome occurs. Thus, a DNA synthesis-based process can create duplications that seal end-to-end fusions, in the absence of Breakage-Fusion-Bridge cycles.
2011-01-21 | GSE26691 | GEO
Project description:Tumors arise and grow despite anti-cancer immune responses. These responses can be stimulated by immunotherapies such as immune checkpoint inhibitors (e.g. anti-PD1 antibodies) and chimeric antigen receptors (CAR). Efficacy of these agents in solid tumors including colorectal cancers (CRC) is limited by immunosuppressive tumor microenvironment (TME) that prevents killing of malignant cells by cytotoxic T lymphocytes (CTL). Understanding the nature of TME-generated immunosuppression is of paramount importance. Here we report that TME elicited immunosuppression via eliminating activated CTL; this elimination required TME stress-induced downregulation of the IFNAR1 chain of type I interferon (IFN) receptor and attenuation of its signaling. Downregulation of IFNAR1 was observed in human colorectal cancers (CRC) and in mouse CRC models where it was required for efficient tumor development and progression. Stabilization of IFNAR1 on CTL improved their survival and increased anti- tumor activities of CAR T cells and PD1 inhibitors thereby providing a rationale for targeting IFNAR1 degradation for immunotherapies optimization. Two genotypes of mice were examined either 9 or 21 days post injection of MC38 colon cancer cells or MC38mRFP cells, respectively. 2-3 replicate mice were analyzed on separate arrays for each condition. 6 conditions in total were analyzed.
2016-01-15 | E-GEOD-76889 | biostudies-arrayexpress
Project description:Bacterial populations face the constant threat of viral predation exerted by bacteriophages (or phages). In response, bacteria have evolved a wide range of defense mechanisms against phage challenges. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection. We observed a broad phage inhibition by aminoglycosides. We demonstrate that aminoglycosides do not prevent the injection of phage DNA into bacterial cells but instead block an early step of the viral life cycle. In this context, we used RNA sequencing of S. venezuelae cells infected with phage Alderaan to comparatively investigate the influence of apramycin on phage DNA tanscription at two different time points after inital infection.
2021-04-30 | GSE171784 | GEO