Using a chemical genetic screen to enhance our understanding of the antimicrobial properties of copper

Metallomics ◽  
2021 ◽  
Author(s):  
Natalie Gugala ◽  
Daniel A Salazar-Alemán ◽  
Gordon Chua ◽  
Raymond J Turner
Keyword(s):  
Essential Gene ◽  
Antimicrobial Properties ◽  
Central Carbon Metabolism ◽  
Copper Resistance ◽  
Copper Tolerance ◽  
Copper Stress ◽  
Bacterial Cells ◽  
Iron Sulfur Cluster ◽  
Mechanisms Of Toxicity ◽  
Chemical Genetic

Abstract The competitive toxic and stress inducing nature of copper necessitates systems that sequester and export this metal from the cytoplasm of bacterial cells. Several predicted mechanisms of toxicity include the production of reactive oxygen species, thiol depletion, DNA and iron-sulfur cluster disruption. Accompanying these mechanisms include pathways of homeostasis such as chelation, oxidation, and transport. Still, the mechanisms of copper resistance and sensitivity are not fully understood. Furthermore, studies fail to recognize that the response to copper is likely a result of numerous mechanisms, as in the case for homeostasis, in which proteins and enzymes work as a collective to maintain appropriate copper concentrations. In this study we used the Keio collection, an array of 3985 Escherichia coli mutants, each with a deleted non-essential gene, to gain a better understanding of prolonged copper exposure. In short, we recovered two copper homeostatic gene and genes involved in transporting and assembling to be involved in mediating prolonged copper stress under the conditions assessed. The gene coding for the protein TolC was uncovered as a sensitive hit and we demonstrated that tolC, an outer membrane efflux channel, is key in mitigating copper sensitivity. Additionally, the activity of tRNA processing was enriched and the deletion of several proteins involved in import generated copper tolerance. Lastly, key genes belonging to central carbon metabolism and nicotinamide adenine dinucleotide biosynthesis were uncovered as tolerant hits. Overall, this study shows that copper sensitivity and tolerance are a result of numerous mechanisms acting in combination within the cell.

scholarly journals Using a Chemical Genetic Screen to Enhance Our Understanding of the Antimicrobial Properties of Gallium against Escherichia coli

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 34 ◽  
Author(s):  
Natalie Gugala ◽  
Kate Chatfield-Reed ◽  
Raymond J. Turner ◽  
Gordon Chua
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Antimicrobial Properties ◽  
Iron Sulfur Cluster ◽  
Nucleotide Biosynthesis ◽  
E Coli ◽  
Chemical Genetic ◽  
Iron Sulfur ◽  
Insight Into ◽  
Mutant Collection

The diagnostic and therapeutic agent gallium offers multiple clinical and commercial uses including the treatment of cancer and the localization of tumors, among others. Further, this metal has been proven to be an effective antimicrobial agent against a number of microbes. Despite the latter, the fundamental mechanisms of gallium action have yet to be fully identified and understood. To further the development of this antimicrobial, it is imperative that we understand the mechanisms by which gallium interacts with cells. As a result, we screened the Escherichia coli Keio mutant collection as a means of identifying the genes that are implicated in prolonged gallium toxicity or resistance and mapped their biological processes to their respective cellular system. We discovered that the deletion of genes functioning in response to oxidative stress, DNA or iron–sulfur cluster repair, and nucleotide biosynthesis were sensitive to gallium, while Ga resistance comprised of genes involved in iron/siderophore import, amino acid biosynthesis and cell envelope maintenance. Altogether, our explanations of these findings offer further insight into the mechanisms of gallium toxicity and resistance in E. coli.

scholarly journals Copper Stress Targets the Rcs System To Induce Multiaggregative Behavior in a Copper-Sensitive Salmonella Strain

2010 ◽  
Vol 192 (23) ◽  
pp. 6287-6290 ◽  
Author(s):  
Lucas B. Pontel ◽  
Alejandro Pezza ◽  
Fernando C. Soncini
Keyword(s):  
Extracellular Polysaccharide ◽  
Copper Resistance ◽  
Copper Stress ◽  
Colanic Acid ◽  
Solid Media

ABSTRACT Salmonella ΔcuiD strains form mucoid colonies on copper-containing solid media. We show here that this multiaggregative behavior is caused by the Rcs-dependent induction of colanic acid extracellular polysaccharide. Deletion of cps operon genes in a ΔcuiD strain increased the sensitivity to copper, indicating a role for colanic acid in copper resistance.

scholarly journals The Role of the CopA Copper Efflux System in Acinetobacter baumannii Virulence

2019 ◽  
Vol 20 (3) ◽  
pp. 575 ◽  
Author(s):  
Saleh Alquethamy ◽  
Marjan Khorvash ◽  
Victoria Pederick ◽  
Jonathan Whittall ◽  
James Paton ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Copper Toxicity ◽  
In Silico Analysis ◽  
Opportunistic Pathogen ◽  
Copper Resistance ◽  
Copper Stress ◽  
Causative Agents ◽  
High Level ◽  
P Type

Acinetobacter baumannii has emerged as one of the leading causative agents of nosocomial infections. Due to its high level of intrinsic and adapted antibiotic resistance, treatment failure rates are high, which allows this opportunistic pathogen to thrive during infection in immune-compromised patients. A. baumannii can cause infections within a broad range of host niches, with pneumonia and bacteraemia being associated with the greatest levels of morbidity and mortality. Although its resistance to antibiotics is widely studied, our understanding of the mechanisms required for dealing with environmental stresses related to virulence and hospital persistence, such as copper toxicity, is limited. Here, we performed an in silico analysis of the A. baumannii copper resistome, examining its regulation under copper stress. Using comparative analyses of bacterial P-type ATPases, we propose that A. baumannii encodes a member of a novel subgroup of P1B-1 ATPases. Analyses of three putative inner membrane copper efflux systems identified the P1B-1 ATPase CopA as the primary mediator of cytoplasmic copper resistance in A. baumannii. Using a murine model of A. baumannii pneumonia, we reveal that CopA contributes to the virulence of A. baumannii. Collectively, this study advances our understanding of how A. baumannii deals with environmental copper toxicity, and it provides novel insights into how A. baumannii combats adversities encountered as part of the host immune defence.

Occurrence of cop-like copper resistance genes among bacteria isolated from a water distribution system

1995 ◽  
Vol 41 (7) ◽  
pp. 642-646 ◽  
Author(s):  
Chuzhao Lin ◽  
Betty H. Olson
Keyword(s):  
Pseudomonas Syringae ◽  
Resistance Genes ◽  
Distribution System ◽  
Water Distribution ◽  
Water Distribution System ◽  
Culture Media ◽  
Copper Resistance ◽  
Copper Tolerance ◽  
Resistant Bacteria ◽  
Copper Resistance Genes

The occurrence of cop-like copper resistance determinants homologous to the cop genes of Pseudomonas syringae among bacteria isolated from a water distribution system experiencing copper corrosion was investigated in this study. It was found that at least 49% of the copper-resistant bacteria and less than 15% of the copper-sensitive isolates possessed a cop homolog. The occurrence of this determinant in the copper-resistant population correlated with the degree of copper tolerance exhibited by the bacteria. The effect of organic substances present in the culture media on the empirical degree of bacterial copper tolerance is also discussed.Key words: copper resistance genes, water distribution system, cop.

scholarly journals Copper Stress Induces a Global Stress Response in Staphylococcus aureus and Represses sae and agr Expression and Biofilm Formation

2009 ◽  
Vol 76 (1) ◽  
pp. 150-160 ◽  
Author(s):  
Jonathan Baker ◽  
Sutthirat Sitthisak ◽  
Mrittika Sengupta ◽  
Miranda Johnson ◽  
R. K. Jayaswal ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Transcriptional Profiling ◽  
Resistance Mechanism ◽  
Growth Conditions ◽  
Copper Resistance ◽  
Transcriptional Analysis ◽  
Copper Stress ◽  
High Copper

ABSTRACT Copper is an important cofactor for many enzymes; however, high levels of copper are toxic. Therefore, bacteria must ensure there is sufficient copper for use as a cofactor but, more importantly, must limit free intracellular levels to prevent toxicity. In this study, we have used DNA microarray to identify Staphylococcus aureus copper-responsive genes. Transcriptional profiling of S. aureus SH1000 grown in excess copper identified a number of genes which fall into four groups, suggesting that S. aureus has four main mechanisms for adapting to high levels of environmental copper, as follows: (i) induction of direct copper homeostasis mechanisms; (ii) increased oxidative stress resistance; (iii) expression of the misfolded protein response; and (iv) repression of a number of transporters and global regulators such as Agr and Sae. Our experimental data confirm that resistance to oxidative stress and particularly to H2O2 scavenging is an important S. aureus copper resistance mechanism. Our previous studies have demonstrated that Eap and Emp proteins, which are positively regulated by Agr and Sae, are required for biofilm formation under low-iron growth conditions. Our transcriptional analysis has confirmed that sae, agr, and eap are repressed under high-copper conditions and that biofilm formation is indeed repressed under high-copper conditions. Therefore, our results may provide an explanation for how copper films can prevent biofilm formation on catheters.

scholarly journals Optimizing Bioremediation: Elucidating Copper Accumulation Mechanisms of Acinetobacter sp. IrC2 Isolated From an Industrial Waste Treatment Center

2021 ◽  
Vol 12 ◽  
Author(s):  
Wahyu Irawati ◽  
Eric Santoso Djojo ◽  
Lucia Kusumawati ◽  
Triwibowo Yuwono ◽  
Reinhard Pinontoan
Keyword(s):  
Membrane Proteins ◽  
Industrial Waste ◽  
Waste Treatment ◽  
Protein Profile ◽  
Copper Resistance ◽  
Copper Stress ◽  
Treatment Center ◽  
Resistant Bacteria ◽  
Cu Ions ◽  
Industrial Waste Treatment

Acinetobacter sp. IrC2 is a copper-resistant bacterium isolated from an industrial waste treatment center in Rungkut, Surabaya. Copper-resistant bacteria are known to accumulate copper inside the cells as a mechanism to adapt to a copper-contaminated environment. Periplasmic and membrane proteins CopA and CopB have been known to incorporate copper as a mechanism of copper resistance. In the present study, protein profile changes in Acinetobacter sp. IrC2 following exposure to copper stress were analyzed to elucidate the copper resistance mechanism. Bacteria were grown in a Luria Bertani agar medium with and without CuSO4 supplementation. Intracellular copper ion accumulation was quantified using atomic absorption spectrophotometry. Changes in protein profile were assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results showed that 6 mM CuSO4 was toxic for Acinetobacter sp. IrC2, and as a response to this copper-stress condition, the lag phase was prolonged to 18 h. It was also found that the bacteria accumulated copper to a level of 508.01 mg/g of cells’ dry weight, marked by a change in colony color to green. The protein profile under copper stress was altered as evidenced by the appearance of five specific protein bands with molecular weights of 68.0, 60.5, 38.5, 24.0, and 20.5 kDa, suggesting the presence of CopA, multicopper oxidase (MCO), CopB, universal stress protein (Usp), and superoxide dismutase (SOD) and/or DNA-binding protein from starved cells, respectively. We proposed that the mechanism of bacterial resistance to copper involves CopA and CopB membrane proteins in binding Cu ions in the periplasm and excreting excess Cu ions as well as involving enzymes that play a role in the detoxification process, namely, SOD, MCO, and Usp to avoid cell damage under copper stress.

scholarly journals Overexpression of mqsR in Xylella fastidiosa Leads to a Priming Effect of Cells to Copper Stress Tolerance

2021 ◽  
Vol 12 ◽  
Author(s):  
Isis Gabriela Barbosa Carvalho ◽  
Marcus Vinicius Merfa ◽  
Natália Sousa Teixeira-Silva ◽  
Paula Maria Moreira Martins ◽  
Marco Aurélio Takita ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Current Knowledge ◽  
Xylella Fastidiosa ◽  
Cell Formation ◽  
Cell Aggregation ◽  
Plant Diseases ◽  
Copper Tolerance ◽  
Copper Stress ◽  
Economic Damage ◽  
Environmental Damage

Copper-based compounds are widely used in agriculture as a chemical strategy to limit the spread of multiple plant diseases; however, the continuous use of this heavy metal has caused environmental damage as well as the development of copper-resistant strains. Thus, it is important to understand how the bacterial phytopathogens evolve to manage with this metal in the field. The MqsRA Toxin–Antitoxin system has been recently described for its function in biofilm formation and copper tolerance in Xylella fastidiosa, a plant-pathogen bacterium responsible for economic damage in several crops worldwide. Here we identified differentially regulated genes by X. fastidiosa MqsRA by assessing changes in global gene expression with and without copper. Results show that mqsR overexpression led to changes in the pattern of cell aggregation, culminating in a global phenotypic heterogeneity, indicative of persister cell formation. This phenotype was also observed in wild-type cells but only in the presence of copper. This suggests that MqsR regulates genes that alter cell behavior in order to prime them to respond to copper stress, which is supported by RNA-Seq analysis. To increase cellular tolerance, proteolysis and efflux pumps and regulator related to multidrug resistance are induced in the presence of copper, in an MqsR-independent response. In this study we show a network of genes modulated by MqsR that is associated with induction of persistence in X. fastidiosa. Persistence in plant-pathogenic bacteria is an important genetic tolerance mechanism still neglected for management of phytopathogens in agriculture, for which this work expands the current knowledge and opens new perspectives for studies aiming for a more efficient control in the field.

scholarly journals The role of copper resistance in Mycobacterium tuberculosis pathogenesis

2021 ◽  
Author(s):  
Jon Mitchell Ambler ◽  
Matthys Gerhardus Potgieter ◽  
Marisa Klopper ◽  
Melanie Grobbelaar ◽  
Margaretha De Vos ◽  
...  
Keyword(s):  
South Africa ◽  
Mycobacterium Tuberculosis ◽  
Molybdenum Cofactor ◽  
Copper Resistance ◽  
New Drugs ◽  
Differentially Expressed ◽  
Copper Stress ◽  
Western Cape ◽  
Severity Of Disease ◽  
Transcriptomic Level

AbstractDespite the development of new drugs and social interventions, tuberculosis remains a leading cause of mortality. This burden falls disproportionately on developing countries, particularly those where the incidence of HIV is high. In the Western Cape, South Africa, we have identified and isolated two Beijing family strains of Mycobacterium tuberculosis that, despite few differences at a genomic level, differ greatly in their severity of disease caused, providing an opportunity to study virulence in this organism. The aim of this study was to identify differences at a genomic and transcriptomic level that may identify the cause of the different virulence levels observed in the two isolates.The isolates were compared at the transcriptome level under four different growth conditions including oxidative stress. In comparing the transcriptome of the two isolates, an operon containing genes involved in the production of molybdenum cofactor that showed consistently lower levels of expression in the hypervirulent isolate was identified. A copper sensing transcriptional regulator was identified as the most probable regulator, and we found that the Cso operon which it is known to regulate was similarly differentially expressed in the strains.The production of molybdenum cofactor is effected in two ways by copper levels. Through the independent insertion of copper into molybdopterin (MPT), and destabilisation of Fe-S clusters. As MoaA3 contains a Fe-S cluster that is known to be destabilised by copper, and a number of copper sensitive genes are likewise found differentially expressed, it is likely that the strains differ in terms of their levels of resistance to copper.It is therefore hypothesised that the differences in virulence are as a result of different levels of resistance to phagosome copper overload, and the mechanism by which copper levels are linked to the production of molybdenum cofactor is described.Author summaryIn this article, we describe the differences in gene expression of two closely related strains of Mycobacterium tuberculosis isolated in the Western Cape of South Africa that differ in the severity of disease that they cause. We compared the strains at a genomic and transcriptomic level, and in doing so, we discovered a set of molybdenum cofactor genes regulated by a copper sensing transcription factor that came up in all datasets. Further genes linked to copper response were identified, providing greater evidence that the difference between the two strains was the manner in which they responded to copper stress. Phagocytes are known to exploit high levels of copper to kill intracellular bacteria, suggesting an important link between copper and disease. We conclude that resistance to copper toxicity is the most probable reason for the relative increase in virulence, and describe the regulatory relationship between copper levels and molybdenum cofactor synthesis.

scholarly journals Emergence of growth and dormancy from a kinetic model of the Escherichia coli central carbon metabolism

2022 ◽  
Author(s):  
Yusuke Himeoka ◽  
Namiko Mitarai
Keyword(s):  
Escherichia Coli ◽  
Kinetic Model ◽  
Carbon Metabolism ◽  
Wide Spectrum ◽  
Central Carbon Metabolism ◽  
Bacterial Cells ◽  
Futile Cycle ◽  
Dormant State ◽  
Dormant Cells ◽  
Central Carbon

Abstract Physiological states of bacterial cells exhibit a wide spectrum of timescale. Under nutrient-rich conditions, most of the cells in an isogenic bacterial population grow at certain rates, while a small subpopulation sometimes stays in a dormant state where the growth rates slow down by orders of magnitude. For revealing the origins of such heterogeneity of timescales, we studied the kinetic model of Escherichia coli central carbon metabolism including the dynamics of the energy currency molecules. We found that the model robustly exhibits both the growing- and the dormant state. In order to unveil the mechanism of distinct behaviours, we developed a recursive method to simplify the model without changing the qualitative feature of the dynamics. Analytical and numerical studies of the 2-variable minimal model revealed the necessary conditions for the distinct behaviour, namely, the depletion of energy due to the futile cycle and its non-uniform impact on the kinetics because of the coexistence of the energy currency-coupled and uncoupled reactions as well as branching of the network. The result is consistent with the experimental reports that the dormant cells commonly exhibit low ATP levels, and provides a possible explanation for the appearance of dormant cells that causes antibiotic persistence.

scholarly journals CpxR/CpxA ControlsscsABCDTranscription To Counteract Copper and Oxidative Stress inSalmonella entericaSerovar Typhimurium

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Carolina López ◽  
Susana K. Checa ◽  
Fernando C. Soncini
Keyword(s):  
Oxidative Stress ◽  
Stress Responses ◽  
Disulfide Bonds ◽  
Redox Balance ◽  
Copper Resistance ◽  
Gene Arrangement ◽  
Copper Stress ◽  
Content Type ◽  
Disulfide Oxidoreductase ◽  
Conserved Gene

ABSTRACTPeriplasmic thiol/disulfide oxidoreductases participate in the formation and isomerization of disulfide bonds and contribute to the virulence of pathogenic microorganisms. Among the systems encoded in theSalmonellagenome, the system encoded by thescsABCDlocus was shown to be required to cope with Cu and H2O2stress. Here we report that this locus forms an operon whose transcription is driven by a promoter upstream ofscsAand depends on CpxR/CpxA and on Cu. Furthermore, genes homologous toscsB,scsC, andscsDare always detected immediately downstream ofscsAand in the same genetic arrangement in allscsA-harboring enterobacterial species. Also, a CpxR-binding site is detected upstream ofscsAin most of those species, providing evidence of evolutionarily conserved function and regulation. Each individualscsgene shows a different role in copper and/or H2O2resistance, indicating hierarchical contributions of these factors in the defense against these intoxicants. A protective effect of Cu preincubation against H2O2toxicity and the increased Cu-mediated activation ofcpxPin the ΔscsABCDmutant suggest that the CpxR/CpxA-controlled transcription of the ScsABCD system contributes to prevent Cu toxicity and to restore the redox balance at theSalmonellaenvelope.IMPORTANCECopper intoxication triggers both specific and nonspecific responses inSalmonella. Thescslocus, which codes for periplasmic thiol/disulfide-oxidoreductase/isomerase-like proteins, has been the focus of attention because it is necessary for copper resistance, oxidative stress responses, and virulence and because it is not present in nonpathogenicEscherichia coli. Still, the conditions under which thescslocus is expressed and the roles of its individual components remain unknown. In this report, we examine the contribution of each Scs factor to survival under H2O2and copper stress. We establish that thescsgenes form a copper-activated operon controlled by the CpxR/CpxA signal transduction system, and we provide evidence of its conserved gene arrangement and regulation in other bacterial pathogens.

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