The copBL operon protects Staphylococcus aureus from copper toxicity: CopL is an extracellular membrane–associated copper-binding protein

Rosario-Cruz, Zuelay; Eletsky, Alexander; Daigham, Nourhan S.; Al-Tameemi, Hassan; Swapna, G.V.T.; Kahn, Peter C.; Szyperski, Thomas; Montelione, Gaetano T.; Boyd, Jeffrey M.

doi:10.1074/jbc.ra118.004723

Cited by 44 publications

(51 citation statements)

References 109 publications

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“…The evolutionary pressure of the main nosocomial lineages is thought to be related to the hospital environment, but other evidence suggests that initial core genome changes in sequence type 2 (ST2) isolates result in increased acquisition of resistance traits (Lee et al, 2018). The important role of horizontal gene transfer in the evolution of S. epidermidis (Méric et al, 2018) and the wellestablished role of Staphylococcus aureus chromosomal cassette elements in bacterial fitness (Purves et al, 2018;Zapotoczna et al, 2018;Rosario-Cruz et al, 2019), suggest that the stepwise evolution of the cassette chromosomal elements and mecA gene is critical to spread of staphylococcal resistance, thus leading to the MRSA and MRSE epidemic (Rolo et al, 2017). The fine balance between acquisition and maintenance of accessory chromosomal elements shapes the evolutionary fitness of S. epidermidis (Zamudio et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Genomic Stability of Composite SCCmec ACME and COMER-Like Genetic Elements in Staphylococcus epidermidis Correlates With Rate of Excision

et al. 2020

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The epidemiological success of methicillin-resistant Staphylococcus aureus USA300 has been associated with the presence of two mobile elements, the arginine catabolic mobile element (ACME) and the copper and mercury resistance (COMER) element. These two mobile elements are associated with resistance to copper, which has been related to host fitness and survival within macrophages. Several studies found that ACME is more prevalent, and exhibits greater diversity, in Staphylococcus epidermidis while COMER has not been identified in S. epidermidis or any other staphylococcal species. We aimed in this study to evaluate the presence and diversity of ACME and COMER-like elements in our S. epidermidis clinical isolates. The genomes of 58 S. epidermidis clinical isolates, collected between 2009 and 2018 in a Scottish hospital, were sequenced. A core-genome phylogenetic tree and genome based MLST typing showed that more than half of the isolates belong to the clinically predominant sequence type2 (ST2) and these isolates have been found to split into two lineages within the phylogenetic tree. Analysis showed the presence of SCCmec in the majority of isolates. Comparative analysis identified a cluster of ACME-positive isolates with most of them belonging to ST48. ACME showed high variation even between isolates of the same ACME type and ST. COMER-like elements have been identified in one of the two major hospital adapted drug resistant ST2 lineages; and showed high stability. This difference in stability at the genomic level correlates well with the up to one hundred times higher excision frequency found for the SCCmec elements in ACME-containing isolates compared to COMER-like element containing isolates. ACME/COMER-like element positive isolates did not show a significant phenotype of decreased copper susceptibility, while resistance to mercury was over-represented in COMER-like element

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Section: Introductionmentioning

confidence: 99%

Genomic Stability of Composite SCCmec ACME and COMER-Like Genetic Elements in Staphylococcus epidermidis Correlates With Rate of Excision

et al. 2020

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“…USA300 LAC ( 40 ) accounts for most MRSA infections in the United States ( 41 ). This S. aureus strain has two systems that protect against Cu toxicity, CopAZ and CopBL ( 42 ). Figure 4H demonstrates that VU0026921 is also growth inhibitory to USA300 LAC and that deletion of the Cu export genes (Δ cop ) increases VU0026921 toxicity in the presence of 10 μM Cu, which is not toxic without VU0026921.…”

Section: Resultsmentioning

confidence: 99%

“…Mutant strains were constructed using the pJB38 allelic exchange as described previously ( 65 ). The Δ copAZ Δ copBL strain ( cop -) was created using the Δ copBL mutant (JMB7901) (ΔSAUSA300_0078-0079) ( 42 ) and pJB38_Δ copAZ .…”

Section: Methodsmentioning

confidence: 99%

A Small-Molecule Modulator of Metal Homeostasis in Gram-Positive Pathogens

Juttukonda

Beavers

Unsihuay

et al. 2020

mBio

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Metals are essential nutrients that all living organisms acquire from their environment. While metals are necessary for life, excess metal uptake can be toxic; therefore, intracellular metal levels are tightly regulated in bacterial cells. Staphylococcus aureus, a Gram-positive bacterium, relies on metal uptake and metabolism to colonize vertebrates. Thus, we hypothesized that an expanded understanding of metal homeostasis in S. aureus will lead to the discovery of pathways that can be targeted with future antimicrobials. We sought to identify small molecules that inhibit S. aureus growth in a metal-dependent manner as a strategy to uncover pathways that maintain metal homeostasis. Here, we demonstrate that VU0026921 kills S. aureus through disruption of metal homeostasis. VU0026921 activity was characterized through cell culture assays, transcriptional sequencing, compound structure-activity relationship, reactive oxygen species (ROS) generation assays, metal binding assays, and metal level analyses. VU0026921 disrupts metal homeostasis in S. aureus, increasing intracellular accumulation of metals and leading to toxicity through mismetalation of enzymes, generation of reactive oxygen species, or disruption of other cellular processes. Antioxidants partially protect S. aureus from VU0026921 killing, emphasizing the role of reactive oxygen species in the mechanism of killing, but VU0026921 also kills S. aureus anaerobically, indicating that the observed toxicity is not solely oxygen dependent. VU0026921 disrupts metal homeostasis in multiple Gram-positive bacteria, leading to increased reactive oxygen species and cell death, demonstrating the broad applicability of these findings. Further, this study validates VU0026921 as a probe to further decipher mechanisms required to maintain metal homeostasis in Gram-positive bacteria. IMPORTANCE Staphylococcus aureus is a leading agent of antibiotic-resistant bacterial infections in the world. S. aureus tightly controls metal homeostasis during infection, and disruption of metal uptake systems impairs staphylococcal virulence. We identified small molecules that interfere with metal handling in S. aureus to develop chemical probes to investigate metallobiology in this organism. Compound VU0026921 was identified as a small molecule that kills S. aureus both aerobically and anaerobically. The activity of VU0026921 is modulated by metal supplementation, is enhanced by genetic inactivation of Mn homeostasis genes, and correlates with increased cellular reactive oxygen species. Treatment with VU0026921 causes accumulation of multiple metals within S. aureus cells and concomitant upregulation of genes involved in metal detoxification. This work defines a small-molecule probe for further defining the role of metal toxicity in S. aureus and validates future antibiotic development targeting metal toxicity pathways.

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“…For other CDIs, we have previously measured a small increase in the effective concentration in some MRSA strains, but this is the first time we observed pre-existing, natural tolerance against a CDI in select MRSA strains. Recent publications have highlighted two previously unknown copper resistant proteins in a MRSA isolate (USA-300/JE2) termed CopX and CopL, in addition to the already reported copper resistance pump CopA and the copper chaperone CopZ 32,33 . CopA and CopZ are found in every S. aureus strain, while the presence of CopX and CopL seems to be limited to MRSA isolates 32,34 , making them candidate genes for the observed APT-6K tolerance phenotype of MRSA-3 and MRSA-4.…”

Section: Antibiotic Effect Of Apt-6k On Multidrug Resistant S Aureusmentioning

confidence: 99%

A copper-dependent compound restores ampicillin sensitivity in multidrug-resistant Staphylococcus aureus

Crawford

Dalecki

Perez

et al. 2020

Sci Rep

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Multi-drug resistant Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), has become a worldwide, major health care problem. While initially restricted to clinical settings, drug resistant S. aureus is now one of the key causative agents of community-acquired infections. We have previously demonstrated that copper dependent inhibitors (cDis), a class of antibiotics that are only active in the presence of copper ions, are effective bactericidal agents against MRSA. A secondgeneration CDI, APT-6K, exerted bactericidal activity at nanomolar concentrations. At sub-bactericidal concentrations, it effectively synergized with ampicillin to reverse drug resistance in multiple MRSA strains. APT-6K had a favorable therapeutic index when tested on eukaryotic cells (TI: > 30) and, unlike some previously reported CDIs, did not affect mitochondrial activity. These results further establish inhibitors that are activated by the binding of transition metal ions as a promising class of antibiotics, and for the first time, describe their ability to reverse existing drug resistance against clinically relevant antibiotics. The rapid increase of antibiotic resistance within bacterial populations is associated with longer hospital stays, increased treatment costs, and more patient deaths 1,2. An estimated 700,000 individuals die each year as a result of infections with antibiotic resistant bacteria, and the amount of deaths are expected to increase if no alternative, effective therapies are developed 3. New antibiotics are essential to avoid a public health crisis. The identification of new antibiotics for Staphylococcus aureus is an especially urgent task, with antibiotic resistance in this bacterium already observed against some of the last line of defense antibiotics such as vancomycin, linezolid, and daptomycin 4. An alternative to the discovery of new antibiotics are drugs that restore the efficacy of available antibiotics and overcome bacterial drug resistance mechanisms. Ideal drugs would be ones that are both effective by themselves and that restore the activity of current antibiotics by reversing antibiotic resistance 5,6. Copper dependent inhibitors (CDIs) are a functionally new type of antibiotic gaining increased appreciation due to their ability to inhibit drug resistant bacteria such as S. aureus, Mycobacterium tuberculosis, Mycoplasma spp., and Neisseria gonorrheae 7-13. These compounds utilize copper for their activities and include the FDA approved drug disulfiram and anti-cancer compounds like 8-hydroxyquinoline (8HQ) 7,9,13-15. Hundreds of new CDIs with antibacterial and antifungal activity have been identified in drug screens against S. aureus, M. tuberculosis, and Cryptococcus neoformans using defined culture medium that contains physiologically relevant concentrations of copper that were previously not identified in these compound libraries when screened under industrial standard conditions (no consideration of transition metal concentrations), demonstrating the untapped potential of CDIs 10,12...

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The copBL operon protects Staphylococcus aureus from copper toxicity: CopL is an extracellular membrane–associated copper-binding protein

Cited by 44 publications

References 109 publications

Genomic Stability of Composite SCCmec ACME and COMER-Like Genetic Elements in Staphylococcus epidermidis Correlates With Rate of Excision

Genomic Stability of Composite SCCmec ACME and COMER-Like Genetic Elements in Staphylococcus epidermidis Correlates With Rate of Excision

A Small-Molecule Modulator of Metal Homeostasis in Gram-Positive Pathogens

A copper-dependent compound restores ampicillin sensitivity in multidrug-resistant Staphylococcus aureus

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