Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

Monk, Ian R.; Tree, Jai J.; Howden, Benjamin P; Stinear, Timothy P.; Foster, Timothy J.

doi:10.1128/mbio.00308-15

Cited by 209 publications

(211 citation statements)

References 45 publications

(58 reference statements)

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“…A chromosomal region encompassing atpA was PCR amplified from WT S. aureus JE2 chromosomal DNA using primer pair: 5′-ATATGAGCTCGAAGAGTTAGATAAGATTGTCAAACTAG-3′/5′-GATACAAGATCTGATGGTTTGTATTGCTACTTGC-3′ and cloned into pBASE6 via SacI/BglII. This plasmid was purified from E. coli IM08B (Monk et al, 2015) and transformed directly into JE2 atpA ::ΦNΣ (NE592) at 30°C followed by chromosomal integration by plating on TSA (10 μg/ml chloramphenicol) at 44°C overnight. Plasmid cross-out was performed by passage at 30°C followed by plating on TSA (500 ng/ml anhydrotetracycline) and successful allelic exchange of the transposon insertion with the intact atpA gene was selected for by replica plating of colonies and screening for sensitivity toward erythromycin and chloramphenicol.…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Identification of Antimicrobial Intrinsic Resistance Determinants in Staphylococcus aureus

et al. 2016

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The emergence of antimicrobial resistance severely threatens our ability to treat bacterial infections. While acquired resistance has received considerable attention, relatively little is known of intrinsic resistance that allows bacteria to naturally withstand antimicrobials. Gene products that confer intrinsic resistance to antimicrobial agents may be explored for alternative antimicrobial therapies, by potentiating the efficacy of existing antimicrobials. In this study, we identified the intrinsic resistome to a broad spectrum of antimicrobials in the human pathogen, Staphylococcus aureus. We screened the Nebraska Transposon Mutant Library of 1920 single-gene inactivations in S. aureus strain JE2, for increased susceptibility to the anti-staphylococcal antimicrobials (ciprofloxacin, oxacillin, linezolid, fosfomycin, daptomycin, mupirocin, vancomycin, and gentamicin). Sixty-eight mutants were confirmed by E-test to display at least twofold increased susceptibility to one or more antimicrobial agents. The majority of the identified genes have not previously been associated with antimicrobial susceptibility in S. aureus. For example, inactivation of genes encoding for subunits of the ATP synthase, atpA, atpB, atpG and atpH, reduced the minimum inhibitory concentration (MIC) of gentamicin 16-fold. To elucidate the potential of the screen, we examined treatment efficacy in the Galleria mellonella infection model. Gentamicin efficacy was significantly improved, when treating larvae infected with the atpA mutant compared to wild type cells with gentamicin at a clinically relevant concentration. Our results demonstrate that many gene products contribute to the intrinsic antimicrobial resistance of S. aureus. Knowledge of these intrinsic resistance determinants provides alternative targets for compounds that may potentiate the efficacy of existing antimicrobial agents against this important pathogen.

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Section: Methodsmentioning

confidence: 99%

Genome-Wide Identification of Antimicrobial Intrinsic Resistance Determinants in Staphylococcus aureus

et al. 2016

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“…Previous work with S. aureus has shown that lineages from different clonal complexes of S. aureus express divergent HsdS enzymes recognizing different TRMs (24). In agreement with that observation, we found that this is also the case with S. epidermidis because RP62A (CC10), NIH051475 (CC89), and NIH04008 (CC2) express divergent hsdS genes that specify different TRMs, while NIH051475 and VCU036, both of which belong to CC89, exhibit identical TRMs for the type I RM system (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis

et al. 2017

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Staphylococcus epidermidis is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical S. epidermidis strains is poorly understood due to the strong genetic barrier that precludes the efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical S. epidermidis isolates is primarily due to the type I and IV restriction-modification systems, which act as genetic barriers. Here, we show that efficient plasmid transformation of clinical S. epidermidis isolates from clonal complexes 2, 10, and 89 can be realized by employing a plasmid artificial modification (PAM) in Escherichia coli DC10B containing a Δdcm mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of S. epidermidis and hence improve our understanding of their pathogenesis in human infections.IMPORTANCE Staphylococcus epidermidis is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of S. epidermidis are poorly understood. Genetic manipulations of clinically relevant strains of S. epidermidis are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation of clinically relevant S. epidermidis isolates has now become possible, thus improving our understanding of S. epidermidis as a pathogen.KEYWORDS Staphylococcus epidermidis, transformation, restriction system, DC10B, clonal complexes, HsdS, efficiency, methylation, restriction barrier S taphylococcus epidermidis is a ubiquitous human commensal that normally colonizes the human skin and mucous membranes (1). S. epidermidis can also assume an invasive lifestyle by colonizing indwelling medical devices, which in some cases can lead to invasive bacteremia. Indeed, due to the common use of implanted medical devices in modern medicine, S. epidermidis is now the number one cause of nosocomial bacteremia in the United States (2). Although these device-related infections are less severe than those due to their Staphylococcus aureus counterparts, these diseases tend to be more persistent, with most being described as subacute or chronic in nature. In many cases, infection cannot be eliminated unless the offending medical devices, such as catheters, prostheses, or pacemakers, are removed, thus leading to high morbidity rates (3). Treatment of S. epidermidis infections is also complicated by the high level of innate antibiotic resistance as well as robust biofilm formation, which can render host defenses and antibiotics ineffective (1).

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“…Transformation of E. coli was performed with a standard heat shock protocol. S. aureus was transformed with electroporation using plasmid DNA isolated from E. coli DC10B (Monk et al, 2012) or IM08B (Monk et al, 2015). Preparation of electrocompetent cells and electroporation were performed essentially as described before (Lofblom et al, 2007).…”

Section: Bacterial Strains Growth Conditions and Transformationmentioning

confidence: 99%

CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus

Stamsås

Myrbråten

Straume

et al. 2018

Molecular Microbiology

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Staphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozE of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the ΔcozEaΔcozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the ΔcozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with and possibly influence localization of the cell division protein EzrA. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozE -depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA.

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Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

Cited by 209 publications

References 45 publications

Genome-Wide Identification of Antimicrobial Intrinsic Resistance Determinants in Staphylococcus aureus

Genome-Wide Identification of Antimicrobial Intrinsic Resistance Determinants in Staphylococcus aureus

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis

CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus

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