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Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci.

ABSTRACT: Antimicrobial resistance in Staphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at the oriT region of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, pSK156 and pCA347 are nonconjugative Staphylococcus aureus plasmids that contain sequences similar to the oriT region of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate the oriT sequences of these nonconjugative plasmids in vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognate oriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variant oriT mimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-like oriT. These data indicate that the conjugative relaxase in trans mechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids.Understanding the mechanism of antimicrobial resistance transfer in bacteria such as Staphylococcus aureus is an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of the Staphylococcus aureus multiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variant oriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase in trans mechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.

SUBMITTER: Pollet RM

PROVIDER: S-EPMC4772599 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci.

Pollet Rebecca M RM Ingle James D JD Hymes Jeff P JP Eakes Thomas C TC Eto Karina Yui KY Kwong Stephen M SM Ramsay Joshua P JP Firth Neville N Redinbo Matthew R MR

Journal of bacteriology 20160104 6

<h4>Unlabelled</h4>Antimicrobial resistance in Staphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, fo ...[more]

PMID: 26728193

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Project description:Klebsiella pneumoniae poses a critical challenge to clinical and public health. Along with conjugative plasmids, nonconjugative resistance or virulence plasmids associated with carbapenem-resistant K. pneumoniae (CRKP), hypervirulent K. pneumoniae (hvKP), and even carbapenem-resistant and hypervirulent K. pneumoniae (CR-hvKP) strains have been spreading globally. In this study, a clinical CRKP strain KP2648 was isolated, and the transferability of its plasmids was assessed using conjugation experiments. The transconjugants were characterized by polymerase chain reaction (PCR) detection, XbaI and S1-pulsed-field gel electrophoresis (PFGE), and/or whole-genome sequencing. Genetically modified IncN3 plasmids were employed to elucidate the self-transferability and the mobilization mechanisms. KP2648 has three natural plasmids: a nonconjugative IncFIB/IncHI3B virulence plasmid, a nonconjugative IncFII/IncR carbapenem-resistant plasmid, and a self-transferable IncN3 plasmid with a high conjugation frequency (7.54 ± 1.06) × 10-1. The IncN3 plasmid could mobilize the coexisting nonconjugative virulence/resistance plasmids either directly or by employing intermediate E. coli with two forms: a hybrid plasmid fused with IncN3 or a cotransfer with the helper plasmid, IncN3. Various mobile genetic elements, including ISKpn74, ISKpn14, IS26, ISShes11, ISAba11, and Tn3, are involved in the genetic transposition of diverse hybrid plasmids and the cotransfer process during the intra/interspecies transmission. IMPORTANCE Nowadays, the underlying mobilization mechanism and evolutionary processes of nonconjugative virulence or resistance plasmids in Klebsiella pneumoniae remain poorly understood. Our study revealed the high conjugation ability of IncN3 plasmid isolated from carbapenem-resistant K. pneumoniae and confirmed its capability to mobilize the nonconjugative virulence or resistance plasmids. The self-transferable IncN3 plasmid could facilitate the transmission of pathogenicity and genetic evolution of carbapenem-resistant and hypervirulent K. pneumoniae, including hv-CRKP (virulence plasmid obtained by carbapenem-resistant K. pneumoniae) and CR-hvKP (resistance plasmid obtained by hypervirulent K. pneumoniae), warranting further monitoring.

Project description:BackgroundPrime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models.ResultspAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors.ConclusionTaken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.

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Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci.

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Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci.

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