Allelic replacement in Staphylococcus aureus with inducible counter-selection

Bae, Taeok; Schneewind, Olaf

doi:10.1016/j.plasmid.2005.05.005

Cited by 547 publications

(634 citation statements)

References 22 publications

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“…Subsequently, we ligated the 1,000-bp regions immediately upstream and downstream of our gene of interest by using PCR overlay and cloned this product into pKOR1-mcs in E. coli DC10B (35). After electroporation into S. aureus Newman, allelic replacement was induced by temperature shift (34). The mutants were generated in a step-wise fashion: We first deleted the eap gene (resulting in MR1811, or Δeap), then eapH1 (MR1852, or ΔeapΔH1) and subsequently eapH2 (MR1860, or ΔeapΔH1ΔH2).…”

Section: Methodsmentioning

confidence: 99%

Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

Stapels

Ramyar

Bischoff

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

128

189

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Neutrophils are indispensable for clearing infections with the prominent human pathogen Staphylococcus aureus. Here, we report that S. aureus secretes a family of proteins that potently inhibits the activity of neutrophil serine proteases (NSPs): neutrophil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not related serine proteases, are specifically blocked by the extracellular adherence protein (Eap) and the functionally orphan Eap homologs EapH1 and EapH2, with inhibitory-constant values in the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal infection in vivo. The crystal structure of the EapH1/NE complex showed that Eap molecules constitute a unique class of noncovalent protease inhibitors that occlude the catalytic cleft of NSPs. These findings increase our insights into the complex pathogenesis of S. aureus infections and create opportunities to design novel treatment strategies for inflammatory conditions related to excessive NSP activity.immune evasion | bacteria | phagocytes

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Section: Methodsmentioning

confidence: 99%

Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

Stapels

Ramyar

Bischoff

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

128

189

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“…We used a representative strain for each of the two clinically significant S. aureus capsule serotypes, CP5 and CP8: Mu50, a known vancomycin-intermediate S. aureus (VISA) isolate that produces capsule serotype 5; and UAMS-1, a vancomycin-sensitive S. aureus (VSSA) osteomyelitis isolate that produces capsule serotype 8. A restriction-deficient laboratory strain of S. aureus, RN4220, and the DH5a strain of Escherichia coli were used to move plasmid constructs into the strains of choice through transformation and phage transduction, as described elsewhere (Bae & Schneewind, 2006;Sahukhal & Elasri, 2014;Samanta & Elasri, 2014). S. aureus strains were grown on tryptic soy agar (TSA) or in tryptic soy broth (TSB).…”

Section: Methodsmentioning

confidence: 99%

“…The allelic replacement method was used to generate the msaABCR and msaB deletion mutants in strains Mu50 and UAMS-1 (Bae & Schneewind, 2006). For trans-complementation, the msaABCR region was cloned into the pCN34 low-copy vector with the modification of changing the kanamycin selectable marker to a chloramphenicolresistance marker as described elsewhere (Samanta & Elasri, 2014;Charpentier et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

MsaB activates capsule production at the transcription level in Staphylococcus aureus

2016

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Staphylococcus aureus produces several virulence factors that allow it to cause a variety of infections. One of the major virulence factors is the capsule, which contributes to the survival of the pathogen within the host as a way to escape phagocytosis. The production of the capsular polysaccharide is encoded in a 16 gene operon, which is regulated in response to several environmental stimuli including nutrient availability. For instance, the capsule is produced in the late-and post-exponential growth phases, but not in the early-or mid-exponential growth phase. Several regulators are involved in capsule production, but the regulation of the cap operon is still poorly understood. In this study, we show that MsaB activates the cap operon by binding directly to a 10 bp repeat in the promoter region. We show that despite the fact that MsaB is expressed throughout four growth phases, it only activates capsule production in the late-and post-exponential growth phases. Furthermore, we find that MsaB does not bind to its target site in the early and mid-exponential growth phases. This correlates with decreased nutrient availability and capsule production. These data suggest either that MsaB binding ability changes in response to nutrients or that other cap operon regulators interfere with the binding of MsaB to its target site. This study increases our understanding of the regulation of capsule production and the mechanism of action of MsaB.

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“…To monitor genome flip-flop reversion, using gene replacement strategy (40,41), a reporter gene gfp with an S/D sequence and tetracycline-inducible promoter…”

Section: Methodsmentioning

confidence: 99%

Coordinated phenotype switching with large-scale chromosome flip-flop inversion observed in bacteria

Cui

Neoh

Iwamoto

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Genome inversions are ubiquitous in organisms ranging from prokaryotes to eukaryotes. Typical examples can be identified by comparing the genomes of two or more closely related organisms, where genome inversion footprints are clearly visible. Although the evolutionary implications of this phenomenon are huge, little is known about the function and biological meaning of this process. Here, we report our findings on a bacterium that generates a reversible, large-scale inversion of its chromosome (about half of its total genome) at high frequencies of up to once every four generations. This inversion switches on or off bacterial phenotypes, including colony morphology, antibiotic susceptibility, hemolytic activity, and expression of dozens of genes. Quantitative measurements and mathematical analyses indicate that this reversible switching is stochastic but self-organized so as to maintain two forms of stable cell populations (i.e., small colony variant, normal colony variant) as a bet-hedging strategy. Thus, this heritable and reversible genome fluctuation seems to govern the bacterial life cycle; it has a profound impact on the course and outcomes of bacterial infections.

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Allelic replacement in Staphylococcus aureus with inducible counter-selection

Cited by 547 publications

References 22 publications

Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

MsaB activates capsule production at the transcription level in Staphylococcus aureus

Coordinated phenotype switching with large-scale chromosome flip-flop inversion observed in bacteria

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