The development of fluorescent protein tracing vectors for multicolor imaging of clinically isolated Staphylococcus aureus

Abstract

Recent advances in fluorescent protein technology provide a wide variety of biological imaging applications; however current tools for bio-imaging in the Gram-positive bacterium Staphylococcus aureus has necessitated further developments for fluorescence intensity and for a multicolor palette of fluorescent proteins. To enhance the expression of multicolor fluorescent proteins in clinical S. aureus strains, we developed new fluorescent protein expression vectors, containing the blaZ/sodp promoter consisting of the β-lactamase gene (blaZ) promoter and the ribosome binding site (RBS) of superoxide dismutase gene (sod). We found S. aureus-adapted GFP (GFPsa) driven by the blaZ/sodp promoter was highly expressed in the S. aureus laboratory strain RN4220, but not in the clinical strains, MW2 and N315, harboring the endogenous blaI gene, a repressor of the blaZ gene promoter. We therefore constructed a constitutively induced blaZ/sodp promoter (blaZ/sodp(Con)) by introducing substitution mutations into the BlaI binding motif, and this modification allowed enhanced expression of the multicolor GFP variants (GFPsa, EGFP, mEmerald, Citrine, Cerulean, and BFP) as well as codon-optimized reef coral fluorescent proteins (mCherry and AmCyan) in the S. aureus clinical strains. These new fluorescent probes provide new tools to enhance expression of multicolor fluorescent proteins and facilitate clear visualization of clinical S. aureus strains.

Recommendations for design and conduct of preclinical in vivo studies of orthopedic device‐related infection

“…Some research groups have now developed a variety of fluorescent reporter plasmids for labelling S. aureus by utilizing plasmids encoding either green fluorescent protein (GFP) or higher wavelength reporter variants for yellow (YFP) and red (mCherry) labeling . These reporters are placed under control of characterized promoters to enable constitutive or inducible expression using antibiotics such as chloramphenicol, erythromycin, or tetracycline . While fluorescent strains are very useful for in vitro studies, they have very limited value for in vivo studies because of the low intensity of the signal and quenching by the host tissue.…”

The Association Between Onset of Staphylococcal Non-menstrual Toxic Shock Syndrome With Inducibility of Toxic Shock Syndrome Toxin-1 Production

“…To confirm whether the number of T repeats in the tst promoter region affects serum induction of TSST-1 production, a reporter assay was performed using green fluorescent protein (GFP) expression vector pKAT ( Kato et al, 2017 ). A set of tst promoter regions containing different number of T repeats (from six to nine) were individually cloned into the upstream of the egfp gene of pKAT to generate a series of eGFP expression plasmids, where the eGFP expression was regulated by the inserts.…”

“…To assess the activity of tst promoter, the tst promoter regions of representative strains (N315, JMUB2909, JMUB3011, and JMUB3038) were amplified by PCR using the primer set pKAT-N315-tst-prom-F2 and egfp-N315-tst-prom-R 1 https://github.com/rrwick/Porechop 2 https://www.ncbi.nlm.nih.gov (Supplementary Table 2). In addition, egfp and pKAT vector (Kato et al, 2017) were amplified with primer sets egfp-F/pKATegfp-R and pKAT-F/pKAT-R, respectively (Supplementary Table 2). The three PCR fragments were assembled by NEBuilder (New England Biolabs, MA, United States) to construct four plasmids: pKAT-9T-tst-promoter-gfp, pKAT-8T-tst-promotergfp, pKAT-7T-tst-promoter-gfp, and pKAT-6T-tst-promoter-gfp.…”

Construction and validation of EGFP-expressingStaphylococcus aureusclinical strains for adhesion and internalization assays on epithelial cells

“…Recently, Kato et al . [16] developed fluorescent protein tracing vectors for multicolour imaging of three clinical isolates of S. aureus (N315, MW2 and TY34). Furthermore, imaging of EGFP-expressing S. aureus avoids traditional immunostaining steps including fixation, permeabilisation and antibody labelling.…”