Selective activation of sar promoters with the use of green fluorescent protein transcriptional fusions as the detection system in the rabbit endocarditis model

Abstract

The global regulatory locus sar is composed of three overlapping transcripts initiated from a triple-promoter system (designated P1, P3, and P2). To explore if the individual sar promoters are differentially expressed in vitro and in vivo, we constructed a shuttle plasmid (pALC1434) containing a promoterless gfpUV gene (a gfp derivative [Clontech]) preceded by a polylinker region. Recombinant shuttle vectors containing individual sar promoters upstream of the gfpUV reporter gene were then introduced into Staphylococcus aureus RN6390. Northern and immunoblot analysis revealed that P1 is stronger than the P2 and P3 promoters in vitro. Additionally, the levels of the gfpUV transcript driven by individual sar promoters also correlated with the growth cycle dependency of these promoters in liquid cultures, thus suggesting the utility of pALC1434 as a vehicle for reporter fusion. Using the rabbit endocarditis model, we examined the expression of these three GFPUV fusions in vivo by fluorescence microscopy of infected cardiac vegetations 24 h after initial intravenous challenge. Similar to the in vitro findings, P1 was activated both in the center and on the surface of the vegetations. In contrast, the P3 promoter was silent both in vivo and in vitro as determined by fluorescence microscopy. Remarkably, P2 was silent in vitro but became highly activated in vivo. In particular, the sar P2 promoter was activated on the surface of the vegetation but not in the center of the lesion. These data imply that in vivo promoter activation of sar differed from that observed in vitro. Moreover, the individual sar promoters may be differentially expressed in different areas within the same anatomic niche, presumably reflecting the microbial physiological response to distinct host microenvironments. As the sar locus controls the synthesis of both extracellular and cell wall virulence determinants, these promoter-gfpUV constructs should be useful to characterize many aspects of S. aureus gene regulation in vivo.

FIG. 1

Excitation of RN6390-derived clones containing sar promoters with a long-range UV light (maximal excitation at 365 nm). Strains ALC1435, ALC1436, and ALC1437 contain pALC1434 with the sar P1, P3, and P2 promoters, respectively. The control strain ALC1440 contains only the vector pALC1434.

FIG. 2

Northern blot of gfp transcripts of pALC1434 driven by the sar P1, P3, and P2 promoters. The control vector with no promoter upstream yielded no transcripts (data not shown). stat, stationary.

FIG. 3

Immunoblot of cell lysates of RN6390 clones with GFP_UV driven by sar promoters. The anti-GFP antibody was used at a 1:5,000 dilution.

FIG. 4

(A) Twenty-four-hour-old vegetations infected with ALC1435 (RN6390 containing the sar P1-gfp_UV construct), in which the infecting strain was preincubated in RPMI 1640 medium for 6 days at 4°C to turn off fluorescence (the half-life of GFP_UV is 24 h [10b]). These bacteria were then injected into catheterized rabbits to induce endocarditis. As seen, bacterial colonies deep within the vegetation now exhibit uniformly bright green fluorescence. Magnification, ∼×300. The area deep within the vegetation is farther from the area of blood flow. (B) Same experiment as in panel A, except that bacterial colonies are on the surfaces of the vegetations. Note brightly fluorescent bacterial colonies (arrowhead). Magnification, ∼×300. This area is proximal to the region of blood flow. (C) Same preparation as in panel B stained with Giemsa stain to show bacterial colonies on the vegetation surface. Magnification, ∼×225. (D) Twenty-four-hour-old vegetations infected with ALC1437 (sar P2-gfp_UV construct). The arrowhead denotes a colony of nonfluorescent bacteria upon excitation with a UV light source. Note that the yellow-stained background represents infected tissue, not green fluorescent bacteria. Magnification, ∼×200. (E) Same experiment as in panel D, except that bacterial colonies are found on the vegetation surface. Note fluorescent P2-gfp_UV-expressing cells (arrowhead) attached to the vegetation surface. Magnification, ∼×300. (F) Same preparation as in panel E stained with Giemsa stain to show bacterial colonies on the vegetation surface. Magnification, ∼×225.