Strains of Staphylococcus aureus were transformed with plasmid DNA containing a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in both gram-positive and gram-negative bacteria. S. aureus cells containing this novel lux construct, downstream of an appropriate promoter sequence, are highly bioluminescent, allowing the detection of fewer than 100 CFU in vitro (direct detection of exponentially dividing cells in liquid culture). Furthermore, these bacteria produce light stably at 37°C and do not require exogenous aldehyde substrate, thus allowing S. aureus infections in living animals to be monitored by bioluminescence. Two strains of S. aureus 8325-4 that produce high levels of constitutive bioluminescence were injected into the thigh muscles of mice, and the animals were then either treated with the antibiotic amoxicillin or left untreated. Bioluminescence from bacteria present in the thighs of the mice was monitored in vivo over a period of 24 h. The effectiveness of the antibiotic in the treated animals could be measured by a decrease in the light signal. At 8 h, the infection in both groups of treated animals had begun to clear, as judged by a decrease in bioluminescence, and by 24 h no light signal could be detected. In contrast, both groups of untreated mice had strong bioluminescent signals at 24 h. Quantification of CFU from bacteria extracted from the thigh muscles of the mice correlated well with the bioluminescence data. This paper shows for the first time that bioluminescence offers a method for monitoring S. aureus infections in vivo that is sensitive and noninvasive and requires fewer animals than conventional methodologies.Many strains of Staphylococcus aureus, which cause a wide variety of diseases ranging from pyoderma to toxic shock syndrome, are methicillin and gentamicin resistant, with some strains also showing limited resistance to vancomycin. Such bacteria are a major problem in nosocomial, or hospital-acquired, infections (3, 9, 18). Without the development of new antibiotics it is possible that, given time, such bacteria will be untreatable by conventional means. In order to combat such bacterial infections, novel and effective drugs will be needed. In addition, new approaches for screening candidate antibiotics both in vitro and in vivo are essential to accelerate the development of new antiinfectives. To this end, bioluminescence offers a method that is sensitive and innocuous and allows only live, or viable, cells to be detected. Furthermore, in vivo detection of bioluminescent bacteria is noninvasive, allowing rapid monitoring of the infective state of eukaryotic cells both in culture and in animals (4, 7). Such a method of monitoring in vivo bioluminescent organisms in living animals has been described for gram-negative bacteria and it was demonstrated to correspond to bacterial CFU data with a correlation coefficient of 0.98

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Animal studies with Streptococcus pneumoniae have provided valuable models for drug development. In order to monitor long-term pneumococcal infections noninvasively in living mice, a novel gram-positive lux transposon cassette, Tn4001 luxABCDE Km r , that allows random integration of lux genes onto the bacterial chromosome was constructed. The cassette was designed so that the luxABCDE and kanamycin resistance genes were linked to form a single promoterless operon. Bioluminescence and kanamycin resistance only occur in a bacterial cell if this operon has transposed downstream of a promoter on the bacterium's chromosome. S. pneumoniae D39 was transformed with plasmid pAUL-A Tn4001 luxABCDE Km r , and a number of highly bioluminescent colonies were recovered. Genomic DNA from the brightest D39 strain was used to transform a number of clinical S. pneumoniae isolates, and several of these strains were tested in animal models, including a pneumococcal lung infection model. Strong bioluminescent signals were seen in the lungs of the animals containing these pneumococci, allowing the course and antibiotic treatment of the infections to be readily monitored in real time in the living animals. Recovery of the bacteria from the animals showed that the bioluminescent signal corresponded to the number of CFU and that the lux construct was highly stable even after several days in vivo. We believe that this lux transposon will greatly expand the ability to evaluate drug efficacy against gram-positive bacteria in living animals using bioluminescence.Streptococcus pneumoniae is the leading cause of invasive bacterial disease in the very young and the elderly and is the bacterium most responsible for community-acquired pneumonia in the developed world (31). It can behave as a transient commensal, colonizing the nasopharynx of 40% of healthy adults and children, with no adverse effects (2). Children carry this pathogen in the nasopharynx asymptomatically for about 4 to 6 weeks, often carrying several serotypes at a time (13, 33). Occasionally, perhaps in conjunction with a viral infection (9), one of these strains gives rise to a symptomatic pneumococcal infection, including sinusitis, otitis media, pneumonia, and meningitis (12, 13, 16).Antibiotic treatment of pneumococcal infections has been less effective in recent years with the increased occurrence of multidrug-resistant strains of S. pneumoniae. About one-third to one-half of pneumococci recovered from humans are at least partially resistant to penicillin, which may occur in addition to resistance to a number of other common antibiotics (1, 3). These factors, plus the ability of the pneumococcus to transfer genes for resistance, encapsulation, and virulence via transformation (12), make it imperative to develop a better understanding of the mechanism by which pneumococci cause disease. Probably the best way to enhance this process is to develop a better animal model.In 1995, Contag et al. (7) showed that it was possible to monitor disease processes in living animals using b...

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Bacterial adherence to host tissue involves specific microbial surface adhesins of which a subfamily termed microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) specifically recognize extracellular matrix components. We now report on the biophysical characterization of recombinant fibronectin binding MSCRAMMs originating from several different species of Gram-positive bacteria. The far-UV CD spectra (190 -250 nm) of recombinant forms of the ligand binding domain of the MSCRAMMs, in a phosphate-buffered saline solution at neutral pH, were characteristic of a protein containing little or no regular secondary structure. The intrinsic viscosity of this domain was found to be the same in the presence or absence of 6 M guanidine hydrochloride, indicating that the native and denatured conformations are indistinguishable. On addition of fibronectin NH 2 terminus as ligand to the recombinant adhesin there is a large change in the resulting far-UV CD difference spectra. At a 4.9 M excess of the NH 2 terminus the difference spectra shifted to what was predominately a ␤-sheet conformation, as judged by comparison with model far-UV CD spectra. The fibronectin NH 2 -terminal domain undergoes a minute but reproducible blue-shift of its intrinsic tryptophan fluorescence on addition of rFNBD-A, which contains no tryptophan residues. Since this result indicates that there is no large change in the environment of the tryptophan residues of the NH 2 terminus on binding, the large shift in secondary structure observed by CD analysis is attributed to induction of a predominately ␤-sheet secondary structure in the adhesin on binding to fibronectin NH 2 terminus.Many pathogenic bacteria have been shown to specifically recognize and bind to various components of the extracellular matrix in an interaction which appears to represent a host tissue colonization mechanism. This adherence involves a group of bacterial proteins termed MSCRAMMs 1 (microbial surface components recognizing adhesive matrix molecules) (1, 2). A number of Gram-positive bacteria have been shown to express fibronectin (Fn) binding MSCRAMMs, and in some cases these proteins have been isolated and the corresponding genes cloned and characterized. The primary Fn binding sites in these MSCRAMMs have been localized to domains present in most Fn binding adhesins. This domain is composed of a unit of 37-48 amino acids, repeated three or four times (Fig. 1A). The repeat regions have been overexpressed as recombinant fusion proteins in Escherichia coli where the recombinant Fn binding domains (rFNBD) are linked to a stretch of histidine residues which are utilized for affinity purification of the rFNBD proteins. These proteins have been designated as rFNBD-D, rFNBD-A, rFNBD-B, and rFNBD-P, respectively (Fig. 1A). The rFNBDs were found to exhibit similar binding kinetics and dissociation constants; for example, the dissociation constants of the four recombinant proteins binding to porcine Fn was determined by biosensor analysis to be in the low nM range...

Many pathogenic Gram-positive bacteria express fibronectin (Fn)-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), most of which have a similar structural organization with a primary ligand-binding domain consisting of 3Ϫ6 repeats of 40Ϫ50 amino-acidresidue motifs. The MSCRAMMs appear to preferentially bind to the N-terminal region of Fn, which is composed of five type-I modules. Here we report that the Fn-binding MSCRAMM FnbpA of Staphylococcus aureus contains a second ligand-binding domain located outside the repeat units. In addition, several sites in the Fn N-terminus presented as recombinant type-I module pairs bind to the repeat domain of the MSCRAMM. All of the MSCRAMMs analyzed, which include FnbpA of Staphylococcus aureus, Sfb of Streptococcus pyogenes, and FnbA and FnbB of Streptococcus dysgalactiae, were shown to bind to multiple sites in the N-terminal domain of Fn. By dissecting the repeat domain of FnbpA using synthetic peptides and recombinant fragments, we show that discrete, different motifs are responsible for the binding to individual sites in Fn, rather than a common motif being able to bind to several pairs of type-I Fn modules. The C-terminal half of many of the MSCRAMM repeat units contain a common motif, which is shown here to bind to the type-I module pair 4 and 5. In addition, some of the repeat units of FnbpA contain N-terminal motifs which bound to the type-I module pairs 1Ϫ2 and 2Ϫ3, respectively. These latter binding motifs appear to be partly overlapping and dependent on flanking sequences. Fluorescence polarization experiments using fluorescein-labeled MSCRAMM peptides and recombinant type-I Fn module pairs revealed dissociation constants of 1Ϫ13 µM. It was also shown that the fluorescein-labeled peptides differed in their primary binding sites on Fn.Keywords : bacterial adhesion; Staphylococcus aureus; Streptococcus pyogenes; Streptococcus dysgalactiae; fibronectin.Fibronectin (Fn), an~450-kDa glycoprotein, is produced by most mammalian cells. It is deposited in the extracellular matrix where it interacts with other matrix components such as proteoglycans, collagen, and fibrin/fibrinogen. A Fn-containing matrix can serve as a substrate for mammalian cell adhesion, a process which involves cell surface receptors of the integrin type. Integrins participate in cell signaling activities which may lead to both an outside-in and an inside-out signal transduction

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We have analyzed antibody reactivity to a fibronectin-binding microbial surface component that recognizes adhesive matrix molecules (MSCRAMM) in blood plasma collected from patients with staphylococcal infections. All patients had elevated levels of anti-MSCRAMM antibodies compared to those of young children who, presumably, had not been exposed to staphylococcal infections. The anti-MSCRAMM antibodies preferentially reacted with the ligand-binding repeat domain of the adhesin. However, these antibodies did not inhibit fibronectin binding. Essentially, all patients had antibodies which specifically recognized the fibronectin-MSCRAMM complex but not the isolated components. Epitopes recognized by these anti-ligand-induced binding sites antibodies were found in each repeat unit of the MSCRAMM. These results demonstrate that staphylococci have bound fibronectin some time during infection and that each repeat unit in the MSCRAMM can engage in ligand binding. Furthermore, our previously proposed model, suggesting that an unordered structure in the MSCRAMM undergoes a conformational change upon ligand binding (