Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon.

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.

Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell beta1 integrins.

Although Staphylococcus aureus is primarily considered an extracellular pathogen, recent evidence suggests that this bacterium can invade a variety of nonprofessional phagocytic cells. Here we investigate the early stages of cellular invasion by S. aureus and determine the bacterial and host components that are required for this process. S. aureus expresses two cell surface-associated fibronectin (FN)-binding proteins (FnbpA and FnbpB) that mediate the interaction of the bacteria with both soluble and solid-phase FN in vitro. Using a mutant of S. aureus that lacks the expression of both Fnbps, we show that the expression of either protein is necessary for efficient uptake by the mouse fibroblast line GD25beta1A. Invasion could be inhibited by soluble recombinant proteins encompassing either the FN-binding D repeat region or the A region (and B repeats) of FnbpA, suggesting that the activities of both regions are important in this process. We demonstrate that FN is also required for invasion of this cell line. In the presence of FN-depleted fetal bovine serum, the invasion level was reduced by approximately 40% compared to in the presence of whole fetal bovine serum. Invasion could be further reduced by the addition of anti-mouse FN antibodies to the assay. Finally, we utilize a mutant mouse fibroblast line, which lacks beta1 integrin expression, to demonstrate that host cell beta1 integrins are necessary for efficient cellular invasion. The level of invasion of the mutant cell line GD25 was reduced by approximately 97% compared to the beta1-expressing complemented cell line GD25beta1A. In addition, invasion of the GD25beta1A cell line could be inhibited by an RGD-containing peptide, further implicating a role for integrins in this process. Based on these observations, we put forward a model of S. aureus invasion in which host FN forms a bridge between the bacterial Fnbps and host cell beta1 integrins, leading to bacterial uptake.

Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct.

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 degrees 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.

Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells.

Most bacterial infections are initiated by the adherence of microorganisms to host tissues. This process involves the interaction of specific bacterial surface structures, called adhesins, with host components. In this review, we discuss a group of microbial adhesins known as Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) which recognize and bind FN. The interaction of bacteria with FN is believed to contribute significantly to the virulence of a number of microorganisms, including staphylococci and streptococci. Several FN-binding MSCRAMMs of staphylococci and streptococci exhibit a similar structural organization and mechanism of ligand recognition. The ligand-binding domain consists of tandem repeats of a approximately 45 amino acid long unit which bind to the 29-kDa N-terminal region of FN. The binding mechanism is unusual in that the repeat units are unstructured and appear to undergo a conformational change upon ligand binding. Apart from supporting bacterial adherence, FN is also involved in bacterial entry into non-phagocytic mammalian cells. A sandwich model has been proposed in which FN forms a molecular bridge between MSCRAMMs on the bacterial surface and integrins on the host cell. However, the precise mechanism of bacterial invasion and the roles of FN and integrins in this process have yet to be fully elucidated.

Antibody response to fibronectin-binding adhesin FnbpA in patients with Staphylococcus aureus infections.

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 (K. House-Pompeo, Y. Xu, D. Joh, P. Speziale, and M. Höök, J. Biol. Chem. 271:1379-1384, 1996), is presumably operational in patients during infections.

Multiple specificities of the staphylococcal and streptococcal fibronectin-binding microbial surface components recognizing adhesive matrix molecules.

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 3-6 repeats of 40-50 amino-acid-residue 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 1-2 and 2-3, 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 1-13 microM. It was also shown that the fluorescein-labeled peptides differed in their primary binding sites on Fn.

A monoclonal antibody enhances ligand binding of fibronectin MSCRAMM (adhesin) from Streptococcus dysgalactiae.

A monoclonal antibody 3A10, generated from a mouse immunized with the Streptococcus dysgalactiae fibronectin (Fn) binding protein FnbA, was isolated, and its effect on ligand binding by the antigen was examined. The epitope for 3A10 was localized to a previously unidentified Fn binding motif (designated An) just N-terminal of the repeat domain which represents the primary ligand binding site on FnbA. Fn binding to Au was enhanced by 3A10 rather than inhibited. This effect was demonstrated in two different assays. First, in the presence of 3A10 the Au-containing proteins and synthetic peptide more effectively competed with bacterial cells for binding to Fn. Second, 3A10 dramatically increased the binding of biotin-labeled forms of the Au-containing proteins to Fn immobilized on a blotting membrane. Pure 3A10 IgG did not recognize the antigen by itself, and Fn was required for the immunological interaction between the antibody and the epitope. This induction effect of Fn was shown in both Western blot and enzyme-linked immunosorbent assay in which immobilized Au-containing molecules were probed with 3A10 in the presence of varying concentrations of Fn. Specificity analyses of 3A10 revealed that the monoclonal also recognized a ligand binding motif in a Streptococcus pyogenes Fn binding MSCRAMM but not the corresponding motifs in two related adhesins from Staphylococcus aureus and S. dysgalactiae. Furthermore, 3A10 stimulated Fn binding by S. pyogenes cells. These results together with subsequent biophysical studies presented in the accompanying paper (House-Pomepeo, K., Xu, Y., Joh, D., Speziale, P., and Höök, M. (1996) J. Biol. Chem. 271, 1379-1384) indicate that the ligand binding sites of Fn binding MSCRAMMs have little or no secondary structure. However, on binding to Fn, they appear to undergo a structural rearrangement resulting in a defined structure rich in beta sheet and expressing a ligand-induced binding site for antibodies such as 3A10.

Conformational changes in the fibronectin binding MSCRAMMs are induced by ligand binding.

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 NH2 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 NH2 terminus the difference spectra shifted to what was predominately a beta-sheet conformation, as judged by comparison with model far-UV CD spectra. The fibronectin NH2-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 NH2 terminus on binding, the large shift in secondary structure observed by CD analysis is attributed to induction of a predominately beta-sheet secondary structure in the adhesin on binding to fibronectin NH2 terminus.