TEXT

Top Abstract Text References

Binding to the intercellular matrix protein fibronectin is a crucial step in the pathogenesis of infections by group A streptococci (GAS) (9). Such binding promotes specific and firm bacterial adherence to human epithelial tissues and, eventually, the internalization into human epithelial cells as well. There are several fibronectin-binding molecules described to be expressed on the GAS surface, such as proteins F1 and SfbI (7, 30), F2 (11), SOF and SfbII (13, 28), FBP54 (1), PFBP (29), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (25).

Among these molecules, protein F1 was found to be present in 50 to 70% of all clinical isolates tested (5, 19, 33) and to be the predominant fibronectin-binding factor in serotype M6 GAS isolates. The structure and function of protein F1 in this specific serotype was well studied. Fibronectin-binding activity was located in two conserved domains of the protein (24, 31). Due to a bridging effect of the bound fibronectin to ₅₁- and _v3-type integrins, the streptococci attached to buccal epithelial cells and various epithelial cell lines (8, 17, 22, 23, 34). As one consequence, the bacteria can internalize into the epithelial cells (10, 18, 21). This feature has been associated with persistent colonization of asymptomatic human carriers by GAS (20), as well as with the capability to cause invasive diseases (16).

Although considerable N-terminal variability of protein F and/or SfbI sequences has been demonstrated (12), the constitutive expression of these proteins could be disadvantageous for the bacteria because of the constant exposure to the immune system. There are also environmental conditions such as nutrient limitation under which attachment to an anatomical compartment is not necessarily beneficial for GAS. Thus, it seemed logical that the expression of protein F1 would respond to environmental conditions. A few stimuli such as the oxygen partial pressure (35) and superoxide levels (4) have been identified. So far, only one regulatory gene, the rofA gene, has been found to be involved in the response to these environmental conditions (2, 3). The corresponding RofA protein was recently described to exert its regulatory activity by directly binding to two conserved boxes in the promoter region of the protein F1-encoding prtF1 gene (6). Quantitative determination of fibronectin binding by serotype M6 GAS mutants harboring an insertional rofA inactivation indicated that RofA acted as a positive regulator of prtF1, and its own gene under decreased O₂ partial pressure (3). However, some of its regulatory activity depended on the genomic background (2).

Analysis of the serotype M1 genome sequence revealed the presence of a rofA gene and, simultaneously, the presence of two genes that were 30 to 50% homologous to rofA (6, 27). In a serotype M49 GAS strain, the nra gene was identified as exhibiting 60% homology to rofA (27). These findings prompted Granok et al. (6) to describe a RofA-like family of proteins (RALPs). Among these genes, the function of nra was determined as that of a negative regulatory factor for several virulence genes (27) and thus as controlling properties necessary for prolonged intracellular persistence (G. Molinari, M. Rohde, S. R. Talay, G. S. Chhatwal, S. Beckert, and A. Podbielski, submitted for publication).

In the present study, the effect of a serotype M6 rofA gene on virulence genes other than prtF1 as well as on eukaryotic cell adherence and internalization was investigated. The results suggest that RALPs are global regulators with a predominantly negative activity and are involved in cell attachment and invasion.

Generation of a serotype M6 GAS rofA insertional mutant. A fragment of the rofA gene from serotype M6 isolate 2/66 of the GAS strain collection from the Prague WHO reference laboratory was amplified by PCR. The fragment corresponding to positions 31 to 980 of the published M6 rofA sequence (3) was cloned into the plasmid vector pSF152 (32). Integration of the plasmid by single cross-over resulted in disruption of the 1,491-bp rofA gene at bp 798.

Effects of rofA inactivation on prtF1 expression and fibronectin binding. Experiments were done to confirm that this mutation of rofA was affecting the protein F1 transcription and fibronectin binding as reported elsewhere (3). Total RNA was prepared according to an established protocol (27) from wild-type bacteria, and the isogenic rofA mutant was grown to mid-log growth phase in a standing culture under a 20% O₂-5% CO₂ atmosphere. A prtF1-specific probe (19) was used for a semiquantitative Northern blot hybridization analysis (Fig. 1). The prtF1 message was found to be decreased at least eightfold in the rofA mutant.

View larger version (112K): [in this window] [in a new window]   FIG. 1.   Quantitation of virulence gene mRNAs in a serotype M6 GAS strain (wt) and its isogenic rofA regulator mutant. Total RNA was isolated from both strains grown to mid-log phase, was serially diluted in twofold increments, separated by denaturing gel electrophoresis, and subjected to Northern blot hybridizations with digoxigenin-labeled virulence gene-specific probes. CSPD (Roche Molecular Biochemicals, Mannheim, Germany) and autoradiography were used to detect hybridization. The amounts of total RNA used in the assays were 20 µg (mga and rofA), 10 µg (prtF1, slo, and speB), and 5 µg (emm6, pel-sagA, and speA).

Effects of rofA inactivation on expression of virulence genes and functions. The rofA homologue nra was found to control the expression of at least a dozen GAS genes and the collagen-binding activity in a serotype M49 GAS strain ((27); Molinari et al., submitted). Our next objective was to determine whether a functional rofA gene has a comparably broad regulatory activity.

Impact of rofA mutation on eukaryotic cell attachment and internalization. In protein F1-expressing GAS strains, this protein was found to play a major role in bacterial interactions with eukaryotic cells. Thus, our rofA mutant was predicted to be less adhesive and invasive than the corresponding wild type.

Eukaryotic cell viability in presence of the rofA mutant. A decreased host cell viability could lead to a reduction of bacterial viability counts when using culture media containing antibiotics.

Conclusion. In this study, we investigated the impact of a mutation in a member of the RALP regulator family, rofA, on virulence gene expression and the concomitant biological behavior of a serotype M6 GAS strain. In agreement with previous publications (3), expression of the protein F1-encoding prtF1 gene and fibronectin binding as well as the truncated rofA gene was decreased in the mutant, confirming the status of RofA as a positive regulator for these virulence traits. However, a functional rofA gene also appeared to act as a negative regulator for the expression of several other virulence genes. These results agreed with many of those obtained for another RALP member, the nra gene (27). Since the promoters of the controlled genes lack the recently identified RofA binding box (6), it needs to be determined whether RofA regulation is indirect for these virulence genes. Because a functional rofA decreased the expression of mga, the global positive virulence regulator in the serotype M6 strain (14), rofA could be the superimposed negative regulator, whose presence was recently predicted in a study on Mga functions in a serotype M6 strain (15). Like the homologous Nra regulator (Molinari et al., submitted), RofA influences the host cell viability once the bacteria are internalized into the eukaryotic cells. This property is obviously associated with control mechanisms exceeding the known regulation of protein F1 expression. Since other RALP members have a similar function (Molinari et al., submitted), these regulators could have an important general role in stabilizing the intracellular status of GAS.