Mutation of luxS affects growth and virulence factor expression in Streptococcus pyogenes.

Adaptive responses of bacteria that involve sensing the presence of other bacteria are often critical for proliferation and the expression of virulence characteristics. The autoinducer II (AI-2) pathway has recently been shown to be a mechanism for sensing other bacteria that is highly conserved among diverse bacterial species, including Gram-positive pathogens. However, a role for this pathway in the regulation of virulence factors in Gram-positive pathogens has yet to be established. In this study, we have inactivated luxS, an essential component of the AI-2 pathway, in the Gram-positive pathogen Streptococcus pyogenes. Analyses of the resulting mutants revealed the aberrant expression of several virulence properties that are regulated in response to growth phase, including enhanced haemolytic activity, and a dramatic reduction in the expression of secreted proteolytic activity. This latter defect was associated with a reduced ability to secrete and process the precursor of the cysteine protease (SpeB) as well as a difference in the timing of expression of the protease. Enhanced haemolytic activity of the luxS strain was also shown to be linked with an increased expression of the haemolysin S-associated gene sagA. Disruptions of luxS in these mutants also produced a media-dependent growth defect. Finally, an allelic replacement analysis of an S. pyogenes strain with a naturally occurring insertion of IS1239 in luxS suggested a mechanism for modulation of virulence during infection. Results from this study suggest that luxS makes an important contribution to the regulation of S. pyogenes virulence factors.

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.

Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes.

Survival in aerobic conditions is critical to the pathogenicity of many bacteria. To investigate the means of aerotolerance and resistance to oxidative stress in the catalase-negative organism Streptococcus pyogenes, we used a genomics-based approach to identify and inactivate homologues of two peroxidase genes, encoding alkyl hydroperoxidase (ahpC) and glutathione peroxidase (gpoA). Single and double mutants survived as well as the wild type under aerobic conditions. However, they were more susceptible than the wild type to growth suppression by paraquat and cumene hydroperoxide. In addition, we show that S. pyogenes demonstrates an inducible peroxide resistance response when treated with sublethal doses of peroxide. This resistance response was intact in ahpC and gpoA mutants but not in mutants lacking PerR, a repressor of several genes including ahpC and catalase (katA) in Bacillus subtilis. Because our data indicate that these peroxidase genes are not essential for aerotolerance or induced resistance to peroxide stress in S. pyogenes, genes for a novel mechanism of managing peroxide stress may be regulated by PerR in streptococci.

The RofA binding site in Streptococcus pyogenes is utilized in multiple transcriptional pathways.

Understanding the regulation of adhesins defines a pathogenic bacterium's interaction with the local environment within the host. In certain strains of Streptococcus pyogenes, transcription of prtF, the gene which encodes the fibronectin-binding adhesin protein F, is activated by RofA under anaerobic conditions. RofA binds specifically to DNA in its target promoters and autoregulates its own expression. In this study, we have used DNase I protection assays to further investigate the interaction of RofA with its target promoters. In the region between rofA and the gene which encodes protein F (prtF), RofA binds to two distinct sites: a smaller site (17 bp) adjacent to the rofA promoter, and a larger site (40 bp) adjacent to the prtF promoter. Analysis of fusions to a novel reporter gene whose product consists of the fusion of the N-terminal secretion domain of protein F with the C-terminal enzymatic domain of the enterococcal alkaline phosphatase (PhoZ) revealed that the small RofA binding site had no direct role in control of prtF transcription but contributed to regulation of rofA. Comparison in several strains representing different patterns of prtF expression indicated that the larger site was required for activation of rofA and of prtF in all strains by both RofA-dependent and -independent pathways. Thus, it would appear that a common recognition sequence provides separate entries to a final common pathway in S. pyogenes virulence gene expression. The identification of multiple RofA-like proteins and promoters with RofA binding sites implies the existence of a widespread interacting regulatory network.

Contribution of NADH oxidase to aerobic metabolism of Streptococcus pyogenes.

An understanding of how the heme-deficient gram-positive bacterium Streptococcus pyogenes establishes infections in O(2)-rich environments requires careful analysis of the gene products important in aerobic metabolism. NADH oxidase (NOXase) is a unique flavoprotein of S. pyogenes and other lactic acid bacteria which directly catalyzes the four-electron reduction of O(2) to H(2)O. To elucidate a putative role for this enzyme in aerobic metabolism, NOXase-deficient mutants were constructed by insertional inactivation of the gene that encodes NOXase. Characterization of the resulting mutants revealed that growth in rich medium under low-O(2) conditions was indistinguishable from that of the wild type. However, the mutants were unable to grow under high-O(2) conditions and demonstrated enhanced sensitivity to the superoxide-generating agent paraquat. Mutants cultured in liquid medium under conditions of carbohydrate limitation and high O(2) tension were characterized by an extended lag phase, a reduction in growth, and a greater accumulation of H(2)O(2) in the growth medium compared to the wild-type strain. All of these mutant phenotypes could be overcome by the addition of glucose. Either the addition of catalase to the culture medium of the mutants or the introduction of a heterologous NADH peroxidase into the mutants eliminated the accumulation of H(2)O(2) and rescued the growth defect of the mutants under high-O(2) conditions in carbohydrate-limited liquid medium. Taken together, these data show that NOXase is important for aerobic metabolism and essential in environments high in O(2) with carbohydrate limitation.

A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes.

The ability of numerous microorganisms to cause disease relies upon the highly regulated expression of secreted proteinases. In this study, mutagenesis with a novel derivative of Tn4001 was used to identify genes required for the expression of the secreted cysteine proteinase (SCP) of the pathogenic Gram-positive bacterium Streptococcus pyogenes. Designated as Rop loci (regulation of proteinase), ropB is a rgg-like transcriptional activator required for transcription of the gene which encodes the proteinase. In contrast, ropA contributes post-transcriptionally to the secretion and processing of SCP and encodes a homologue of Trigger Factor, a peptidyl-prolyl isomerase and putative chaparone which is highly conserved in most bacterial species, but of unknown function. Analysis of additional ropA mutants demonstrated that RopA acts both to assist in targeting SCP to the secretory pathway and to promote the ability of the proprotein to establish an active conformation upon secretion. This latter function was dependent upon the peptidyl-prolyl isomerase domain of RopA and mutants that lacked this domain exhibited a bipartite deficiency manifested as a kinetic defect in autologous processing of the proprotein to the mature proteinase, and as a catalytic defect in the mature proteinase. These results provide insight into the function of Trigger Factor, the regulation of proteinase activity and the mechanism of secretion in Gram-positive bacteria.

Constitutive expression of fibronectin binding in Streptococcus pyogenes as a result of anaerobic activation of rofA.

Protein F is a fibronectin-binding surface protein of Streptococcus pyogenes (group A streptococcus) that mediates adherence to host cells. A gene product encoded by rofA activates transcription of the gene that encodes protein F (prtF) and was identified in a strain of S. pyogenes that expressed high levels of protein F under all conditions tested. Insertional inactivation of rofA in this strain results in a phenotype similar to that of other strains where high-level transcription of prtF occurs only in response to increased oxygen tension. In this study, we have compared the regulation of prtF and rofA in O2-regulated and constitutive strains in order to gain further insight into the function of rofA. Comparison of the prtF and rofA transcripts by S1 nuclease and primer extension assays indicated that the same promoters for each transcript are used in both O2-regulated and constitutive strains. However, analyses of rofA-lacZ reporter alleles revealed that a key difference between strains involves regulation of rofA itself. In O2-regulated strains, expression of rofA was elevated following culture under conditions of reduced O2 tension. However, a much more robust activation of rofA expression was observed when constitutive strains were grown under similar conditions. Exchange of reporter and rofA alleles between strains demonstrated that host genetic background, and not the sequence of the respective rofA allele or regulatory region, dictates the expression phenotype. Activation of rofA required RofA, and RofA was shown to bind specifically to DNA containing the promoters for rofA and prtF. Finally, overexpression of either allele of rofA caused constitutive expression of prtF regardless of host background. These data suggest a model where anaerobic expression of prtF in constitutive hosts is controlled at the level of transcription of rofA and implicate additional factors in this regulatory pathway.

Insertional inactivation of Streptococcus pyogenes sod suggests that prtF is regulated in response to a superoxide signal.

In establishing an infection, Streptococcus pyogenes has the capacity to bind to the host extracellular matrix protein fibronectin via its protein F adhesin. Previous studies have suggested that the expression of protein F is stimulated during aerobic growth or upon addition of superoxide-generating agents to the culture under O2-limited conditions. To further explore the role of superoxide, we have examined the transcription of the gene which encodes protein F (prtF), as well as the expression of superoxide dismutase (SOD) under conditions which promote or repress protein F expression. These studies show that prtF transcription is regulated in response to superoxide concentration and that SOD is regulated in different environments in a manner which directly parallels the expression of protein F. A mutant deficient in SOD activity was constructed by insertional mutation into the gene which encodes SOD (sod). The resulting mutant was sensitive to superoxide and aerobic conditions, showed hypersensitive induction of prtF in response to superoxide, and expressed prtF under normally unfavorable O2-limited conditions. These findings suggest that a streptococcal signal transduction system which senses superoxide may coordinately control expression of prtF and sod.

Protein F2, a novel fibronectin-binding protein from Streptococcus pyogenes, possesses two binding domains.

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)-binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn-binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene (prtF2) from GAS strain 100076 encoding a novel Fn-binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high-affinity Fn binding. prtF2-related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn-binding protein in GAS. Protein F2 is highly homologous to Fn-binding proteins from Streptococcus dysgalactiae and Streptococcus equisimilis, particularly in its carboxy-terminal portion. Two domains are responsible for Fn binding by protein F2. One domains (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non-repeated stretch of approximately 100 amino acids and is located 100 amino acids aminoterminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.

A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix.

Streptococcus pyogenes binds to the extracellular matrix (ECM) and a variety of host cells and tissues, causing diverse human diseases. Protein F, a S.pyogenes adhesin that binds fibronectin (Fn), contains two binding domains. A repeated domain (RD2) and an additional domain (UR), located immediately N-terminal to RD2. Both domains are required for maximal Fn binding. In this study, we characterize RD2 and UR precisely and compare their functions and binding sites in Fn. The minimal functional unit of RD2 is of 44 amino acids, with contributions from two adjacent RD2 repeats flanked by a novel 'MGGQSES' motif. RD2 binds to the N-terminal fibrin binding domain of Fn. UR contains 49 amino acids, of which six are from the first repeat of RD2. It binds to Fn with higher affinity than RD2, and recognizes a larger fragment that contains fibrin and collagen binding domains. Expression of UR and RD2 independently on the surface-exposed region of unrelated streptococcal protein demonstrates that both mediate adherence of the bacteria to the ECM. We describe here a mechanism of adherence of a pathogen that involves two pairs of sites located on a single adhesin molecule and directed at the same host receptor.

Biological properties of a Streptococcus pyogenes mutant generated by Tn916 insertion in mga.

The mga regulon of Streptococcus pyogenes contains genes which contribute to the pathogenicity and virulence of this significant human pathogen. Transposon insertional inactivation of the regulatory mga gene in a S. pyogenes strain of the clinically important M1 serotype, blocked the expression of four genes located downstream of mga. These genes encode the M1 protein, the IgG-binding protein H, protein SIC which is an extracellular inhibitor of complement, and the C5a peptidase which interferes with granulocyte migration. The wild-type strain is resistant to phagocytosis and adheres to human skin tissue sections; properties that were lost in the transposon mutant. Moreover, the mutant was less virulent to mice but more cytolytic to human lymphocytes, the latter due to an increased activity of streptolysin S, whereas the production of streptolysin O, another toxin of S. pyogenes, was not affected. The mga mutation was complemented in trans with an intact mga gene which restored the phenotype of the wild-type strain.

Distribution of fibronectin-binding proteins among group A streptococci of different M types.

Binding of fibronectin by group A streptococci (GAS) promotes adherence to epithelial cells. The fibronectin-binding activity and the presence of prtF, a gene encoding a fibronectin-binding protein, were studied among 109 strains. Fifty-six strains of 42 different M types possessed prtF-related genes, and 89% of these strains bound fibronectin at high levels. The prtF-related genes varied in the number of repeats that constitute one of its two fibronectin-binding domains. Fifty-three strains of 21 different M types lacked prtF. Thirty-nine of these (74%), representing 13 different M types, bound fibronectin at very low levels. However, 9 (17%), of 5 different M types, bound fibronectin at high levels. The presence of prtF and the capacity to bind fibronectin correlated strongly with the M type of various strains of GAS. This correlation may suggest the existence of a relationship between fibronectin binding and the pathogenic potential of GAS.

Role of the conserved C-repeat region of the M protein of Streptococcus pyogenes.

The surface-located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C-repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M-protein-mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C-repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C-repeat domain, a region of the M-protein molecule distinct from the C-repeat domain confers upon S. pyogenes its ability to resist phagocytosis.

M protein and protein F act as important determinants of cell-specific tropism of Streptococcus pyogenes in skin tissue.

The pathogenic gram-positive bacterium Streptococcus pyogenes (group A streptococcus) causes numerous diseases of cutaneous tissue, each of which is initiated after the interaction of the bacterium with the cells of the epidermis. In this study, we show that different surface proteins of S. pyogenes play important roles in determining the cell-specific tropism of the bacterium in skin. Using streptococcal strains with defined mutations in the genes which encode surface proteins in combination with primary cultures of human skin and an in situ adherence assay which uses histological sections of human skin, we show that the M protein of S. pyogenes mediates the binding of the bacterium to keratinocytes, while a second streptococcal surface protein, protein F, directs the adherence of the organism to Langerhans' cells. Characterization of binding revealed that adherence was inhibited by purified streptococcal proteins and pretreatment of both host cells with the protease trypsin. Adherence was only slightly affected by the state of keratinocyte differentiation in vitro, but was considerably modulated in response to environmental conditions known to regulate expression of M protein and protein F, suggesting that the interaction between these bacterial cell-surface structures/adhesins and keratinocytes and Langerhans' cells may play an important role in streptococcal skin disease.

The identification of rofA, a positive-acting regulatory component of prtF expression: use of an m gamma delta-based shuttle mutagenesis strategy in Streptococcus pyogenes.

Binding of the Gram-positive pathogenic bacterium Streptococcus pyogenes (group A streptococcus) to respiratory epithelium is mediated by the fibronectin-binding adhesin, protein F. Most strains of streptococci regulate the expression of protein F in response to oxygen levels and redox potential; however, JRS4 constitutively binds high levels of fibronectin under all environmental conditions. In this study, we have examined the regulation of protein F expression in JRS4 using a shuttle mutagenesis strategy novel to S. pyogenes. Cloned DNA representing the chromosomal loci adjacent to the gene which encodes protein F (prtF) was subjected to transposon mutagenesis in Escherichia coli using a derivative of transposon m gamma delta that was modified to contain a streptococcal antibiotic-resistance gene. mutagenized DNA was then returned to the streptococcal chromosome by allelic replacement. Analysis of the resulting fibronectin-binding phenotypes revealed that insertions in a region upstream of prtF abolished the constitutive phenotype. However, these mutants now demonstrated regulation in response to both oxygen levels and redox potential. Because these insertions define a locus responsible for the constitutive phenotype, it has been designated rofA (regulator of F). Chromosomal interruption studies using integrational plasmids together with complementation data from a previous study (VanHeyningen et al., 1993) suggested that rofA acts as a positive trans-acting regulator of prtF. Construction of prtF-lacZ fusions indicated that transcription of prtF is constitutive in JRS4 but is regulated in rofA mutants. Analysis of the DNA sequence defined by the rofA insertions revealed a 1495 bp open reading frame, whose predicted product (RofA) possessed both a putative helix-turn-helix motif and limited homology to two other transcriptional activators (Mry, PrgR) of Gram-positive surface proteins. Sequences homologous to rofA were found in regulated strains of S. pyogenes, which suggests that rofA may act as an activator of prtF in response to an unidentified environmental signal. We speculate that the allele reported here contains a mutation that renders it constitutively active.

Analysis of Streptococcus pyogenes promoters by using novel Tn916-based shuttle vectors for the construction of transcriptional fusions to chloramphenicol acetyltransferase.

We have developed a series of shuttle vectors based on the conjugative transposon Tn916 that have been designed for the analysis of transcriptional regulation in Streptococcus pyogenes and other gram-positive bacteria. Designated the pVIT vectors (vectors for integration into Tn916), the vectors are small, stable plasmids in Escherichia coli to facilitate the fusion of promoters from cloned S. pyogenes genes to a promoterless gene which encodes chloramphenicol acetyltransferase. The vectors each contain one or more small regions of Tn916 to direct the integration of the transcriptional fusion into the transposon via homologous recombination following transformation of S. pyogenes or other suitable gram-positive hosts. Integration can be monitored by the inactivation or replacement of an antibiotic resistance determinant in modified derivatives of Tn916. Promoter activity can then be quantitated by the determination of chloramphenicol acetyltransferase-specific activity. In addition, since integration is into loci that do not disrupt the conjugative transpositional functions of Tn916, the vectors are useful for analysis of regulation in strains that are difficult or impossible to transform and can be introduced into these strains by conjugation following transformation of an intermediate host. The promoters for the genes which encode both the M protein and protein F of S. pyogenes were active in pVIT vectors, as was the region which controls transcription of mry, a trans-acting positive regulator of M protein expression. However, neither of the two characterized promoters for mry demonstrated activity when independently analyzed in pVIT-generated partial diploid strains, suggesting that regulation of mry is more complex than predicted by current models. The broad host range of Tn916 should make the pVIT vectors useful for analysis of regulation in numerous other bacterial species.

Protein F: an adhesin of Streptococcus pyogenes binds fibronectin via two distinct domains.

The binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C-terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the N-terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N-terminal domain was mixed in a binding reaction with a protein extract containing the C-terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C-terminal domain, blocked the binding partially. A clone exclusively expressing the C-terminal domain, completely blocked the binding of the 30 kDa N-terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N-terminal domain failed to block the binding of this FN fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Positive transcriptional control of mry regulates virulence in the group A streptococcus.

Transcription of the antiphagocytic M protein in the group A streptococcus (Streptococcus pyogenes) is environmentally regulated in response to CO2 and requires Mry, a trans-acting positive regulatory protein. We have examined the role of Mry in environmental regulation by analysing the factors that regulate expression of the gene that encodes Mry (mry). By employing a strategy that utilizes integrational plasmids, it was found that expression of mry requires the participation of DNA sequences that extend 473 base pairs upstream of the Mry coding region. Transcription of mry, as analysed in S1 nuclease protection assays, is initiated from two separate promoters located within this extended regulatory region. Construction and analysis of transcriptional fusions between the mry promoters and a promoterless chloramphenicol acetyltransferase gene demonstrated that mry is autoregulated and environmentally regulated in response to the level of CO2. These data suggest a model for the regulation of virulence in S. pyogenes where positive transcriptional control of mry in response to environmental stimuli regulates the expression of the M protein.

Expression of protein F, the fibronectin-binding protein of Streptococcus pyogenes JRS4, in heterologous streptococcal and enterococcal strains promotes their adherence to respiratory epithelial cells.

In a previous study we reported the identification of protein F, a fibronectin-binding protein that was essential to the ability of Streptococcus pyogenes JRS4 to adhere to respiratory epithelial cells (E. Hanski and M. Caparon, Proc. Natl. Acad. Sci. USA, 89:6172-6176, 1992). To further evaluate the role of protein F in the adherence of the group A streptococci, we screened other group A streptococcal strains, including six recent clinical isolates, and one strain of Enterococcus faecalis for their capacity to bind fibronectin and for the presence of the gene encoding protein F (prtF). Seven of eight group A streptococcal strains analyzed, including all recent clinical isolates, both bound fibronectin at high affinity and contained DNA sequences that hybridized with a prtF-specific probe. One group A streptococcal isolate and the strain of E. faecalis examined neither contained a prtF-related gene nor bound fibronectin. These two strains also could not efficiently adhere to respiratory epithelial cells. However, upon the introduction of the cloned prtF gene, both of these strains gained the capacity to bind fibronectin and to adhere to respiratory epithelial cells. These results suggest that protein F is an important adhesin, which may have a general role in the virulence of the group A streptococci.