The collagens are a family of animal proteins containing segments of repeated Gly-Xaa-Yaa (GXY) motifs that form a characteristic triple-helical structure. Genes encoding proteins with repeated GXY motifs have also been reported in bacteria and phages; however, it is unclear whether these prokaryotic proteins can form a collagen-like triple-helical structure. Here we used two recently identified streptococcal proteins, Scl1 and Scl2, containing extended GXY sequence repeats as model proteins. First we observed that prior to heat denaturation recombinant Scl proteins migrated as homotrimers in gel electrophoresis with and without SDS. We next showed that the collagen-like domain of Scl is resistant to proteolysis by trypsin. We further showed that circular dichroism spectra of the Scl proteins contained features characteristic of collagen triple helices, including a positive maximum of ellipticity at 220 nm. FurthermorethetriplehelicesofScl1andScl2showedatemperature-dependent unfolding with melting temperatures of 36.4 and 37.6°C, respectively, which resembles those seen for collagens. We finally demonstrated by electron microscopy that the Scl proteins are organized into "lollipoplike" structures, similar to those seen in human proteins with collagenous domains. This implies that the repeated GXY tripeptide motif is a structural indicator of collagenlike triple helices in proteins from such phylogenetically distant sources as bacteria and humans.The collagens comprise a large family of structurally related proteins that are essential building elements of connective tissues in animals (1). In addition to contributing to the structural integrity of the tissue, collagens may also affect cell behavior, e.g. by interacting with specific cellular receptors. All collagens contain a triple-helical domain composed of three left-handed polyproline II-type chains wound around the central axis to form a right-handed superhelix (2). The segments of the collagen polypeptide forming a triple helix contain the repeating amino acid sequence Gly-Xaa-Yaa (GXY) in which a glycine residue occupies every third position, since only glycine is small enough to be accommodated at the center of the triple helix. The three chains are linked by hydrogen bonds between the backbone group, -NH (Gly) and the backbone carbonyl group (X position) of another chain, and by water-mediated interactions. The X and Y positions are frequently occupied by the imino acids proline and hydroxyproline, respectively. Hydroxyprolines are formed by post-translational hydroxylation of proline residues and are important in stabilizing the collagen triple helix. In humans, mutations that affect collagen triple helix formation and fiber assembly have serious pathological consequences often leading to death (3).The vertebrate collagens are classified into 19 types that are grouped in two major categories known as fibrillar (types I-III, V, and XI) and nonfibrillar (IV, VI-X, and XII-XIX) collagens. In addition, a variety of other mammalian proteins have been identif...

Group A Streptococcus (GAS) expresses cell surface proteins that mediate important biological functions such as resistance to phagocytosis, adherence to plasma and extracellular matrix proteins, and degradation of host proteins. An open reading frame encoding a protein of 348 amino acid residues was identified by analysis of the genome sequence available for a serotype M1 strain. The protein has an LPATGE sequence located near the carboxy terminus that matches the consensus sequence (LPXTGX) present in many gram-positive cell wall-anchored molecules. Importantly, the central region of this protein contains 50 contiguous Gly-X-X triplet amino acid motifs characteristic of the structure of human collagen. The structural gene (designated scl for streptococcal collagen-like) was present in all 50 GAS isolates tested, which together express 21 different M protein types and represent the breadth of genomic diversity in the species. DNA sequence analysis of the gene in these 50 isolates found that the number of contiguous Gly-X-X motifs ranged from 14 in serotype M6 isolates to 62 in a serotype M41 organism. M1 and M18 organisms had the identical allele, which indicates very recent horizontal gene transfer. The gene was transcribed abundantly in the logarithmic but not stationary phase of growth, a result consistent with the occurrence of a DNA sequence with substantial homology with a consensus Mga binding site immediately upstream of the scl open reading frame. Two isogenic mutant M1 strains created by nonpolar mutagenesis of the scl structural gene were not attenuated for mouse virulence as assessed by intraperitoneal inoculation. In contrast, the isogenic mutant derivative made from the M1 strain representative of the subclone most frequently causing human infections was significantly less virulent when inoculated subcutaneously into mice. In addition, both isogenic mutant strains had significantly reduced adherence to human A549 epithelial cells grown in culture. These studies identify a new extracellular GAS virulence factor that is widely distributed in the species and participates in adherence to host cells and soft tissue pathology.

Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme assays confirmed that the mutant derivatives were deficient in expression of enzymatically active SpeB cysteine protease. To test the hypothesis that the cysteine protease participates in host mortality, we assessed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intraperitoneal inoculation. Compared to wild-type parental organisms, the serotype M3 speB mutant lost virtually all ability to cause mouse death ( P Ͻ 0.00001), and similarly, the virulence of the M49 mutant was detrimentally altered ( P Ͻ 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide additional evidence that microbial cysteine proteases are critical in host-pathogen interactions.

The Streptococcus pyogenes cell-surface protein Scl2 contains a globular N-terminal domain and a collagen-like domain, (GlyXaa-Xaa) 79 , which forms a triple helix with a thermal stability close to that seen for mammalian collagens. Hyp is a major contributor to triple-helix stability in animal collagens, but is not present in bacteria, which lack prolyl hydroxylase. To explore the basis of bacterial collagen triple-helix stability in the absence of Hyp, biophysical studies were carried out on recombinant Scl2 protein, the isolated collagen-like domain from Scl2, and a set of peptides modeling the Scl2 highly charged repetitive (GlyXaa-Xaa) n sequences. At pH 7, CD spectroscopy, dynamic light scattering, and differential scanning calorimetry of the Scl2 protein all showed a very sharp thermal transition near 36°C, indicating a highly cooperative unfolding of both the globular and triple-helix domains. The collagen-like domain isolated by trypsin digestion showed a sharp transition at the same temperature, with an enthalpy of 12.5 kJ/mol of tripeptide. At low pH, Scl2 and its isolated collagen-like domain showed substantial destabilization from the neutral pH value, with two thermal transitions at 24 and 27°C. A similar destabilization at low pH was seen for Scl2 charged model peptides, and the degree of destabilization was consistent with the strong pH dependence arising from the GKD tripeptide unit. The Scl2 protein contained twice as much charge as human fibril-forming collagens, and the degree of electrostatic stabilization observed for Scl2 was similar to the contribution Hyp makes to the stability of mammalian collagens. The high enthalpic contribution to the stability of the Scl2 collagenous domain supports the presence of a hydration network in the absence of Hyp.Collagens are considered to be the characteristic structural molecules of the extracellular matrix of multicellular animals. Fibril-forming collagens and basement membrane collagens are ubiquitous in vertebrates and invertebrates, whereas families of more specialized collagens have developed in different organisms such as the 28 distinct collagen types found in vertebrates (1-3) and the ϳ100 cuticle collagen genes in Caenorhabditis elegans (4). In recent years, the range of occurrence of collagen-like sequences with Gly as every 3rd residue and a high Pro content has been extended from metazoans to Ͼ100 proteins in bacteria and bacteriophage (5). An understanding of the structure and stabilization of such bacterial collagens presents new challenges because they lack the Hyp post-translational modification characteristic of animal collagens.A high content of Hyp is a unique stabilizing feature of animal collagens. The characteristic structural motif of all collagens is the triple helix, composed of three left-handed polyproline II-type chains (3 residues/turn) wound around the central axis to form a right-handed superhelix (6 -8). The close packing of each chain near the central axis constrains every 3rd residue of the amino acid sequence to be G...

Extracellular proteins made by group A Streptococcus (GAS) play critical roles in the pathogenesis of human infections caused by this bacterium. Although many extracellular GAS proteins have been identified and characterized, there has been no systematic analysis of culture supernatant proteins. Proteins present in the culture supernatant of strains of serotype M1 (MGAS 5005) and M3 (MGAS 315) mutants lacking production of the major extracellular cysteine protease were separated by two-dimensional gel electrophoresis and identified by amino-terminal amino acid sequencing and interrogation of available databases, including a serotype M1 genome sequence. In the aggregate, amino-terminal amino acid sequence data for 66 protein spots were generated, 53 unique sequences were obtained, and 44 distinct proteins were identified. Sixteen of the 44 proteins had apparent secretion signal sequences and 27 proteins did not. Eight of the 16 proteins with apparent secretion signal sequences have not been previously described for GAS. Antibodies against most of the apparently secreted proteins were present in sera from mice infected subcutaneously with MGAS 5005 or MGAS 315. Humans with documented GAS infections (pharyngitis, acute rheumatic fever, and severe invasive disease) also had serum antibodies reacting with many of the apparently secreted proteins, indicating that they were synthesized in the course of GAS-human interaction. The genes encoding four of the eight previously undescribed and apparently secreted culture supernatant proteins were cloned, and the proteins were overexpressed in Escherichia coli. Western blot analysis with these recombinant proteins and sera from GASinfected mice and humans confirmed the immunogenicity of these proteins. Taken together, the data provide new information about the molecular aspects of GAS-host interactions.

To test this idea, a new nonpolar mutagenesis method employing a spectinomycin resistance cassette was used to inactivate the sic gene in an M1 GAS strain. The isogenic Sic-negative mutant strain was significantly (P < 0.019) impaired in ability to colonize the mouse mucosal surface after intranasal infection. These results support the hypothesis that the predominance of M1 strains in human infections is related, in part, to a Sic-mediated enhanced colonization ability.

A recent study found that group A Streptococcus (GAS) expresses a cell surface protein with similarity to human collagen (S. Lukomski, K. Nakashima, I. Abdi, V. J. Cipriano, R. M. Ireland, S. R. Reid, G. G. Adams, and J. M. Musser, Infect. Immun. 68:6542-6553, 2000). This streptococcal collagen-like protein (Scl) contains a long region of Gly-X-X motifs and was produced by serotype M1 GAS strains. In the present study, a second member of the scl gene family was identified and designated scl2. The Scl2 protein also has a collagen-like region, which in M1 strains is composed of 38 contiguous Gly-X-X triplet motifs. The scl2 gene was present in all 50 genetically diverse GAS strains studied. The Scl2 protein is highly polymorphic, and the number of Gly-X-X motifs in the 50 strains studied ranged from 31 in one serotype M1 strain to 79 in serotype M28 and M77 isolates. The scl1 and scl2 genes were simultaneously transcribed in the exponential phase, and the Scl proteins were also produced. Scl1 and Scl2 were identified in a cell-associated form and free in culture supernatants. Production of Scl1 is regulated by Mga, a positive transcriptional regulator that controls expression of several GAS virulence factors. In contrast, production of Scl2 is controlled at the level of translation by variation in the number of short-sequence pentanucleotide repeats (CAAAA) located immediately downstream of the GTG (Val) start codon. Control of protein production by this molecular mechanism has not been identified previously in GAS. Together, the data indicate that GAS simultaneously produces two extracellular human collagen-like proteins in a regulated fashion.Group A Streptococcus (GAS) causes human infections of the throat and soft tissue and systemic diseases (39). This broad spectrum of infected tissues indicates that GAS can adapt to changing environments during pathogen-host interactions. In addition, the wide range of infections caused by GAS suggests that virulence factor expression is a very complex regulated process. Indeed, transcriptional and posttranscriptional mechanisms that control expression of virulence genes have been described (1,9,17,21,31). For example, several streptococcal cell surface proteins expressed in exponential growth are regulated by Mga, a positive transcriptional activator protein (3,23,27,32).Microbial extracellular molecules interact with host proteins and often mediate adherence (26). Several cell surface proteins of gram-positive bacteria have structural similarities that include a variable amino terminus, a central region composed of repeating units, and a carboxy-terminal cell-associated region with an LPXTG cell wall anchor motif (8). GAS cell surface proteins have been identified as proven or potential virulence factors and include M protein (7), immunoglobulin (14) and fibronectin (13) binding proteins, serum opacity factor (4), C5a peptidase (44), and GRAB (34).Recently, we identified a new GAS cell surface protein that contains a central region composed of variable numbers of Gly-X-X ...

Serotype M1 group A Streptococcus strains cause epidemic waves of human infections long thought to be mono- or pauciclonal. The gene encoding an extracellular group A Streptococcus protein (streptococcal inhibitor of complement) that inhibits human complement was sequenced in 1,132 M1 strains recovered from population-based surveillance of infections in Canada, Finland and the United States. Epidemic waves are composed of strains expressing a remarkably heterogeneous array of variants of streptococcal inhibitor of complement that arise very rapidly by natural selection on mucosal surfaces. Thus, our results enhance the understanding of pathogen population dynamics in epidemic waves and infectious disease reemergence.