Bacteriophage endolysins (lysins) bind to a cell wall substrate and cleave peptidoglycan, resulting in hypotonic lysis of the phage-infected bacteria. When purified lysins are added externally to Gram-positive bacteria they mediate rapid death by the same mechanism. For this reason, novel therapeutic strategies have been developed using such enzybiotics. However, like other proteins introduced into mammalian organisms, they are quickly cleared from systemic circulation. PEGylation has been used successfully to increase the in vivo half-life of many biological molecules and was therefore applied to Cpl-1, a lysin specific for S. pneumoniae. Cysteine-specific PEGylation with either PEG 10K or 40K was achieved on Cpl-1 mutants, each containing an additional cysteine residue at different locations To the best of our knowledge, this is the first report of the PEGylation of bacteriophage lysin. Compared to the native enzyme, none of the PEGylated conjugates retained significant in vitro anti-pneumococcal lytic activity that would have justified further in vivo studies. Since the anti-microbial activity of the mutant enzymes used in this study was not affected by the introduction of the cysteine residue, our results implied that the presence of the PEG molecule was responsible for the inhibition. As most endolysins exhibit a similar modular structure, we believe that our work emphasizes the inability to improve the in vivo half-life of this class of enzybiotics using a cysteine-specific PEGylation strategy.
Bacteriophage; S. pneumoniae; Cpl-1; PEGylation; Endolysin; Enzybiotic
Staphylococcus aureus is a major human pathogen responsible for a number of serious and sometimes fatal infections. One of its reservoirs on the human body is the skin, which is known to be a source of invasive infection. The potential for an engineered staphylococcus-specific phage lysin (ClyS) to be used for topical decolonization is presented. We formulated ClyS into an ointment and applied it to a mouse model of skin colonization/infection with S. aureus. Unlike the standard topical antibacterial agent mupirocin, ClyS eradicated a significantly greater number of methicillin-susceptible S. aureus (MSSA) and -resistant S. aureus (MRSA) bacteria: a 3-log reduction with ClyS as opposed to a 2-log reduction with mupirocin in our model. The use of ClyS also demonstrated a decreased potential for the development of resistance by MRSA and MSSA organisms compared to that from the use of mupirocin in vitro. Because antibodies may affect enzyme function, we tested antibodies developed after repeated ClyS exposure for their effect on ClyS killing ability. Our results showed no inhibition of ClyS activity at various antibody titers. These data demonstrate the potential of developing ClyS as a novel class of topical antimicrobial agents specific to staphylococcus.
In the billion years that bacteriophage (or phage) have existed together with bacteria the phage have evolved systems that may be exploited for our benefit. One of these is the lytic system used by the phage to release their progeny from an infected bacterium. Endolysins (or lysins) are highly evolved enzymes in the lytic system produced to cleave essential bonds in the bacterial cell wall peptidoglycan for progeny release. Small quantities of purified recombinant lysin added externally to gram-positive bacteria results in immediate lysis causing log-fold death of the target bacterium. Lysins have now been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and in infected tissues. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Lysins therefore, may be a much-needed anti-infective (or enzybiotic) in an age of mounting antibiotic resistance.
bacteriophage; endolysin; gram-positive bacteria; lytic enzymes; mucosal colonization; phage; prophylaxis; therapeutic
Recent metagenomic sequencing studies of uncultured viral populations have provided novel insights into the ecology of environmental bacteriophage. At the same time, viral metagenomes could also represent a potential source of recombinant proteins with biotechnological value. In order to identify such proteins, a novel two-step screening technique was devised for cloning phage lytic enzymes from uncultured viral DNA. This plasmid-based approach first involves a primary screen in which transformed Escherichia coli clones that demonstrate colony lysis following exposure to inducing agent are identified. This effect, which can be due to the expression of membrane-permeabilizing phage holins, is discerned by the development a hemolytic effect in surrounding blood agar. In a secondary step, the clones identified in the primary screen are overlaid with autoclaved Gram-negative bacteria (specifically Pseudomonas aeruginosa) to assay directly for recombinant expression of lytic enzymes, which are often encoded proximally to holins in phage genomes. As proof-of-principle, the method was applied to a viral metagenomic library constructed from mixed animal feces, and 26 actively expressed lytic enzymes were cloned. These proteins include both Gram-positive-like and Gram-negative-like enzymes, as well as several atypical lysins whose predicted structures are less common among known phage. Overall, this study represents one of the first functional screens of a viral metagenomic population, and it provides a general approach for characterizing lysins from uncultured phage.
Bacillus cereus spores are assembled with a series of concentric layers that protect them from a wide range of environmental stresses. The outermost layer, or exosporium, is a bag-like structure that interacts with the environment and is composed of more than 20 proteins and glycoproteins. Here, we identified a new spore protein, ExsM, from a β-mercaptoethanol extract of B. cereus ATCC 4342 spores. Subcellular localization of an ExsM-green fluorescent protein (GFP) protein revealed a dynamic pattern of fluorescence that follows the site of formation of the exosporium around the forespore. Under scanning electron microscopy, exsM null mutant spores were smaller and rounder than wild-type spores, which had an extended exosporium (spore length for the wt, 2.40 ± 0.56 μm, versus that for the exsM mutant, 1.66 ± 0.38 μm [P < 0.001]). Thin-section electron microscopy revealed that exsM mutant spores were encased by a double-layer exosporium, both layers of which were composed of a basal layer and a hair-like nap. Mutant exsM spores were more resistant to lysozyme treatment and germinated with higher efficiency than wild-type spores, and they had a delay in outgrowth. Insertional mutagenesis of exsM in Bacillus anthracis ΔSterne resulted in a partial second exosporium and in smaller spores. In all, these findings suggest that ExsM plays a critical role in the formation of the exosporium.
Staphylococcus aureus is the causative agent of several serious infectious diseases. The emergence of antibiotic-resistant S. aureus strains has resulted in significant treatment difficulties, intensifying the need for new antimicrobial agents. Toward this end, we have developed a novel chimeric bacteriophage (phage) lysin that is active against staphylococci, including methicillin-resistant S. aureus (MRSA). The chimeric lysin (called ClyS) was obtained by fusing the N-terminal catalytic domain of the S. aureus Twort phage lysin with the C-terminal cell wall-targeting domain from another S. aureus phage lysin (phiNM3), which displayed Staphylococcus-specific binding. ClyS was expressed in Escherichia coli, and the purified protein lysed MRSA, vancomycin-intermediate strains of S. aureus (VISA), and methicillin-sensitive (MSSA) strains of S. aureus in vitro. In a mouse nasal decolonization model, a 2-log reduction in the viability of MRSA cells was seen 1 h following a single treatment with ClyS. One intraperitoneal dose of ClyS also protected against death by MRSA in a mouse septicemia model. ClyS showed a typical pattern of synergistic interactions with both vancomycin and oxacillin in vitro. More importantly, ClyS and oxacillin at doses that were not protective individually protected synergistically against MRSA septic death in a mouse model. These results strongly support the development of ClyS as an attractive addition to the current treatment options of multidrug-resistant S. aureus infections and would allow for the reinstatement of antibiotics shelved because of mounting resistance.
Lysins are highly evolved enzymes produced by bacteriophage ( phage for short) to digest the bacterial cell wall for phage progeny release. In gram-positive bacteria, small quantities of purified recombinant lysin added externally results in immediate lysis causing log-fold death of the target bacterium. Lysins have been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and infected tissues. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Thus, lysins may be a much needed anti-infective in an age of mounting antibiotic resistance.
Phage; Bacteriophage; Cell wall; Gram-positive bacteria; Infection; Lysin; Lytic enzymes; Mucosal colonization; Pathogens; Peptidoglycan
Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.
A rapid protocol was developed for constructing plasmid libraries from small quantities of genomic/metagenomic DNA. The technique utilizes linker amplification with topoisomerase cloning and allows for inducible transcription in Escherichia coli. As proof of principle, several anti-Bacillus lysins were cloned from bacteriophage genomes and an aerolysin was cloned from a metagenomic sample.
Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes.
Bacteria–host interactions are dynamic processes, and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization. We used oligonucleotide microarrays to monitor (in vitro) differential gene expression in group A streptococci during pharyngeal cell adherence, the first overt infection stage. We present neighbor clustering, a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes. We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application. We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data. Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence.
Microarray technology is commonly used to reveal genome-wide transcriptional changes in bacterial pathogens during interactions with the host. Clustering algorithms, which group genes with similar expression patterns, facilitate microarray data organization and are based on assumptions that co-expressed genes share common function or regulation; however, clustering solely by co-expression may not reveal all of the information contained in bacterial array data. We introduce neighbor clustering, a new tool for analyzing bacterial gene expression profiles, which distinguishes itself from other programs by incorporating details unique to the architecture of bacterial chromosomes into the analysis. Neighbor clustering combines two informative characteristics of bacterial genes that share common function or regulation—(1) similar expression profiles and (2) physical proximity on the chromosome—and extracts statistically significant clusters of gene neighbors that are potentially related by function or regulation. We present the analysis of microarray data from group A streptococci during adherence to human pharyngeal cells, the first overt infection step. We show that neighbor clustering identifies more differentially expressed genes than rigorous statistical analyses alone, and can provide functional clues about unknown genes. We extended the analysis to include a previously published streptococcal array study to demonstrate the applicability of the method.
Staphylococcus epidermidis is an important opportunistic pathogen causing nosocomial infections and is often associated with infections in patients with implanted prosthetic devices. A number of virulence determinants have been identified in S. epidermidis, which are typically acquired through horizontal gene transfer. Due to the high recombination potential, bacteriophages play an important role in these transfer events. Knowledge of phage genome sequences provides insights into phage-host biology and evolution. We present the complete genome sequence and a molecular characterization of two S. epidermidis phages, φPH15 (PH15) and φCNPH82 (CNPH82). Both phages belonged to the Siphoviridae family and produced stable lysogens. The PH15 and CNPH82 genomes displayed high sequence homology; however, our analyses also revealed important functional differences. The PH15 genome contained two introns, and in vivo splicing of phage mRNAs was demonstrated for both introns. Secondary structures for both introns were also predicted and showed high similarity to those of Streptococcus thermophilus phage 2972 introns. An additional finding was differential superinfection inhibition between the two phages that corresponded with differences in nucleotide sequence and overall gene content within the lysogeny module. We conducted phylogenetic analyses on all known Siphoviridae, which showed PH15 and CNPH82 clustering with Staphylococcus aureus, creating a novel clade within the S. aureus group and providing a higher overall resolution of the siphophage branch of the phage proteomic tree than previous studies. Until now, no S. epidermidis phage genome sequences have been reported in the literature, and thus this study represents the first complete genomic and molecular description of two S. epidermidis phages.
While screening the clonality of Streptococcus pyogenes isolates from an outbreak of erythromycin-resistant pharyngitis in Pittsburgh, PA, we found a correlation between the presence of the chimeric element Φ10394.4 (carrying the macrolide efflux gene, mefA) and genomic DNA being resistant to cleavage by SmaI restriction endonuclease. A search of the open reading frames in Φ10394.4 identified a putative type II restriction-modification (R-M) cassette containing a cytosine methyltransferase gene (spyIM). Heterologous expression of the cloned spyIM gene, as well as allelic-replacement experiments, showed that the action of this methyltransferase (M.SpyI) was responsible for the inhibition of SmaI digestion of genomic DNA in the Φ10394.4-containing isolates. Analysis of the methylation patterns of streptococcal genomic DNA from spyIM-positive strains, a spyIM deletion mutant, and a spyIM-negative strain determined that M.SpyI specifically recognized and methylated the DNA sequence to generate 5′-CmCNGG. To our knowledge, this is the first methyltransferase gene from S. pyogenes to be cloned and to have its activity characterized. These results reveal why pulsed field gel electrophoresis analysis of SmaI-digested genomic DNA cannot be used to analyze the clonality of some streptococci containing Φ10394.4 and may explain the inability of previous epidemiological studies to use SmaI to analyze DNAs from macrolide-resistant streptococci. The presence of the SpyI R-M cassette in Φ10394.4 could impart a selective advantage to host strain survival and may provide another explanation for the observed increase in macrolide-resistant streptococci.
Pneumococcal prophages are extremely frequent, but no role in pathogenesis has so far been attributed to them. We isolated a variant of phage MM1, named MM1-1998, from a serotype 24 strain of Streptococcus pneumoniae. We created three isogenic strain pairs (serotypes 3, 4, and 24) that differed only by the lysogenic presence of the MM1-1998 phage and did a phenotypic comparison. Lysogeny led to improved adherence to inert surfaces and pharyngeal cells compared to that with the cured variants of the strains. We found that lysogeny with MM1-1998 coincided with a more transparent phenotype and phage curing with more opaque colonies in all strain pairs, and we discovered that transparency was associated with more successful and stable lysogeny. Since transparency alone was possibly responsible for the adherence difference, we further compared the TIGR4 lysogen with an equally transparent variant of TIGR4 in order to reassess the role of phage or transparency separately. The results revealed that improved adherence was independently associated with lysogeny with the MM1-1998 phage. Other phenotypic differences such as faster growth, increased autolysis, and decreased intracellular hemolytic activity were more likely due to transparency. By improving the adherence of pneumococci, this prophage may contribute to their fitness and possibly to their persistence in humans.
Our laboratory has developed phage lytic enzymes to prevent infection by specifically destroying disease bacteria on mucous membranes and in blood. Enzymes specific for S. pneumoniae and S. pyogenes have been developed to be used nasally and orally to control these organisms in environments such as hospitals and nursing homes to prevent or markedly reduce serious infections by these pathogens. In addition, a B. anthracis-specific enzyme was developed to kill the vegetative forms of these bacteria in the blood of infected individuals. In animal studies, >80% of mice colonized mucosally or infected intravenously with pathogenic bacteria were decolonized or survived after a single enzyme treatment delivered to the same site of colonization or infection.
Bacterial surface proteins are important molecules in the infectivity and survival of pathogens. Surface proteins on gram-positive bacteria have been shown to attach via a transpeptidase, termed sortase, that cleaves an LPXTG sequence found close to the C termini of nearly all surface proteins on these bacteria. We previously identified a unique enzyme (LPXTGase) from Streptococcus pyogenes that also cleaves the LPXTG motif with a catalytic activity higher than that of sortase, suggesting that it plays an important role in the attachment process. We have now purified and characterized an LPXTGase from Staphylococcus aureus and found that it has both similar and unique features compared to the S. pyogenes enzyme. The S. aureus enzyme is glycosylated and contains unusual amino acids, like its streptococcal counterpart. Like the streptococcal enzyme, staphylococcal LPXTGase has an overrepresentation of amino acids found in the peptidoglycan, i.e., glutamine/glutamic acid, glycine, alanine, and lysine, and furthermore, we find that these amino acids are present in the enzyme at precisely the same ratio at which they are found in the peptidoglycan for the respective organism. This suggests that enzymes responsible for wall assembly may also play a role in the construction of LPXTGase.
Phage-mediated lysis has been an essential laboratory tool for rapidly identifying Bacillus anthracis for more than 40 years, relying on the γ phage derivative of a Bacillus cereus prophage called W. The complete genomic sequences of the temperate W phage, referred to as Wβ, and its lytic variant γ were determined and found to encode 53 open reading frames each, spanning 40,864 bp and 37,373 bp, respectively. Direct comparison of the genomes showed that γ evolved through mutations at key loci controlling host recognition, lysogenic growth, and possibly host phenotypic modification. Included are a cluster of point mutations at the gp14 tail fiber locus of γ, encoding a protein that, when fused to green fluorescent protein, binds specifically to B. anthracis. A large 2,003-bp deletion was also identified at the γ lysogeny module, explaining its shift from a temperate to a lytic lifestyle. Finally, evidence of recombination was observed at a dicistronic Wβ locus, encoding putative bacterial cell surface-modifying proteins, replaced in γ with a locus, likely obtained from a B. anthracis prophage, encoding demonstrable fosfomycin resistance. Reverse transcriptase PCR analysis confirmed strong induction at the dicistronic Wβ locus and at four other phage loci in B. anthracis and/or B. cereus lysogens. In all, this study represents the first genomic and functional description of two historically important phages and is part of a broader investigation into contributions of phage to the B. anthracis life cycle. Initial findings suggest that lysogeny of B. anthracis promotes ecological adaptation, rather than virulence, as with other gram-positive pathogens.
We have cloned a lytic enzyme, PlyPH, with a specific lytic effect on Bacillus anthracis strains. PlyPH remains active between pH 4 and 10.5, and a single dose rescued a significant percentage of mice infected intraperitoneally with an attenuated B. anthracis strain. We propose PlyPH as a novel therapeutic agent.
On January 2, 2005, the scientific community lost a valued colleague and friend. Maclyn McCarty, or “Mac,” as he was better known, was perhaps most recognized for his part in the discovery of DNA as the carrier of genetic information. But McCarty's scientific career was long and fruitful, and his contributions to science were vast. This retrospective offers a look at some of Mac's other notable scientific achievements.
Streptococcus gordonii shows promise as a live mucosal vaccine vector for immunization against respiratory pathogens. In preparation for clinical trials to evaluate S. gordonii engineered to express group A streptococcal M protein antigens, we characterized the responses of 150 healthy volunteers to combined nasal and oral inoculation with approximately 1.5 × 109 CFU of SP204(1-1), an S. gordonii strain not bearing vaccine antigens. SP204(1-1) was selected for resistance to streptomycin and 5-fluoro-2-deoxyuridine to distinguish it from indigenous flora. In two antibiotic treatment studies, we performed serial culturing of nose, mouth, and saliva samples from 120 subjects treated with azithromycin beginning 5 days after inoculation to determine whether SP204(1-1) could be rapidly eliminated should safety concerns arise. A natural history study was performed to assess the time until spontaneous eradication in the remaining 30 subjects, who did not receive the antibiotic and who were monitored with repeated culturing for 14 weeks after inoculation. SP204(1-1) was generally well tolerated. Symptoms reported most often within 5 days of inoculation were nasal congestion (36%), headache (30%), and sore throat (19%). The strain was detected by culturing in 98% of subjects. A single dose of azithromycin eliminated colonization in 95% of subjects; all subjects receiving a 5-day course of an antibiotic showed clearance by day 11. Without the antibiotic, 82% of subjects showed spontaneous eradication of the implanted strain within 7 days, and all showed clearance by 35 days. The results of these clinical trials provide encouragement that the use of S. gordonii as a live mucosal vaccine vector is a feasible strategy.
A combination of Cpl-1, a bacteriophage lytic enzyme, and penicillin, gentamicin, levofloxacin, or azithromycin was tested against Streptococcus pneumoniae strains with various susceptibilities to penicillin. Activities of Cpl-1 and gentamicin were increasingly synergistic with a decreasing penicillin MIC, while Cpl-1 and penicillin showed synergy against an extremely penicillin-resistant strain.
Group B streptococci (GBS) are the leading cause of neonatal meningitis and sepsis worldwide. The current treatment strategy is limited to intrapartum antibiotic prophylaxis in pregnant women to prevent early-onset neonatal diseases, but considering the potential for antibiotic resistance, the risk of losing control over the disease is high. To approach this problem, we have developed a bacteriophage (phage) lytic enzyme to remove colonizing GBS. Bacteriophage muralytic enzymes, termed lysins, are highly evolved molecules designed to degrade the cell wall of host bacteria to release phage particles from the bacterial cytoplasm. Several different lysins have been developed to specifically kill bacterial pathogens both on mucosal surfaces and in blood and represent a novel approach to control infection. A lysin cloned from a phage infecting GBS was found to contain two putative catalytic domains and one putative binding domain, which is similar to the domain organization of some staphylococcal phage lysins. The lysin (named PlyGBS) was recombinantly expressed in Escherichia coli, and purified PlyGBS efficiently killed all tested GBS serotypes in vitro. In a mouse model, a single dose of PlyGBS significantly reduced bacterial colonization in both the vagina and oropharynx. As an alternative strategy for intrapartum antibiotic prophylaxis, this approach may be used to reduce vaginal GBS colonization in pregnant women before delivery or to decontaminate newborns, thus reducing the incidence of GBS-associated neonatal meningitis and sepsis.
Enterococcus faecalis and Enterococcus faecium infections are increasingly difficult to treat due to high levels of resistance to antibiotics. PlyV12, a bacteriophage lytic enzyme, was isolated and shown to effectively kill both E. faecalis and E. faecium (including vancomycin-resistant strains), as well as other human pathogens. We propose its development and use as an alternative therapeutic tool.
Streptococcal surface enolase (SEN) is a major plasminogen-binding protein of group A streptococci. Our earlier biochemical studies have suggested that the region responsible for this property is likely located at the C-terminal end of the SEN molecule. In the present study, the gene encoding SEN was cloned from group A streptococci M6 isolate D471. A series of mutations in the sen gene corresponding to the C-terminal region (428KSFYNLKK435) of the SEN molecule were created by either deleting one or more terminal lysine residues or replacing them with leucine. All purified recombinant SEN proteins with altered C-terminal ends were found to be enzymatically active and were analyzed for their Glu- and Lys-plasminogen-binding activities. Wild-type SEN bound to Lys-plasminogen with almost three times more affinity than to Glu-plasminogen. However, the recombinant mutant SEN proteins with a deletion of Lys434-435 or with K435L and K434-435L replacements showed a significant decrease in Glu- and Lys-plasminogen-binding activities. Accordingly, a streptococcal mutant expressing SEN-K434-435L showed a significant decrease in Glu- and Lys-plasminogen-binding activities. Biochemical and functional analyses of the isogenic mutant strain revealed a significant decrease in its abilities to cleave a chromogenic tripeptide substrate, acquire plasminogen from human plasma, and penetrate the extracellular matrix. Together, these data indicate that the last two C-terminal lysine residues of surface-exposed SEN contribute significantly to the plasminogen-binding activity of intact group A streptococci and hence to their ability to exploit host properties to their own advantage in tissue invasion.