Myosin Cross-reactive Antigen Research Articles

Deciphering the mechanisms of Limosilactobacillus fermentum L1 involved in conjugated linoleic acid regulated by luxS/AI-2 quorum sensing

Conjugated linoleic acid (CLA) is an unsaturated fatty acid with many active functions. Here, we demonstrate quorum sensing (QS) to study mechanisms involved in CLA production of L. fermentum L1. Addition of linoleic acid (LA) with increased concentration results in promising CLA production of 2.22–10.36 mg/mL, cell density and autoinducer-2 (AI-2) involved in QS of L1. Further, proteomics analysis quantified up-expression of 185 differentially expressed proteins (DEPs) upon addition substrate of 8% LA, including myosin cross-reactive antigen protein, enolase (eno), and S-ribosylhomocysteine lyase (luxS), while 174 DEPs were down-regulated. Furthermore, S-Adenosyl-l-methionine metabolic pathway involved in luxS/AI-2 QS system was up-regulated. Additionally, protein-protein interaction network leverage key proteins, luxS and eno responsible for CLA production. Finally, furanone inhibitor block luxS/AI-2 QS system to allow AI-2 content, CLA, and cell density of L1 to decrease level. Western blot analysis confirmed, furanone successfully inhibited expression of luxS and eno. Whereas, luxS/AI-2 QS system was activated with LA results in increase expression of eno and production of CLA. Collectively, this study provides a new mechanism of CLA production by lactic acid bacteria.

Genetic determinates for conjugated linolenic acid production in Lactobacillus plantarum ZS2058.

To investigate the genetic determinates for conjugated linolenic acid (CLNA) production in Lactobacillus plantarum ZS2058, a high CLNA producer. After culturing with α-linolenic acid (ALA) in the medium, the fatty acid compositions of supernatant fluid and cell pellets were analysed via GC-MS. cis9,trans11,cis15-CLNA was identified to be the predominant isomer. And during CLNA production, 10-hydroxy-cis12-cis15-octadecenoic acid (10-HOEA) and 10-oxo-cis12-cis15-octadecenoic acid (10-OXOA) were accumulated. The E. coli recombinants harbouring genes encoding myosin-cross-reactive antigen (MCRA), short-chain dehydrogenase/oxidoreductase (DH) and acetoacetate decarboxylase (DC), respectively, were analysed for their roles in CLNA production. The results indicated that MCRA converted ALA to 10-HOEA, following converted to 10-OXOA by DH. While with the combination of three recombinants, ALA could be transformed into CLNA plus 10-HOEA and 10-OXOA. When the three genes were deleted, none of the L. plantarum ZS2058 knockout mutants could produce any CLNA, after complementation, and all the complementary mutants recovered the CLNA-production ability at similar levels as the wild strain. Lactobacillus plantarum ZS2058 produced CLNA from ALA with 10-HOEA and 10-OXOA as intermediates. The triple-component isomerase of MCRA, DH and DC was the unique genetic determinant for CLNA generation. The current results firstly provided conclusive evidence that the triple-component isomerase complex was shared by both CLA and CLNA production in lactobacilli.

Prevention of enteric bacterial infections and modulation of gut microbiota with conjugated linoleic acids producing Lactobacillus in mice

ABSTRACT Probiotics are recognized for outcompeting pathogenic bacteria by competitive receptor-mediated colonization and secretion of functional metabolites which are antimicrobial against certain microbes as well as improving host’s gut health and immunity. Recently, we have constructed a bioactive Lactobacillus casei (LC) strain, LC+mcra , by inserting mcra (myosin cross-reactive antigen) gene, which stimulates the conversion of conjugated linoleic acids. In this study, we evaluated the modulation of gut microbiome and protective roles of LC+mcra against pathogenic Salmonella enterica serovar Typhimurium (ST) and enterohemorrhagic E. coli (EHEC) infections in BALB/cJ mice. We observed that LC+mcra colonized efficiently in mice gut intestine and competitively reduced the infection with ST and EHEC in various locations of small and large intestine, specifically cecum, jejunum, and ileum (p < 0.05). Positive modulation of the cecal microbiota, for example, higher relative abundances of Firmicutes, lower relative abundances of Proteobacteria, and increased bacterial species diversity/richness, was detected in ST-challenged mice pretreated with LC+mcra based on 16S metagenomic sequencing. Cytokine gene expression analysis indicated that mice pretreated with LC+mcra associated with attenuated bacterial pathogen-induced gut inflammation. Furthermore, mice fed daily with LC+mcra for one week could protect themselves from the impairments caused by enteric infections with ST or EHEC. These impairments include weight loss, negative hematological changes, intestinal histological alterations, and potential death. This in vivo study suggests that daily consumption of novel conjugated linoleic acids over-producing probiotic effectively improves intestinal microbiota composition and prevents/combats foodborne enteric bacterial infections with pathogenic Salmonella and diarrheagenic E. coli.

Synbiotic-like effect of linoleic acid overproducing Lactobacillus casei with berry phenolic extracts against pathogenesis of enterohemorrhagic Escherichia coli

BackgroundMajority of enteric infections are foodborne and antimicrobials including antibiotics have been used for their control and treatment. However, probiotics or prebiotics or their combination offer a potential alternative intervention strategy for improving the host health and preventing foodborne pathogen colonization/infections in reservoir. Further, bioengineered probiotics expressing bioactive products to achieve specific function is highly desirable. Recently, we over-expressed mcra (myosin cross-reactive antigen) gene in Lactobacillus casei (Lc) and developed a bioengineered probiotics Lc + CLA which produce higher amounts of metabolites including conjugated linoleic acid (CLA). Furthermore, we also reported that prebiotic like components such as berry pomace (byproduct) phenolic extracts (BPEs) can enhance the growth of probiotics and improved the beneficial effects of probiotics. In this study, we evaluated the antimicrobial effect of modified Lc + CLA in combination of BPEs on growth, survival and pathogenesis of enterohemorrhagic Escherichia coli (EHEC).ResultsIn mixed culture condition, the growth of EHEC was significantly reduced in the presence Lc + CLA and/or BPEs. Cell-free cultural supernatant (CFCS) collected from Lc or Lc + CLA strain also inhibited the growth and survival of EHEC and the inhibitory effects of CFCSs against EHEC were enhanced in the presence of BPEs in concentration dependent manner. Interaction between EHEC and intestinal epithelial INT-407 cells were also altered significantly in the presence of either Lc or Lc + CLA strain or their CFCSs with or without BPEs. The expression of multiple virulence genes and physicochemical properties of EHEC were also altered when the bacterial cells were pretreated with CFCSs and/or BPEs.ConclusionsThese results showed that diet containing bioactive Lc + CLA and natural prebiotic like component such as BPEs might be an effective way to prevent foodborne infections with EHEC.

Role of 10-hydroxy-cis-12-octadecenic acid in transforming linoleic acid into conjugated linoleic acid by bifidobacteria.

10-hydroxy-cis-12 octadecenoic acid (10-HOE) is a type of octadecenoic acid with a hydroxyl on the C10 carbon. It is generated from linoleic acid (LA) catalyzed by linoleate hydratase in lactobacilli, which was initially named as myosin-cross-reactive antigen (MCRA). In lactobacilli, 10-HOE is the first intermediate in the production of conjugated LA (CLA). Although MCRA from bifidobacteria can generate 10-HOE, the precise role of 10-HOE in CLA production in bifidobacteria remains unknown. In the current work, 10-HOE and LA were added to the medium as the substrate both separately and synchronously toanalyze their influence on CLA production. Using 10-HOE as the substrate, bifidobacteria were able to generate CLA by first converting it to LA, followed by CLA accumulation. Recombinant MCRA catalyzed the conversion of 10-HOE to LA, indicating that bifidobacterial MCRA can account for the reversible conversion between LA and 10-HOE. This is the first report to demonstrate the precise role of 10-HOE in the process of CLA production among bifidobacteria.

Linoleic Acids Overproducing Lactobacillus casei Limits Growth, Survival, and Virulence of Salmonella Typhimurium and Enterohaemorrhagic Escherichia coli.

Probiotics, particularly lactic acid bacteria, are biologic agents which limit the growth, virulence, and survival/colonization of various enteric bacterial pathogens and serve as potential alternatives to antibiotics. Mechanisms that contribute to this antimicrobial effect include producing bioactive metabolites/acids, increasing nutrient and receptor-mediated competition, and modulating gut microbiome ecology. However, these functions of common probiotic strains are limited due to the finite quantity of metabolites they produce and their total number in the gut ecosystem. Conjugated linoleic acids (CLAs), critical metabolites of Lactobacillus, have multiple beneficial effects on human health including anti-carcinogenesis, anti-inflammation, anti-oxidation, and anti-pathogenicity. In this study, we aim to overexpress the myosin cross-reactive antigen gene (mcra) in Lactobacillus casei (LC) to enhance the production of CLA and investigate its effectiveness against enteric bacterial pathogens, specifically Salmonella enterica serovar Typhimurium (ST) and enterohaemorrhagic Escherichia coli (EHEC). By inserting mcra in L. casei, we generated LC-CLA and found the total linoleic acid production by an individual bacterial cell was raised by 21-fold. The adherence ability of LC-CLA on human epithelial cells increased significantly and LC-CLA competitively excluded both ST and EHEC in a mixed-culture condition. Furthermore, LC-CLA significantly altered the physicochemical properties, biofilm formation abilities, interactions with host cells of both ST and EHEC, and triggered anti-inflammatory activities of host cells. These findings offer insights on applying a genetically engineered probiotic to control gut intestinal infections caused by ST and EHEC and prevent foodborne enteric illness in human.

Characterization of the triple-component linoleic acid isomerase in Lactobacillus plantarum ZS2058 by genetic manipulation.

To assess the mechanism for conjugated linoleic acid (CLA) production in Lactobacillus plantarum ZS2058. CLA has attracted great interests for decades due to its health-associated benefits including anticancer, anti-atherogenic, anti-obesity and modulation of the immune system. A number of microbial CLA producers were widely reported including lactic acid bacteria. Lactobacillus plantarum ZS2058, an isolate from Chinese traditional fermented food, could convert LA to CLA with various intermediates. To characterize the genetic determinants for generating CLA, a cre-lox-based system was utilized to delete the genes encoding myosin cross-reactive antigen (MCRA), short-chain dehydrogenase/oxidoreductase (DH) and acetoacetate decarboxylase (DC) in Lact. plantarum ZS2058, respectively. Neither intermediate was detected in the corresponding gene deletion mutant. Meanwhile all those mutants could recover the ability to convert linoleic acid to CLA when the corresponding gene was completed. The results indicated that CLA production was a multiple-step reaction catalysed by triple-component linoleate isomerase system encoded by mcra, dh and dc. Multicomponent linoleic acid isomerase provided important results for illustration unique mechanism for CLA production in Lact. plantarum ZS2058. Lactobacilli with CLA production ability offer novel opportunities for functional food development.

Conjugated Linoleic Acid Production by Bifidobacteria: Screening, Kinetic, and Composition.

Conjugated linoleic acids (CLA) are positional and geometric isomers of linoleic acid involved in a number of health aspects. In humans, CLA production is performed by gut microbiota, including some species of potential probiotic bifidobacteria. 128 strains of 31 Bifidobacterium species were screened with a spectrophotometric assay to identify novel CLA producers. Most species were nonproducers, while producers belonged to B. breve and B. pseudocatenulatum. GC-MS revealed that CLA producer strains yielded 9cis,11trans-CLA and 9trans,11trans-CLA, without any production of other isomers. Hydroxylated forms of LA were absent in producer strains, suggesting that the myosin-cross-reactive antigen (MCRA) protein that exerts hydratase activity is not involved in LA isomerization. Moreover, both CLA producer and nonproducer species bear a MCRA homologue. The strain B. breve WC 0421 was the best CLA producer, converting LA into 68.8% 9cis,11trans-CLA and 25.1% 9trans,11trans-CLA. Production occurred mostly during the lag and the exponential phase. For the first time, production and incorporation of CLA in biomass were assessed. B. breve WC 0421 stored CLA in the form of free fatty acids, without changing the composition of the esterified fatty acids, which mainly occurred in the plasmatic membrane.

Identification and characterization of an oleate hydratase-encoding gene from Bifidobacterium breve

Bifidobacteria are common commensals of the mammalian gastrointestinal tract. Previous studies have suggested that a bifidobacterial myosin cross reactive antigen (MCRA) protein plays a role in bacterial stress tolerance, while this protein has also been linked to the biosynthesis of conjugated linoleic acid (CLA) in bifidobacteria. In order to increase our understanding on the role of MCRA in bifidobacteria we created and analyzed an insertion mutant of the MCRA-encoding gene of B. breve NCFB 2258. Our results demonstrate that the MCRA protein of B. breve NCFB 2258 does not appear to play a role in CLA production, yet is an oleate hydratase, which contributes to bifidobacterial solvent stress protection.

Crystal structure analysis of a fatty acid double-bond hydratase fromLactobacillus acidophilus

Bacteria have evolved mechanisms for the hydrogenation of unsaturated fatty acids. Hydroxy fatty acid formation may be the first step in such a process; however, knowledge of the structural and mechanistic aspects of this reaction is scarce. Recently, myosin cross-reactive antigen was shown to be a bacterial FAD-containing hydratase which acts on the 9Z and 12Z double bonds of C16 and C18 non-esterified fatty acids, with the formation of 10-hydroxy and 10,13-dihydroxy fatty acids. These fatty acid hydratases form a large protein family which is conserved across Gram-positive and Gram-negative bacteria with no sequence similarity to any known protein apart from the FAD-binding motif. In order to shed light on the substrate recognition and the mechanism of the hydratase reaction, the crystal structure of the hydratase from Lactobacillus acidophilus (LAH) was determined by single-wavelength anomalous dispersion. Crystal structures of apo LAH and of LAH with bound linoleic acid were refined at resolutions of 2.3 and 1.8 Å, respectively. LAH is a homodimer; each protomer consists of four intricately connected domains. Three of them form the FAD-binding and substrate-binding sites and reveal structural similarity to three domains of several flavin-dependent enzymes, including amine oxidoreductases. The additional fourth domain of LAH is located at the C-terminus and consists of three α-helices. It covers the entrance to the hydrophobic substrate channel leading from the protein surface to the active site. In the presence of linoleic acid, the fourth domain of one protomer undergoes conformational changes and opens the entrance to the substrate-binding channel of the other protomer of the LAH homodimer. The linoleic acid molecule is bound at the entrance to the substrate channel, suggesting movement of the lid domain triggered by substrate recognition.

Myosin-cross-reactive antigens from four different lactic acid bacteria are fatty acid hydratases

The 67kDa myosin-cross-reactive antigen (MCRA) is a member of the MCRA family of proteins present in a wide range of bacteria and was predicted to have fatty acid isomerase function. We have now characterised the catalytic activity of MCRAs from four LAB stains, including Lactobacillus rhamnosus LGG, L. plantarum ST-III, L. acidophilus NCFM and Bifidobacterium animalis subsp. lactis BB-12. MCRA genes from these strains were cloned and expressed in Escherichia coli, and the recombinant protein function was analysed with lipid profiles by GC-MS. The four MCRAs catalysed the conversion of linoleic acid and oleic acid to their respective 10-hydroxy derivatives, which suggests that MCRA proteins catalyse the first step in conjugated linoleic acid production. This is the first report of MCRA from L. rhamnosus with such catalytic function.

Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB)

Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which “holdfasts” and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract.

Myosin-cross-reactive antigen (MCRA) protein from Bifidobacterium breve is a FAD-dependent fatty acid hydratase which has a function in stress protection

BackgroundThe aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates.ResultsMCRA catalysed the conversion of palmitoleic, oleic and linoleic acids to the corresponding 10-hydroxy fatty acids, but shorter chain fatty acids were not used as substrates, while the presence of trans-double bonds and double bonds beyond the position C12 abolished hydratase activity. The hydroxy fatty acids produced were not metabolised further. We also found that heterologous Lactococcus and Corynebacterium expressing MCRA accumulated increasing amounts of 10-HOA and 10-HOE in the culture medium. Furthermore, the heterologous cultures exhibited less sensitivity to heat and solvent stresses compared to corresponding controls.ConclusionsMCRA protein in B. breve can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, which may be catalysing the first step in conjugated linoleic acid (CLA) production, and this protein has an additional function in bacterial stress protection.

Characterization of aStaphylococcus aureusSurface Virulence Factor That Promotes Resistance to Oxidative Killing and Infectious Endocarditis

Staphylococcus aureus is a prominent human pathogen and a leading cause of community- and hospital-acquired bacterial infections worldwide. Herein, we describe the identification and characterization of the S. aureus 67.6-kDa hypothetical protein, named for the surface factor promoting resistance to oxidative killing (SOK) in this study. Sequence analysis showed that the SOK gene is conserved in all sequenced S. aureus strains and homologous to the myosin cross-reactive antigen of Streptococcus pyogenes. Immunoblotting and immunofluorescence analysis showed that SOK was copurified with membrane fractions and was exposed on the surface of S. aureus Newman and RN4220. Comparative analysis of wild-type S. aureus and an isogenic deletion strain indicated that SOK contributes to both resistance to killing by human neutrophils and to oxidative stress. In addition, the S. aureus sok deletion strain showed dramatically reduced aortic valve vegetation and bacterial cell number in a rabbit endocarditis model. These results, plus the suspected role of the streptococcal homologue in certain diseases such as acute rheumatic fever, suggest that SOK plays an important role in cardiovascular and other staphylococcal infections.

Conjugated linoleic acid‐producing enzymes: A bioinformatics study

AbstractLinoleic acid isomerases (LAI) are enzymes responsible for conversion of linoleic acid (LA) to conjugated linoleic acids (CLA) in different isomeric forms. CLAs are well known for numerous beneficial effects as functional foods. Despite identification of several LAI producing‐bacteria and release of their LAI nucleotide sequences to Bio‐Banks, no related bioinformatics study on these important enzymes is addressed yet. To gain insights into the structural/functional and phylogentic relations of LAIs as well as recombinant production of the desired enzyme, we employed several bioinformatical tools to analyze their primary structure and physicochemical properties. The results indicated that LAIs produced in different bacterial strains might be divided in two distinct families (Propionibacterium acnes and myosin cross‐reactive antigen (MCRA)‐like LAIs) with specific isomerase activities. In another part of the study, physicochemical properties, solubility upon over expression in E. coli, disulfide bond formation, and potential glycosylation sites in LAI sequences were predicted using bioinformatics tools and the most appropriate strategy for recombinant production of each LAI was determined. Our predicted data may be useful for further experimental studies toward production of the desired LAI.

Functional and phenotypic characterization of a protein from Lactobacillus acidophilus involved in cell morphology, stress tolerance and adherence to intestinal cells

Structural components of the cell surface have an impact on some of the beneficial attributes of probiotic bacteria. In silico analysis of the L. acidophilus NCFM genome sequence revealed the presence of a putative cell surface protein that was predicted to be a myosin cross-reactive antigen (MCRA). As MCRAs are conserved among many probiotic bacteria, we used the upp-based counterselective gene replacement system, designed recently for use in L. acidophilus, to determine the functional role of this gene (LBA649) in L. acidophilus NCFM. Phenotypic assays were undertaken with the parent strain (NCK1909) and deletion mutant (NCK2015) to assign a function for this gene. The growth of NCK2015 (ΔLBA649) was reduced in the presence of lactate, acetate, porcine bile and salt. Adhesion of NCK2015 to Caco-2 cells was substantially reduced for both stationary-phase (∼45 % reduction) and exponential-phase cells (∼50 % reduction). Analysis of NCK2015 by scanning electron microscopy revealed a longer cell morphology after growth in MRS broth compared to NCK1909. These results indicate a role for LBA649 in stress tolerance, cell wall division and adherence to Caco-2 cells.

Myosin Cross-reactive Antigen of Streptococcus pyogenes M49 Encodes a Fatty Acid Double Bond Hydratase That Plays a Role in Oleic Acid Detoxification and Bacterial Virulence

The myosin cross-reactive antigen (MCRA) protein family is highly conserved among different bacterial species ranging from Gram-positive to Gram-negative bacteria. Besides their ubiquitous occurrence, knowledge about the biochemical and physiological function of MCRA proteins is scarce. Here, we show that MCRA protein from Streptococcus pyogenes M49 is a FAD enzyme, which acts as hydratase on (9Z)- and (12Z)-double bonds of C-16, C-18 non-esterified fatty acids. Products are 10-hydroxy and 10,13-dihydroxy fatty acids. Kinetic analysis suggests that FAD rather stabilizes the active conformation of the enzyme and is not directly involved in catalysis. Analysis of S. pyogenes M49 grown in the presence of either oleic or linoleic acid showed that 10-hydroxy and 10,13-dihydroxy derivatives were the only products. No further metabolism of these hydroxy fatty acids was detected. Deletion of the hydratase gene caused a 2-fold decrease in minimum inhibitory concentration against oleic acid but increased survival of the mutant strain in whole blood. Adherence and internalization properties to human keratinocytes were reduced in comparison with the wild type. Based on these results, we conclude that the previously identified MCRA protein can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, that plays a role in virulence of at least S. pyogenes M49.

Cloning and sequence analysis of a gene encoding a 67-kilodalton myosin-cross-reactive antigen of Streptococcus pyogenes reveals its similarity with class II major histocompatibility antigens.

The group A streptococcal sequela acute rheumatic fever (ARF) has been associated with immunological cross-reactivity between streptococcal and heart proteins. To identify Streptococcus pyogenes genes that encode a myosin cross-reactive antigen(s) recognized by ARF sera, a genomic library from an emm deletion strain (T28/51/4) was screened with a single ARF serum. A positively identified lambda EMBL3 clone (T.2.18) produced a protein which reacted with myosin-specific antibodies affinity purified from individual ARF sera. The recombinant protein was initially estimated to be 60 kDa in size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, upon sequence analysis it had a molecular mass equivalent to 67 kDa. Sera from patients with streptococcal infections, acute glomerulonephritis, and ARF were reactive with the recombinant 67-kDa protein. However, individual sera from healthy persons were negative or demonstrated low levels of reactivity with the 67-kDa antigen. The gene encoding the 67-kDa myosin-cross-reactive antigen was subcloned, and its nucleotide sequence was determined by using a combined strategy of DNA sequencing of the cloned gene and N-terminal amino acid sequencing of the protein expressed in Escherichia coli. The amino-terminal sequence deduced from the nucleotide sequence of an open reading frame was identical to that determined from the 67-kDa protein expressed in E. coli. The gene encoded 590 amino acids with a calculated molecular weight of 67,381. No cleavable signal peptide was detected with the 67-kDa protein expressed in E. coli. The deduced amino acid sequence of the 67-kDa protein did not exhibit significant similarity to any known streptococcal proteins. However, it was found to be 19% identical and 62% similar over 151 amino acid residues to the beta chain of mouse major histocompatibility complex class II antigen (I-Au). Similar degrees of homology to the beta chains of other murine and human class II haplotypes were found. Mouse anti-IA sera reacted with the recombinant 67-kDa protein about five times more strongly than normal mouse sera in the enzyme-linked immunosorbent assay. Southern hybridization experiments using a probe for the gene encoding the 67-kDa protein showed that the gene was present and conserved among pathogenic groups A, C, and G of streptococci. These data suggest that the streptococcal protein, which is distinct from the M protein, may have structural features in common with the beta chain of the class II antigens, as well as myosin, and may play an important role in the pathogenesis of streptococcal infections.