Streptococcal M protein extracted by nonionic detergent. I. Properties of the antiphagocytic and type-specific molecules.

Group A streptococcal M protein was extracted with nonionic detergent and subjected to a number of physical, chemical, and immunological tests. M protein thus extracted was composed of multiple protein bands, ranging from 35,000 down to 6,000 daltons, all having type-specific precipitating activity. The anti-phagocytic proteins, however, were limited to three molecular species having mol wt of 28,000, 31,000, and 35,000 daltons, and could be separated from those proteins that had only type specificity. Physical studies indicated that these proteins existed as individual asymmetrical molecules which were not aggregated. By radiolabeling M protein on living streptococci, it was determined that these protein bands were found on the streptococcal cell wall in this multiple form. Also, by pulse chase experiments supported by chemical and immunological data, evidence was obtained strongly suggesting that the smaller, type-specific molecules are used to assemble the larger, antiphagocytic proteins.

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Size variation of the M protein in group A streptococci.

Journal of Experimental Medicine ◽

10.1084/jem.161.6.1384 ◽

1985 ◽

Vol 161 (6) ◽

pp. 1384-1401 ◽

Cited By ~ 64

Author(s):

V A Fischetti ◽

K F Jones ◽

J R Scott

Keyword(s):

Cell Wall ◽

Antigenic Variation ◽

Size Variation ◽

Molecular Size ◽

Immunoblot Analysis ◽

M Protein ◽

Size Change ◽

Streptococcal Cell Wall ◽

Protein Molecules ◽

Group A

In addition to the type-specific antigenic variation that is a well-known characteristic for the group A streptococcal M protein, we have now found that the M molecules vary with respect to their molecular size, both between M types and within an M type. By the use of an M6 monoclonal antibody, which crossreacts with 20 different M protein types, and antibodies to the N-acetyl glucosamine determinant of the cell wall, we have been able to identify the M protein molecules released from the streptococcal cell wall with muralytic enzymes, particularly group C phage-associated lysin. Immunoblot analysis of the cell extract identified M protein molecules bound to various cell wall fragments, suggesting a peptidoglycan linkage for the M molecule. M protein extracted from 20 different streptococcal serotypes revealed size variations from 41,000 to 80,000 in molecular weight. This extreme variation is unusual for related proteins. Similar size variations in the M molecule were also found in random clinical isolates of type 6 streptococci. No size change was seen in M6 protein isolated from: (a) strains within a limited epidemic, (b) a strain passaged in mice 192 times, and (c) a strain passaged in the laboratory for 156 generations, suggesting that the observed variation is not a rapid process. The results indicate that, within the broad limits observed in this study, the size of the M protein may not be critical to the antiphagocytic activity of the molecule.

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Identification of an endogenous membrane anchor-cleaving enzyme for group A streptococcal M protein. Its implication for the attachment of surface proteins in gram-positive bacteria.

Journal of Experimental Medicine ◽

10.1084/jem.170.6.2119 ◽

1989 ◽

Vol 170 (6) ◽

pp. 2119-2133 ◽

Cited By ~ 36

Author(s):

V Pancholi ◽

V A Fischetti

Keyword(s):

Cell Wall ◽

Sequence Similarity ◽

Surface Proteins ◽

M Protein ◽

Membrane Anchor ◽

Gram Positive ◽

Streptococcal Cell Wall ◽

Gram Positive Bacteria ◽

Streptococcal M Protein ◽

Triton X

How streptococcal M protein or other surface proteins of gram-positive bacteria are anchored to the cell is poorly understood. Previously, we reported that M protein released after cell wall removal with a muralytic enzyme lacked the COOH terminal hydrophobic amino acids and charged tail predicted from DNA sequence. An endogenous membrane anchor-cleaving enzyme has now been identified with the ability to release M protein from isolated streptococcal protoplasts. At pH 5.5 in the presence of 30% raffinose, the streptococcal cell wall may be removed with a muralytic enzyme without releasing M protein from the resulting protoplasts indicating that the M molecule is attached through the bacterial cytoplasmic membrane. Release of M molecules occurs when the M protein-charged protoplasts are placed in raffinose buffer at pH 7.4. Although Zn2+, Cd2+, Ca2+, PHMB, and pHMPS inhibit the activity of the releasing enzyme, the blocking activity of Zn2+, Cd2+, and Ca2+ are reversible while PHMB and pHMPS are irreversible. PHMB-treated protoplasts are unable to release M protein at pH 7.4. However, M protein is liberated from these protoplasts when mixed with those prepared from M- streptococci serving as an enzyme source. The supernatant from M- protoplasts is unable to release M protein from PHMB-inactivated M+ protoplasts, confirming that the anchor-cleaving enzyme is membrane bound. Thus, the M protein releasing activity appears to be the result of a thiol-dependent anchor-cleaving enzyme. Streptococcal membranes treated with sodium carbonate and Triton X-114 still retain the M protein verifying that it is an integral membrane molecule. Evidence also is presented indicating significant sequence similarity between M protein and certain GPI-anchored proteins in the region responsible for protein anchoring.

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THE LYSIS OF GROUP A HEMOLYTIC STREPTOCOCCI BY EXTRACELLULAR ENZYMES OF STREPTOMYCES ALBUS

Journal of Experimental Medicine ◽

10.1084/jem.96.6.569 ◽

1952 ◽

Vol 96 (6) ◽

pp. 569-580 ◽

Cited By ~ 63

Author(s):

Maclyn McCarty

Keyword(s):

Cell Wall ◽

Extracellular Enzymes ◽

Group A Streptococci ◽

Carbohydrate Component ◽

Streptococcal Cell Wall ◽

Intact Cells ◽

Enzymatic Lysis ◽

Streptomyces Albus ◽

Group A ◽

Isolated Cell

Cell wall preparations of uniform chemical constitution have been obtained from several strains of group A streptococci. The isolated cell walls are dissolved by the same fractions of the Streptomyces albus enzymes that are effective in the lysis of intact cells, and it is likely that enzymatic lysis of group A streptococci is effected by an attack on the cell wall. The streptococcal cell wall, as prepared in this study, consists of approximately two-thirds carbohydrate and one-third protein. Small amounts of other components may be present. The carbohydrate component, which is composed primarily of N-acetyl-glucosamine and rhamnose, is the group-specific C carbohydrate. The evidence indicates that one of the streptomyces enzymes is directed toward the carbohydrate component of the cell wall.

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Group A streptococcal M protein activates the NLRP3 inflammasome

Nature Microbiology ◽

10.1038/s41564-017-0005-6 ◽

2017 ◽

Vol 2 (10) ◽

pp. 1425-1434 ◽

Cited By ~ 27

Author(s):

J. Andrés Valderrama ◽

Angelica M. Riestra ◽

Nina J. Gao ◽

Christopher N. LaRock ◽

Naveen Gupta ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

M Protein ◽

Streptococcal M Protein ◽

Group A

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Induction of human gamma interferon by structurally defined polypeptide fragments of group A streptococcal M protein.

Infection and Immunity ◽

10.1128/iai.43.1.122-126.1984 ◽

1984 ◽

Vol 43 (1) ◽

pp. 122-126 ◽

Cited By ~ 4

Author(s):

D A Weigent ◽

E H Beachey ◽

T Huff ◽

J W Peterson ◽

G J Stanton ◽

...

Keyword(s):

Gamma Interferon ◽

M Protein ◽

Streptococcal M Protein ◽

Group A

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Inhibition of the alternative C3 convertase and classical C5 convertase of complement by group A streptococcal M protein.

Infection and Immunity ◽

10.1128/iai.58.8.2535-2541.1990 ◽

1990 ◽

Vol 58 (8) ◽

pp. 2535-2541 ◽

Cited By ~ 15

Author(s):

K Hong ◽

T Kinoshita ◽

J Takeda ◽

H Kozono ◽

P Pramoonjago ◽

...

Keyword(s):

M Protein ◽

Streptococcal M Protein ◽

Group A

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Streptococcal cell walls and synovial cell activation. Stimulation of synovial fibroblast plasminogen activator activity by monocytes treated with group A streptococcal cell wall sonicates and muramyl dipeptide.

Journal of Experimental Medicine ◽

10.1084/jem.155.6.1702 ◽

1982 ◽

Vol 155 (6) ◽

pp. 1702-1718 ◽

Cited By ~ 32

Author(s):

J A Hamilton ◽

J B Zabriskie ◽

L B Lachman ◽

Y S Chen

Keyword(s):

Cell Wall ◽

Plasminogen Activator ◽

Synovial Fibroblast ◽

Cell Activation ◽

Interleukin 2 ◽

Muramyl Dipeptide ◽

Cellular Interactions ◽

Human Monocytes ◽

Streptococcal Cell Wall ◽

Group A

Group A streptococcal peptidoglycan has previously been shown to be arthritogenic in rats and has been implicated as a structure present in a class of possible etiologic agents for rheumatoid arthritis. The present study reports that conditioned medium from human monocytes, after interaction with cell wall sonicates of four group A streptococcal strains, stimulates the plasminogen activator (PA) activity of nonrheumatoid synovial fibroblasts. Low concentrations of N-acetylmuramyl-L-alanyl-D isoglutamine (muramyl dipeptide) can also generate this synovial activator (SA) activity from human monocytes. Preliminary biochemical data suggest that the SA activity is distinct from interferon-gamma, interleukin 1, and interleukin 2. These results indicate that agents that are arthritogenic in rats can modulate human synovial fibroblast functions via monocytes. The findings are proposed to have possible significance for an understanding of the cellular interactions involved in the formation and function of the rheumatoid pannus, because PA has been invoked as possibly being generally important for the processes of cell migration, tissue remodeling, and inflammation.

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Structure-based group A streptococcal vaccine design: Helical wheel homology predicts antibody cross-reactivity among streptococcal M protein–derived peptides

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011258 ◽

2020 ◽

Vol 295 (12) ◽

pp. 3826-3836 ◽

Cited By ~ 1

Author(s):

Michelle P. Aranha ◽

Thomas A. Penfound ◽

Jay A. Spencer ◽

Rupesh Agarwal ◽

Jerome Baudry ◽

...

Keyword(s):

Group A Streptococcus ◽

Coiled Coil ◽

Cross Reactivity ◽

M Protein ◽

High Sequence Identity ◽

Sequence Identity ◽

Streptococcal M Protein ◽

Helical Wheel ◽

Group A ◽

Immunological Assays

Group A streptococcus (Strep A) surface M protein, an α-helical coiled-coil dimer, is a vaccine target and a major determinant of streptococcal virulence. The sequence-variable N-terminal region of the M protein defines the M type and also contains epitopes that promote opsonophagocytic killing of streptococci. Recent reports have reported considerable cross-reactivity among different M types, suggesting the prospect of identifying cross-protective epitopes that would constitute a broadly protective multivalent vaccine against Strep A isolates. Here, we have used a combination of immunological assays, structural biology, and cheminformatics to construct a recombinant M protein–based vaccine that included six Strep A M peptides that were predicted to elicit antisera that would cross-react with an additional 15 nonvaccine M types of Strep A. Rabbit antisera against this recombinant vaccine cross-reacted with 10 of the 15 nonvaccine M peptides. Two of the five nonvaccine M peptides that did not cross-react shared high sequence identity (≥50%) with the vaccine peptides, implying that high sequence identity alone was insufficient for cross-reactivity among the M peptides. Additional structural analyses revealed that the sequence identity at corresponding polar helical-wheel heptad sites between vaccine and nonvaccine peptides accurately distinguishes cross-reactive from non–cross-reactive peptides. On the basis of these observations, we developed a scoring algorithm based on the sequence identity at polar heptad sites. When applied to all epidemiologically important M types, this algorithm should enable the selection of a minimal number of M peptide–based vaccine candidates that elicit broadly protective immunity against Strep A.

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