Streptoccal inhibitor of complement-mediated lysis, protein SIC

Details Streptoccal inhibitor of complement-mediated lysis, protein SIC Streptoccal inhibitor of complement-mediated lysis, protein SIC

1998-09-08

2003-06-10

Allen, Marianne P. (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Reexamination Certificate

active

06576741

ABSTRACT:

The present invention relates to a new protein, protein SIC, that can be derived from
Streptococcus pyogenes
strains of serotypes M1 and M57. Methods for analysis and purification and pharmaceutical preparations including vaccine compositions related to the protein as well as specific antibodies are also claimed.
Streptococcus pyogenes
is an important human pathogen causing a number of acute suppurative infections such as erysipelas, necrotizing fasciitis, and pharyngitis. These Gram-positive bacteria also cause a serious toxic shock-like syndrome, whereas glomreulonephritis and rheumatic fever are serious poststreptococcal sequelae.
To elude the host defence and establish an infection,
S. pyogenes
has developed multiple molecular mechanisms. Some of these are dependent on genes located in a chromosomal region designated the mga locus according to a recent agreement, which is under the control of the positive regulator gene mga, previously called mry (Caparon and Scott, 1987: Proc. Natl. Acad. Sci. U. S. A. 84, 8677-8681) or virR (Simpson et al., 1990: J. Bacteriol. 172, 696-700).
Since the late 1980's unusually severe
S. pyogenes
infections have been reported world-wide. These hyperacute and often lethal infections have frequently been associated with the M1 serotype (Musser et al., 1993: J. Infect. Dis. 167, 337-346). This serotype is also connected with glomerulonephritis and rheumatic fever.
Based on structural variations in the antiphagocytic M protein (Fischetti, 1989: Clin. Microbiol. Rev. 2, 285-314; and Kehoe, 1994: New Compr. Biochem. 27, 217-261)
S. pyogenes
can be divided into more than 80 different serotypes. Most of these serotypes are rather harmless. However, during the last years, a lot of lethal streptococcal infections have been caused by strain belonging to the M1 serotype. Presently, because of the large amount of serotypes and the great similarity between these types, it is time-consuming and labourious to determine the serotype of a sample of
Streptococcus pyogenes
. Consequently, there is a need for simpler methods for determining whether a particular sample of
Streptococcus pyogenes
is virulent. There is also a need for better vaccines against virulent
Streptococcus pyogenes
strains.
SUMMARY OF THE INVENTION
A new protein from
Streptococcus pyogenes
, serotype M1, fulfilling the above mentioned needs has now been discovered and characterized. The protein and its corresponding gene has been obtained from strain AP1, which is of the M1 serotype.
In the mga locus of AP1, the regulatory gene mga is followed by emm1, the gene encoding the M1 protein. Immediately down-stream of emm1 is sph ({dot over (A)}kesson et al., 1994: Biochem. J. 300, 877-886), the gene encoding an IgGFc-binding M protein-related molecule called protein H ({dot over (A)}kesson et al., 1990: Mol. Immunol. 27, 523-531; Gomi et al., 1990: J. Immunol. 144, 4046-4052; WO 91/19740; EP 0 371 199). Located adjacent to sph is a previously uncharacterized gene. This gene constitutes one of the objects of the present invention.
The protein shows some variability and M1 serotype strains of
Streptococcus pyogenes
producing variants of protein SIC have also been isolated. When comparing the amino acid sequences of three different protein SIC variants, some conserved regions were found.
DETAILED DESCRIPTION OF THE INVENTION
The protein encoded by the uncharacterized gene as well as variants, subfragments and multiples of the protein having essentially the same antigenic and/or binding characteristics also constitutes an object of the present invention. The data obtained implicate that this extracellular protein plays a role in
S. pyogenes
pathogenicity and virulence through previously unknown molecular mechanisms. The new protein is referred to as protein SIC, Streptococcal Inhibitor of Complement-mediated lysis.
“SIC proteins” as utilized herein refers to streptococcal proteins which inhibit hemolysis by interacting with the plasma proteins clusterin and members of the cystatin protein superfamily, such as HRG, as demonstrated by affinity chromatography on a protein SIC Sepharose column or by indirect ELISA. SIC proteins may be distinguished from other proteins based upon criteria such as specific binding to the above mentioned plasma proteins and sequence homology. For example, SIC proteins of the present invention should comprise at least one of the following partial amino acid sequences:
a) glu thr tyr thr ser arg asn phe (SEQ ID NO:7);
b) asp trp ser gly asp asp trp pro glu asp asp trp (SEQ ID NO:8);
c) arg ser gly val gly leu ser gln tyr gly trp ser (SEQ ID NO:9);
d) trp ser ser asp lys lys asp glu thr glu asp lys thr (SEQ ID NO:10);
e) gly thr gly tyr glu lys arg asp asp trp gly gly pro gly (SEQ ID NO:11);
f) lys arg asp asp trp arg gly pro gly his ile pro lys pro (SEQ ID NO:12);
preferably the amino acid sequence of the SIC proteins should be at least 70% homologous, more preferably at least 85% homologous, still more preferably at least 90% homolgous and most preferably at least 95% homologous to anyone of the amino acid sequences disclosed in SEQ. ID. NOS 2, 3 and 4.
As already mentioned and suggested above, different variants of protein SIC have been isolated. Most isolates produced a variant showing close resemblance to protein SIC isolated from strain AP1, whose amino acid sequence is disclosed in SEQ. ID. NO. 2. However, two more variants having the amino acid sequences according to SEQ. ID. NO. 3 and SEQ. ID. NO. 4, respectively, have also been discovered. After aligning the sequences of the different variants the conserved partial sequences a)-f) above were found. Regions a)-e) are present in all known protein SIC variants. Protein SIC from one of the isolates only comprised a part of the f) region. Consequently the above regions are considered to have important functions in the protein.
By “subfragment” is meant a part-fragment of the given protein having essentially the same antigenic and/or binding characteristics. By “variants” is meant proteins or peptides in which the original amino acid sequence has been modified or changed by insertion, addition, substitution, inversion or exclusion of one or more amino acids. By “multiples” is meant those proteins containing multiples of the whole original protein or those protein containing multiples of subfragments and/or variants thereof.
The present invention also relates to nucleic acid sequences encoding protein SIC. As utilized within the context of the present invention, nucleic acid sequences which encode protein SIC are deemed to be substantially similar to those disclosed herein if: (a) the nuclcic acid sequence is derived from the coding region of a native protein SIC gene (including, for example, variations of the sequences disclosed herein); (b) the nucleic acid sequcnce is capable of hybridization to nucleic acid sequences of the present invention under conditions of either moderate or high stringency (hybridization in 5×SSPE containing 0.1% SDS and 0.1 mg/ml ssDNA, at 50-65° C. dependent on the probe length, or 10-20° C. below the T
m
of the probe; washing in 1×SSPE, 0.1% SDS at 15-20° C. below the T
m
of the probe for moderate stringency, and in 0.1×SSPE, 0.1% at 10° C. below the T
m
of the probe for high stringency conditions) (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY, 1989); or (c) nucleic acid sequences are degenerate as a result of the genetic code to the nucleic acid sequences defined in (a) or (b). Furthermore, although nucleic acid molecules are primarily referred to herein, as should be evident to one of skill in the art given the disclosure provided herein, a wide variety of related nucleic acid molecules may also be utilized in various embodiments described herein, including for example, RNA, nucleic acid analogues, as well as chimeric nucleic acid molecules which may be composed of more than one type of nucleic acid.
Within another aspect of the present invention, probes and primers are provided for d

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