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Journal of Bacteriology, April 2007, p. 2933-2935, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01619-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

DRS Is Far Less Divergent than Streptococcal Inhibitor of Complement of Group A Streptococcus{triangledown}

Vivek Sagar,1 Rajesh Kumar,2 Nirmal K. Ganguly,3 Thangam Menon,4 and Anuradha Chakraborti1*

Departments of Experimental Medicine and Biotechnology,1 Community Medicine, PGIMER, Chandigarh,2 ICMR, New Delhi,3 Department of Microbiology, University of Madras, Chennai, India4

Received 18 October 2006/ Accepted 11 January 2007


   ABSTRACT
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When 100 group A streptococcus isolates were screened, drs, a variant of sic, was identified in emm12 and emm55 isolates. Molecular characterization showed that the drs gene sequence is highly conserved, unlike the sic gene sequence. However, the variation in gene size observed was due to the presence of extra internal repeat sequences.


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Group A beta-hemolytic streptococcus (GAS; Streptococcus pyogenes) causes various human diseases and is responsible for major health problems in developing countries like India. A wide variety of virulence factors are associated with GAS (5), and streptococcal inhibitor of complement (SIC) is one of them (1). Originally, SIC was characterized as an inhibitor of complement function that inhibits the cytolytic activity of the membrane attack complex. Subsequently, SIC was also shown to inhibit the antimicrobial activity of lysozyme, secretory leukocyte proteinase inhibitor, {alpha}- and ß-defensins, and cathelicidin LL-37, which are the components of the innate immune system (1, 3, 7, 9). The sic gene was initially found in serotypes M1 and M57 (1, 10); later, a variant of sic, distantly related sic (drs), was isolated from emm12 and emm55 strains (10). DRS (for "distantly related to SIC") is known to exhibit sequence homology with SIC in the signal sequence and in a proline-rich region (2) and also to share some biological functions with SIC (8); therefore, it is considered to be an equally important virulence factor. For DRS, only a few alleles have been described thus far (4). Recently, information regarding heterogeneous emm type prevalence in India has been reported (6, 12, 13), but there are no data on the virulence status of GAS from this region. Here we report the distribution of the drs gene among Indian isolates and describe the sequence heterogeneity.

This study included 100 GAS isolates; 94 of these isolates were from North India, and the remainder were from South India. The 94 strains from North India, collected from community, as well as from the hospital (Postgraduate Institute of Medical Education and Research) in Chandigarh, were categorized into 37 emm types (unpublished data). The six strains from South India, belonging to emm type 12, were obtained from Chennai (Dr. ALM PG Institute of Basic Medical Sciences, University of Madras). These six emm12 strains were collected over a 6-month period from different patients residing in different parts of Chennai and belonged to different biotypes. All these isolates were screened for the drs gene by PCR analysis using previously described primers (10). A PCR product corresponding to the full gene, amplified by specific primers (3), was purified with a QIAGEN kit (United States), and sequencing was performed using internal primers (4). Full-length drs55 was first cloned in the TOPO/pBAD vector by using an Invitrogen cloning kit (United States) and then was sequenced. Sequencing was carried out with an automated DNA sequencer (model 310; Applied Biosystems, United States). The sequences of the drs gene from Indian isolates were compared with previously deposited gene bank data by using the Clustal X program (11). Secretory protein was isolated by trichloroacetic acid precipitation, and DRS-specific protein was detected using a specific antibody as described by Binks and Sriprakash (2).

In the present study a ~760-bp drs-specific fragment was isolated (Fig. 1A) from all eight emm type 12 (drs12) isolates (two isolates from North India and six isolates from South India). The single emm55 isolate obtained in this study was also positive for the drs gene; however, the size of the drs amplicon (drs55) was found to be ~1.0 kb.


Figure 1
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  		FIG. 1. Characterization of DRS. (A) PCR analysis of drs. Lane M, 100-bp ladder (NEB); lane 1, M12 reference strain used as a positive control; lane 2, representative emm12 clinical isolate; lane 3, emm55 clinical isolate; lane (–), negative control with no template. (B) Comparison of the amino acid sequences of Indian DRS12 (I) and DRS55 (I) with Australian DRS12 (A) and DRS55 (A) by multiple-sequence alignment. Asterisks indicate a conserved sequence; underlining indicates an additional repeat sequence; and a sequence not available in the gene bank database is indicated by question marks. (C) Western blot analysis showing expression of DRS. Lane 1, M12 reference strain used as a positive control; lane 2, DRS negative control; lane 3, representative emm12 isolate; lane 4, emm55 isolate.

 
The full-length (942-bp) sequences corresponding to the complete gene from all eight drs12 isolates were identical (accession numbers EF182584 to EF182591). The sequence was also conserved compared to drs12 alleles found in Germany (accession number AJ300679) and Australia (accession number AF060762) except that variable numbers of internal repeats were often found. Furthermore, we report here the full-length sequence for drs55 (accession number EF182592), and we found that the sequence was also highly conserved compared to a single entry (accession number AF060763) in the database (Fig. 1B). A repeat sequence consisting of 74 amino acids inserted at position 70 was observed in DRS12 from India compared to DRS12 and DRS55 sequences found in Australia (10) (Fig. 1B). Similarly, two 74-amino-acid insertional repeat sequences (148 amino acids) exhibiting 100% internal homology were observed in Indian DRS55 in comparison to Australian DRS12 and DRS55 (Fig. 1B). The size variation in DRS55 at the protein level was also confirmed when secretory proteins from the isolates were analyzed. A 34-kDa protein and a 37-kDa protein corresponding to DRS12 and DRS55, respectively, were detected using specific antibody (Fig. 1C).

Thus, most of the DRS sequence was conserved, and the heterogeneity observed was due to only large internal repeat sequences, whereas in the case of SIC, variations persist throughout the whole sequence even for geographically related isolates having the same emm type (14). The conserved nature of DRS may be due to less intense immune pressure.


   ACKNOWLEDGMENTS
 
We acknowledge financial support from ICMR, New Delhi, India.

We thank K. S. Sriprakash and Michael Binks of the Queensland Institute of Medical Research, Queensland, Australia, for providing DRS-specific antibody.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Experimental Medicine and Biotechnology, PGIMER, Chandigarh, 160012 India. Phone: 91 172 2746277. Fax: 91 172 2744401. E-mail: superoxide{at}sify.com. Back

{triangledown} Published ahead of print on 19 January 2007. Back


   REFERENCES
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Journal of Bacteriology, April 2007, p. 2933-2935, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01619-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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