Identification of a hypervariable region in Streptococcus agalactiae ATG GTA CGA ATA CTTGTG ATT TACGGA GCC ATT CATGTT AAT GTG CTCTTG CTT AGCCTG M V R I LVI Y G A I HV NV L L L S L TTTTTACTT GGA TACGTAGATGTG GGA ATTCTG ATG ATG CTTCAA GTAGCC TTTCTAGCC F L LG Y V D V G I L M M L Q V AFLA ATC ATCAGC TGG GATTGG ATA CGCTAT GGC AAA GGT ATT GTC AGC TTG TCT TTA AAA AAG I ISWD W I R YG K GI V S L S L K K CGA GCT TAT TGG CTGTTT GGA AGT TTGATG GGA ATG GTT TCT TTGACTCTTCTC ATG AGC RAY W L F G S L MG M V SLTLLM S TTG CTT TTT CCA GTT GGTTCA AGA AAC CAA GAA ATA TTACTA ACT GTTCAA CGA CAAATAL L F P V GS R N Q E I L LT V Q R Q I CCAAACTACTCT TTT ATTCTC TTCTTG CTC AAT GCC AGT GTGGTC GAG GAATAT GTT TAT P NYS F I L F L L N A S VV E E Y V Y CGG CAGCTAATT TGG GAA CGA TTG AAA CAA CCAGCT ATA CAGTTA TTT GTA ACG AGC TTT R Q L I W E R L KQ P AI Q LF V T S F CTT TTT ACC CTA TCT CAT CAC CCAGTCAGC TTG GTT AAT TGG TGT ATC TAT GGC GGATTG L F T L S H H P V S L VN W C I Y G GL GGTCTG AGT TTA GGGTTCATT AGG TTG AAA ACG GAT TCCCTG ACATCA ACC TTG TTA CAT G L S L GF I R L K T D SL T S T L L H ATCACT TGG AAC AGCCTT GTT TTCCTTCTA ACT TTT TTG TGA I T W N S L V FLL T F L Works Cited Introduction Conclusions Study Questions Future Directions Results Abstract Results Acknowledgements Figure 2: DNA and amino acid sequence of hyv1 in the sequenced serotype III isolate, NEM316. All changes are shown relative to the hvy1 sequence in the sequenced serotype V isolate, 2603V/R. Key: Non-synonymous changes Synonymous changes Not present in SAG1273 Tara C. Smith, PhD and Brenda Boese Center for Emerging Infectious Diseases, Department of Epidemiology, University of Iowa College of Public Health, Iowa City, IA Background: Streptococcus agalactiae (group B streptococcus, GBS) is a gram-positive bacterium and a leading infectious cause of neonatal morbidity and mortality. More recently, the role of this organism as a cause of infection in nonpregnant adults has been described. GBS is a frequent inhabitant of the gastrointestinal tract in both males and females, and women can also be colonized in the genital tract.Methods: An in silico analysis was performed comparing two sequenced isolates of Streptococcus agalactiae. BAA-611 (2603 V/R) is a serotype V isolate and 12043 (NEM316) is a serotype III isolate. Both strains were taken from invasive infections and their entire genome sequences are available at TIGR (www.tigr.org). An area of the S. agalactiae genome was identified which contained a stretch of 24 hypervariable genes (termed the GBS hypervariable region, HVR). DNA and predicted protein sequences were compared between the two sequenced isolates in order to estimate the ratio of non-synonymous (Ka) to synonymous (Ks) base pair changes. This ratio (Ka/Ks), termed w, provides a quantitative measure of selection; the higher the ratio, the more likely it is that positive (diversifying) selection has affected the evolution of the locus in question.Results: One of these genes (SAG1293/GBS1366; collectively termed hvy1) was chosen for further analysis. This gene codes for a putative protease approximately 200 amino acids in length. The hvy1 genes in BAA-611 and 12043 were approximately 50% identical at the sequence level. Preliminary sequencing of the gene in 10 isolates (5 serotype III, 5 serotype V) revealed 2 new alleles in addition to the alleles in the sequenced isolates. A calculation of w in each case gave a Ka/Ks ratio greater than 2, indicating positive selection was a force driving the evolution of the locusConclusions: This hypervariable region identified in S. agalactiae has undergone diversifying selection, and may potentially play a role in virulence. Additional studies are underway to test that hypothesis. • A hypervariable genetic region is present in the group B streptococcus • Several genes in this region appear to have undergone positive selection • Does S. agalactiae contain a region of hypervariable genes? • What is the genetic diversity in this region? • Are any alleles associated with invasive disease? DNA and predicted protein sequences were compared between the two sequenced isolates in order to estimate the ratio of non-synonymous (Ka) to synonymous (Ks) base pair changes. This ratio (Ka/Ks), termed ω, provides a quantitative measure of selection (7); the higher the ratio, the more likely it is that positive selection has affected the evolution of the locus in question. One of these genes (SAG1273/GBS1345; collectively termed hvy1) was chosen for further analysis. This gene codes for a hydrophilic hypothetical protein approximately 100 amino acids in length. The hvy1 genes in 2603V/R and NEM316 were approximately 50% identical at the sequence level. A calculation of ω in each case gave a Ka/Ks ratio greater than 2, indicating positive selection was a force driving the evolution of the locus (see Figure 2). We anticipate future research leading in several directions. It is possible that this region may be useful as a molecular typing scheme, similar to that use for the emm gene in Streptococcus pyogenes (1). The current method of typing Streptococcus agalactiae is based on properties of the capsule, and is of limited usefulness. One reason for this is the relatively few number of different serotypes it yields; yet studies have shown that organisms which group together within these serotypes are far from homogeneous (reviewed in 5). Additionally, capsular typing is labor-intensive and few laboratories have the reagents necessary to perform it (2). A typing scheme based on one or more of these genes would be reproducible, inexpensive, and portable from laboratory to laboratory. It is predicted that it will also provide far more than 8 groups of GBS, elucidating more information on the epidemiology of these organisms than the current serotype grouping does. These genes could also be further examined to determine whether they play any role in bacterial pathogenesis. Initial studies may focus on the expression of these genes under various circumstances, both in vitro (by cells grown in broth media or on agar plates) or in vivo (using either a tissue culture model or an animal model of infection). Knockouts of these genes could be made and tested in an animal infection model. Additionally, the genes can be cloned and expressed in E. coli and used as antigens to screen sera from patients who have experienced a GBS infection in order to determine whether these are indeed expressed in vivo and are antigenic. Knowing the population structure and diversity of these genes beforehand will facilitate this last project, as it will allow primers to be more efficiently designed. An in silico analysis was performed comparing two sequenced isolates of Streptococcus agalactiae. 2603V/R is a serotype V isolate (6) and NEM316 is a serotype III isolate (3). Both strains were taken from invasive infections and their entire genome sequences are available at TIGR (www.tigr.org). An area of the S. agalactiae genome was identified which contained a stretch of 24 hypervariable genes (collectively termed the GBS hypervariable region, HVR). This region was found in cluster XIII of NEM316 and and cluster 16 of 2603V/R. • One of the most challenging of host defenses is adaptive immunity. Therefore, many species of microbes have evolved a strategy that allows for variation of antigens which are exposed to host defenses (generally, proteins or portions of proteins which are either exposed on the surface of the pathogen, or secreted proteins). These genes mutate at a high rate and undergo “positive” or “diversifying” selection—that is, there is a selection for mutations that change the amino acid sequence of the protein, and thus, result in the generation of a novel antigen which the host immune system has not targeted previously. Therefore, at the population level, the pathogen will show a large variability in gene sequences for these antigens. • In Streptococcus agalactiae, a common cause of sepsis and meningitis in newborns, a hypervariable gene or region has not been previously characterized. Most of the epidemiological studies in this organism have focused on the capsular serotype, of which 8 varieties have been identified (reviewed in 5). The capsule has also been targeted as a possible vaccine candidate, although as a polysaccharide, it does not induce an immune response as well as many protein antigens (4). A handful of other putative virulence factors have been identified in this organism (5), but all are fairly conserved at the genetic level. Thus, this study will be the first to systemically analyze a potential hypervariable region in this bacterium. It is hoped that this will lead to further insights into not only the virulence of this pathogen, but the natural history and evolution of the organism as well. As this bacterium causes severe invasive disease, particularly in newborns and in the elderly, it merits further study. The Streptococcus agalactiae hypervariable region (HVR) is an area of genes which show this diversifying selection, and as such, may be targets of the human immune system. 1. Beall, B; Facklam, R; Thompson, T. 1996. J Clin Microbiol. 34: 953-8. 2. Borchardt SM, Foxman B, Chaffin DO, Rubens CE, Tallman PA, Manning SD, Baker CJ, Marrs CF. 2004. J. Clin. M icrobiol. 42: 146-50. 3. Glaser,P., Rusniok,C., Chevalier,F., et al. 2002. Mol. Microbiol. 45:1499-1513. 4. Kelly, DF; Moxon, ER; Pollard, AJ. 2004. Immunology. 113: 163-74. 5. Manning, SD. 2003. Front Biosci. 8:s1-18. 6. Tettelin H, Masignani V, Cieslewicz MJ, et al. 2002. PNAS. 99: 12391-6. 7. Yang, Z; Bielawski, JP. 2000. TREE. 15:496-502. Table 1: summary of genes present in HVR 1Conserved hypothetical protein 2First third of protein is absent in type V; no homology found in 2603V/R. 3GBS 1359-1362 and 1364, and SAG 1286-1289 and 1291 correspond to proteins from Tn5252. *SAG1277: also some similarity to type III 1132 This work was supported by a University of Iowa New Investigator Grant (TCS).