Abstract:We report a closed genome sequence of group A Streptococcus genotype emm3 (GAS M/emm3) strain STAB902, isolated from a superficial pyodermatitis. The genome is composed of 1,892,124 bp, 6 integrated prophages, and has 1,858 identified coding sequences (CDSs). It has been fitted with the two available invasive GAS M/emm3 strains.
“…A) identified the skg ‐encoded‐SKG88 as SK2a (SK2a G88 ) which is in complete agreement with prior reports on clustering of skcg alleles . Accordingly, STAB902 and ALAB49 SKs were subclustered as SK2a (SK2a STAB902 ) and SK2b (SK2b ALAB49 ), respectively . PCR amplification of all three parental SKs produced the expected 1250‐bp amplicon (Fig.…”
Streptokinase (SK) is a plasminogen activator which converts inactive plasminogen (Pg) to active plasmin (Pm), which cleaves fibrin clots. SK secreted by groups A, C, and G
Streptococcus
(SKA/SKC/SKG) is composed of three domains: SKα, SKβ and SKγ. Previous domain‐swapping studies between SK1/SK2b‐cluster variants revealed that SKβ plays a major role in the activation of human Pg. Here, we carried out domain‐swapping between
skcg
‐SK/SK2‐cluster variants to determine the involvement of SKβ in several SK functionalities, including specific/proteolytic activity kinetics, fibrinogen‐bound Pg activation and α
2
‐antiplasmin resistance. Our results indicate that SKβ has a minor to determining role in these diverse functionalities for
skcg
‐SK and SK2b variants, which might potentially be accompanied by few critical residues acting as hot spots. Our findings enhance our understanding of the roles of SKβ and hot spots in different functional characteristics of SK clusters and may aid in the engineering of fibrin‐specific variants of SK for breaking down blood clots with potentially higher efficacy and safety.
“…A) identified the skg ‐encoded‐SKG88 as SK2a (SK2a G88 ) which is in complete agreement with prior reports on clustering of skcg alleles . Accordingly, STAB902 and ALAB49 SKs were subclustered as SK2a (SK2a STAB902 ) and SK2b (SK2b ALAB49 ), respectively . PCR amplification of all three parental SKs produced the expected 1250‐bp amplicon (Fig.…”
Streptokinase (SK) is a plasminogen activator which converts inactive plasminogen (Pg) to active plasmin (Pm), which cleaves fibrin clots. SK secreted by groups A, C, and G
Streptococcus
(SKA/SKC/SKG) is composed of three domains: SKα, SKβ and SKγ. Previous domain‐swapping studies between SK1/SK2b‐cluster variants revealed that SKβ plays a major role in the activation of human Pg. Here, we carried out domain‐swapping between
skcg
‐SK/SK2‐cluster variants to determine the involvement of SKβ in several SK functionalities, including specific/proteolytic activity kinetics, fibrinogen‐bound Pg activation and α
2
‐antiplasmin resistance. Our results indicate that SKβ has a minor to determining role in these diverse functionalities for
skcg
‐SK and SK2b variants, which might potentially be accompanied by few critical residues acting as hot spots. Our findings enhance our understanding of the roles of SKβ and hot spots in different functional characteristics of SK clusters and may aid in the engineering of fibrin‐specific variants of SK for breaking down blood clots with potentially higher efficacy and safety.
“…However, the Gps did not correlate with STSS. STAB902 isolated from a non-invasive superficial cutaneous infection [18] harbored the same set of prophages as MGAS315 and SSI-1. By contrast, there were no prophages in the STSS strains, H293 and MGAS27061.Fig.…”
BackgroundGroup A Streptococcus (GAS) is a major human pathogen, which is associated with a wide spectrum of invasive diseases, such as pharyngitis, scarlet fever, rheumatic fever, and streptococcal toxic shock syndrome (STSS). It is hypothesized that differences in GAS pathogenicity are related to the acquisition of diverse bacteriophages (phages). Nevertheless, the GAS genome also harbors clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (cas) genes, which play an important role in eliminating foreign DNA, including those of phages. However, the structure of prophages in GAS strains is mosaic, and the phylogenetic relationship between prophages and CRISPR is not clear. In this study, we analyzed CRISPR and prophage structure using 118 complete genome sequences of GAS strains to elucidate the relationship between two genomic elements. Additionally, phylogenetic and M-type analyses were performed.ResultsOf the 118 GAS strains, 80 harbored type I-C and/or II-A CRISPR/cas loci. A total of 553 spacer sequences were identified from CRISPR/cas loci and sorted into 229 patterns. We identified and classified 373 prophages into 14 groups. Some prophage groups shared a common integration site, and were related to M-type. We further investigated the correlation between spacer sequences and prophages. Of the 229 spacer sequence patterns, 203 were similar to that of other GAS prophages. No spacer showed similarity with that of a specific prophage group with mutL integration site. Moreover, the average number of prophages in strains with type II-A CRISPR was significantly less than that in type I-C CRISPR and non-CRISPR strains. However, there was no statistical difference between the average number of prophages in type I-C strains and that in non-CRISPR strains.ConclusionsOur results indicated that type II-A CRISPR may play an important role in eliminating phages and that the prophage integration site may be an important criterion for the acceptance of foreign DNA by GAS. M type, spacer sequence, and prophage group data were correlated with the phylogenetic relationships of GAS. Therefore, we hypothesize that genetic characteristics and/or phylogenetic relationships of GAS may be estimated by analyzing its spacer sequences.Electronic supplementary materialThe online version of this article (10.1186/s12866-019-1393-y) contains supplementary material, which is available to authorized users.
“…GAS (STAB902) and GGS (G88) with accession numbers CP007041.1 and HM390000.1, respectively were selected as the sources of SK for β-domain exchange. Based on DNA sequences of the variable region of SKβ, SK STAB902 and SK G88 have been reported as SK2a and SK2a co-clustered- skcg alleles, respectively [ 15 , 20 , 21 ] .…”
Section: Methodsmentioning
confidence: 99%
“…We have recently reported the isolation of SK ( skg ) with high PA activity from a GGS (SKG88) [ 20 ] . In the present study, using SKG88 (with high PA activity) and a well-known SK2a variants from GAS (SK STAB902 ) with low PA activity [ 21 ] , we evaluated the contribution of SK domains in kinetics and FG-bound-Plg activation via “Molecular (SKβ) domain-exchange strategy” between SK genes of these two groups of streptococci.…”
Background:SK, a heterogeneous PA protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SK). Based on the variable region of SKβ, GAS-SK (ska) are clustered as SK1 and SK2 (including SK2a/SK2b), which show low and high FG-dependent Plg activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SK (skcg) variants display properties similar to SK1. Herein, by SKβ exchange between GGS (G88) and GAS-SK2a (STAB902) variants, the potential roles of SK domains in amidolytic/proteolytic activity and FG-bound-Plg activation are represented. Methods:Two parental SKG88 and SKSTAB902 genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SKβ-exchanged constructs (SKC1: αG88-βSTAB-γG88 and SKC2; αSTAB-βG88-γSTAB) were constructed by BstEII/BsiWI digestion/cross-ligation in parental plasmids. SK were expressed in E. coli and purified by Ni-NTA chromatography. PA potencies of SK were measured by colorimetric assay. Results:SDS-PAGE and Western-blot analyses confirmed the proper expression of 47-kDa SK. Analyses indicated that the catalytic efficiency (Kcat/Km) for amidolytic and proteolytic activity were less and moderately dependent on SKβ, respectively. The increase of FG-bound-Plg activation for SKSTAB902/SKC1 containing SK2aβ was around six times, whereas for SKG88/SKC2 containing skcgβ, it was four times. Conclusion:Although SKβ has noticeable contribution in FG-bound-Plg activation activity, it had minor contribution in fibrin-independent, amidolytic activity. These data might be of interest for engineering fibrin-specific versions of SK.
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