scholarly journals Identification of the cpsA gene as a specific marker for the discrimination of Streptococcus pneumoniae from viridans group streptococci

2010 ◽  
Vol 59 (10) ◽  
pp. 1146-1152 ◽  
Author(s):  
Hee Kuk Park ◽  
Sang-Jae Lee ◽  
Jang Won Yoon ◽  
Jong Wook Shin ◽  
Hyoung-Shik Shin ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Capsular Polysaccharide ◽  
Subtractive Hybridization ◽  
Rapid Identification ◽  
Specific Marker ◽  
Viridans Streptococci ◽  
Polysaccharide Biosynthesis ◽  
Viridans Group Streptococci

Streptococcus pneumoniae, the aetiological agent of pneumonia and non-gonococcal urethritis, shares a high degree of DNA sequence identity with the viridans group of streptococci, particularly Streptococcus mitis and Streptococcus oralis. Although their clinical and pathological manifestations are different, discrimination between S. pneumoniae and its close viridans cocci relatives is still quite difficult. Suppression subtractive hybridization was performed to identify the genomic differences between S. pneumoniae and S. mitis. Thirty-four resulting S. pneumoniae-specific clones were examined by sequence determination and comparative DNA sequence analysis using blast. S. pneumoniae-specific primers were subsequently designed from one of the clonal DNA sequences containing the cps gene (coding for capsular polysaccharide biosynthesis). The primer specificities were evaluated using 49 viridans streptococci including 26 S. pneumoniae, 54 other streptococci, 14 Lactococcus species, 14 Enterococcus species and three Vagococcus species, and compared with the specificities of previously described autolysin (lytA), pneumolysin (ply), Spn9802 and Spn9828 primers. The newly designed cpsA-specific primer set was highly specific to S. pneumoniae and was even better than the existing primers. These findings may help improve the rapid identification and differentiation of S. pneumoniae from closely related members of the viridans group streptococci.

Identification of Streptococcus pneumoniae genes specifically induced in mouse lung tissues

2008 ◽  
Vol 54 (1) ◽  
pp. 58-65 ◽  
Author(s):  
Jiang-Ping Meng ◽  
Yi-Bing Yin ◽  
Xue-Mei Zhang ◽  
Yuan-Shuai Huang ◽  
Kai Lan ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Mouse Lung ◽  
Selection System ◽  
Polysaccharide Biosynthesis ◽  
Dual Reporter ◽  
Promoter Trap ◽  
Substance Transport ◽  
Adherence Energy

To identify Streptococcus pneumoniae genes expressed specifically during infections, a selection system based on the in vivo expression technology (IVET) was established. galU, which is critical for capsular polysaccharide biosynthesis, and lacZY encoding β-galactosidase were employed as dual reporter genes to screen in-vivo-induced (ivi) genes of S. pneumoniae. The galU-deficient mutant of S. pneumoniae is incapable of utilizing galactose, thus failing to synthesize capsular polysaccharide, and therefore loses its ability to survive in the host. A promoter-trap library was constructed in S. pneumoniae, which was used to infect BALB/c mice in an intranostril model. Those strains recovered from lung tissue of mice and exhibiting a white colony phenotype on tryptic soy agar containing X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) were collected and identificated. A total of 15 unique sequences were obtained through in vivo screening. The ivi genes of S. pneumoniae are involved in many processes, such as colonization and adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism, and cell wall synthesis. There are some hypothetical proteins whose functions are not clear. This novel IVET is a useful tool for identifying ivi genes in S. pneumoniae.

scholarly journals The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H)

2005 ◽  
Vol 389 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Nehmé SAKSOUK ◽  
Ludovic PELOSI ◽  
Pierre COLIN-MOREL ◽  
Manel BOUMEDIENNE ◽  
Patricia L. ABDIAN ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Biochemical Characterization ◽  
Repeat Unit ◽  
Sugar Residue ◽  
Functional Identification ◽  
Polysaccharide Biosynthesis ◽  
Sugar Addition ◽  
Galactose Residue

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one α-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([→4)-α-D-Glcp-(1→4)-α-D-Galp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→]n). Expression of WciS–His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/ΔgalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [β-D-GlcAp-(1→4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by α-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS–His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

scholarly journals Distribution of Subclasses mefA and mefE of the mefA Gene among Clinical Isolates of Macrolide-Resistant (M-Phenotype) Streptococcus pneumoniae, Viridans Group Streptococci, and Streptococcus pyogenes

2005 ◽  
Vol 49 (2) ◽  
pp. 827-829 ◽  
Author(s):  
Carmen Ardanuy ◽  
Fe Tubau ◽  
Josefina Liñares ◽  
María Angeles Domínguez ◽  
Román Pallarés ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Streptococcus Pyogenes ◽  
Clinical Isolates ◽  
Viridans Streptococci ◽  
Viridans Group Streptococci

ABSTRACT The distribution of subclasses mefA and mefE of the mefA gene among 116 M-phenotype streptococci was as follows: pneumococci (38 strains had mefE and 4 mefA), viridans streptococci (49 mefE and 1 mefA), and Streptococcus pyogenes (24 mefA). Spain9V-3-14 and England14-9 clones of serotype 14 were dominant among pneumococci.

scholarly journals Analysis of the 5′ Portion of the Type 19A Capsule Locus Identifies Two Classes of cpsC, cpsD, andcpsE Genes in Streptococcus pneumoniae

1999 ◽  
Vol 181 (11) ◽  
pp. 3599-3605 ◽  
Author(s):  
Judy K. Morona ◽  
Renato Morona ◽  
James C. Paton
Keyword(s):  
Streptococcus Pneumoniae ◽  
Southern Hybridization ◽  
Capsular Polysaccharide ◽  
Sequence Data ◽  
Direct Sequencing ◽  
Southern Hybridization Analysis ◽  
Polysaccharide Biosynthesis ◽  
Capsule Locus ◽  
Pcr Products ◽  
Long Range Pcr

ABSTRACT Analysis of the sequence data obtained from the 5′ portion of theStreptococcus pneumoniae type 19A capsular polysaccharide biosynthesis locus (cps19a) revealed that the first seven genes are homologous to the first seven genes in the type 19F (cps19f) locus. The former genes were designatedcps19aA to -G and were 70 to 90% identical to their cps19f counterparts. Southern hybridization analysis of the cps loci from various S. pneumoniaeserotypes with probes specific for the cps19aC,cps19aD, and cps19aE genes indicated a hybridization pattern complementary to that previously reported forcps19fC, cps19fD, and cps19fE. That is, all serotypes tested contained high-stringency homologues of either the cps19aC to -E genes or thecps19fC to -E genes, but not both. On this basis S. pneumoniae cps loci can be divided into two distinct classes. Long-range PCR was used to amplify thecps regions between cpsB and aliAfrom a variety of pneumococcal serotypes. Direct sequencing of the 5′ end of these PCR products, and phylogenetic analysis of the sequence data, confirmed the presence of the two distinct classes ofcpsC. Whereas members within one class are greater than 95% identical to each other, the DNA sequence identity between the two classes is only approximately 70%.

scholarly journals Detection of Bacterial Virulence Genes by Subtractive Hybridization: Identification of Capsular Polysaccharide ofBurkholderia pseudomallei as a Major Virulence Determinant

2001 ◽  
Vol 69 (1) ◽  
pp. 34-44 ◽  
Author(s):  
Shauna L. Reckseidler ◽  
David DeShazer ◽  
Pamela A. Sokol ◽  
Donald E. Woods
Keyword(s):  
Immunoelectron Microscopy ◽  
Dna Sequences ◽  
Capsular Polysaccharide ◽  
Lethal Dose ◽  
Subtractive Hybridization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virulence Determinants ◽  
Wild Type ◽  
Virulence Determinant ◽  
Insertional Inactivation

ABSTRACT Burkholderia pseudomallei, the etiologic agent of melioidosis, is responsible for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic. Burkholderia thailandensisis a nonpathogenic environmental organism closely related to B. pseudomallei. Subtractive hybridization was carried out between these two species to identify genes encoding virulence determinants in B. pseudomallei. Screening of the subtraction library revealed A-T-rich DNA sequences unique toB. pseudomallei, suggesting they may have been acquired by horizontal transfer. One of the subtraction clones, pDD1015, encoded a protein with homology to a glycosyltransferase fromPseudomonas aeruginosa. This gene was insertionally inactivated in wild-type B. pseudomallei to create SR1015. It was determined by enzyme-linked immunosorbent assay and immunoelectron microscopy that the inactivated gene was involved in the production of a major surface polysaccharide. The 50% lethal dose (LD50) for wild-type B. pseudomallei is <10 CFU; the LD50 for SR1015 was determined to be 3.5 × 105 CFU, similar to that of B. thailandensis (6.8 × 105CFU). DNA sequencing of the region flanking the glycosyltransferase gene revealed open reading frames similar to capsular polysaccharide genes in Haemophilus influenzae,Escherichia coli, and Neisseria meningitidis. In addition, DNA from Burkholderia mallei andBurkholderia stabilis hybridized to a glycosyltransferase fragment probe, and a capsular structure was identified on the surface of B. stabilis via immunoelectron microscopy. Thus, the combination of PCR-based subtractive hybridization, insertional inactivation, and animal virulence studies has facilitated the identification of an important virulence determinant in B. pseudomallei.

Sign in / Sign up

Export Citation Format

Share Document