scholarly journals Structure determination of the CAMP factor of Streptococcus agalactiae with the aid of an MBP tag and insights into membrane-surface attachment

2019 ◽  
Vol 75 (8) ◽  
pp. 772-781 ◽  
Author(s):  
Yajuan Li ◽  
Weihong Zeng ◽  
Yuelong Li ◽  
Weirong Fan ◽  
Huan Ma ◽  
...  
Keyword(s):  
Structure Determination ◽  
Streptococcus Agalactiae ◽  
Membrane Surface ◽  
Heavy Atom ◽  
Group B Streptococcus ◽  
Bacterial Toxin ◽  
Camp Factor ◽  
Interaction Interface ◽  
Group B

CAMP factor is a unique α-helical bacterial toxin that is known for its co-hemolytic activity in combination with staphylococcal sphingomyelinase. It was first discovered in the human pathogen Streptococcus agalactiae (also known as group B streptococcus), but homologous genes have been found in many other Gram-positive pathogens. In this study, the efforts that led to the determination of the first structure of a CAMP-family toxin are reported. Initially, it was possible to produce crystals of the native protein which diffracted to near 2.45 Å resolution. However, a series of technical obstacles were encountered on the way to structure determination. Over a period of more than five years, many methods, including selenomethionine labeling, mutations, crystallization chaperones and heavy-atom soaking, were attempted, but these attempts resulted in limited progress. The structure was finally solved using a combination of iodine soaking and molecular replacement using the crystallization chaperone maltose-binding protein (MBP) as a search model. Analysis of native and MBP-tagged CAMP-factor structures identified a conserved interaction interface in the C-terminal domain (CTD). The positively charged surface may be critical for binding to acidic ligands. Furthermore, mutations on the interaction interface at the CTD completely abolished its co-hemolytic activities. This study provides novel insights into the mechanism of the membrane-permeabilizing activity of CAMP factor.

scholarly journals Genetic Basis Underlying the Hyperhemolytic Phenotype of Streptococcus agalactiae Strain CNCTC10/84

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Luchang Zhu ◽  
Stephen B. Beres ◽  
Prasanti Yerramilli ◽  
Layne Pruitt ◽  
Concepcion C. Cantu ◽  
...  
Keyword(s):  
Streptococcus Agalactiae ◽  
Hemolytic Activity ◽  
Genetic Basis ◽  
Group B Streptococcus ◽  
Single Nucleotide ◽  
Altered Expression ◽  
Homopolymeric Tract ◽  
Camp Factor ◽  
Nucleotide Insertion ◽  
Group B

ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) is a major cause of infections in newborns, pregnant women, and immunocompromised patients. GBS strain CNCTC10/84 is a clinical isolate that has high virulence in animal models of infection and has been used extensively to study GBS pathogenesis. Two unusual features of this strain are hyperhemolytic activity and hypo-CAMP factor activity. These two phenotypes are typical of GBS strains that are functionally deficient in the CovR-CovS two-component regulatory system. A previous whole-genome sequencing study found that strain CNCTC10/84 has intact covR and covS regulatory genes. We investigated CovR-CovS regulation in CNCTC10/84 and discovered that a single-nucleotide insertion in a homopolymeric tract in the covR promoter region underlies the strong hemolytic activity and weak CAMP activity of this strain. Using isogenic mutant strains, we demonstrate that this single-nucleotide insertion confers significantly decreased expression of covR and covS and altered expression of CovR-CovS-regulated genes, including that of genes encoding β-hemolysin and CAMP factor. This single-nucleotide insertion also confers significantly increased GBS survival in human whole blood ex vivo. IMPORTANCE Group B streptococcus (GBS) is the leading cause of neonatal sepsis, pneumonia, and meningitis. GBS strain CNCTC10/84 is a highly virulent blood isolate that has been used extensively to study GBS pathogenesis for over 20 years. Strain CNCTC10/84 has an unusually strong hemolytic activity, but the genetic basis is unknown. In this study, we discovered that a single-nucleotide insertion in an intergenic homopolymeric tract is responsible for the elevated hemolytic activity of CNCTC10/84.

CAMP test v1

2021 ◽  
Author(s):  
lydiariver not provided
Keyword(s):  
Staphylococcus Aureus ◽  
Streptococcus Agalactiae ◽  
Group B Streptococcus ◽  
Blood Agar ◽  
Camp Factor ◽  
Camp Test ◽  
Group B

Group B Streptococcus agalactiae has CAMP factor which allow it to hemolized zones when it is grown on blood agar plates near to Staphylococcus aureus ATCC 25293 colonies, this effect is brought about by Staphylococcus aureus sphingomyelinase.

scholarly journals In VitroActivity of Solithromycin against Erythromycin-Resistant Streptococcus agalactiae

2013 ◽  
Vol 58 (3) ◽  
pp. 1693-1698 ◽  
Author(s):  
Giorgio Piccinelli ◽  
Prabhavathi Fernandes ◽  
Carlo Bonfanti ◽  
Francesca Caccuri ◽  
Arnaldo Caruso ◽  
...  
Keyword(s):  
Streptococcus Agalactiae ◽  
Group B Streptococcus ◽  
Phenotypic Characterization ◽  
Content Type ◽  
Resistant Strains ◽  
Group B ◽  
Microdilution Broth

ABSTRACTThein vitroantibacterial activity of solithromycin (CEM-101) against macrolide-resistant isolates (n= 62) ofStreptococcus agalactiae(group B streptococcus [GBS]) was determined. Phenotypic characterization of macrolide-resistant strains was performed by double-disc diffusion testing. A multiplex PCR was used to identify theerm(B),erm(TR), andmef(A/E) genes, capsular genotypes, and alpha-like (Alp) protein genes from the GBS strains. Determination of MIC was carried out using the microdilution broth method. The Etest method was used for penicillin, azithromycin, clarithromycin, and erythromycin. Solithromycin had a MIC50of ≤0.008 μg/ml and a MIC90of 0.015 μg/ml against macrolide-susceptibleS. agalactiae. These MICs were lower than those displayed by penicillin (MIC50of 0.032 μg/ml and MIC90of 0.047 μg/ml), the antibiotic agent of choice for prophylaxis and treatment of GBS infections. Against macrolide-resistantS. agalactiae, solithromycin had a MIC50of 0.03 μg/ml and a MIC90of 0.125 μg/ml. Againsterm(B) strains, solithromycin had a MIC50of 0.03 μg/ml and a MIC90of 0.06 μg/ml, while againstmef(A) strains, it had a MIC50of 0.03 μg/ml and a MIC90of 0.125 μg/ml. Most erythromycin-resistant GBS strains were of serotype V (64.5%) and associated significantly withalp2-3. Moreover, a statistically significant association was observed between the constitutive macrolide-lincosamide-streptogramin B resistance (cMLSB) phenotype and theerm(B) gene-carrying strains, thealp2-3gene and the M phenotype, and themef(A/E) gene andepsilon. Overall, our results show that solithromycin had lower or similar MICs than penicillin and potent activity against macrolide-resistant strains independent of their genotype or phenotype, representing a valid therapeutic alternative where β-lactams cannot be used.

scholarly journals Specificity of the hyaluronate lyase of group-B streptococcus toward unsulphated regions of chondroitin sulphate

1997 ◽  
Vol 327 (1) ◽  
pp. 65-71 ◽  
Author(s):  
John R. BAKER ◽  
Hao YU ◽  
Kelly MORRISON ◽  
William F. AVERETT ◽  
David G. PRITCHARD
Keyword(s):  
Streptococcus Agalactiae ◽  
Chondroitin Sulphate ◽  
Group B Streptococcus ◽  
Hyaluronate Lyase ◽  
Purification And Properties ◽  
The Subject ◽  
Group B ◽  
Host Tissues ◽  
Testicular Hyaluronidase

The purification and properties of a hyaluronate lyase secreted by Streptococcus agalactiae, which is believed to facilitate the invasion of host tissues by the organism, have been described previously [Pritchard, Lin, Willingham and Baker (1994) Arch. Biochem. Biophys. 315, 431–436]. The specificity of the limited cleavage of chondroitin sulphate by the enzyme is the subject of this report. To simplify the task, a chondroitin sulphate from the Swarm rat chondrosarcoma, which contains only 4-sulphated and unsulphated disaccharide repeats, was used in this study. Tetrasaccharides from an ovine testicular hyaluronidase digest of the chondroitin sulphate were isolated, identified and tested as substrates of the streptococcal hyaluronate lyase. Only tetrasaccharides with an unsulphated disaccharide at the reducing end were cleaved (by elimination at the N-acetylgalactosaminidic bond). Thus chondroitin sulphate chains are cleaved by the action of this lyase at every unsulphated disaccharide repeat, but release of unsaturated unsulphated disaccharides only occurs from sites where two or more sequential unsulphated disaccharide repeats are present. Analysis of the chondrosarcoma chondroitin sulphate showed that of approximately five unsulphated disaccharide repeats per chain, two are clustered. The ability of group-B streptococcal hyaluronate lyase to cleave chondroitin sulphate may allow the organisms to invade tissues more efficiently. The demonstrated specific and highly limited cleavage of chondroitin sulphate by this bacterial lyase promises to be a useful tool in the determination of chondroitin sulphate structure and variability.

An Emerging Infection: Streptococcal Toxic Shock-Like Syndrome Caused By Group B Streptococcus (GBS), Streptococcus Agalactiae

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S140-S140
Author(s):  
F Rajack ◽  
A Afsari ◽  
A M Ramadan ◽  
T J Naab
Keyword(s):  
Soft Tissue ◽  
Necrotizing Fasciitis ◽  
Streptococcus Agalactiae ◽  
Group A Streptococcus ◽  
Multiorgan Failure ◽  
Group B Streptococcus ◽  
Stage Iii ◽  
Streptococcal Toxic Shock Syndrome ◽  
Toxic Shock ◽  
Group B

Abstract Introduction/Objective Streptococcus agalactiae, Group B Streptococcus (GBS), is a major cause of neonatal sepsis and infections in pregnant women. However, incidence of invasive GBS infections has more than doubled in the last two decades with highest risk in adults 65 years or older. Other risk factors are diabetes, malignancy, and immunocompromised state. Bacteremia and skin soft tissue infections are the most common invasive infections in nonpregnant adults. Rarely GBS infection has a fulminating pyrogenic exotoxin-mediated course characterized by acute onset, multiorgan failure, shock, and sometimes death, referred to as toxic shock-like syndrome. Methods A 77-year-old hypertensive female with uncontrolled type 2 diabetes mellitus and a history of bilateral foot ulcers presented to the hospital in probable septic shock. Clinical diagnosis of necrotizing fasciitis was made and she underwent bilateral lower limb amputations. Results Grossly soft tissue appeared gray. Microscopically fascia was necrotic without neutrophils present and Gram stain revealed sheets of Gram positive cocci. These findings reflected histopathologic Stage III necrotizing fasciitis, which is associated with 47% mortality. Autopsy showed a similar histology of Stage III necrotizing fasciitis involving the surgical stump. Erythema and desquamation of the upper limbs bilaterally and multi-organ failure met the clinical picture of Streptococcal Toxic Shock Syndrome (STSS) and fulfilled the criteria for TSS due to Group A Streptococcus (GAS), defined by The Working Group on Severe Streptococcal Infections. Conclusion Group B Streptococcal Toxic Shock-Like Syndrome may have a similar outcome to STSS caused by GAS and other pathogens and, in limited studies, mortality has been 30% or greater.

scholarly journals Prevalence and Serotype Determination of Streptococcus agalactiae Isolated From Non-Pregnant Women in Tehran, Iran

2020 ◽  
Author(s):  
Leila Goudarzi ◽  
Mohammad Bagher Khalili ◽  
Mahmood Vakili ◽  
Maryam Sadeh
Keyword(s):  
Pregnant Women ◽  
Streptococcus Agalactiae ◽  
Group B Streptococcus ◽  
Cross Sectional Study ◽  
Chi Square ◽  
Cross Sectional ◽  
Specific Pcr ◽  
Pcr Method ◽  
Group B ◽  
Tehran Area

Consequence of Streptococcus agalactiae, Group B Streptococcus (GBS) relating infant’s diseases are well documented. Although many women carry this bacterium in their vagina, they may transfer to their infant during delivery and may result in different neonatal invasive diseases. The aim of this study was to determine the prevalence of GBS and serotyping the isolated species among un-selective non-pregnant women who attended two gynecology clinics in Tehran. In this cross-sectional study, a total of 560 vaginal samples collected from non-pregnant women. Following inoculation of the specimen on Blood Agar, the standard technology was applied for the final identification of GBS. Detected GBS species were further confirmed using specific PCR directed on dlts gene. Capsular serotyping was done by using the multiplex PCR method. The chi-square method was used for statistical analysis. Fifty (8.9%) out of 560 non-pregnant women were carriers of GBS. The most common types were III (36%), followed by type II (32%), Ia (26%), and Ib (6%), respectively. Results represent that the prevalence rate of GBS in non-pregnant women was reliable and similar to what obtained from pregnant women. In addition, the serotype III was found the most dominant types, as well as other investigations in the Tehran area. Therefore, vaccine designation based on type III is recommended.

scholarly journals Combining solution wide-angle X-ray scattering and crystallography: determination of molecular envelope and heavy-atom sites

2009 ◽  
Vol 42 (2) ◽  
pp. 259-264 ◽  
Author(s):  
Xinguo Hong ◽  
Quan Hao
Keyword(s):  
Structure Determination ◽  
Heavy Atom ◽  
Unit Cell ◽  
Wide Angle ◽  
X Ray ◽  
X Ray Scattering ◽  
Molecular Envelope ◽  
Waxs Data ◽  
Ray Scattering

Solving the phase problem remains central to crystallographic structure determination. A six-dimensional search method of molecular replacement (FSEARCH) can be used to locate a low-resolution molecular envelope determined from small-angle X-ray scattering (SAXS) within the crystallographic unit cell. This method has now been applied using the higher-resolution envelope provided by combining SAXS and WAXS (wide-angle X-ray scattering) data. The method was tested on horse hemoglobin, using the most probable model selected from a set of a dozen bead models constructed from SAXS/WAXS data using the programGASBORat 5 Å resolution (qmax= 1.25 Å−1) to phase a set of single-crystal diffraction data. It was found that inclusion of WAXS data is essential for correctly locating the molecular envelope in the crystal unit cell, as well as for locating heavy-atom sites. An anomalous difference map was calculated using phases out to 8 Å resolution from the correctly positioned envelope; four distinct peaks at the 3.2σ level were identified, which agree well with the four iron sites of the known structure (Protein Data Bank code 1ns9). In contrast, no peaks could be found close to the iron sites if the molecular envelope was constructed using the data from SAXS alone (qmax= 0.25 Å−1). The initial phases can be used as a starting point for a variety of phase-extension techniques, successful application of which will result in complete phasing of a crystallographic data set and determination of the internal structure of a macromolecule to atomic resolution. It is anticipated that the combination ofFSEARCHand WAXS techniques will facilitate the initial structure determination of proteins and provide a good foundation for further structure refinement.

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