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K.b. Ramachandran - One of the best experts on this subject based on the ideXlab platform.

  • Ratio of intracellular precursors concentration and their flux influences hyaluronic acid molecular weight in Streptococcus zooepidemicus and recombinant Lactococcus lactis
    Bioresource technology, 2014
    Co-Authors: Sneh Sanjay Badle, Guhan Jayaraman, K.b. Ramachandran
    Abstract:

    HA molecular weight variation in Streptococcus zooepidemicus and two recombinant Lactococcus lactis strains were investigated by chemostat experiments and metabolic flux analysis (MFA). The study showed that intracellular flux ratio of UDP-GlcUA to UDP-GlcNAc correlated directly with HA molecular weight, for all the three strains. The ratio of intracellular concentration of these HA precursors also exhibited a similar trend. Phosphoglucoisomerase activity and glucose flux towards lactic acid formation were found to be the major bottlenecks for HA production in all the three strains. The study suggests that environmental conditions and genetic manipulations that balance the intracellular flux and HA precursors concentrations will result in increased molecular weight.

  • Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis.
    Applied microbiology and biotechnology, 2012
    Co-Authors: Shashi Bala Prasad, K.b. Ramachandran, Guhan Jayaraman
    Abstract:

    The has operon genes in the hyaluronan (HA) producer, Streptococcus zooepidemicus, encode for some of the critical enzymes in the HA biosynthetic pathway. Heterologous expression of different combinations of multiple has genes has resulted in increasing HA production to varying degrees in different recombinant strains. In this work, a recombinant Lactococcus lactis strain (SJR6) was constructed, with insertion of three has operon genes (hasABD) from S. zooepidemicus. The SJR6 strain was found to be a better HA producer than two previously constructed recombinant L. lactis strains (SJR2 and SJR3), containing hasAB and hasABC genes, respectively, but exhibited lower HA production than the native HA producer S. zooepidemicus. To understand the differences in HA yield between the various strains, transcriptions of the HA biosynthesis genes (has genes and their homologues) were compared at different phases of exponential growth of the L. lactis and S. zooepidemicus cultures. The mRNA levels of all the heterologous has genes were expectedly far higher than their corresponding homologues in the L. lactis strains. The relative mRNA level of the hasB-homologue, viz. ugd (encoding UDP-glucose dehydrogenase), was found to be much lower than that of other homologues, corroborating earlier reports which indicate tight transcriptional regulation of the ugd gene in L. lactis. Interestingly, all the has gene homologues were found to be up-regulated in all the recombinant L. lactis strains, when compared with the corresponding genes in the untransformed strain, L. lactis NZ9000. A transcription analysis of S. zooepidemicus cultures revealed that the has operon was down-regulated in the mid-exponential growth phase in comparison to the early- and late-exponential growth phases. The transcription analyses in this study have provided insights for the design of recombinant strains with higher HA productivity.

  • Influence of competing metabolic processes on the molecular weight of hyaluronic acid synthesized by Streptococcus zooepidemicus
    Biochemical Engineering Journal, 2010
    Co-Authors: Swaminathan Jagannath, K.b. Ramachandran
    Abstract:

    Abstract Hyaluronic acid (HA) is a linear high molecular weight glycosaminoglycan polymer with its molecular weight determining its physiological role, rheological properties and applications. The commercially used microbial source—Streptococcus zooepidemicus is incapable of synthesizing very high molecular weight polymer with low polydispersity, an essential niche in the HA market. In this organism, three important metabolic processes—glycolysis, hyaluronic acid synthesis and biomass formation compete for carbon source, nitrogen source, energy and precursors to determine the molecular weight of the synthesized polymer. After studying the role of culture conditions on these competing processes, it was found that the best strategy for enhancing molecular weight was to weaken the glycolytic process, strengthen HA synthesis process and maintain nitrogen under limiting condition to reduce the rate of biomass formation. Based on this newly developed strategy—temperature switches, addition of precursor N-acetylglucosamine and addition of pyruvate experiments enhanced the molecular weight of the polymer by 23%, 74% and 64%, respectively. Improving precursor levels, particularly UDP-N-acetylglucosamine and thereby strengthening the HA flux plays a direct role in improving molecular weight of hyaluronic acid.

  • Sensitivity to Hydrogen Peroxide of Growth and Hyaluronic Acid Production by Streptococcus zooepidemicus ATCC 39920
    Developments in Chemical Engineering and Mineral Processing, 2008
    Co-Authors: M.d. Mashitah, H. Masitah, K.b. Ramachandran
    Abstract:

    The sensitivity to hydrogen peroxide (H2O2) of growth and hyaluronic acid (HA) production by Streptococcus zooepidemicus ATCC 39920 was studied under various conditions. In sheep blood agar-plates, no detectable zone was observed even when the concentration of H2O2 was increased to 0.15 mM. With brain heart infusion-agar and chemically defined medium-agar plates, a profound zone was detected at 0.015 mM concentration of H2O2. To determine the minimal inhibitory concentration (MIC) of the strain in culture broth, various concentrations of H2O2 (0-200 mM) were maintained in the medium prior to fermentation. The result showed that for higher concentrations of H2O2 in the medium, the greater was the inhibition. Streptococcus is catalase-negative and known to produce H2O2 which may affect growth, HA production and glucose utilization. In order to determine at which growth phase H2O2 had the maximum inhibitory activity, a batch fermentation of S. zooepidemicus was conducted in shake flask culture. It was found that H2O2 production took place during the growth phase, and HA production started after the growth had reach late exponential phase when H2O2 in the culture media was depleted. This indicates that H2O2 produced by the cells did not affect cell growth but influenced HA production.

Bhushan L. Chaudhari - One of the best experts on this subject based on the ideXlab platform.

  • Optimization of medium components for hyaluronic acid production by Streptococcus zooepidemicus MTCC 3523 using a statistical approach
    Carbohydrate Polymers, 2011
    Co-Authors: Kanchankumar P. Patil, Kirtee K. Kamalja, Bhushan L. Chaudhari
    Abstract:

    Abstract Hyaluronic acid (HA), produced from Streptococcus sp., has raised interest in the medical and cosmetics industries because of the various biological functions of HA. Optimization of medium components used for HA production in Streptococcus zooepidemicus MTCC 3523 performed by two-step optimization. A 2 4 full factorial design was employed to study the effect of crucial factors on HA production. To get optimized levels of these factors further, a central composite design was conducted. Through these two phase experiments carried out for optimization of HA production, the medium that yielded maximum average HA comprises 4.05% of glucose, 5.12% of soyapeptone, 0.075% of MgSO 4 ·7H 2 O, and 0.25% of K 2 HPO 4 approximately. The maximum average productivity of HA for this composed medium was 0.798 g/L on fermentation in shake flask. The yield of HA was increased by about 65% using these statistical techniques of media optimization.

  • Production of hyaluronic acid from Streptococcus zooepidemicus MTCC 3523 and its wound healing activity.
    Journal of bioscience and bioengineering, 2010
    Co-Authors: Kanchankumar P. Patil, Deepak K. Patil, Bhushan L. Chaudhari, S. B. Chincholkar
    Abstract:

    Exopolysaccharide produced and purified from Streptococcus zooepidemicus MTCC 3523 was identified as hyaluronic acid (HA) based on IR and NMR spectroscopy while its Mw was found to be 5.38 × 10(5)Da. HA produced passed bacterial endotoxin test and showed significant wound healing activity in Wistar rats on 12th and 16th day.

Kanchankumar P. Patil - One of the best experts on this subject based on the ideXlab platform.

  • Optimization of medium components for hyaluronic acid production by Streptococcus zooepidemicus MTCC 3523 using a statistical approach
    Carbohydrate Polymers, 2011
    Co-Authors: Kanchankumar P. Patil, Kirtee K. Kamalja, Bhushan L. Chaudhari
    Abstract:

    Abstract Hyaluronic acid (HA), produced from Streptococcus sp., has raised interest in the medical and cosmetics industries because of the various biological functions of HA. Optimization of medium components used for HA production in Streptococcus zooepidemicus MTCC 3523 performed by two-step optimization. A 2 4 full factorial design was employed to study the effect of crucial factors on HA production. To get optimized levels of these factors further, a central composite design was conducted. Through these two phase experiments carried out for optimization of HA production, the medium that yielded maximum average HA comprises 4.05% of glucose, 5.12% of soyapeptone, 0.075% of MgSO 4 ·7H 2 O, and 0.25% of K 2 HPO 4 approximately. The maximum average productivity of HA for this composed medium was 0.798 g/L on fermentation in shake flask. The yield of HA was increased by about 65% using these statistical techniques of media optimization.

  • Production of hyaluronic acid from Streptococcus zooepidemicus MTCC 3523 and its wound healing activity.
    Journal of bioscience and bioengineering, 2010
    Co-Authors: Kanchankumar P. Patil, Deepak K. Patil, Bhushan L. Chaudhari, S. B. Chincholkar
    Abstract:

    Exopolysaccharide produced and purified from Streptococcus zooepidemicus MTCC 3523 was identified as hyaluronic acid (HA) based on IR and NMR spectroscopy while its Mw was found to be 5.38 × 10(5)Da. HA produced passed bacterial endotoxin test and showed significant wound healing activity in Wistar rats on 12th and 16th day.

John F. Timoney - One of the best experts on this subject based on the ideXlab platform.

  • Influence of serum antibody on occurrence of Streptococcus zooepidemicus in the nasopharynx of weanling foals
    Journal of Equine Veterinary Science, 2016
    Co-Authors: Sridhar Velineni, John F. Timoney
    Abstract:

    Abstract Streptococcus zooepidemicus (Sz) is a tonsillar commensal of horses that opportunistically invades the respiratory tract of weanling foals causing purulent rhinitis and tracheobronchitis. Ten weanling foals in an isolated herd with a history of Sz-associated respiratory disease in recent years were immunized with recombinant proteins SzPNC78, endopeptidase, and hyaluronidase prior to weaning to determine the effect of serum antibodies on populations of nasopharyngeal Sz and mucopurulent nasal discharges. Levels of serum antibodies to all three proteins were significantly ( P ≤ .01) increased compared to levels in 10 nonimmunized control foals at weaning (day 0). Numbers of Sz in nasopharyngeal washes of control foals were initially higher on day 35 than in washes of immunized foals. However, this difference was not maintained, and counts in washes of both groups were similar over the following 7 weeks. By day 104 postweaning, Sz was no longer detected in washes of six foals in each group. Streptococcus zooepidemicus (ST-45) expressing a specific protective protein (SzP) phenotype dominated from days 35 to 104 in both groups. A second clone, ST-10, was detected intermittently in five immunized and two control foals. Mucopurulent nasal discharges of seven immunized and eight control foals on days 78 and 85 were associated with counts of Sz that ranged as high as 10 6  colony-forming units/mL and with significantly increased levels of SzP-specific IgG in washes. Increase in specific IgG but not IgA was temporary suggesting transudation through inflamed mucosa. This increase was generally greater in washes of immunized (mean enzyme-linked immunosorbent assay optical density [ELISA OD] = 1.73) than control (mean ELISA OD = 1.08) foals suggesting a benefit of systemic immunization may be therapeutic by enhancing clearance of Sz following inflammation provoked by local invasion.

  • Clones of Streptococcus zooepidemicus from outbreaks of hemorrhagic canine pneumonia and associated immune responses.
    Clinical and vaccine immunology : CVI, 2014
    Co-Authors: Sridhar Velineni, John F. Timoney, Kim Russell, Heidi J. Hamlen, Patricia A. Pesavento, William D. Fortney, P. Cynda Crawford
    Abstract:

    Acute hemorrhagic pneumonia caused by Streptococcus zooepidemicus has emerged as a major disease of shelter dogs and greyhounds. S. zooepidemicus strains differing in multilocus sequence typing (MLST), protective protein (SzP), and M-like protein (SzM) sequences were identified from 9 outbreaks in Texas, Kansas, Florida, Nevada, New Mexico, and Pennsylvania. Clonality based on 2 or more isolates was evident for 7 of these outbreaks. The Pennsylvania and Nevada outbreaks also involved cats. Goat antisera against acutely infected lung tissue as well as convalescent-phase sera reacted with a mucinase (Sz115), hyaluronidase (HylC), InlA domain-containing cell surface-anchored protein (INLA), membrane-anchored protein (MAP), SzP, SzM, and extracellular oligopeptide-binding protein (OppA). The amino acid sequences of SzP and SzM of the isolates varied greatly. The szp and szm alleles of the closely related Kansas clone (sequence type 129 [ST-129]) and United Kingdom isolate BHS5 (ST-123) were different, indicating that MLST was unreliable as a predictor of virulence phenotype. Combinations of conserved HylC and serine protease (ScpC) and variable SzM and SzP proteins of S. zooepidemicus strain NC78 were protectively immunogenic for mice challenged with a virulent canine strain. Thus, although canine pneumonia outbreaks are caused by different strains of S. zooepidemicus, protective immune responses were elicited in mice by combinations of conserved or variable S. zooepidemicus proteins from a single strain.

  • Identification of novel immunoreactive proteins of Streptococcus zooepidemicus with potential as vaccine components.
    Vaccine, 2013
    Co-Authors: Sridhar Velineni, John F. Timoney
    Abstract:

    Abstract Background Streptococcus zooepidemicus is an important opportunistic pathogen of the equine respiratory and reproductive tracts. A normal tonsillar and mucosal commensal, it becomes invasive under conditions of stress such as virus infection, weaning, high temperature, prolonged transportation and failure of uterine involution. The aim of this study was to evaluate the vaccine potential of several surface exposed and secreted proteins of a novel mucoid clone of SzNC78 (ST-307) from an epizootic of equine respiratory disease. Methods An expression gene library of SzNC78 was probed with a pool of convalescent equine sera from a clonal epizootic of respiratory disease. Eleven proteins were selected and purified based on putative function, surface expression or secretion and possible importance as virulence factors. Three additional proteins (AhpC, GAPDH and enolase) were also included based on their putative virulence function. Groups of ICR mice were vaccinated subcutaneously with each recombinant antigen and QuilA and later challenged with SzNC78. Results SzM protected 100% mice ( P P  = 0.01). MAP, SzMAC and ScpC each protected 63% ( P P  = 0.05), followed by HylC + MAP (50%; P  = 0.06) and ScpC + MAP (40%; P  = 0.1). Serum antibody responses of horses recently recovered from Sz respiratory infection were highest against ScpC, MAP and SzP. A combination of SKC and Sz115 stimulated no protection against challenge with SzW60. Conclusion A subset of Sz proteins reactive with convalescent equine antibody have potential as components of experimental vaccines to aid in prevention of opportunistic Sz infections.

  • Polymerase chain reaction-restriction fragment length polymorphism analysis of the SzP gene of Streptococcus zooepidemicus isolated from the respiratory tract of horses
    American journal of veterinary research, 2002
    Co-Authors: Toru Anzai, John F. Timoney, Yasushi Kuwamoto, Ryuichi Wada, Msaaki Oikawa, Tohru Higuchi
    Abstract:

    Objective—To develop polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for molecular typing of strains of Streptococcus zooepidemicus and to use the new typing method to analyze a collection of isolates from the respiratory tract of Thoroughbreds. Sample Population—10 strains of S zooepidemicus, 65 isolates from the respiratory tract of 9 yearlings following long distance transportation, and 89 isolates from tracheal aspirates of 20 foals with pneumonia. Procedure—Phenotypic variations in the SzP protein were detected by western immunoblot analysis. Using PCR-RFLP analysis, genotypes were obtained with primer sets from the SzP gene, followed by restriction endonuclease digestion of the amplicons. Results—Unique genotypic patterns were obtained with a primer set designed from both ends of the structural gene and the restriction endonuclease Dde I. Forty-five isolates from the lymphoid tissue within the pharyngeal recess (ie, pharyngeal tonsil) of yearlings included 10 S...

  • Comparison of the phenotypes of Streptococcus zooepidemicus isolated from tonsils of healthy horses and specimens obtained from foals and donkeys with pneumonia.
    American journal of veterinary research, 2000
    Co-Authors: Tohru Anzai, John A. Walker, Matthew B. Blair, T. M. Chambers, John F. Timoney
    Abstract:

    Objective—To determine whether streptococcal pneumonia is caused by strains of Streptococcus zooepidemicus similar to those obtained from the tonsils of healthy horses. Sample Population—5 tonsils from healthy horses, 8 tracheal washes and 6 lung specimens from foals with pneumonia, and 5 nasopharyngeal swab specimens from donkeys with acute bronchopneumonia. Procedure—Variable M-like protectively immunogenic SzP proteins of 5 isolates of S zooepidemicus from each tonsil and clinical specimen were compared, using immunoblots. The SzP gene of 13 isolates representative of various SzP immunoblot phenotypes from 1 healthy horse and 9 horses and donkeys with pneumonia were sequenced and compared. Cell-associated hyaluronic acid concentration and resistance to phagocytosis of some isolates were measured. Results—Tonsils of each healthy horse were colonized by several SzP phenotypes similar to those of foals or donkeys with pneumonia. In contrast, multiple isolates from animals with pneumonia had the same SzP p...

S. B. Chincholkar - One of the best experts on this subject based on the ideXlab platform.