Publications by authors named "Prakash Narayana Reddy"

9 Publications

  • Page 1 of 1

Application of IgY antibodies against staphylococcal protein A (SpA) of Staphylococcus aureus for detection and prophylactic functions.

Appl Microbiol Biotechnol 2020 Nov 22;104(21):9387-9398. Epub 2020 Sep 22.

Department of Biotechnology, Vignan's Foundation for Science, Technology and Research (Deemed to be University), Guntur-Tenali Highway Vadlamudi, Guntur District, Andhra Pradesh, 522 213, India.

In the present study, immunoglobulin Y (IgY) antibodies were raised in hens against the surface staphylococcal protein A (SpA) of Staphylococcus aureus. Anti-SpA IgY were tested in vitro for diagnostic applications, bacteriostatic, and biofilm inhibition effects. A specific and sensitive immunocapture PCR (IPCR) was developed to detect S. aureus from food, clinical, and environmental samples. Anti-SpA IgY were used for capturing S. aureus cells from different matrices. Chicken antibodies were chosen over mammalian antibodies based on its inertness to immunoglobulin (Ig)-binding property of SpA protein. No cross-reactivity was encountered with closely related Gram-positive and Gram-negative food pathogens. Inter-assay variation is < 10%. The assay was found suitable for testing on solid and liquid food samples, skin, and nasal swabs. The assay showed limit of detection of ≥ 10 CFU/mL from broth cultures and 10 to 10 CFU/ml from diverse natural samples. This assay overcomes the false positives commonly encountered while using mammalian immunoglobulins (IgG). Anti-SpA IgY antibodies were tested for their bacteriostatic effect on the growth of S. aureus. IgY antibodies at a concentration of 150 μg/ml inhibited the growth of S. aureus completely indicating the potential of IgY antibodies in neutralization of infectious pathogens. Similarly, anti-SpA IgY at MIC50 concentration reduced biofilm formation by ~ 45%. In view of advantages offered by IgY antibodies for specific detection of S. aureus in immunocapture PCR (IPCR) assay and in vitro neutralization potential of S. aureus, we recommend using IgY over conventional IgG of mammals involving S. aureus and its antigens. KEY POINTS: • IPCR with anti-SpA IgY for S. aureus was specific and sensitive for natural samples. • Anti-SpA IgY at 150 ug/ml displayed growth inhibition of S. aureus strains temporarily. • Anti-SpA IgY at MIC50 concentrations inhibited the biofilm formation partially.
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http://dx.doi.org/10.1007/s00253-020-10912-5DOI Listing
November 2020

Functional characterization of a broad and potent neutralizing monoclonal antibody directed against outer membrane protein (OMP) of Salmonella typhimurium.

Appl Microbiol Biotechnol 2020 Mar 29;104(6):2651-2661. Epub 2020 Jan 29.

Department of Microbiology, Defence Research & Development Establishment (DRDE - DRDO), Gwalior, Madhya Pradesh, 474 002, India.

In the present study, we have generated a murine monoclonal antibody (mAb) named Sal-06 by using the crude outer membrane protein preparation of Salmonella enteric subsp. enterica serovar Typhimurium ATCC 14028 strain as antigen. Sal-06mAb belonging to IgG1 isotype demonstrated broad cross-reactivity to standard and isolated strains of genus Salmonella and others such as Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. Cross-reactivity across several bacterial genera indicated that the epitopes reactive to Sal-06mAb are conserved among these members. Neutralizing effects of Sal-06mAb on Salmonella growth and survival was evaluated in vitro using bacteriostatic and bactericidal activity with and without complement and bacterial invasion inhibition assay. Sal-06mAb demonstrated a bacteriostatic effect on the growth of S. typhimurium ATCC 14028 strain which is both time and concentration (of mAb) dependent. It was also found that the bacterial growth inhibition was complement independent. When the bacterial cells were preincubated with Sal-06mAb, it reduced the adherence and invasion of bacterial cells into A549 epithelial cell line. This was confirmed by CFU count analysis, phase contrast, and fluorescence microscopy. Scanning electron microscope (SEM) imaging confirmed the antimicrobial effects of Sal-06mAb on S. typhimurium ATCC 14028. The development of broadly reactive and cross protective Sal-06mAb opens new possibilities for immunotherapy of sepsis caused by Gram-negative Enterobacteriaceae members.
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http://dx.doi.org/10.1007/s00253-020-10394-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080182PMC
March 2020

A novel tandem repeat cloning technique for creation of multiple short peptide repeats to differentiate closely related antigens.

J Immunol Methods 2019 06 17;469:11-17. Epub 2019 Jan 17.

Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka 570011, India.

Antibody cross-reactivity is a problem often associated with closely related antigens. This study was aimed to develop a method enabling differentiation of closely related toxins based on antigen designing strategy. The method involves identification of disparate amino acids (AA) confined to target antigen in comparison with two or more closely related antigens, their assembly into a DNA oligomer and further cloning as six tandem repeats (TR) using restriction and ligation strategy into a desired vector and finally generation of antigen specific antibodies. The practical utility of this method was demonstrated by generating and testing the specificity of polyclonal antibodies against staphylococcal enterotoxin C (SEC). Cross-reactivity is a problem often associated with SEC in immunoassays due to its amino acid sequence identity with staphylococcal enterotoxin B (SEB) (40-60%). To circumvent the same, the above-mentioned strategy was applied. Unique AA of SEC (36 AA) in comparison to SEB were selected, reassembled and with deduced corresponding nucleotides, an oligomer of 117 bases was designed. Using primers with restriction overhangs, three constructs were created each with two repeats using a common restriction site. The resulting three constructs were sequentially cloned into alternating restriction sites of pRSET A vector in directional orientation, expressed in E. coli for rTR/SEC protein which was used to generate specific polyclonal antibodies against SEC. Specificity was compared with antibody raised against whole SEC recombinant protein using Western blot and dot blot assays. High specificity was achieved through the developed strategy signifying its possible application to address cross-reactivity problem associated with closely related antigens.
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http://dx.doi.org/10.1016/j.jim.2019.01.008DOI Listing
June 2019

Recent Advances in Probiotics as Live Biotherapeutics Against Gastrointestinal Diseases.

Curr Pharm Des 2018 ;24(27):3162-3171

Department of Biotechnology, Vignan's Foundation for Science, Technology and Research, Vadlamudi, Guntur-522213, Andhra Pradesh, India.

Background: Gastrointestinal (GI) diseases are a major cause of emergency department visits requiring hospitalizations leading to considerable burden on global economy. Several factors contribute to the onset of gastrointestinal diseases such as pathogens (parasites, bacteria, virus, toxins etc.), autoimmune disorders and severe inflammation of intestine.

Objective: One common feature among all these diseases is the dysentery and alteration of gut microbiota composition (gut dysbiosis). Apart from conventional therapies such as antibiotics and ORS supplementation, gut microbiota modulation with probiotic supplementation has emerged as a successful and healthy alternative in mitigating GI diseases. In this review our goal is to discuss the causes of gastrointestinal diseases and the present state of various therapeutic strategies such as probiotics as live biotherapeutics and Fecal Microbial Transplants (FMT's).

Conclusion: Several reports and clinical trials point out to the beneficial effects of probiotics in modulating the gut microbiota and improving the side effects of gastrointestinal diseases. Live biotherapeutics and FMT's could be suitable and successful alternatives to conventional therapies in mitigating the gastrointestinal pathogens.
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http://dx.doi.org/10.2174/1381612824666180717105128DOI Listing
November 2019

An efficient method for integration of PCR fragments into adjacent or overlapping restriction sites during gene cloning.

3 Biotech 2018 Apr 24;8(4):197. Epub 2018 Mar 24.

Department of Biotechnology, Vignan's Foundation for Science, Technology and Research (VFSTR), Vadlamudi, Guntur District, Andhra Pradesh 522213 India.

In the present work, a simple and straightforward method was developed to clone any PCR-amplified products into restriction sites that are very close, adjacent or overlapping in the expression vector. The novelty of the methodology involves a crucial primer-designing step by adding appropriate overhangs to the 5' ends of primers based on the multiple cloning sites (MCS) (polylinker) region of expression vector. After PCR amplification, actual cloning is performed not in adjacent RE sites, but in sites that are little distant in the MCS. However, the sites lost during this cloning step are maintained intact since they are provided by the cloned PCR product (through the primer overhangs). Gene for green fluorescent protein (GFP) was cloned and expressed employing this strategy to demonstrate its simplicity. This method is highly useful for vector modification without losing the restriction sites present in the MCS.
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http://dx.doi.org/10.1007/s13205-018-1214-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866252PMC
April 2018

An Update on Clinical Burden, Diagnostic Tools, and Therapeutic Options of .

Infect Dis (Auckl) 2017 22;10:1179916117703999. Epub 2017 May 22.

Department of Biotechnology, Vignan's University, Guntur, India.

is an important pathogen responsible for a variety of diseases ranging from mild skin and soft tissue infections, food poisoning to highly serious diseases such as osteomyelitis, endocarditis, and toxic shock syndrome. Proper diagnosis of pathogen and virulence factors is important for providing timely intervention in the therapy. Owing to the invasive nature of infections and the limited treatment options due to rampant spread of antibiotic-resistant strains, the trend for development of vaccines and antibody therapy is increasing at rapid rate than development of new antibiotics. In this article, we have discussed elaborately about the host-pathogen interactions, clinical burden due to infections, status of diagnostic tools, and treatment options in terms of prophylaxis and therapy.
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http://dx.doi.org/10.1177/1179916117703999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443039PMC
May 2017

Congenital Hypothyroidism: Facts, Facets & Therapy.

Curr Pharm Des 2017 ;23(16):2308-2313

Department of Biotechnology, Vignan's Foundation for Science, Technology and Research University (VFSTRU), Guntur-5222, Andhra Pradesh. India.

Background: Thyroid hormone (T3) is essential for normal development of children enabling brain development and somatic growth. However, certain individuals are genetically predisposed with insufficient or no thyroid hormones. Such a condition is termed congenital hypothyroidism (CH).

Objective: In the present review, a brief back ground about congenital hypothyroidism, factors associated with CH leading to thyroid dysgenesis and thyroid dyshormonogenesis is elaborated. Additionally, the guidelines for available treatment options, management and follow-up required for patients diagnosed with CH are discussed. Treatment options in terms of treatment initiation and dosage of hormone replacement are discussed.

Conclusion: Though CH is considered as the most common neonatal metabolic disorder, it is also easily treatable compared to other metabolic or hereditary diseases. The outcome of CH treatment depends on the compliance of parents early in life and by patients themselves during later part of life.
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http://dx.doi.org/10.2174/1381612823666170206124255DOI Listing
April 2018

Evaluation of recombinant SEA-TSST fusion toxoid for protection against superantigen induced toxicity in mouse model.

Toxicon 2015 Sep 16;103:106-13. Epub 2015 Jun 16.

Department of Microbiology, Defence Food Research Laboratory, Mysore 570011, India. Electronic address:

Treatment of Staphylococcus aureus infections has become complicated owing to growing antibiotic resistance mechanisms and due to the multitude of virulence factors secreted by this organism. Failures with traditional monovalent vaccines or toxoids have brought a shift towards the use of multivalent formulas and neutralizing antibodies to combat and prevent range of staphylococcal infections. In this study, we evaluated the efficacy of a fusion protein (r-ET) comprising truncated regions of staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST-1) in generating neutralizing antibodies against superantigen induced toxicity in murine model. Serum antibodies showed specific reactivity to both SEA and TSST-1 native toxins. Hyperimmune serum from immunized animals protected cultured splenocytes from non-specific superantigen induced proliferation completely. Passive antibody administration prevented tissue damage from acute inflammation associated with superantigen challenge from S. aureus cell free culture supernatants. Approximately 80% and 50% of actively and passively immunized mice respectively were protected from lethal dose against S. aureus toxin challenge. This study revealed that r-ET protein is non-toxic and a strong immunogen which generated neutralizing antibodies and memory immune response against superantigen induced toxic effects in mice model.
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http://dx.doi.org/10.1016/j.toxicon.2015.06.008DOI Listing
September 2015

Application of a Chimeric Protein Construct having Enterotoxin B and Toxic Shock Syndrome Toxin Domains of S. aureus in Immunodiagnostics.

Indian J Microbiol 2012 Sep 31;52(3):449-55. Epub 2012 Mar 31.

Department of Microbiology, Defence Food Research Laboratory, Siddharthanagar, Mysore, 570011 India.

Staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 are the super antigens responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. At low serum concentrations, SEB can trigger toxic shock, profound hypotension and multi organ failure and hence is recognized as biowarfare molecule. In this study, a multidomain fusion protein (r-TE) was generated with specificity for SEB and toxic shock syndrome toxin (Tsst-1). The fusion gene comprising the conserved regions of seb and the tsst genes was codon-optimized for expression in Escherichia coli and encoded a 26 kDa recombinant multidomain chimeric protein (r-TE). Hyperimmune antiserum raised against r-TE specifically reacted with SEB (~28 kDa) and Tsst-1 (~22 kDa) components during Western blot analysis and by plate ELISA in confirmed toxin producing strains of S. aureus. The antigenicity of the SEB component of the r-TE protein was also confirmed using TECRA kit. The described procedure of creating a single protein molecule carrying components of two different toxins whilst still retaining the original antigenic determinants of individual toxins proved highly advantageous in the development of rapid, reliable and cost effective immunoassays and may also have the potential to serve as candidate molecule for vaccine studies.
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http://dx.doi.org/10.1007/s12088-012-0269-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460130PMC
September 2012