Abstract
Helicobacter pylori (H. pylori) is one of most commonly found pathogen in the stomach. In spite of emergence of different treatment strategies, H. pylori infection remains difficult to treat. The bioengineered probiotic lactobacilli that could displace H. pylori and simultaneously present immunogenic peptides such as heparan sulphate binding protein (Hsbp) to elicit immune response could emerge as a potential therapeutic agent. The aim of this study was to discover the anti-H. pylori activities and faster exclusion of H. pylori from host cells by the recombinant strain of Lactobacillus expressing the immunogenic Hsbp protein. The results were promising and showed a 65% reduction in H. pylori adhesion after two hours of pre-incubation with recombinant-LGG and HeLa S3 cells, followed by the adhesion of H. pylori pathogen (P < 0.002). Additionally, 36% and 39% reduction were examined in co-incubation and post-incubation with recombinant-LGG, respectively. When challenged with H. pylori, the proinflammatory cytokine expression was also down regulated in recombinant-LGG treated HeLa S3 cells. This promising result provides a new insight of bioengineered probiotic lactobacilli which could displace H. pylori and simultaneously has immunogenic properties thereby may be useful to prevent H. pylori infection.
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Funding
This research work was financially supported by the Indian Council of Medical Research (ICMR), New Delhi, under the ICMR-PDF (3/1/3/PDF(7)/2013-HRD) program and Science and Engineering Research Board, Government of India, under Early Carrier Research Award (No. ECR/2017/000270) Grant.
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AKY received funds, conducted experiments, and wrote the manuscript. SVR conduct experiments of qPCR and analysed data. ND, AK and MK analysed data of the manuscript. RH thoroughly revised the manuscript for its intellectual content and gave final approval for the corrected version of the manuscript. All authors have read and agreed to the published version of the manuscript.
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13205_2022_3428_MOESM1_ESM.pptx
Supplementary file1 Fig. S1 Growth curve of (A) recombinant-LGG and (B) non-recombinant LGG at different pH-2.5, 4.0 and 6.5. Growth pattern of recombinant-LGG and non-recombinant-LGG at different pH on various time intervals were non-significant (P>0.05). Fig. S2 Agarose gel showing PCR amplification of Signal and anchor sequences. (A) 1. Csp signal from genomic DNA of LP 21, 2. PrtR Anchor from Genomic DNA of LGG, M. 50 bp DNA Ladder, (B) Overlap PCR amplification fusion product using Csp_F_Nco1/PrtR_R_HindIII, M. 1kb DNA Ladder. Fig. S3 Expression study of recombinant LGG expressing green fluorescent protein. (A) Control: LGG cells harbouring pSIP503CMR (B) Fluorescence detection of recombinant-LGG cells harbouring pSIP503CMR-Gfp induced with 50 ng/ml of nisin using fluorescent microscope. Fig. S4 SDS-PAGE and western blot of Hsbp protein isolated from recombinant-LGG. SDS-PAGE showing the expression of Hsbp protein induced with nisin, Lane: 1. Un-induced LGG culture harbouring pSIP503CMR-Hsbp cell lysate, 2. LGG culture harbouring LGG-pSIP503CMR-Hsbp cell lysate induced with 25 ng/ml of nisin, 3. LGG culture harbouring LGG-pSIP503CMR-Hsbp cell lysate induced with 50 ng/ml of nisin. B. Western blot showing recombinant Hsbp protein using Novex ECL Chemiluminescent detection system. Fig. S5 Nucleotide sequence of the fusion construct. 1-6: NcoI, 7-71: Csp (signal sequence), 72-107 NEXT (NdeI, EcoRI, XhoI, Thrombin), 108-638: prtR (Anchor sequence) and 638-644: HindIII (PPTX 1272 KB)
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Yadav, A.K., Varikuti, S.R., Kumar, A. et al. Expression of heterologous heparan sulphate binding protein of Helicobacter pylori on the surface of Lactobacillus rhamnosus GG. 3 Biotech 13, 19 (2023). https://doi.org/10.1007/s13205-022-03428-4
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DOI: https://doi.org/10.1007/s13205-022-03428-4