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Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
Published on: July 1, 2011
Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein
Cathy X Y Zhang1, Brian W Brooks2, Hongsheng Huang2
1Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, Ottawa, Ontario, Canada Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
Unlabelled:
The Gram-positive bacterium Listeria monocytogenes causes a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range of L. monocytogenes serotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of the L. monocytogenes serotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains of L. monocytogenes which are variable among other Listeria species. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46 L. monocytogenes lineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17 L. monocytogenes lineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some other Listeria species grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker of L. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples.
Importance:
L. monocytogenes is traditionally divided into at least 12 serotypes. Currently, there are no monoclonal antibodies (MAbs) available that are capable of binding to the surface of L. monocytogenes strains representing all 12 serotypes. Such antibodies would be useful and are needed for the development of methods to detect and isolate L. monocytogenes from food samples. In our study, we aimed to identify surface proteins that possess regions of well-conserved amino acid sequences among various serotypes and then to employ them as antigen targets (biomarkers) for the development of MAbs. Through bioinformatics and protein expression analysis, we identified one of the four putative surface protein candidates, LMOf2365_0639, encoded by the genome of the L. monocytogenes serotype 4b strain F2365, as a useful surface biomarker. Extensive assessment of 35 MAbs raised against LMOf2365_0639 in our study revealed three MAbs (M3643, M3644, and M3651) that recognized a wide range of L. monocytogenes isolates.
Insights
Researchers identified LMOf2365_0639 as a promising surface biomarker for Listeria monocytogenes detection. Specific monoclonal antibodies targeting this protein show high reactivity across various L. monocytogenes serotypes, aiding in pathogen identification.
Area of Science:
- Microbiology
- Immunology
- Biotechnology
Background:
- Listeria monocytogenes is a major cause of fatal foodborne illnesses.
- Effective detection and isolation methods are crucial for public health.
- Current diagnostic tools lack antibodies that target all L. monocytogenes serotypes.
Purpose of the Study:
- To identify novel surface protein biomarkers for L. monocytogenes.
- To develop specific monoclonal antibodies (MAbs) for pathogen detection.
- To assess the efficacy of identified biomarkers and MAbs across diverse L. monocytogenes strains.
Main Methods:
- Genomic analysis of L. monocytogenes serotype 4b strain F2365 to identify putative surface proteins.
- Expression analysis and antibody generation against candidate proteins.
- Screening of monoclonal antibodies for specific binding to L. monocytogenes isolates.
- Testing antibody reactivity against various Listeria species and under different culture conditions.
Main Results:
- LMOf2365_0639 was identified as a surface protein expressed across L. monocytogenes strains.
- Three monoclonal antibodies (MAbs M3643, M3644, M3651) demonstrated high specificity and reactivity.
- These MAbs recognized a significant percentage (up to 91.7%) of L. monocytogenes isolates from different lineages and culture media.
- Minimal cross-reactivity was observed with other Listeria species.
Conclusions:
- LMOf2365_0639 is a validated surface biomarker for L. monocytogenes.
- The developed MAbs offer a promising tool for sensitive and specific pathogen detection.
- This approach can enhance the isolation and identification of L. monocytogenes in clinical, food, and environmental samples.
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