Identification and Expression Characterization of Surface Proteins for the Detection and Isolation of Listeria monocytogenes Cathy Xin Yue Zhang Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Doctorate in Philosophy degree in Microbiology and Immunology Department of Biochemistry, Microbiology and Immunology Faculty of Medicine University of Ottawa © Cathy Xin Yue Zhang, Ottawa, Canada, 2015 Abstract Listeria monocytogenes causes a serious foodborne illness (listeriosis) with a fatality rate of about 30% in susceptible individuals (1). Timely identification of foods and food processing environments carrying this deadly bacterium is crucial for implementing effective interventions but remains a practical challenge due to the complexity of test samples, low level of bacterial contamination, and the ubiquity and the genetic diversity of Listeria isolates. The purpose of this work was to identify and assess surface proteins of L. monocytogenes that can serve as diagnostic biomarkers for pathogen isolation and detection using antibody-based methods. Bioinformatics analysis of 130 putative surface proteins encoded by the genome of L. monocytogenes F2365 (serotype 4b) revealed four uncharacterized proteins with extensive amino acid sequences unique to L. monocytogenes. These proteins did not contain identifiable PrfA-controlled promoter elements. The four proteins were expressed at the transcriptional level in vitro, as demonstrated by RT-PCR, but only one of the four proteins, LMOf2365_0639, was detected on the cell surface by immunofluorescence microscopy (IFM) using rabbit polyclonal antibodies (PAbs) raised against corresponding recombinant proteins. Transcription start site mapping and promoter prediction analysis provided evidence that the LMOf2365_0639 gene was expressed under the control of a sigma B factor-dependent promoter, an alternative sigma factor involved in stress response. Non-gel based proteomics analysis of L. monocytogenes surface proteins identified 36 surface proteins in at least one of the three trials performed. IFM with PAbs raised against each of the five candidate surface proteins identified from the proteomics study revealed a strong fluorescence signal on the surface of live L. monocytogenes cells with LMOf2365_0148 specific PAbs, indicating a good level of expression of this protein. These results suggested the potential of the surface proteins LMOf2365_0639 and LMOf2365_0148 as diagnostic biomarkers for L. monocytogenes. ii Thirty-five and 24 monoclonal antibodies (MAbs) were developed against purified recombinant LMOf2365_0639 and LMOf2365_0148, respectively. Three MAbs against LMOf2365_0639 and five MAbs against LMOf2365_0148 were selected and evaluated for their potential in L. monocytogenes detection and isolation based on the observation that these MAbs recognized the highest number of the 53 L. monocytogenes isolates and the lowest number of the 10 other Listeria species isolates tested. None of these MAbs reacted with the four foodborne pathogens (Campylobacter jejuni, Samonella enterica serovar Typhimurium, Escherichia coli O157:H7 and Bacillus cereus) tested. All three MAbs to LMOf2365_0639 were specific for lineage I and II isolates of L. monocytogenes commonly found in clinical and food isolates respectively and recognized the N-terminal region of LMOf2365_0639. Anti-LMOf2365_0148 MAbs were reactive to lineage I and lineages III L. monocytogenes isolates commonly found in clinical and animal isolates respectively. Both LMOf2365_0639 and LMOf2365_0148 were expressed in standard enrichment culture conditions according to Health Canada’s MFHPB-30 and MFHPB-07 methods. In addition, MAbs against LMOf2365_0148 could specifically isolate live L. monocytogenes by immunomagnetic separation even in a mixture of L. monocytogenes and non-target L. innocua. The dissociation constants of the MAbs capable of capturing L. monocytogenes ranged from 2.58 x 10-8 M to 8.87 x 10-10 M. In conclusion, two novel surface proteins LMOf2365_0639 and LMOf2365_0148 were identified, were shown to be expressed in L. monocytogenes grown in standard selective enrichment cultures, and can be explored as surface biomarkers for the isolation and detection of L. monocytogenes with specific MAbs developed in this study. iii List of Tables Page Table 2-1. Major invasive listeriosis outbreaks from 1981 to 2010. 13 Table 3-1. Oligonucleotide primers used for RT-PCR. 44 Table 3-2. Oligonucleotide primers used in 5’RACE. 45 Table 3-3. Oligonucleotide primers for cloning surface protein candidates identified by sequence comparison. 46 Table 3-4. Oligonucleotide primers for cloning GST-rLMOf2365_0639 peptide fusion. 47 Table 3-5. Oligonucleotide primers for cloning surface protein candidates identified by proteomics. 45 Table 3-6. Bacterial isolates used in indirect ELISA. 52 Table 3-7. Bacterial strains used for immunomagnetic separation. 56 Table 3-8. Oligonucleotide primers used for epitope mapping of LMOf2365_0639 MAbs 61 Table 3-9. Oligonucleotide primers used for epitope mapping of LMOf2365_0148 MAbs 62 Table 5-1. Proteins of L. monocytogenes strain LI0521 (serotype 4b) identified in untreated and trypsin-treated cells. 114 Table 5-2. Proteins identifications observed in only one trial. 127 Table 6-1. OD414 readings of highly reactive L. monocytogenes isolates. 154 Table 7-1. Capture efficiency at various input cell numbers for five selected MAbs. 174 Table 7-2. Capture ability of MAbs M3686, M3697, M3699 and M3700 to other L monocytogenes serotypes. 176 Table 7-3. Capture ability of MAbs M3686, M3697, M3699 and M3700 to other Listeria species. 179 Table 7-4. Capture ability of MAbs M3686, M3697, M3699 and M3700 to Salmonella enterica serovar Typhimurium and E.coli O157:H7. 180 Table 7-5. Capture ability of MAbs M3686, M3697, M3699 and M3700 to L. monocytogenes after abbreviated culture in selective enrichment media. 185 Table 7-6. Association (ka) and dissociation (kd) rate constants and equilibrium dissociation (KD) constants for M3644, M3686, M3692, M3697, M3699 and M3700. 196 iv List of Tables Page Table S1. Proteins of F2365 L. monocytogenes containing LysM domain. 228 Table S2. Proteins of F2365 L. monocytogenes containing p60 domain. 228 Table S3. Proteins of F2365 L. monocytogenes containing GW modules. 229 Table S4. Proteins of F2365 L. monocytogenes containing LPXTG sorting motif. 230 Table S5. Lipoproteins of F2365 L. monocytogenes. 232 Table S6. Proteins of F2365 L. monocytogenes containing C-terminal hydrophobic tail. 237 Table S7. Summary of surface proteins identified in L. monocytogenes strain F2365. 237 v List of Figures Page Figure 2-1. Schematic of current Listeria detection methods employed by CFIA. 35 Figure 4-1. RT-PCR detection of candidate genes: LMOf2365_2117, LMOf2365_0639, LMOf2365_0581 and LMOf2365_0578. 68 Figure 4-2. PCR screening of recombinant inducible plasmids for LMOf2365_0578, LMOf2365_0581, LMOf2365_0639 and LMOf2365_2117 expression. 69 Figure 4-3. Coomassie Blue and western blot of purified recombinant surface protein candidates. 70 Figure 4-4. Western blot analysis of LMOf2365_0578, LMOf2365_0581, LMOf2365_0639 and LMOf2365_2117 proteins in whole cell extracts of L. monocytogenes strain LI0521. 71 Figure 4-5. Surface localization assessment of LMOf2365_0639, LMOf2365_0578, LMOf2365_0581 and LMOf2365_2117 proteins on live L. monocytogenes strain LI0521 by immunofluorescence microscopy. 73 Figure 4-6. Analysis of LMOf2365_0639 expression on the cell surface of L. ivanovii, L. seeligeri, L. welshimeri and L. innocua by immunofluorescence microscopy. 76 Figure 4-7. Sequence comparison of sequenced Listeria strains. 79 Figure 4-8. SDS-PAGE Coomassie Blue stain of purified His-GST and His-GST-peptide. 81 Figure 4-9. MAbs are reactive to live L. monocytogenes and purified recombinant LMOf2365_0639. 83 Figure 4-10. Detection of lineage I, II and III isolates of L. monocytogenes and other Listeria species and bacteria cultured in BHI by indirect ELISA. 85 Figure 4-11. Detection of Listeria isolates cultured according to the MFHPB-07 method by indirect ELISA. 90 Figure 4-12. Detection of Listeria isolates cultured according to the MFHPB-30 method by indirect ELISA. 93 Figure 4-13. Epitope mapping for M3651, M3644 and M3643 using four overlapping protein fragments (A, B, C and D) that span the full-length protein of LMOf2365_0639 protein. 97 Figure 4-14. Comparison of N-terminal residues (40 to 60) of the LMO2365_0639 protein with the corresponding region in L. monocytogenes and other Listeria species homolog protein by multiple alignment. 98 vi List of Figures Page Figure 4-15. Transcription start site of LMOf2365_0639. 100 Figure 5-1. SDS-PAGE and silver staining of supernatant after incubation in digestion buffer. 109 Figure 5-2. Colony-forming units (CFUs) of L. monocytogenes cells after incubation in digestion buffer. 110 Figure 5-3. Colony-forming units (CFUs) of trypsin treated and untreated L. monocytogenes cells. 111 Figure 6-1. PCR screen of recombinant plasmids of LMOf2365_0148, LMOf2365_0312, LMOf2365_0546, LMOf2365_2111 and LMOf2365_2742. 138 Figure 6-2. Coomassie blue stained SDS-PAGE gel and western blot analysis of purified recombinant surface protein candidates. 139 Figure 6-3. Western blot analysis of LMOf2365_0148, LMOf2365_0312, LMOf2365_0546, LMOf2365_1883, LMOf2365_2111 and LMOf2365_2742 proteins in whole cell extracts of L. monocytogenes strain LI0521. 143 Figure 6-4. Surface localization assessment