Proteomic Analysis of In Vivo Expressed and Immunogenic Proteins of Vibrio cholerae Regina C. LaRocque, Jason B. Harris, Bryan Krastins, Edward T. Ryan, David Sarracino, Firdausi Qadri, Stephen B. Calderwood Massachusetts General Hospital Harvard Medical School Harvard-Partners Center for Genetics and Genomics International Center for Diarrheal Disease Research, Bangladesh
Obstacles to Cholera Vaccine Development Natural infection with V. cholerae induces long-lived immunity, but mechanism is poorly understood Specific bacterial targets of mucosal and systemic immune responses are undefined
Approaches to Identifying Bacterial Targets of Human Immune Responses Plausible targets (e.g. cholera toxin) Functional genomics of organism in animal models in vivo expression technology signature tagged mutagenesis in human samples in vivo induced antigen technology gene microarray Proteomics
Transcriptional Profiling of V. cholerae during Human Infection LaRocque et al, I&I, 2005
TcpA is Immunogenic during Cholera Infection Asaduzzaman et al, I&I, 2004
Proteomic Approaches to Identifying Bacterial Targets Mass spectrometry 1. characterization of V. cholerae proteins present in patient samples 2. identification of immunogenic proteins among the pool identified in (1)
Methods: Proteome of Human- Derived V. cholerae Stool and vomitus collected from 6 cholera patients presenting to ICDDR,B prior to antibiotics/ors Bacteria collected by differential centrifugation & lyophilized Bacterial pellet resuspended in urea/sds/dtt Fractionated on Tris-glycine gel
Representative Gels Patient 1 Stool Patient 1 Vomit
Protein Digestion Proteins In gel Digestion of the proteins with an amino acid specific protease (trypsin) Peptide mixture
Liquid Chromatography Ion Trap Mass Spectrometry Time
Peptide Characterization and Database Matching Doubly charged peptide NH 3+ -Gly-Leu-Asp-Pro-Trp-Tyr-Arg + -CO 2 H NH 3+ -Gly -Leu-Asp-Pro-Trp-Tyr-Arg + -CO 2 H NH 3+ -Gly-Leu -Asp-Pro-Trp-Tyr-Arg + -CO 2 H NH 3+ -Gly-Leu-Asp -Pro-Trp-Tyr-Arg + -CO 2 H NH 3+ -Gly-Leu-Asp-Pro -Trp-Tyr-Arg + -CO 2 H NH 3+ -Gly-Leu-Asp-Pro-Trp -Tyr-Arg + -CO 2 H NH 3+ -Gly-Leu-Asp-Pro-Trp-Tyr -Arg + -CO 2 H
V. cholerae Proteins Identified in Six Paired Patient Samples Patient Stool Vomit 1 330 27 2 40 146 3 101 2 4 35 1 5 236 28 6 21 2
Functional Classification of 445 V. cholerae Proteins in Stool protein fate pathogenesis chemotaxis & motility protein synthesis hypothetical energy metabolism
V. cholerae Proteins Identified 1. Pathogenesis in Stool cholera toxin A & B subunits toxin coregulated pilin TcpF proteins in virulence cascade (TagD, AcfA) 2. Cell envelope outer membrane proteins 3. Energy metabolism iron regulation proteins 4. Chemotaxis and motility flagellar proteins methyl accepting chemotaxis proteins
Functional Classification of 169 V. cholerae Proteins in Vomit protein fate pathogenesis chemotaxis & motility protein synthesis hypothetical energy metabolism
V. cholerae Proteins Identified in Vomit 1. Regulatory functions LuxP (quorum sensing) 2. Pathogenesis TcpF other proteins in virulence cascade 3. Not identified cholera toxin toxin coregulated pilin
Which V. cholerae proteins expressed in vivo are immunogenic?
Methods: Identification of Immunogenic Proteins Acute and convalescent serum samples from 18 patients (Inaba, Ogawa, O139) were pooled IgG from pooled acute and convalescent serum was purified with HiTrap Protein G column (Amersham) Purified IgG bound to HiTrap NHS-activated column Tryptic digests of individual patient stool and vomit samples were bound to and eluted from IgG-charged acute & convalescent columns Eluates evaluated by mass spec
25 Proteins Recognized Uniquely by Convalescent Serum Protein Threonine dehydratase Thiazole biosynthesis protein Ubiquinone biosynthesis methyltransferase Glycerol 3-phosphate acyltransferase Thioredoxin LysE family protein Cholera enterotoxin, beta chain Cholera enterotoxin, alpha chain Response regulator Aldehyde dehydrogenase PTS system, nitrogen regulatory OmpS OmpU Histidine kinase rrna methylase Protein Hypothetical protein Hypothetical protein Hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein Hypothetical protein
Strengths & Limitations Strengths high-throughput no a priori assumptions Limitations not quantitative limit of detection unknown and may be affected by presence of non-microbial proteins method of isolating peptides may influence results tryptic digest mass/charge ratio vs. potential (electrical) only protein antigens are identified
Conclusions Mass spectrometry can be used to characterize V. cholerae proteins within a complex patient sample pathogenesis proteins are identified in vivo proteome of V. cholerae is similar between different patient samples Probing such patient samples using human antibodies is a promising approach to identifying bacterial targets of human immune responses
Future Plans Develop more quantitative approaches to assessing V. cholerae protein abundance Use acute and convalescent antibodies to probe an expression pool of sequenced V. cholerae clones representing entire genome 96-well plate format
Mass General Jason Harris Regina LaRocque Ed Ryan Steve Calderwood ICDDR,B (Dhaka) CIRS workers & participants Ashraful Khan Fahima Chowdhury ASG Faruque Firdausi Qadri HPCGG Bryan Krastins David Sarracino Funding: NIAID/ICTDR, Fogarty International Center International Research Scientist Development Award, ICDDR,B