MIIM30011 2024 L11-Workshop_1_virulence_factors-1.pdf

 MIIM30011 – Medical Microbiology: Bacteriology Workshop 1 – Identification of virulence factors  Workshop aims To learn how scientists identify virulence factors using molecular methods To work with colleagues to provide and receive constructive feedback on writ...

 MIIM30011 – Medical Microbiology: Bacteriology Workshop 1 – Identification of virulence factors  Workshop aims To learn how scientists identify virulence factors using molecular methods To work with colleagues to provide and receive constructive feedback on written work How do we identify virulence factors? Experimentation!! Koch’s Postulates – rules to experiment by? 1. The microbe must be found in all organisms suffering from the disease and not in healthy organisms. 2. The microbe must be isolated from the diseased organism and grown as a pure culture. 3. A pure culture of the microbe, when inoculated into a susceptible and healthy host, must reproduce the disease. 4. The microbe must be reisolated in pure culture from the experimentally infected host. Can you see anything wrong with Koch’s postulates? Koch’s Postulates in the molecular era 1. "The phenotype or property under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species.” 2. "Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence." 3. "Reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity." Identifying virulence genes 1. What was the phenotype we were interested in understanding? 2. What was our model? 3. What was our method? Think it over by yourself → choose a partner → share your answer with them Phenotype and model Method Transformation Antibiotic selection Array into 96-well tray Results Carey and Newton, PLoS Pathog, 2011  Screening… Can you think of a better way to screen? What are some problems with this approach? What are some benefits?  Interesting findings Phenotypes associated with disruption of some Dot/Icm effectors Similar studies Martinez et al., PLoS Pathog, 2014 cig2::Tn Higher throughput → TnSeq Chao et al, 2016, Nat Rev Micro TnSeq results http://www.cureffi.org/2014/10/01/genetics-11/  TnSeq advantages Capacity to test very large numbers of mutants all at once – statistically very powerful If using an animal model – significant reduction in number of animals used  Trial short answer questions A) There are two recognised mechanisms for bacteria to invade host cells. Describe the key features of each of these mechanisms and include in your description an example of a pathogen that uses each mechanism. (4 marks) B) Once inside a host cell, an intracellular bacterial pathogen will establish a replicative niche that is either cytosolic, intravacuolar or intralysosomal. Select a bacterial pathogen that establishes a cytosolic niche and describe the virulence traits that the pathogen uses to thrive in this niche. (4 marks)