1. The Evolution of Virulence Lecture Outline Introduction to virulence theory Transmission mode experiment Transmission timing experiment Metapopulation experiment Summary

2. A Model Host-Pathogen System We used Escherichia coli (host) and phage T4 (pathogen) to study the dynamics of a large host-pathogen metapopulation. Bacteria and virus are grown in microtiter plates, which impose a metapopulation structure. E. coli Bacteria and phage do not coexist in a well. There are three types of wells: empty, bacteria-filled, and phage-filled, exhibiting “rock-paper-scissors” The transitions in the state of any well (due to dilution or immigration) can be gauged empirically and organized into a transition matrix. reproduction infectiondilution T4

3. Unrestricted Restricted Stochastic Cellular Automata density (  10 7 /mL) time phage bacteria density (  10 7 /mL) time phage bacteria F F Migration PatternEcological DynamicsSpatial Dynamics Predictions: Under restricted migration, (1) metapopulation dynamics are more stable (2) phage mean density is lower and bacterial mean density is higher

4. Restricted Unrestricted True Cellular Automata Migration Pattern m m metapopulationssterile shell arm Ecological DynamicsSpatial Dynamics Predictions: Under restricted migration, (1) metapopulation dynamics are more stable (2) phage mean density is lower and bacterial mean density is higher

5. For each evolved isolate, we measured: -productivity: the average number of progeny phage per parent -competitive ability: how well the evolved isolate does in head-to-head competition with a marked mutant In both cases, we controlled for the initial ratio of phage to bacteria (called the “multiplicity of infection”) Restricted phage were significantly more productive Unrestricted phage were significantly more competitive After pooling the data, we found (for 2 of 3 MOI levels) a significant negative correlation between productivity and competitive ability. Evolved Phage Properties

6. productivity competitive ability A Microbial ‘Tragedy’ Different migration treatments have evolutionarily favored different strategies: -“Rapacious” phage in the Unrestricted treatment -“Prudent” phage in the Restricted treatment. We have a tragedy of the commons: rapacious prudent phage evolve in the Unrestricted treatment phage persist in the Restricted treatment dilution reproduction -Rapacious phage outcompete prudent phage in a mixed population -Pure wells of prudent phage have higher progeny outputs than pure wells of rapacious phage. Why are rapacious phage found in the Unrestricted treatment? -As rapacious mutants are generated, they take over, lowering productivity -Less productive phage are less persistent. -The probability of migration to hosts is lower in the Restricted treatment -This limited host access in the Restricted treatment makes rapacious phage extinction-prone reproduction & competition

7. Rapacious phage fare better in the Unrestricted treatment for two reasons: 1) Mixing of phage types is more likely (leading to more tragedies) 2) Persistence is less important (any well’s tragedy is less severe) Averting the Tragedy of the Commons Restricted Migration with Evolution density (  10 7 /mL) prudent phage bacteria time Unrestricted: Tragedy Realized rapacious phage prudent phage bacteria density (  10 7 /mL) time Restricted: Tragedy Averted rapacious phage Take 3 minutes to talk to your neighbor about the following: So far the description of rapacious and prudent phage has been at the population level. What would you want to know about the phage itself in terms of its evolution? What would you want to know phenotypically? Genetically?

8. Lytic phage life cycle The Evolution of Phage Life History adsoption & injection progeny production host lysis phage (pathogen) bacteria (host) The life cycle of lytic phage: -Adsorption to host and injection of phage genome -Production of progeny particles in the host -Lysis of the host and progeny release Phage evolved under Unrestricted Migration are more infective, virulent, and tend to be shorter- lived outside their host.

9. From Demes to Genes TTTT inner membrane outer membrane periplasmic space RI T EEEEEEEEE TAAAAAT wild-type TAAAAT rI mutant OUTSIDE HOST CELL INSIDE HOST CELL rI A mutant rI B mutant wild type Current working model ( Tran et al. 2005 ): -At a specific time, holins disrupt the inner membrane allowing endolysin to pass. -Cell wall is degraded and the cell lyses. -Progeny phage are released. Gene t is an attractive candidate locus: -Non-synonymous mutations in holins produce different latent periods. -Null mutants overproduce progeny without lytic release (‘t’ from Tithonus) We found no mutations in gene t. Gene rI codes for an antiholin that forms a complex with the holin; mutations in rI can hasten lysis (shorten latent period). We found two unique deletions in rI: -More rI mutants were found in the Unrestricted treatment. -The rI mutations are sufficient to have visible effects on the host population.

10. A Genetic Basis for the Tragedy of the Commons Relative to wild type, the engineered rI mutants have: -A shorter latent period -A smaller burst size Relative to wild type, the engineered rI mutants are: -More competitive for hosts -Less productive when alone Mutations at rI are sufficient to generate a tradeoff between competitive ability and productivity. Thus, we have rapacious and prudent alleles at the rI locus: a genetic basis of the tragedy of the commons. adsoption & injection progeny production host lysis phage (pathogen) bacteria (host)

11. Acknowledgements Yen Nhan Dang Roxy Vouk Mily Gualu Josh Nahum Kelsea Laegreid Jodi Stewart Stacy Schneider Christal Eshelman Beth Halsne Jake Cooper Brandon Rogers Spencer Smith Sterling Sawaya Chris Shyue Kelsey Hobbs Sara Drescher Shawn Decew

12. The Evolution of Virulence Lecture Outline Introduction to virulence theory Transmission mode experiment Transmission timing experiment Metapopulation experiment Summary

13. Virulence is the damage to a host caused by an inhabiting pathogen (increased mortality, decreased reproduction, etc.) Virulence varies between and within pathogen species (both naturally and in the laboratory). The conventional wisdom is that virulence should decrease evolutionarily, but it is sometimes predicted to increase if it trades off with transmission or within host competition. Many factors (host density, superinfection frequency, environmental reservoirs) will affect the predicted level of virulence and some of these factors have been experimentally tested: - Bull et al. found higher virulence in a phage pathogen under horizontal transmission. - Cooper et al. found higher virulence in an insect pathogen under early transmission. - The pattern of migration in a metapopulation can affect the evolution of virulence