The Red Queen's race. Source
There was a time when constructive discussions about microbial pathogens had reached their limits when someone brought up evolution. Students giving thesis defenses or preliminary exams knew they were home free when a committee member asked “so, how do you think this system may have evolved?” Speculation, hand-waving, and just-so storytelling ensued, and everyone joined in because of course none of us knew what we were talking about, and data to support or refute any particular hypothesis was scant. Well, those days are gone. An emerging body of work—aided by advances in next-generation sequencing—is making a frontal attack on mechanisms of evolution, including its role in shaping host-microbe interactions. These studies are uncovering the continual efforts through which pathogens and their hosts jockey to withstand each other’s attempts to survive over time (which is my clumsy way of recasting the Red Queen Hypothesis).
How do we know about these evolutionary battles? A standard approach to understanding evolution of protein function is to carry out a BLAST search. We often do these to uncover areas of homology between that protein and others in the database. Conserved areas might tell us about constraints on critical regions of the protein during evolution. These might be binding sites for small-molecule cofactors or substrates. Such areas of high conservation are said to have undergone “purifying selection,” eliminating many codon options (and, consequently, amino acids) that do not enable these important molecular interactions. But selection leads to enhanced fitness, which requires more than just conserving traits through purifying selection. There is also diversifying selection, in which fitness is enhanced by rapid change—as opposed to constraints—in particular sequences. How is “rapid” measured in evolutionary time? When base changes lead to codon changes more frequently than the overall changes that occur within a coding sequence, those codons are undergoing rapid, positive selection.
Conflict associated with host-pathogen interactions can be a cause of rapid evolution. In the Red Queen model of the battle for survival, pathogens and their hosts keep the stakes high by fighting back when the other gains a fitness advantage. Think of an escalating arms race: weapons developed by one side are rapidly, and specifically, countered with defenses by the other. Spears lead to shields; bullets lead to armor; missiles lead to…well, anti-missiles. In these conflict situations, the phrase “adapt or die” has a literal connotation. A neat example of this comes from research on how pathogens and hosts wage a war for iron. Because iron is so precious, the host tries to keep a tight grip on it and uses proteins with very high avidity for iron called transferrin and lactoferrin. Microbes in turn have evolved mechanisms to harvest low concentrations of iron, including producing transferrin or lactoferrin-binding proteins that enable acquisition of iron from those host molecules.
A region of transferrin, it turns out, is under positive selection, meaning it is evolving rapidly. This region is where the microbial transferrin-binding protein TbpA binds, suggesting that a driving force behind the rapid evolution of transferrin is its interaction with TbpA. TbpA from two host-restricted microbes—Neisseria gonorrhoeae and Haemophilus influenzae—was tested for the ability to bind either human or nonhuman transferrin. Both molecules bound well to transferrin from some species, such as humans and gorillas, but poorly to transferrin of other species including chimpanzees, orangutans, and baboons. And—here’s the Red Queen notion at work—TbpA is also evolving: its protein sequence in different strains of N. gonorrhoeae, N. meningitidis, and H. influenzae shows evidence of variation precisely within the regions of the protein that bind transferrin, making the variants either more or less likely to recognize the two major transferrin variants found in human populations.
These types of tradeoffs and point-counterpoint variations are found in many other host-pathogen genetic conflicts. They often occur in interactions with the innate immune system, that earliest apparatus for recognizing and responding to danger. For example, variation in a virus capsid structure to avoid detection is mirrored by variation in a host protein that recognizes the capsid to keep the virus from replicating. These narrow battlefields of evolution tell us about molecular mechanisms in the host-pathogen biology, and also define the much broader nature of host tropism by specific pathogens. Work in this general area of genetic conflict and evolution is of course being carried out in many labs, and a recognized leader is Harmit Malik of the Fred Hutchinson Cancer Center in Seattle, who will receive the prestigious Eli Lilly Award at the ASM Microbe meeting in June; if you attend Microbe, make sure to attend that Award lecture.
There are other areas where experimental biology makes those hand-waving discussions about evolution relics of the past. With advances in deep sequencing technology, it is possible to gain insight into evolving populations of bacteria within a single patient! Cystic fibrosis patients suffer from debilitating, life-threatening lung infections from Pseudomonas aeruginosa, Burkholderia spp., and other microbes. A recent study determined the genome sequences of 22 B. multivorans isolates recovered from a single patient over 20 years. Over this period, the original isolate evolved through numerous single-nucleotide polymorphisms and indels into four distinct clades, the latest of which included a mismatch repair mutation that significantly increased the overall mutation frequency in that clade. A few mutations were fixed very early as the clades evolved, and multiple codon mutations within specific genes suggested that these were under selection during the 20-year evolutionary scale being observed. Regulatory/signaling systems, lipid metabolism, and a few other physiological traits appeared to be the target of many mutations, and one early mutation that remained in three of the four clades led to elevated expression of the second messenger cyclic di-GMP, associated with reduced motility and increased biofilm formation.
A close-up view showing the Ara-3 population and two others from Richard Lenski's long-term evolution experiment with E. coli. The middle Ara-3 population is more turbid because it evolved the capacity to use the citrate in the DM25 liquid medium. Source
Examining how these strains evolved over time in a single patient is more than just a captivating exercise in deep-sequence analysis. Connecting the evolution of the pathogen with the status of the patient—showing that the rise of clade three was associated with the greatest decrease in lung capacity—suggests that the phenotypic traits of this clade might be appropriate targets for therapeutic development. Applications of evolution in medicine are emerging as a promising approaches to understanding and fighting disease.
A discussion of the modern study of evolution in microbial sciences would be criminally negligent without noting the Long Term Evolution Experiment (LTEE) carried out by my Michigan State colleague Richard Lenski and his laboratory (Rich blogs here). Initiated in 1988, this experiment is based on 12 parallel cultures of Escherichia coli, growing in a minimal glucose medium and subcultured every day for the past 29 years. Every 500 generations (about 75 days), a sample is frozen away, providing a fossil record of evolutionary change extending to well over 65,000 generations (which amounts to more than a million people-years of evolution).
Malcolm Gladwell famously proposed that it takes 10,000 hours of practice to master something (a conclusion that has not gone unchallenged,) The LTEE has extended now for over 10,500 days (see video below). If Gladwell is right, and assuming at least an hour a day spent subculturing alone, it is safe to say that Lenski and his colleagues have mastered the study of evolution with this experiment. Through the LTEE, they have tested the reproducibility of evolution and examined questions of ecological specialization in populations, the dynamics of adaptation over time, the rationale underlying evolution of high mutation rates, and other Big Questions. The LTEE has also stimulated other investigators to apply experimental evolution in more complex host-pathogen systems.
So at your next thesis committee meeting, let’s have no more storytelling and speculation after the question of “how did this evolve?” The tools and concepts for studying evolution in microbial systems are out there, so adapt your thinking appropriately. To help you get up to speed, spend some time with the thoroughly enjoyable ASM podcast, This Week in Evolution, hosted by Vincent Racaniello and Nels Elde (listen to an episode below).