Sometimes it’s amazing just how little we know about the microbes around us. For precious few microbes, we know a good deal about virulence factors–genes and proteins that, when present, increase the severity of disease either in animal models or in humans (or both). However, much of this research has been done investigating acute infectious diseases, where one is infected, becomes ill, and gets better in the course of a few weeks to a month. Much less is known about factors that affect long-term (or chronic) infection. A recent study addressed one gap in this research, examining what happened in patients with cystic fibrosis who were chronically infected with the bacterium Pseudomonas aeruginosa.
Cystic fibrosis (CF) is a genetic disease that currently affects approximately 35,000 Americans, with about 1,000 new diagnoses every year. The average lifespan of CF patients is currently around 30 years; patients eventually succumb to the chronic lung infections that they suffer from during much of their lives. Common pathogens are Burkholderia cepacia and Pseudomonas aeruginosa. Both are gram negative soil bacteria. In humans, they’re opportunistic pathogens, infecting those with compromised immune systems. Both of these species are notoriously difficult to treat, having high levels of antibiotic resistance. In this study, they examined the changing population of P. aeruginosa in chronically infected cystic fibrosis patients.
The basic question they asked was this: how does the bacterial population evolve during the months and years of chronic infection?
Though P. aeruginosa is common in CF patients, studies have found that patients generally acquire the bacteria from environmental reservoirs, rather than other patients. Therefore, each patient will start out with a “wild type” isolate, adapted to an environment quite different from that found in the lung. In this study, Smith et al collected an isolate of P. aeruginosa early in a patient’s infection (6 months) and then a second 96 months into the infection–then they sequenced both of them and compared the two, detecting 68 mutations, including one that removed 188 kb of DNA (~139 genes). Approximately a quarter of the other mutations caused a stop codon, a frameshift mutation, a transposon insertion or a gene deletion. A number of genes previously reported to play a role in virulence, as well as antibiotic resistance genes, were also mutated. Interestingly, the mutated genes caused the 96-month isolate to have reduced biofilm formation. This was unexpected, as it’s been thought that biofilm formation was one reason that P. aeruginosa was such a successful pathogen in these types of infections.
Additionally, while some antibiotic resistance genes were mutated, the 96-month isolate still had increased resistance to several aminoglycosides. (This was as expected, as the patient had received this class of antibiotics). It was also found to have a mutation in the mutS gene, which encodes for a protein involved in DNA mismatch repair. Mutations in this gene, therefore, result in an increased mutation rate–again, this fits with their other results.
Interesting stuff by itself, and then they toss in a bit of extras. They also looked at an isolate collected from this patient at 30 and 60 months into the infection to see if the mutations in the 96-month isolate were present (and if so, how many). They found almost half of the mutations (26/68) in the 60-month isolate, and half again (13/26) in 30-month isolates. They also discovered several mutations that weren’t present in the 96 month isolate, allowing them to get an estimate of heterogeneity in the population at that point in time. They note:
Viewed together, the known mutations in patient 1 isolates indicate that parallel lineages existed during the early years of the infection and coexisted, in some cases, for several years. For example, mutations in mucA commonly occur during CF infections, and mucA mutations arose independently in three different lineages during the patient 1 infection. Isolates from a single time point are also heterogeneous, e.g., five of the six isolates collected at 36 months had detectably different genotypes.
Again, interesting, but are the mutations unique to this individual patient? Or are some of them common to other CF patients who acquire P. aeruginosa? That was their next question, where they examined other P. aeruginosa isolates from 29 additional CF patients that had matched P. aeruginosa isolates (from early infection to late infection). In these isolates, they sequenced 24 of the genes that were mutated in patient 1′s isolates, along with 10 genes that other studies had shown were frequently mutated. Most of them weren’t found to have a large amount of mutations, but they did find a few that were hotbeds of change. In these, mutations were selected for: 5 synonymous (silent) mutations were found in these genes, versus a whopping 103 non-synonymous mutations (that is, mutations that change the amino acid sequence of the encoded protein). They again saw a high number of loss-of-function mutations in these genes as well.
One limitation of this part of the analysis, however, is that they were starting from the genes that had already been identified in patient 1′s bacteria. It’s likely there are other genes mutated in these additional patient isolates that weren’t found in the isolates from Patient 1, so they’re missing some measure of the diversity (and, therefore, missing some genes that may play a role in this transition from acute to chronic infection). What would have been ideal would be to have the complete genetic sequences of all isolates–which, for now, remains cost-prohibitive.
A fascinating portion of this study showed that, in these chronic P. aeruginosa infections, many virulence factors were selected against. Now, virulence factors are generally called as such because they’ve been shown to be critical for a pathogen to establish an infection, or because when they’re lost, the lethality of the pathogen decreases substantially, as I mentioned above. But these analyses are generally based on acute infections, where infection is established (often by an artifical means, such as injection) and then either resolves, or the animal dies. As I mentioned in the first paragraph, much less is known about factors which are important for chronic infections. So-called “antivirulence factors” have also been identified: genes that, when knocked out or mutated, make the organism more virulent instead of less virulent. (Another example I’ve described previously is a mutation in a regulatory gene in Streptococcus pyogenes, where a frameshift mutation increased the virulence of isolates which possessed it). These are nice examples of how an organism can evolve due to loss-of-function mutations. Creationists like to crow about how “information can’t be added” and suggest that evolution is uni-directional, as if proceeding up some ladder. Of course, that’s simply not the case. In addition to this study, there are many examples of a population of organisms evolving–and losing unnecessary genes along the way. Carl Zimmer (in his new digs at Scienceblogs) gives two more examples, describing how a plant microbe and an animal pathogen (another species of Streptococcus, as a matter of fact) lost genes for functions that were no longer necessary for their new life in yogurt–genes encoding proteins responsible for metabolizing some sugars, or genes associated with virulence. Examples like this are so common that a new ASM book, Evolution of Microbial Pathogens, devotes an entire chapter to the phenomenon. How sad that all of this is dismissed as “mere microevolution” and waved away by creationists–they’re missing all the good stuff.
Smith et al. 2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. PNAS. 103:8487-8492. Link.
Image from http://www.ikp-stuttgart.de/ps-aerug.gif