Legionella pneumophila is the bacteria behind legionnaires disease, a severe disease with high mortality rates, but it infects you accidentally… But what do they mean when they say “accidental”? Is it like when I accidentally ate that whole pack of biscuits, or is it actually sincere? Well it turns out that L. pneumophila is a pathogen of single cell organisms named protozoa but when it is inhaled into the lungs it spots an immune cell called a macrophage. Now normally the bacteria wouldn’t infect the macrophage but just like gingernut biscuits, if there are no better options you have to settle for it. But now I bet you’re thinking “what do the bacteria do when they’re only given macrophages to survive in”? Well luckily for you a paper published in 2012 will answer all of your questions using experimental evolution methodology and I will try to make it a bit easier to understand.
So let’s start with what we already know. Within the vacuole of the mouse macrophage, which is where the bacteria replicate, lysine is present. It just so happens that most L. pneumophila can synthesize lysine and those which don’t have trouble replicating in lysine devoid environments (1). We also know that whilst the bacteria has many protozoan targets (~15) there are no natural mammalian reservoirs in which they replicate (2).
Methods and Results
In order to select for adaptions to improved survival and growth in a mammalian environment the researchers used mouse macrophages in minimal media as growth conditions and used genome sequencing to identify any mutations in the DNA that these adaptions may be attributed to.
Figure 1: Growth conditions for the four bacterial lineages of L. pneumophila in bone marrow-derived macrophages. Bacteria were extracted and used to infect new macrophages in three day intervals at an MOI of 0.05. Insertion of a lux operon allowed for cell counts using luminescence experiments (1).
Through whole genome sequencing the researchers were able identify the where the mutations were in the bacterial genome (figure 2). Multiple different mutations occurred in the genome and persistence of these mutations occurs due to a selective advantage or chance, so those mutations which decreased back to 0% of the population genotype after reaching relatively high levels such as lpg0981 likely had a negligible effect on cell growth or had a deleterious effect when coupled with another mutation. There are a few common pathways or structures which these mutations appeared to effect including flagellar structure (used for mobility in bacteria) and also in lysine synthesis.
Figure 2: Mutations present and population genotype in the four L. pneumophila lineages identified using illumine sequencing (1).
Interestingly enough we can also see that many of the mutant strains were able to out-compete the wild type strain (no mutations) in competition experiments. Competition experiments were conducted by infecting macrophages with equal amounts of wild type and a mutant and looking at the ratio after one growth cycle (3 days). If the mutant scores above one on the competitive index we can say that it had a selective advantage, or was able to infect and replicate at a higher rate than the wild type.
Figure 3: Competition experiments between wild type and mutants synthesized in the experiments (1).
Furthermore, when single mutant strains are used to infect protozoa they appeared to have a slower replication rate than that of the wild type (figure 4). In particular, we can see the lysine mutants in Figure 4 d, e and f have a very low growth rate in comparison to the wild type and falls well below one on the competitive index.
Figure 4: Comparative growth in single mutant strains compared to wild type in a variety of protozoa (1).
Additionally, whilst single mutations appeared to slightly increase the ability of L. pneumophila to survive an accumulation of those same mutation resulted in a far higher score on the competitive index, indicating the mutations have a synergistic effect with each other (figure 5a). These mutations may have resulted in some L. pneumophila strains which were grown in the experiment to become lysine auxotroph, which require lysine within the environment to grow (figure 5b).
Figure 5: (A) Accumulation of mutations appears to correlate with a higher competitive index than when the mutations are present alone. (B) Mutation in the macrophage experiments led to what appears to be lysine auxotrophy (reliant on the presence of lysine to grow) in L. pneumophila (1).
Well I guess that means L. pneumophila can adapt to have preferential growth in mammalian macrophages in a year. These experiments have shown that these clonal strains have a superior ability to grow in mammalian macrophages in comparison to wild type as well as a decrease in its competitive index score when growing in protozoa. This may be due to a loss of lysine synthesis as there is already lysine present in the vacuoles of mouse macrophage in addition to changes in flagellar genes. Overall it appears that wild type L. pneumophila isn’t directly targeting mammalian macrophage as a host, however we can see that given the have no other choice they will readily adapt.
- Ensminger AW, Yassin Y, Miron A, & Isberg RR (2012) Experimental Evolution of Legionella pneumophila in Mouse Macrophages Leads to Strains with Altered Determinants of Environmental Survival. PLoS Pathog. 8(5):12.
- Fields BS (1996) The molecular ecology of legionellae. Trends Microbiol. 4(7):286-290.