“It’s close to midnight, something evil’s lurking from the dark under the moonlight. You see a sight that almost stops your heart-- you try to scream, but terror takes the sound before you make it. You start to freeze as horror looks you right between your eyes…” Michael Jackson’s album “Thriller” hit shelves on November 30, 1982. Little did he know at the time that he wrote the perfect monologue of one of humankind’s most successful bacterial pathogens discovered that same year. In the scientific world, the beast about to strike was Helicobacter pylori, the cause of most peptic ulcers and gastritis, was uncovered. 1982 was most definitely a thrilling year.
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In a time when doctors believed that peptic ulcer disease was caused by excessive stress and a problematic lifestyle, this discovery was, at first, laughable. How could something so small be such a huge problem? Scientists Barry Marshall (left) and Robin Warren (right) had to fight their way through the clinical community to get their voices heard; Marshall even performed an experiment on himself, ingesting the microbes after proving his digestive system was healthy. Two weeks after he planted the microbes in his body, an infection in his GI tract started to develop. If it weren’t for Marshall’s conviction, though, one of the biggest breakthroughs in the modern scientific community would never have been accepted.
Since Marshall’s gutsy experiment, the infection by Helicobacter pylori, the zombie of the bacterial world, has become one of the best-studied examples of pathogen biology. The way the microbe spreads infection and causes disease in the human digestive tract has become a hot topic of research; so much so that it got its very own academic journal! Marshall and Warren were awarded the Nobel Prize for Physiology or Medicine in 2005. For something so small, H. pylori sure had a massive impact!
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| An artistic rendering of what H. pylori does once it gets into the lining of its host’s GI tract. Source. |
Now, how exactly the microbes cause peptic ulcers and/or gastritis has not been completely ironed out. But in a way, they act as creatures crawling in search of blood to terrorize y’all’s neighborhood, just as Michael Jackson describes… Except the “ neighborhood” in this case is your gut. It is known that after ingestion of the microbes, they advance through the mucous lining of the GI tract and divide in close proximity to epithelial cells on the surface of the stomach. There, they produce an acidic chemical, which eventually triggers specific inflammatory and widespread immune responses in the body. After a while, this results in peptic ulcers or gastritis, sometimes even leading to gastric cancer.
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| An image of H. pylori taken by a transmission electron microscope. Source. |
Not all sequences available for recombination are created equal: some are far more similar to the microbe’s native genome than others, and the team speculated that the microbes might only “like” to take up DNA that is already quite similar to their own. They refer to this kind of DNA as “homeologous,” and performed experiments comparing this and heterologous recombination events. Before we get to what they did, though, we have to understand that bacteria have bookkeeping methods in place to decipher the differences between DNA that is sufficiently similar or different from their own. One such example is the restriction-modification (R-M) system. R-M systems provide protection against heterologous DNA and rely on a series of enzymes called “restriction endonucleases,” or REases for short. REases are the cell’s “molecular scissors” and are able to recognize and cut out certain sequences of double-stranded DNA after replication if they do not match up with the native sequence. The “match” is denoted by methylation. Think of it as a sort of colorful jacket that the microbe’s own DNA is wearing, but the new DNA is not-- it makes it easy for the REase to tell which is which. This method is displayed in a diagram below; note that the pink lines signify methyl groups.
With this knowledge, Bubendorfer and his colleagues explored how H. pylori’s R-M systems might react to different levels of methylation in imported DNA to influence what kind of DNA recombination takes place, if at all. The team first acquainted themselves with H. pylori’s genome and what its import patterns are. To do this, they looked at a particular strain and performed a series of transformations on it, then sequenced whole genomes at random. They found that it was able to incorporate both short and long stretches of DNA in their lab, and also in a study of chronically-infected humans. Then, they got to the nitty-gritty: this next section assumes that you are familiar with the process of DNA replication. If you are not, click HERE, and then come back!
Bubendorfer and his colleagues proposed that homeologous and heterologous DNA behave differently at the point of replication, because that is when REases get involved. Both types of DNA can be easily transported into the cell, but when the DNA goes to replicate, whether or not the sequence is methylated matters. This is how the REases are able to recognize the DNA as “native.” If, after replication, there is a double-stranded sequence of nucleotides that is not at all methylated, then it susceptible to be chopped out by REases. However, in the case of homeologous recombination, the imported DNA could be similar enough to the recipient to successfully incorporate and pass through replication unscathed. This is because the homeologous-native sequence pairing would always be at least partially methylated, even after replication, so it would fly under the REases’ radar and be passed to a daughter cell with the new gene intact. The team investigated this idea by manipulating different levels of methylation in import sequences that were resistant to the common antibiotic, chloramphenicol. Their reasoning was that if the imported antibiotic-resistant DNA was not at all methylated, then it would not be taken up by the recipient strain. Therefore, it wouldn’t grow on a plate treated with the antibiotic. However, if it was methylated in some way, then it had a higher potential to be taken up and successfully recombined with the recipient’s native DNA, and it would survive in the presence of chloramphenicol.
For a particular series of experiments, they selected for resistance to chloramphenicol by introducing a “CAT” gene cassette which was heterologous to the wild-type host strain of H. pylori they were using, which was called “26695.” Introducing the cassette included a couple of caveats to the design of the experiment. The first was that this CAT cassette was flanked on either side by homeologous stretches of the gene they were to be incorporated with, meaning it had the potential to be recognized by the recipient strain as similar-enough to its own DNA to successfully replicate. The second caveat was that the cassette contained sites for two common REases, GATC and GANTC in between the homeologous border sequences on either side. They were able to control whether these two sites were methylated or not, which served as the basis for the experiment. Remember: if a sequence is methylated and similar enough to native DNA to pair and then replicate, then the imported gene has been successfully incorporated into the genome because it was not cut out by REases.
Figure 5 (Bubendorfer et al. 2016):
While this figure looks intimidating, it conveys exactly the results the team was looking to confirm. Essentially, it compares the growth of the recipient H. pylori strain 26695 on a chloramphenicol-containing medium with different import events. The higher up the data points are, the more successfully the imports integrated into the recipient chromosome. Panel a shows a comparison between the wild-type 26695 strain that was exposed to donor DNA coding for different methylation patterns of GATC and GANTC REases-- the ∆ means that there was no methylation on that particular site. Notice the subtle decrease in abundance from left to right: this confirms the postulate that less- or un-methylated DNA is less easily incorporated than methylated DNA, since there was less growth in the presence of less methylation. Panel b shows a comparison between some amount of and absolutely no methylation in the recipient genome, as denoted on the x-axis. The ∆∆∆∆ REase is a quadruple mutant, meaning that the recipient strain has absolutely no REase activity whatsoever. We can see here from the huge jump between the data on the left and right of panel b that the difference between the uptake of methylated and non-methylated DNA disappears in the absence of REases. The quadruple mutant without any REases was able to recombine DNA regardless of whether or not it was methylated. This suggests that REase activity, methylation status, and the likelihood of recombination occurring are all interconnected in the ways described above.
Implications and Conclusions
Bubendorfer et al.’s findings indicate that REases inhibit heterologous DNA from successfully combining into the recipient’s native chromosome. This supports the previously-proposed model of recombination they were operating under, as well as provided a new direction of study. They were the first researchers to differentiate between heterologous, homologous, and homeologous imports before, and by doing so, they also appear to have proposed an explanation to the paradox of how H. pylori as a species is able to undergo recombination at a high rate but also conserve distinctive parts of its genome over time.
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| An immunostain of H. pylori (brown) associating with cells in the gastric mucus (blue). Source. |
Perhaps the significance of this study traces back to Bubendorfer et al.’s brief study of import patterns in chronically-infected individuals. H. pylori as a species is becoming increasingly resistant to antibiotics, but with this new knowledge, clinicians can work to keep the Helicobacter-pocalypse at bay for a while longer. The R-M system could be an effective target, which could plausibly lead to a couple different outcomes. Researchers could try to deactivate the R-M system and then introduce heterologous DNA that is incredibly susceptible to antibiotics. The bacteria wouldn’t be able to put up a counter-attack, and would eventually die, thus eradicating the infection. This could potentially exterminate human infection faster and more effectively than ever before. Ultimately, Bubendorder et al.’s research allows scientists to develop a more nuanced understanding of one of the most successful bacterial pathogens, and provides a basis for future directions of study, manipulation, and treatment. Whatever direction scientists decide to take next, they are turning the tables on the “Thriller” soundtrack: humans are attacking the bacteria rather than the other way around.
About the Author:
Delany Berry '19
Delany is a junior at Mount Holyoke studying biology and psychology and intends to pursue a career in primary care after graduation. Outside of classes, she enjoys swimming on MHC's varsity team, hiking, and dogspotting. Her favorite microbes are magnetotactic bacteria because they can use the earth's magnetic field to navigate!
References:
Bubendorfer et al. Genome-wide analysis of chromosomal import patterns after natural transformation of Helicobacter pylori. Nature Communications. 7 (2016). doi:10.1038/ncomms11995
Johnston, C., Martin, B., Polard, P. & Claverys, J. P. Postreplication targeting of transformants by bacterial immune systems? Trends Microbiol. 21, 516–521 (2013).
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