The Pathogen's Playbook

When comparing pathogenic bacteria with non-pathogenic species of the same genus or family, we often find a common pattern. In the pathogen:

• The genome is often reduced in size (particularly in endosymbionts, but also in others).
• The genome is often shifted in the direction of higher A+T content (lower G+C content).
• Many pseudogenes are present.
• Often, the pathogen is a slow-grower in pure culture (if it can be cultured at all).
• The pathogen has special nutritional needs.

An extreme case that illustrates all of these points is Mycobacterium leprae, the leprosy bacterium. It has fewer genes than its cousin, M. tuberculosis (which in turn has fewer genes than non-pathogenic Mycobacteria); its genomic G+C content is 8% lower than most other Mycobacteria; it contains over 1100 pseudogenes; it has a doubling time of two weeks; and it cannot be grown in pure culture (presumably because of fastidious nutritional requirements).

M. tuberculosis can be grown in the laboratory, but it, and its M. avium-group cousins, are very slow growers, taking anywhere from four days to two weeks to develop colonies on solid media.

It seems likely that some pathogens (certainly members of the Mycobacteria, but also the tiny Tenericutes, e.g. Mycoplasma, among many others) have evolved slow growth as a survival strategy. Certainly, organisms that have evolved an intracellular parasitic lifestyle need to be careful not to out-grow the host, if the relationship is to be a long one.

All of the factors listed above suggest a certain scenario, a "pathogen's playbook," if you will, which can be summarized as follows:

1. The organism invades a warm-booded host.
2. Phagocytes (white blood cells) ingest the organism.
3. The phagocytes undergo a respiratory burst, flooding the microbe(s) with peroxides, hypochlorites, nitrous oxide, and other noxious oxidants.
4. The flood of reactive oxygenated species triggers an SOS response in the microbe.
5. The microbe's DNA undergoes massive damage. 
6. Any surviving microbial cells are now pathogenic.

The SOS response is known to trigger mutagenicity. In Mycobacterium, for example, peroxides (as well as UV light) can induce up-regulation of dnaE, an error-prone polymerase. Since Mycobacteria are known to lack a MutS mismatch repair system, SOS-induced errors in DNA replication will almost certainly include uncorrected frameshift errors leading to the creation of pseudogenes. But that's a good thing, if you're a Mycobacterium interested in forming a longterm relationship with a host cell. The loss of certain genes (as long as they're not essential!) will likely slow your metabolism and make you dependent on host nutrients. Truly non-essential pseudogenes will simply be jettisoned over time, reducing the footprint of the remaining genome. Any pseudogenes that survive will likely have done so because they're now playing an essential gene-silencing role.

Let's expand on that last part. Take the dnaE gene, for example. M leprae has two copies of this gene, only one of which is functional. Suppose both copies were functional at the time of the massive pseudogenization event that converted so many of M. leprae's genes to pseudogenes 9 to 20 million years ago. After the pseudogenization event (probably a phagocytic respiratory burst), one copy of dnaE became a pseudogene. But continued transcription of the pseudogene in the forward direction means the pseudo-mRNA competes with the "normal" dnaE transcript for ribosomal attention. Transcription of the antisense strand of the disabled gene would, of course, create a messenger RNA product that could silence the normal transcript by doublestranded interaction. Either way, once the pseudogenization event is over, dnaE expression is attenuated—as it should be, once pathogenicity has been established.

Is it realistic to think M. leprae transcribes antisense strands of its pseudogenes? Given that E. coli has been found to contain ~1000 antisense transcripts, and given that we know M. leprae transcribes many of its pseudogenes, I think the answer has to be yes.

So the pattern is: infection, respiratory burst, massive mutation, silencing of many genes, and (oh by the way) creation of many brand-new gene products, some of them no doubt quite toxic to the host, as the result of gene truncation and pseudogene expression.

Whooping Cough Genomics

Pertussis, also known as whooping cough, is a highly contagious respiratory infection caused by Bordetella pertussis, a small aerobic bacterium that secretes numerous toxins capable of disrupting a normal immune response. The disease is rarely fatal but leaves victims with a nasty cough that can last weeks. In 2012, in the U.S., some 48,277 cases of pertussis were reported to the CDC. Of those cases, only 20 were fatal. By contrast, 28 Americans were killed by lightning the same year.

Bordetella pertussis

Unlike tuberculosis (which has been with us for 3 million years), Bordetella shows evidence of being a fairly new (and still rapidly evolving) pathogen, although in this case "fairly new" could still mean 700,000 years.

The complete DNA sequence of B. pertussis has been available for several years. It shows a moderate-size genome (of 4 million base pairs) encoding 3,447 genes, with a substantial number (360) of pseudogenes. The latter represent genes that have (by one means or another) been inactivated, whether through the appearance of premature stop codons in the gene, loss of a promoter region, random deletions, or what have you.

What makes Bordetella's pseudogenes interesting is that they're in remarkably good shape, as pseudogenes go. Usually, once a gene gets inactivated (goes pseudo), it begins to accumulate random point mutations, deletions, insertions, etc. at a substantial rate. In other words it deteriorates, since (supposedly) it's no longer under selection pressure. But when Australian researchers looked at 358 pseudogenes in B. pertussis Tohama I strain, they were shocked to find that the rate of nucleotide polymorphisms (i.e., changes to individual base-pairs in the DNA) was actually lower in pseudogenes than in regular genes (4.7E-5 per site versus 5.1). That's exactly the opposite of what's expected. The researchers commented, somewhat laconically: "This suggests that most pseudogenes in B. pertussis were formed in the recent past and are yet to accumulate more mutations than functional genes."

What other explanation is there? Well, the most obvious alternative explanation is that the genes are still under selection pressure, even though they're turned off. How can that be? I can think of any number of scenarios; perhaps that'll be a future blog post. Suffice it for now to say, ribosomes are not totally unforgiving of missing stop codons (read up on tmRNA) nor are they unforgiving, in all cases, of frameshifts (read about programmed frameshifts), and if an open reading frame should appear on a pseudogene's antisense strand, you now have an RNA silencer (potentially) for the remaining good copy or copies of the gene, with attendant gene-modulation possibilities.

It's worth pointing out that pseudogenes in M. leprae (the leprosy bacterium) are not only conserved and ancient but continue to show strong homology to working orthologues in M. tuberculosis (and even more distantly related organisms such as Gordonia, Corynebacterium, and Nocardia) after millions of years. More of which, in a later post.

For now, I thought it might be worth looking at the base composition of B. pertussis pseudogenes to see if they're riddled with frameshift errors (as is the case with M. leprae's pseudogenes). When I analyzed all 1,125,521 codons for all normal (not pseudo) genes in B. pertussis Tohama I strain, the resulting "paintball diagram" of base composition came up looking like this:

Paintball diagram for normal genes in B. pertussis Tohama I (click to enlarge). Red dots are for codon base one, gold represents the composition at codon base two, blue is "wobble" (third) base composition. Every dot represents statistics for one gene (n=3447). See text for discussion.

Here, we're looking at purine (A+G) content versus G+C content for each base position in the codons. Every dot represents a gene's worth of data. Not unexpectedly, the most extreme G+C values occur in the third ("wobble") base. Codon base one (red dots) is purine-rich, centering on y=0.58. This is typical of most codons in most genes, in most organisms. Notice the "breakaway cloud" of gold points underneath the main gold cloud (at y<0.4). These points represent genes in which the second codon base is mostly a pyrimidine (C or T). Codons with a pyrimidine in base two tend to code for nonpolar amino acids. Thus, the breakaway cloud of gold points represents membrane-associated proteins. In this case, we're looking at about 558 genes falling in that category.

Now look at the paintball diagram for the organism's 360 pseudogenes:

Base composition for "codons" in 360 pseudogenes of B. pertussis Tohama I. (Click to enlarge.) In this graph, as in the one above, dots are rendered with an opacity of 60% (so that overlapping points are less likely to obscure each other). See text for discussion.

In this case, there's a considerable amount of random statistical splay, but some of that is due simply to the fact that pseudogenes are a good deal shorter than normal genes, giving rise to more noise in the signal. (In this case, the average length of a pseudogene is 482 bases, vs. 982 for the 3,447 "normal" genes.) Even with considerable noise, though, it's apparent that the dot clusters tend to center on different parts of the graph, corresponding to the expected locations for normal genes. (Contrast this with the situation in M. leprae, where pseudogenes are riddled with frameshifts, rendering the concept of "codon base position" moot. Refer to the second paintball graph on this page.) Thus, we can say with some confidence that frameshifts are not so rampant in B. pertussis pseudogenes as to have rendered the concept of codons irrelevant. In fact, compared to M. leprae, pseudogenes in B. pertussis are comparatively unaffected by frameshifts. This tends to support the view of the Australian researchers (mentioned earlier) that pseudogenes in B. pertussis have not had enough time to accumulate very many mutations. But it can also be hypothesized that B. pertussis has had plenty of time (700,000 years, in fact) in which to accumulate mutations in its pseudogenes, yet has not done so. The evidence suggests that if anything, Bordetella repairs pseudogenes even more faithfully than regular genes.

At this point it might be relevant to interject that while M. leprae (like other members of the Mycobacteria) lacks the MutS/MutL mismatch repair system, Bordetella does, in fact, have a MutS/MutL mismatch repair system, and this may explain the relative paucity of frameshift errors in Bordetella pseudogenes. But it also implies (rather queerly) that Bordetella goes out of its way to repair its pseudogenes.

Interestingly, 234 out of 360 pseudogenes have a AG1 (purine, base one) content greater than 55%, which means they're probably still "in frame." Of these 234, some 69 (30%) have AG2 less than 40%, meaning they're most likely genes for membrane-associated proteins. If we look at the 2,456 normal genes that have AG1 greater than 55%, only 398 (16%) are putative membrane-associated proteins (with AG2 less than 40%). Bottom line: Pseudogenes for putative membrane-associated proteins are twice as likely to still be in-frame. While this could be a statistical fluke, it could also be that membrane proteins are somehow "spared" preferentially when it comes to leaving pseudogenes translatable. To put it differently: Pseudogenes for non-membrane-associated proteins are less likely to remain in-frame. This makes sense, in that much of Bordetella's pathogenicity can be ascribed to proteins that make up cell-surface antigens or that transport toxins to the outside world. Some of the toxic surface proteins may, in fact, be nonsense (or partial-nonsense) proteins—products of pseudogenes.