Sunday, April 13, 2014

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.


  1. Will u please elaborate how to find psedogene sequence for my genome.I have psedogene no from NCBI.But how to find sequences?

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