Why are bacterial capsules so diverse?

Biology is full of complex problems to solve. One of these problems is the remarkable diversity of some bacterial capsules, like in Streptococcus pneumoniae, Klebsiella pneumoniae or Escherichia coli (100 or more types in each species alone). It's not merely a philosophical question: capsules, so ubiquitous in the bacterial world, are highly medically relevant being the target of polysaccharide conjugate vaccines, and they are more and more often mentioned in the context of phage therapy as phages recognise specific capsule types. As large antigenic diversity poses a problem for eradication of a bacterial disease, it is important to understand what drives it in the first place.

So what drives capsule diversity in bacteria? To address this question, I and Kat Holt have just published a review of this problem in Trends in Microbiology, which is open access (click HERE to read it!). In this piece:

  • We review the literature for many different bacterial species, though focusing the discussion on the "WHO priority pathogens for R&D of new antibiotics" (but see note below*). Many of these bacteria are encapsulated. (This is not a coincidence as capsules are important virulence factors, thus often appear as pathogens.)

  • We talk about "polysaccharide antigens" (PA), not just capsules, as PA can also include diverse O-antigens for example, like in Salmonella enterica or Vibrio cholerae.

  • We discuss evolutionary drivers of diversity and medical implications of PA diversity.

Here's a short breakdown of the three key points made in the review.

1. PA genetic loci are diversity-generating machines.
PA, like capsules, are densely packed "forests" of long chains of polysaccharides, and each polysaccharide consists of a combination of several different sugars (monomer), combined to form a chain (polymer; see figure below). Monomers can combine different sugars in different bacteria, which permits a huge number of combinations. Interestingly, polysaccharides are synthesised by genetic loci which have a remarkably consistent architecture across the bacterial kingdom. In the middle one typically finds genes which synthesise enzymes specialising in linking sugar of one type with sugar of another type. Thus exchanging a gene usually means altering the antigen. As expected, these genes are highly variable across the population, but they are flanked by conserved genes with regulatory functions. This in turn permits exchange of the entire locus between bacteria via homologous recombination, which requires homologies at the flanks, but not the middle. Here's a scheme of a typical PA locus.

Finally, unlike proteins, sugars do not alter structure easily, and thus are much more tolerant to mutations. This makes them less conserved in an evolutionary sense, much like an armour (in fact, capsules are in many ways bacterial armours).

Conclusion: loci encoding PA are evolutionarily optimised to rapidly diversify in the face of environmental conditions, making them great diversity-generating machines.

2. There is no single, universal PA-diversifying force, but...
there are a small number of very strong candidates.

Immunity. This one has been argued as THE diversifying force behind capsules by some, perhaps as the immune system plays an important role in shaping the population structure of many respiratory pathogens, like S. pneumoniae, Haemophilus influenzae or Neisseria meningitidis. But research has shown that PA are more common in non-clinical/environmental strains of bacteria than in pathogens, and commensal bacteria (like Streptococcus mitis) also exhibit large PA diversity. So immunity is likely important but not the only driver.

  • Phages. PA, like capsules, constitute obstacles for phages, which in turn have evolved diverse enzymes to decompose bacterial sugars. Their role in driving PA diversity has been underappreciated, to say the least.

  • Cell-cell interactions. Importance of glycans-protein interactions has only become clear relatively recently, and this has huge consequences for bacteria. Such interactions become relevant for pathogenic bacteria when they encounter host tissue surfaces surrounded by glycans, other commensals, or even eukaryotic predators living in animal intestines – all of which can ultimately shape the PA diversity.

These ecological factors will clearly act on different timescales (e.g., phages have somewhat shorter generation times than humans), but all of them are likely to drive the evolution of diversity of bacterial PA in the long term.

Conclusion: PA interact and thus coevolve with many other living entities, and these interactions will drive their diversity. Understanding the relative importance of these interactions in a given bacterial species should give a better idea of the likely diversifying force candidates behind PA in that species.

3. PA evolution is medically relevant.
Take H. influenzae and S. pneumoniae, both major disease causes in children, both had vaccines introduced in around two decades ago, which have been pretty successful in reducing disease burden. The difference? S. pneumoniae has around 100 capsule serotypes, while H. influenzae has 6. As a result S. pneumoniae, unlike H. influenzae, has been slowly bouncing back since the introduction of the original PCV7, and major effort has gone into introducing new vaccines with broader serotype range. This is obviously very difficult and costly (if you don't believe me just go to ISPPD). In addition, genomic sequencing has demonstrated the power of evolution in the pneumococcus, with vaccine-targeted strains acquiring non-vaccine serotypes via recombination. With such high standing diversity, we can only speculate what new types/strains emerge at low frequencies, and what will happen once the new, broader vaccines clear currently occupied ecological niches. So, given the great potential of PA loci to evolve new types, we do not fully understand the implications of new therapies on the emergence of previously unseen serotypes. This of course applies to other encapsulated bacteria, for example gram-negative Enterobacteriaceae, like K. pneumoniae or Shigella, which are becoming untreatable due to antibiotic resistance, and development of new, polysaccharide-based vaccines is being discussed. It also becomes relevant for phages, which can kill bacteria with a specific capsule type and may be used therapeutically (either live or for isolation of antimicrobials).

Conclusion: In all of these cases, it is important to predict the outcome of a treatment using mathematical models, and such models can only be developed with a good understanding of the underlying biology and ecology. In many cases, we are still lacking such understanding.

In summary...

Bacterial PA, like capsules, are absolutely fascinating, but more often than not they have been looked at in one species at a time. A cross-species look only confirms what some people have suspected: they are powerful diversity-generating weapons, which have likely coevolved in interactions with other biological entities. With the rising threat of antimicrobial resistance and slow turn towards other medical interventions, we need a deeper understanding of how these sugars work and evolve in different bacteria to better predict outcomes of PA-based interventions (like vaccines or phage-based therapies).

*NOTE about the WHO list.
In the paper we have used the "WHO priority pathogens list for R&D of new antibiotics", which you can find here. However, we have referred to this list as "WHO priority pathogens". This of course is a mistake, as was pointed out to me, as it may give the wrong impression that the list includes the most important bacterial pathogens. Well, it does not because it excludes TB for example. The use of that list is still very justified in the context of that article because PA-based treatments are becoming  highly relevant in bacteria which do not respond to antibiotics. But readers should take note that we used it to guide the discussion of PA, and not to point out which bacterial pathogens are most important.