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Close relations – how sequencing revealed the secrets of the strangles bug

30 Jun, 2009

Dr Matt Holden has sequenced many bacterial genomes in his role at the Wellcome Trust Sanger Institute yet, for him, one bug in particular – responsible for a horse disease called strangles – held more than a couple of surprises in its genes.

Streptococcus is a genus of bacteria that’s packed full of harmful species: from bugs that cause tooth decay to those responsible for the flesh-eating disease necrotising fasciitis. It’s not only humans who are prey to these bacteria; a number of animals can also act as hosts.

Two so-called Group C streptococci (see below) have been under the close scrutiny of Dr Matt Holden from the Wellcome Trust Sanger Institute, near Cambridge, UK. He and colleagues have sequenced the genomes of two animal pathogens, ‘Streptococcus zooepidemicus’ and ‘S. equi’, and have been comparing them to each other and to the genome of ‘S. pyogenes’, which causes a number of infections in humans.

As the name suggests, ‘S. equi’ targets horses, donkeys and other members of the genus ‘Equus’, causing a disease called equine strangles. The strain sequenced was isolated from a New Forest pony suffering respiratory problems in 1990. On the other hand, ‘S. zooepidemicus’ can cause diseases in a number of animals, from mastitis in cows to meningitis in humans.

“It’s known that ‘S. equi’ has evolved relatively recently from a common ancestor shared with ‘S. zooepidemicus’,” Dr Holden says. “It has probably developed from a version of ‘S. zooepidemicus’ to become a specialised pathogen.”

‘S. equi’ isn’t just restricted in terms of the host it targets, but the tissues too. On infection, ‘S. equi’ heads straight to the lymph nodes while ‘S. zooepidemicus’ colonises the horse’s respiratory or urogenitary tract waiting for the opportunity to invade. Dr Holden and colleagues compared the genomes of the two bugs to try to help explain the differences in host preference and mechanisms of infection.

Get evolved

Researchers at the Sanger Institute completed the ‘S. equi’ sequence in 2006 and the ‘S. zooepidemicus’ sequence in 2008. When the researchers compared the two, several main differences leapt out at them. Firstly, compared to ‘S. zooepidemicus’, ‘S. equi’ has, in effect, streamlined its genome, doing away with the genes it doesn’t need.

“It seems to have lost a lot of its ancestral capacities, such as the ability to use certain sugars or produce certain surface proteins,” says Dr Holden. “It can now infect only horses and related species, and doesn’t occupy as many different tissues in its host.”

Secondly, Dr Holden says, ‘S. equi’ has “accessorised its genome”. He explains that the bacterium seems to be really good at horizontal gene transfer, where bacteria swap genetic material without reproduction. The strangles bug appears to have gained genetic material from bacteriophages – viruses that infect bacteria.

Surprisingly, many of the viral genes carried within the ‘S. equi’ genome have also been seen in the human pathogen ‘S. pyogenes’. The researchers found that the viral sequences in ‘S. equi’ were more similar to those in ‘S. pyogenes’ than to each other. “It seems like ‘S. equi’ has been dipping into the same pool of virus as ‘S. pyogenes’,” says Dr Holden. While there are genetic traces of four phages in ‘S. equi’, there are none in the ‘S. zooepidemicus’ sequence examined.

These phages can carry cargo genes that can alter the virulence of the receiving bacterial cell. There is also evidence of ‘S. equi’ acting as a reservoir of viral sequence itself. The gain of three virus-associated cargo genes by strains of ‘S. pyogenes’ in the mid-1980s is associated with more serious human infections.

The same regions have been found in a strain of ‘S. equi’ isolated from a horse in 1981, earlier than they have been found in the human pathogen. This raises the possibility that ‘S. pyogenes’ could have gained the region from ‘S. equi’ – an example, in effect, of how a horse-only pathogen can influence the severity of human disease. “Potentially, animal pathogens could be acting as a reservoir for some of the genes that cause problems in the human pathogens,” says Dr Holden.

Image: Schematic circular diagrams of the ‘S. equi’ Se4047 (A) genome and the ‘S. zooepidemicus’ SzH70 genome (B). From: Holden MTG et al. Genomic evidence for the evolution of Streptococcus equi: host restriction, increased virulence, and genetic exchange with human pathogens. PLoS Pathog 2009;5(3):e1000346.

Iron age

The way ‘S. equi’ handles iron is also thought to be key to its emergence as an independent species. All ‘S. equi’ strains studied contained genes that encode the machinery needed to produce a compound involved in iron uptake. ‘Yersinia pestis’, the bacterium that causes plague, makes an iron-grabbing compound called yersiniabactin, the genes for which share similarity with those in ‘S. equi’.

This is the first time that the production of these iron-grabbing compounds has been seen in any streptococci. Dr Holden suggests that this “virulence determinant” shared by the two bacteria could assist them to survive in the lymph glands, and helps to explain why often strangles is referred to as ‘equine plague’.

Both ‘Y. pestis’ and ‘S. equi’ target the lymph nodes. “When ‘Y. pestis’ infects a human, it makes buboes [swellings] at the lymph nodes, and replicates there. The disease shows close parallels with strangles, both pathogens target and grow in the lymph glands,” says Dr Holden.

Does this suggest that ‘S. equi’ and ‘Y. pestis’ share a common ancestor? No, says Dr Holden. “There’s a very distant relationship, and no suggestion they’re from the same place. If anything, this is probably an example of convergent evolution: the targeting of the same type of tissue using the same strategies, rather than the same genes.”

Advances in sequencing technologies mean that researchers are now able to sequence genomes faster and at a higher resolution than ever before. “The initial project involving ‘S. equi’ involved sequencing a single genome. Now, thanks to high-throughput genome sequencing, we can study a wide set of strains to build up a phylogeny, and see how they’re related,” says Dr Holden.

“We will be able to study how strains from different parts of the world compare, and follow how a particular strain changes over the course of an outbreak,” says Dr Holden.

The ability to understand the structure of the population of the strangles bug will be particularly important when newly developed vaccines and diagnostics are put into use. “To understand how effective these things are we need to have an idea of the population structure and how that’s varying over time.”

He is positive about the potential success of such interventions in reducing strangles cases, and even in eradicating the disease altogether. “You could argue that the pathogen has become very vulnerable as it has nowhere else to go – it’s host-restricted,” says Dr Holden. “The question is, can we now make it extinct from that niche or has it got a couple more tricks up its sleeve?”

Top image: A horse with strangles. Severe swelling under the jaw or neck is characteristic of strangles infection. Credit: Professor Derek C Knottenbelt, University of Liverpool.

More information

Holden MTG et al. Genomic evidence for the evolution of Streptococcus equi: host restriction, increased virulence, and genetic exchange with human pathogens. PLoS Pathog 2009;5(3):e1000346.

The Wellcome Trust Sanger Institute was funded by the Horse Trust to sequence the genome of ‘Streptococcus equi’ strain 4047 in collaboration with researchers from the Newcastle University, the University of Cambridge and the Animal Health Trust. ‘S. zooepidemicus’ strain H70 was sequenced with support from the Horserace Betting Levy Board in collaboration with researchers from the Animal Health Trust and the University of Cambridge.

Strepto… what?

Streptococcal species are spherical gram-positive bacteria that grow in pairs or chains. They are broadly classified by their haemolytic properties that is, how well they break down red blood cells.

Alpha-haemolytic streptococci cause partial haemolysis. This group includes ‘S. pneumoniae’, which causes pneumonia and meningitis, and the ‘viridans streptococci’, including ‘S. mutans’, which causes tooth decay.

Beta-haemolytic streptococci cause complete haemolysis. They include:

  • Group A streptococci (‘S. pyogenes’) cause many human diseases, from throat infections (‘Strep throat’) to the skin infection impertigo and the potentially fatal flesh-eating disease necrotising fasciitis.
  • Group B streptococci (‘S. agalactiae’) can pass to babies born to infected mothers during labour and birth. Infection in newborns can cause blood poisoning, meningitis and pneumonia, and can prove fatal.
  • Group C streptococci include ‘S. equi’ and ‘S. zooepidemicus’.
  • Group D streptococci include ‘S. bovis’, thought to cause endocarditis (inflammation of the heart lining) in humans. It is also linked to chronic liver disease and gastric cancer. Note: Group D show variable haemolysis.
  • Group G streptococci are frequently found in the airways of healthy people. They can cause a range of infections in humans and animals.

Many non-haemolytic streptococcal species live in bodies of healthy humans and animals; few cause disease.

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