Cholera- it’s all about the phage

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If, like me, you’ve been reading about the unusually horrific outbreak of cholera in Yemen, you may be wondering how it got so bad. While an understanding of ecology is central to fighting any disease, if feels especially important when discussing cholera, as the current Yemen outbreak is being almost entirely blamed on war resulting in collapsing infrastructure resulting in millions of people losing access to clean drinking water. On top of that, the malnutrition of many children in the area results in them being more susceptible to Vibrio infection.

To add to that, access to rehydration therapy (the common treatment for cholera when intravenous fluids and antibiotics aren’t an option) is low, and the vaccine campaign has been dropped with the justification being that the limited amounts of vaccine would not be as effective in Yemen as they would in areas where less people are infected.

The varieties of Vibrio

The most important vibrio species to human disease are Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae. Vibrio species have flagella and pili which are important virulence factors–notably the toxin co-regulated pilus. The cell walls of Vibrio contain lipopolysaccharides consisting of lipid A (endotoxin), core polysaccharide, and an O polysaccharide side chain. Vibrio can then be divided into serogroups based on this O polysaccharide (200 serogroups in V. cholerae’s case).

V. cholerae O1 and V. cholerae O139 both produce cholera toxin (which causes a rise in cAMP resulting in the cell losing nutrients, which is why you not only need tons of water, but also need to replenish lost electrolytes). These are the serogroups associated with cholera epidemics. Many strains of V. cholerae do not have this toxin and do not cause epidemics though they may still cause illness.

The O1 serogroup is further subdivided into three serotypes: Inaba, Ogawa, and Hikojima. There are then two “biotypes” of V. cholera O1: Classical and El Tor. These biotypes can be further subdivided but let’s just stop here.

The cholera that were famous for killing lots of people in the 1800s were all of the Classical type. The cholera that is responsible for today’s pandemic is of the El Tor biotype.

The CTXφ phage

V. cholerae secretes cholera toxin. This is the toxin that causes the “rice-water” stool (not diarrhea really, as it’s just mucus and water), resulting in dehydration of the host. Colonization of the small intestine required the toxin co-regulated pilus (coded by the vibrio pathogenicity island).PMC3282888_TOMICROJ-6-14_F2.png

The genes for cholera toxin are not in the Vibrio genome unless the bacteria has been infected by a CTXphi (CTXφ) filamentous phage, which inserts it’s genome into the V. cholerae genome. The CTXφ can transmit cholera toxin genes from one V. cholerae sstrain to another (via horizontal gene transfer).

Infectious CTXφ particles are produced when V. cholerae infects humans. Phages are then secreted from the infected bacteria without lysing the cell.

Seasonal epidemics inversely correlated with environmental cholera phage presence

Cholera seasons usually make sense as they tend to coincide with monsoon season. But perhaps less obvious (or totally obvious if you’re into viruses) cholera phages have a very dramatic influence on seasonality.

The presence of viruses infecting V. cholerae O1 or O139 inversely correlates with the occurrence of viable V. cholerae in the environment and the number of cholera cases. Both epidemic and nonepidemic serogroups have been shown to sometimes carry lysogenic phages which reproduce and kill epidemic strains. Lysogenic phages integrate into the genome so it replicates with every reproduction of the bacteria

One common O1 phage can use several V. cholerae non-O1/non-O139 strains as alternative hosts.

Having alternative hosts present combined with the lysogenic V. cholerae strains can result in a cholera phage “bloom,” thus lowering the transmission of phage-sensitive, more virulent cholerae strains.

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Concentration of lytic vibriophages in the aquatic environment of Dhaka, Bangladesh, and the estimated number of cholera cases. From Faruque et al 2004.
Phage and Vibrio waves controlling epidemics

 Cholera outbreaks occur in waves with different serogroups dominating at different times.

The absence of one phage specific for one cholerae type provides an opportunity for that serogroup to begin the seasonal epidemic. However, the phages for that serotype will eventually amplify in the environment and attack this serogroup, ending that epidemic.

A different cholerae serogroup would then be resistant to that first phage or carry it as a prophage in the genome—so it isn’t killing that bacteria. A second epidemic wave from the new dominating serogroup can now occur until phages specific to this new serotype bloom, thus ending the epidemic.

Some serotypes will be resistant to all the phages that were killing the virulent phages in the environment, and these serotypes will occupy the interepidemic periods. These strains usually lack typical virulence factors that would make them particularly good pathogens, but are instead more environmentally adapted than the other more virulent strains.

These resistant serotypes may ALSO harbor prophages—phages integrated in the genome—which kill virulent serogroups and may pick up virulence factors via horizontal gene transfer.
If this happens, new serotypes that were previously not very virulent may emerge as the new epidemic serotype.

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Vibriophage
Self-limiting seasonal epidemic also probably caused by phage

A naturally occurring lytic phage, JSF4 (lytic meaning it simply lyses the cell), infects and kills Vibrio that are sensitive to it.

In a study from 2005, it was shown that the peak of cholera season was preceded by a peak in V. cholerae presence which was then followed with a peak in JSF4 phage presence as the epidemic ended. JSF4 phages would then also be excreted in the diarrhea of sick cholera patients. So the patients at the end of the epidemic end up ingesting both a lot of V. cholerae as well as JSF4 phage which kills the bacteria. The increase of phage results in the decrease of V. cholerae and the epidemic ends.

This is likely why outbreaks are self-limiting.

V. cholerae O139 spread by turtles

Screen Shot 2017-07-14 at 12.56.28 AM.pngWhile O1 causes the majority of outbreaks, O139 is confined to Southeast Asia. Recently however, it’s been discovered that soft-shelled turtles in China are big carriers of O139. While a lot of aquatic animals spread cholera, the soft-shelled turtles have been definitively linked to human disease and make excellent hosts as they are unaffected by the bacteria which clings to many of their surfaces and intestines. These turtles are then consumed by people, to spread more cholera to new unsuspecting hosts. If turtles being cute wasn’t a good enough reason to stop eating them, maybe this is?

Sources:

Jiazheng Wang, Meiying Yan, He Gao, Xin Lu, Biao Kan. Colonization of Vibrio cholerae on the Soft-shelled Turtle. Applied and Environmental Microbiology, 2017

Faruque, S. M., I. B. Naser, M. J. Islam, A. S. G. Faruque, A. N. Ghosh, G. B. Nair, D. A. Sack, and J. J. Mekalanos. “Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages.” Proceedings of the National Academy of Sciences 102.5 (2005)

Faruque, S. M., M. J. Islam, Q. S. Ahmad, A. S. G. Faruque, D. A. Sack, G. B. Nair, and J. J. Mekalanos. “Self-limiting nature of seasonal cholera epidemics: Role of host-mediated amplification of phage.” Proceedings of the National Academy of Sciences 102.17 (2005)

Murray, Patrick R., Ken S. Rosenthal, and Michael A. Pfaller. Medical microbiology. Philadelphia, PA: Mosby/Elsevier, 2016.