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.

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A microbe manipulating sex- and how it can fight Zika

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A fan favorite, and probably the most successful genus on the planet–at least on land—the bacteria, Wolbachia infects an estimate between 40 to 65% of all arthropod and nematode species. This microbe is constantly drifting across the line between mutualist and parasite to it’s host. Some hosts are unable to survive and reproduce without a Wolbachia infection whilst others are killed by it.

Wolbachia as a mutualist

Plenty of species have become reliant on this microbe. The caterpillar of the spotted tentiform leaf miner uses Wolbachia to create green islands on yellowing leaves which remain fresh for munching on.

It provides a benefit to certain nematode worms, such as Brugia malayi and Wuchereria bancrofti which cause elephantiasis, and which cannot survive without a Wolbachia infection. Image1.jpg

Some Wolbachia bacteria provide metabolic advantages to their hosts such as in bed bugs who use it to synthesize B-vitamins that are absent in their blood meals. Wolbachia can even mediate iron metabolism in Drosophila.

But most exciting given the recent explosion of flavivirus infections (Zika traveling farther and farther north every summer for example), Wolbachia provides flies with resistance to many RNA viruses.

Wolbachia as a sex-determinator

In leafhoppers, Zyginidia pullula, females have two X chromosomes while males have only one X chromosome, yet when infected with Wolbachia, the X0 genetic males appeared to be female.

Some females of the Japanese butterfly, Eurema mandarin have a sex chromosome system where the males are (ZZ) and the females are (ZWEurema_blanda_on_flower_by_kadavoor.JPG). This incongruence between chromosomal and phenotypic sex can be explained by feminization of genetic males induced by Wolbachia. Two strains of WolbachiawCI and wFem, have been found in E. mandarina and the females having male chromosomes (ZZ) are consistently infected with both wCI and wFem. However females with only wCI are true females (ZW). Despite having male chromosomes, ZZ females are physically female and fully fertile.

A similar thing happens in woodlice (pillbugs? Roly-polys?), where all the ZZ males infected with Wolbachia develop as female. The W chromosome is sometimes lost entirely in these populations and sex is entirely determined by presence or absence of Wolbachia.

Who needs males?

Wolbachia has evolved into an intracellular parasite, and while it can infect many different organs, it is most famous for infecting the testes and the ovaries. Wolbachia are too large for sperm, but fit nicely into mature eggs so the infection is inherited maternally through the eggs.

So now the evolutionary dilemma that keeps Wolbachia on the balance between parasite and mutualist is, if males are an evolutionary dead-end, how does this intracellular parasite that needs its host to survive and reproduce, and it’s host species to continue thriving, evolve to both spread throughout populations but not allow evolutionary cheater strains to ruin everything? Wolbachia has developed numerous ways of targeting males to help itself spread such as:

  • Male killing- infected male larvae die, so more infected females are born
  • Feminization- where infected males develop as females or infertile pseudofemales
  • Parthenogenesis- when females reproduce without males
  • Cytoplasmic incompatibility (CI)- when Wolbachia-infected males can’t successfully reproduce with uninfected females or females infected with another Wolbachia strain.CI-causing Wolbachia interferes with the chromosomes during mitosis so they no longer divide in sync.
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Warren et al, 2008, Nature Reviews, Microbiology.

Mosquitos carrying Wolbachia have a higher reproductive success when present in a population with mosquitoes not carrying Wolbachia. When a male mosquito carrying Wolbachia tries to mate with a female who is not carrying Wolbachia, the female’s eggs won’t hatch. However females with Wolbachia do not have this issue and produce perfectly healthy offspring which are all also carriers of Wolbachia. So you can imagine how the Wolbachia is able to sweep through a mosquito population. The female Wolbachia carriers have a much higher fitness than the non-carriers.

Scientists have taken advantage of this evolutionary strategy in fighting mosquito-transmitted viruses such as Dengue and Zika. The Aedes aegypti, a black-and-white striped species of mosquito infects people with Dengue virus which has no vaccine or real treatment and causes pains, fevers, rashes, and headaches. A plan (credited to evolution/ecology biologist, Scott O’Neill) to release Wolbachia infected mosquitos into the wild to lower dengue spread is becoming more and more popular. Wolbachia stops Aedes mosquitoes from carrying degue virus. Wolbachia carrying females have a selective advantage and should sweep through the population.

Wolbachia to rescue us from Dengue (and others?)- The original plan

An unusually virulent strain of Wolbachia the ‘popcorn’ strain, essentially halves the mosquito lifespand (it’s pretty gruesome, the bacteria essentially reproduce like crazy in the brains, eyes, and muscles filling up neurons). Dengue takes a long time to be able to reproduce and make it to the salivary glands so it can be transmitted, so only older mosquitos can transmit it.

Unfortunately Aedes (and Anopheles which transmist malaria) are not natural hosts of Wolbachia infections, so Scott O’Neill carefully developed a new symbiosis by injecting eggs. This took forever to work, until one lucky grad student was able to make it a success. Finally, an egg was stably infected and a line of Wolbachia-carrying Aedes was created. But after all that work, the strain was too virulent and the females did NOT have a selective advantage and actually had lower numbers of eggs with lower viabilities (honestly, they should have seen this coming really).

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But none of that even ended up mattering because some other scientists figured out that Wolbachia stops dengue virus from replicating. Simply the presence of a nonvirulent strain of Wolbachia in the population was enough to stop the spread of dengue. So the team switched to a less virulent strain, wMel, and successfully started a line of wMel carrying Aedes mosquitos.

wMelwAlbB update and some critiques of the data

As amazing as Wolbachia is, it will take at least a few years to get significant results and many years to eliminate any particular mosquito-transmitted pathogen with this method. Some papers say Wolbachia was able to make not only dengue, but also Plasmodium and other flaviviruses less able to replicate, but others said the opposite. It would be kind of important to figure out how the Wolbachia is inhibiting the virus because no one seems to agree if it’s general or specific. But now with significant selective pressures on all these diseases I’d suspect it’d be easy to switch host vectors because mosquitos bite hosts, geographically spreading infections very far (potentially) exposing the virus to a wide variety of new potential hosts vector species.

In a 2016 paper looking at the progress of the wMel strains, they collected Ae. Aegypti after one year and it continued to have low levels of dengue (which implies it was passed around through tons of mosquitos and not a whole lot of apparent evolution has taken place in terms of resistance) but what if the some of the dengue viruses switched species?

They could try passing the dengue through many mosquitos perhaps in a mixed host population. Or basically just try to provide the virus with as many opportunities as you can for it to evolve in the hopes that you may understand potential mechanisms the different diseases may have to get around this one Wolbachia infected species of vectors.

While the paper shows wMelwAlbB, the superinfection, that strain of Wolbachia actually doesn’t appear to inhibit DENV very much. But later in the paper, it’s very dramatically different so it’s difficult to say if the data is actually supporting that the superinfection sweep will work.

As far as how they ensure the right strain is dominating all the time given selective pressures towards different mosquito sex alteration methods, that remains unanswered. Combined male-killing CI strains readily become extinct following invasion so CI strains are more selected for but sex-ratio distortion decreases male infection and therefore reduces the occurrence of CI meaning that you might expect selective pressure for evolution from CI to CI/sex ratio distortion to sex ratio distortion only.

Wolbachia’s potential

Using Wolbachia’s ability to stop mosquitos from carrying Zika, Dengue, Chikungunya, Plasmodium—the parasite responsible for malaria, may mean the eventual elimination of these diseases in humans.

It may even be used to someday stop nematode worms from causing blindness, disability, elephantitis etc in many millions of people every year.

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Particularly inspiring about this story is how ecologists and evolutionary biologists ended up being the ones to figure out a way to eliminate viral infections. Yet more evidence that pre-med students or medical researcher hopefuls shouldn’t blow off biodiversity/ecology/evolution classes in college.

Sources

Kageyama, Daisuke, Satoko Narita, and Masaya Watanabe. “Insect Sex Determination Manipulated by Their Endosymbionts: Incidences, Mechanisms and Implications.” Insects 3.4 (2012): 161-99.

I contain multitudes: the microbes within us and a grander view of life. Ed Yong, 2016

Bacterium offers way to control dengue fever. Natasha Gilbert – Nature – 2011.

Establishment of a Wolbachia Superinfection in Aedes aegypti Mosquitoes as a Potential Approach for Future Resistance Management. D. Joubert-Thomas Walker-Lauren Carrington-Jyotika Bruyne-Duong Kien-Nhat Hoang-Nguyen Chau-Iñaki Iturbe-Ormaetxe-Cameron Simmons-Scott O’Neill – PLOS Pathogens – 2016

 

 

The struggle against antibiotic resistance

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“Animals may be evolution’s icing, but bacteria are the cake.” –Andrew Knoll, Life on a Young Planet

Rapid evolution of multi-drug resistant bacteria due to overuse of antibiotics is one of the biggest threats facing humans today. Luckily scientists, motivated by terror of a superbug killing us all, have been working pretty hard recently on trying to solve this problem.

Teixobactin

A new class of antibiotics was discovered with the help of a new device, the iChip. Humans have been unable to figure out how to isolate and culture most (99%) of the Earth’s bacteria, but the iChip was successfully used to culture previously unculturable soil bacteria, notably Eleftheria terrae. This bacteria was found to produce Teixobactin, an antibiotic that micorbes have an exceptionally difficult time evolving resistance to (like, so difficult none have been able to do it yet). Because most antibiotics are usually discovered by accident via fungi or other microbes, being unable to culture most of what was in the soil has been a pretty big barrier in our search. Teixobactin is active against gram-positive bacteria and works by inhibiting cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). Peptidoglycan is what makes up the thick walls of Gram-positive bacteria. Gram-negative bacteria also have peptidoglycan but in much smaller amounts and are protected by a second outer membrane which Gram positive bacteria do not have.

Screen Shot 2017-06-06 at 8.38.59 PM.pngStaphylococcus aureus (MRSA/VRSA) and Mycobacterium tuberculosis (TB), two ofthe biggest names in the antibiotic resistance crisis, were both killed by and unable to develop resistance to, Teixobactin.

Teixobactin is probably more robust against mutations of pathogens, because of its antibiotic mechanism is so unusual. Most antibiotics involve binding to relatively mutatable proteins. Teixobactin however, binds to much less mutatable fatty molecules.

Vancomycin 3.0

Vancomycin has been in use since 1958 and was once considered a last-resort drug because it seemed bacteria were not very good at developing resistance to it but it’s a pretty serious drug. It has a higher toxicity level than most antibiotics you’d usually be prescribed so it is used only for the treatment of life-threatening Gram-positive infections. It kills bacteria by preventing cell wall synthesis by binding to peptides ending in two copies of D-alanine. Unfortunately bacteria were able to replace a D-alanine with a D-lactate (D-lac). Scientists decided to fix this by creating a new vancomycin that binds to peptides ending in D-ala and D-lalc. Other scientists came up with ways to manipulate vancomycin to kill cells by stopping cell wall synthesis in a new way or causing the outer wall membrane to leak.

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The new vancomycin, vancomycin 3.0, has three antimicrobial targets in one antibiotic. It was shown to be effective against vancomycin resistant Enterococcus (VRE) and vancomycin resistant Staphylococcus (VRS). A three-pronged approach presumably means that for a bacterium to be resistant, it would have to have three non-lethal mutations to get around this drug.

Phage-therapy

Bacteriophages, viruses that infect bacteria, are also being used as a method to treat bacterial infections. While this concept is not especially new, it’s been relatively neglected by the U.S.. Phage therapy would take a long time to get FDA approval and it still needs a lot more research. The coevolution of bacteria and bacteriophages, while certainly studied a lot in model organisms, is an important factor to consider when blindly expecting phage therapy to work. The nice thing about phages over antibiotics, is that antibiotics can sometimes have unpleasant side-effects, cause allergic reactions, or have high toxicity to the people/animals taking them. Phage are harmless and extremely specific to their targets, unlike broad-spectrum antibiotics. However phage are huge spreaders of antibiotic resistant genes as they are relatively important engagers in horizontal gene transfer (where genes, instead of being inherited, are picked up and incorporated into a genome via viral infection or collection of raw DNA).

Phage therapy saves man with multidrug-resistant infection
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credit:  Dr Graham Beards
 

In 2015, a four-phage cocktail was administered to target a man with a serious Acinetobacter baumannii infection which had been causing hallucinations and was killing his kidneys. This was the first instance of phages being used intravenously to treat someone almost dead due to an infection caused by a drug-resistant bacteria. Luckily he was married to a genius woman who happened to be an infectious-disease specialist and decided a phage-cocktail was his best bet. It was difficult trying to find the right bacteriophages but it worked in the end.

The Gram-negatives

One of the biggest struggles in finding new antibiotics is Gram-negative bacteria are often intrinsically resistant to antibiotics because their outer membrane is impermeable to large glycopeptide molecules, and so many antibiotics seem to target the cell wall.

Some Gram-negative bacteria that can cause pretty serious infections include Klebsiella (pneumonia, blood, wound), Acinetobacter (pneumonia, blood or wound infections), Pseudomonas aeruginosa (burns, wounds, respiratory), E. coli, Vibrio (Cholera), Yersinia pestis (“the plague”), and Neisseria gonorrhoeae (gonorrhea).

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Neisseria gonorrhoeae

Luckily, a lot of these don’t usually infect people (the first three basically never infect healthy people), however antibiotic resistant sexually transmitted diseases are actually on the rise in the United States and could become a very serious and difficult to control problem pretty rapidly. Gonorrhea’s not really a big deal if you catch it early and treat it, but it’s asymptomatic in many people (making them untreated, unknowing carriers) and left ignored, causes infertility and pelvic inflammatory disease.

So even though we might feel relief at these new discoveries, keep in mind that no antibiotic will work on all bacterial pathogens, and allergies to antibiotics are very common so we can’t just be left with a single last-resort. We kind of need a lot of options.

Sources:

  1. Okano, Akinori, Nicholas A. Isley, and Dale L. Boger. “Peripheral modifications of [Ψ[CH 2 NH]Tpg 4 ]vancomycin with added synergistic mechanisms of action provide durable and potent antibiotics.” Proceedings of the National Academy of Sciences (2017)
  2. “A new antibiotic kills pathogens without detectable resistance.” Ling et al (2015)