Cryptococcus — (r)evolutionary genius

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If you’re in the market for a particularly fun fungus to tell your friends about, I’d suggest the pathogenic yeast, Cryptococcus.

Cryptococcus is primarily known for causing Cryptococcal meningitis—a leading cause of death in AIDS patients. This is Cryptococcus neoformans, which, while more common really only infects the immunocompromised. Its cousin, Cryptococcus gatti, frequently infects immunocompetent, healthy individuals and is an important emerging pathogen. Weirdly enough, C. gatti infections all seem to come from trees. The fungal pathogen has dispersed all over the world surprisingly, scientists believe, due to continental drift. In the past few years, there’s been large outbreaks of the less common Cryptococcus gatti in the Pacific Northwest/Canada and South Africa.

Sex has something to do with it

Cryptococcus needs a lot of sugar to reproduce, particularly inositol which is all over the human brain and spinal cord which is probably why Cryptococcus is known for causing meningitis. Cryptococcus (depending on the species) has between about 6 to 12 genes involved in inositol transport while most fungi have only about two. While inositol is needed for reproduction and results in higher virulence, which mating type the fungus is may also play a large role in pathogenicity.

In yeasts there are two mating types, MATa and MATα. MATα strains are able to produce an extensive hyphen phase in the haploid state called monokaryotic fruiting. All the clonal C. gattii VGII (or C. deuterogatti) that have been infecting people have notably been from the same mating type: Matα. MATα strains are capable of same-sex mating which could be the origin for the outbreak around Vancouver. In C. neoformans, MATα strains which have their own genes specific to that mating type, are associated with higher virulence (but there’s no evidence of this with C. gattii). 

From Springer 2010, isolations of C. gatti from human clinical, veterinary, environmental sources. Underestimation of actual prevalence.
Hypermutator

Researchers found that several isolated C. deuterogatti strains contained mutations in MSH2, one of the genes involved in mismatch repair (genes that fix DNA replication errors). The human homologous MSH2 gene has the same effect where humans with mutations in MSH2 can have Lynch Syndrome, where they’re prone to various cancers.

The mutations in MSH2 in fungi were tested and resulted in their genomes mutating at a faster pace but growing at the same rate as wildtype Cryptococcus strains. When exposed to stressful conditions however, such as antifungals like rapamycin and FK506, the mutant strains rapidly took over as they could quickly acquire resistance to drugs. In the process of acquiring drug resistance the strains actually decreased in virulence and were significantly weakened compared to the outbreak strains.

So the hypermutators are much more fit in stressful conditions and can rapidly outcompete the wildtype strains, however they make a less fit pathogen without significant selective pressures present. This brings up an interesting question in host-pathogen evolution—what kind of genome makes for an ideal pathogen? Fungi in general are lousy pathogens, especially in people. Certainly white-nose syndrome in bats and chytrid fungus in amphibians have devastated populations but there aren’t a whole lot of “in between” fungal pathogens. Having a lower mutation rate than bacteria or viruses contributes to their easiness to treat. While in bacteria a hypermutator strain rapidly evolving drug resistance usually sounds like a bad thing, in this case hypermutator strains taking over would at least lower the virulence and fitness of the fungus as a pathogen.

It’s even worse in men

As is the case with many pathogens, there is an increased incidence of the disease in men and when it does appear mortality rates are significantly higher in men. C. neoformans isolates from females had a slower growth rate and released more capsular glucoronoxylomannan (GXM), (a virulence factor and immunosuppressant). Testosterone was associated with higher levels of GXM release while 17-β estradiol was associated with lower levels and slower growth rate. Furthermore, macrophages from females were better at fighting off C. neoformans than macrophages from males which were more damaged from the infection. This may explain why infections are more common in men.

While Cryptococcus neoformans has been a common issue for a while, Cryptococcus gatti has only recently emerging as a prominent pathogen, which scientists believe could be the result of climate change. These Cryptococcus infections infect healthy people and are fatal if left untreated.

Sources:

  1. The second STE12 homologue of Cryptococcus neoformans is MATa-specific and plays an important role in virulence. Y. Chang-L. Penoyer-K. Kwon-Chung – Proceedings of the National Academy of Sciences – 2001
  2. The Role of Host Gender in the Pathogenesis of Cryptococcus neoformans Infections. Erin Mcclelland-Letizia Hobbs-Johanna Rivera-Arturo Casadevall-Wayne Potts-Jennifer Smith-Jeramia Ory – PLoS ONE – 2013
  3. Highly Recombinant VGII Cryptococcus gattii Population Develops Clonal Outbreak Clusters through both Sexual Macroevolution and Asexual Microevolution. R. Billmyre-D. Croll-W. Li-P. Mieczkowski-D. Carter-C. Cuomo-J. Kronstad-J. Heitman – mBio – 2014

 

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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