How bacteria might win the fight against malaria, dengue and Zika

Have you heard of the most prevalent parasitic microbe in the world? 

Capable of infecting up to 70% of all insects? 

With a preference for murdering males in cold blood? 

Known as “Wolbachia”, this genus of bacteria is found in many arthropods, including those responsible for the transmission of many human pathogens; such as malaria, dengue fever, and the Zika virus. It gets transmitted vertically, meaning that infected females pass it directly to their offspring. Different strains of Wolbachia infect different hosts and can have many different effects. In filiarial nematodes Wolbachia plays a mutualistic role, with elimination of the symbiotic bacteria causing host sterility and sometimes death. In mosquitos however, Wolbachia has a more sinister role - causing something known as “cytoplasmic incompatibility”. I’ll explain what exactly that means in a minute, but the end result is that infected females will always produce infected offspring, and uninfected females have half the chance of producing offspring. This is a case where it’s easier to show, than tell.

This picture, taken from the Werren lab Wolbachia biology page, shows how cytoplasmic incompatibility leads to an increase in the proportion of infected hosts.

The exact mechanism for this cytoplasmic incompatibility (CI) isn’t yet clear. What is known is that some kind of modification must take place in the infected male during spermatogenesis, as mature sperm cells do not contain Wolbachia. We also know that rescue must occur at some point in the fertilised infected egg, as the presence of Wolbachia prevents CI from occurring, but uninfected eggs don’t produce offspring. The main consequence of CI is that the male pronucleus enters mitosis later than the female pronucleus, which means the genetic information from the male does not segregate properly during the first mitosis. This leads to the production of haploid daughter cells which is embryonically lethal, preventing any offspring from being formed.

In May last year, a paper by Daniel LePage et al., was published which found that just two genes in the Wolbachia genome were needed to cause CI. The sequences for these genes were found to originate from viral DNA, integrated from the bacteriophage WO genome at some distant point in Wolbachia’s evolutionary history. These two genes, named cifA and cifB (cytoplasmic incompatibility factors A and B), were initially identified as part of a pool of 113 genes shared between CI-causing strains of Wolbachia. This pool was narrowed down by removing proteins known to be very different or absent in a non-CI-causing strain, wAu, and by removing proteins known to be expressed by infected ovarian tissue as these might be generally expressed and not play a direct role in CI. This left just two genes, WD0631 and WD0632.

In section a, a Venn diagram shows 113 genes are shared between four common CI-causing strains of Wolbachia. In section b, a Venn diagram shows how these 113 genes were narrowed down to just two candidates for CI-causing genes – WD0631 and WD0632.

To investigate if these two were indeed the genes responsible for CI, transgenic lines of Drosophila melanogaster were created with both genes under the control of a promoter that is active in germ line cells. Males of this line were crossed with infected and uninfected females and the relative hatch rate calculated. It can be seen in the graph below that having both genes caused a significant change in the embryo hatch rate when crossed with uninfected females, and that this was rescued when crossed with infected females – as would be expected if these were the culprits for CI.

A comparison of relative embryo hatch rate in D. melanogaster. White symbols indicate uninfected males and females, black symbols indicate those infected with a CI-causing strain of Wolbachia.

Further to this the team investigated whether the cytological appearance was the same between Wolbachia infected embryos and transgenic WD0631⁺/WD0632⁺ lines, further strengthening their case that these were the responsible genes.

Panels a-f show different classifications of cytological appearance in recently fertilised ovaries of D. melanogaster. a) indicates unfertilised eggs, b) shows normal nucleated cells at 1h of development, c) shows normal embryos at 2h of development, d) shows failure of nuclear division after two to three mitoses, e) shows chromatin bridging and f) shows regional mitotic failure. In section g) the relative abundance of each cytological appearance is shown in different crossing scenarios.

The paper doesn’t go on to speculate on or provide any investigation into the mechanism of action of these two genes, but sets the stage for further work into this system and tantalisingly closes with the statement 

“Finally, cifA and cifB are important for arthropod pest and vector control strategies, as they could be an alternative or adjunct to current Wolbachia-based efforts aimed at controlling agricultural pests or curbing arthropod-borne transmission of infectious diseases”

This brings me on to the conclusion of this post and refers all the way back to the title – how can bacteria be used to fight insect-vector based disease?

There are already programs in place that are attempting to use Wolbachia-based methods of eradicating the insect vector of the dengue fever virus, the Aedes aegypti mosquito. By introducing large numbers of infected males into an uninfected population, the population can be reduced without the use of pesticides. The work done by LePage could provide the means to supplement Wolbachia in insect systems or possibly induce CI in other systems. An alternative to herbicides, or maybe maintenance-free pest control – can you think of any other applications? Let me know in the comments.