The brain-eating amoeba Naegleria fowleri is an emerging and deadly protist pathogen whose natural range appears to be rapidly expanding, says Lillian Fritz-Laylin, biology, but it has been “largely unstudied” because it is so dangerous and scientists lack adequate tools for gene manipulation.
Now she has a two-year, $433,600 grant from the NIH’s National Institute of Allergy and Infectious Diseases to develop genetic tools in a non-infectious relative, N. gruberi, to create an easy and safe system in which to test the leading hypotheses about pathogenesis in the deadlier species. This research will allow direct testing of potential molecular mechanisms of how the organism destroys brain tissue.
Fritz-Laylin says, “In many Naegleria species, we can look at what genes are there and when they are turned on. There have been successes in figuring out what genes are turned on in pathogenic contexts or situations, but it doesn’t necessarily mean that those genes are important to the pathogenesis itself. Until we can actually turn genes down or turn them off and see how that changes pathogenic behaviors, we can’t tell for sure which are necessary. That’s what I’m trying to do.”
Not all pond-dwelling Naegleria are pathogenic, but as water temperature rises, the non-pathogenic species die off and the pathogenic ones take over, Fritz-Laylin explains. The amoebas get inside the noses of swimmers, crawl up the olfactory nerve and into the brain where they destroy tissue. “Sadly, victims are often children,” she says. Reported cases appear to be moving northward in recent years; it is thought to be more common in tropical regions and few have been seen in the United States.
A cell biologist who is interested in how cells move, Fritz-Laylin has studied Naegleria for many years because at different life cycle stages, these protists can switch behaviors to either crawl or swim. Her research is “helping to tell us about the evolution of cell movement,” she notes, “and its relationship to other eukaryotes can tell us what was going on in early eukaryotic cell history.”
Fritz-Laylin and colleagues including Vincent Rotello, chemistry, will use two approaches in this investigation, RNA-interference (RNAi) and gene-editing CRISPRcas9 technology. RNAi works with organisms that eat bacteria, she notes, “and luckily for me, this organism is a voracious bacterial predator.”
She will feed Naegleria bacteria that make double-stranded RNA with the same sequence she intends to disrupt. The method will induce Naegleria to destroy some of its own RNA, turning genes off. Also, “CRISPR technology has been used for specific and efficient genome engineering of diverse protist pathogens,” she adds. “Each method has benefits and uses different pathways, so if one fails we hope the other will work.”
Both approaches will target three genes, each of which builds flagella in Naegleria. Disrupting any of them should cause problems that are easy to spot, Fritz-Laylin says. Being able to interfere with Naegleria gruberi genes “will allow the first direct testing of the roles of putative virulence factors in the molecular mechanisms driving pathogenic behaviors,” offering new tools for studying the disease and potentially new drugs for treating it.