Washington, D.C.—Pneumocystis is an unwieldy genus of fungal pathogens that cause severe pneumonia, particularly in immunocompromised people like those with HIV/AIDs or who have received organ transplants. However, the mechanisms by which it infects a host organism—and how it acquires resistance to known treatments—remain largely unknown, which makes devising new therapeutics a formidable challenge.
A new tool offers a way forward. In a recent paper in mBio , researchers at the University of Cincinnati College of Medicine in Ohio reported success in genetically modifying Pneumocystis murina, a species of the fungus that infects mice. Their approach uses extracellular vesicles, or EVs, from mouse lungs to deliver gene-modifying molecules inside the fungal cells. Results from both lab and animal tests showed that the modified fungus expressed the introduced genomic modifications.
"This really is the first use of host EVs as a transport mechanism to introduce DNA and nucleic acid material into pathogenic organisms," said A. George Smulian, M.D., an infectious disease researcher and senior author on the study who has studied the genetic machinery of Pneumocystis for decades.
Pneumocystis has long been poorly understood and difficult to study, in part because the fungus only replicates in its mammalian host—which makes it difficult to culture in a lab—and in part because individual species infect specific hosts. The Pneumocystis that infect a mouse are not the same species as one that infects a person. Many microbes share this limitation, leaving key genetic and mechanistic questions unanswered, and Pneumocystis has long represented a broader class of medically important pathogens that are difficult to study experimentally.
Scientists have long known that EVs act like tiny messengers, ferrying lipids, proteins and genetic material between cells. Prior to this study, molecular biologist Steve Sayson, Ph.D., who led the study, had been studying EVs within the host environment where Pneumocystis lives, trying to determine what specific nutrients transferred from EVs to Pneumocystis. That work led him to suspect that EVs might also be used to transfer gene-editing tools like CRISPR-Cas9, a complex of molecules that can be used to edit specific sections of the genome, like a Trojan Horse delivering hidden cargo to the pathogen.
Smulian's earlier research on the fungus, combined with Sayson's work on EVs, led the researchers to identify and verify successful genetic targets and transformations. The new tool, said Sayson, allows researchers to use mouse models to understand the genetic workings of the fungi, particularly those related to infection. The researchers say the strategy could extend beyond Pneumocystis to other obligate fungal and host-restricted pathogens. Because all mammalian tissues feature EVs, the approach provides a potential framework for delivering genetic tools into organisms that have resisted traditional laboratory manipulation.
One of the mutations they targeted, said Sayson, has been connected to the development of resistance to a common prophylactic drug among immunocompromised people. "So now we're able to interrogate that process to say what's causing resistance," he said. "Maybe we can develop a better drug." That would be particularly useful in parts of the world where people with HIV/AIDs lack access to high quality health care and may be severely immunocompromised, said Smulian.
The next step, said Sayson, is to find ways to better understand the genetic transformation initiated by the EVs. The new work showed how to change a single gene in a single region, but Sayson said it should be possible to control more genes and the level of expression. "And there's a lot more we can do," he said.
The researchers were supported financially by a grant from the National Institutes of Health that encourages the exploration and development of new tools used to study difficult pathogens like Pneumocystis. The grant provided funding for [JU1.1]experimental proposals with out-of-the-box thinking for new tools, rather than testing hypotheses about the pathogens.
"There are big possibilities with this molecular toolbox," Sayson said.
The grant is unique in that it encourages research on the development of fundamental tools and techniques to further the understanding of pathogens that are difficult to study, which is key to expanding capability to research those pathogens. Supporting further study of microbial interactions, fundamental processes, dynamic relationships and microbial evolutionary mechanisms is the focus of ASM Mechanism Discovery.
