When the mammalian immune system detects a pathogen, a kind of immunological call to arms is precipitated by the release of a cytokine known as interferon-gamma, which induces the transcription of tons of host genes in cells throughout the body, not just in immune cells. But the identities of all those genes and what they do to protect the host aren’t well understood. In a study published today (July 15) in Science, researchers found that one gene stimulated by interferon-gamma, apoplipoprotein L3 (APOL3), produces a protein that can destroy bacteria that sneak into host cells by acting like a detergent—surrounding the lipids integral to the pathogens’ cell membranes and causing them to break apart.
The paper is “important for a new understanding of how nonimmune cells like epithelial cells have antibacterial responses,” says Christina Faherty, a bacterial geneticist at Harvard Medical School and Massachusetts General Hospital who was not involved in the work. It’s a powerful example of “that constant warfare between bacteria and hosts of fighting and countering and just trying to figure out who can survive.”
In the new study, John MacMicking, a Howard Hughes Medical Institute investigator and immunologist at Yale University, and colleagues set out to explore the idea “that immune responses to infection not only enlist the classical immune response . . . but they also need to communicate or talk with the nonimmune cells to bring about restriction of infection,” he says. “The infection is actually taking place in cells that are often not part of the immune system and are the first to be breached by different invading pathogens, particularly intracellular organisms.”
MacMicking and his team used CRISPR in human epithelial cells to systematically mutate each of the thousands of genes that could change activity in response to interferon-gamma. They then exposed these cells to interferon-gamma and found that cells with mutations in APOL3 were not able to tamp down rapid replication of a bacterium, Salmonella enterica serovar Typhimurium, one cause of food poisoning in people. The team determined that when cells receive the signal from interferon-gamma warning of pathogens on the loose, they activate the expression of APOL3.
Apolipoproteins are typically secreted proteins found in extracellular spaces, and their main function is thought to be the transportation of lipids, such as cholesterol, throughout the body. But previous work had demonstrated that APOL3 protein and some of its family members are found inside several types of mammalian cells, where they could potentially defend against bacteria that sneak in by similarly binding to lipids, in this case, bacterial lipid membranes. More evidence that this protein family can work in immune defense is that another member, APOL1, had been shown by other groups to provide protection against an extracellular pathogenic protist called a trypanosome.
To figure out how cells use APOL3 to handle the invaders, the researchers looked at what fluorescently tagged APOL3 does in response to fluorescently tagged bacteria, both in cells and with the components mixed together in a petri dish. They saw that APOL3 cooperates with another protein induced by interferon-gamma, guanylate-binding protein 1, to penetrate the outer bacterial membrane. Then APOL3 binds specifically to the lipids in the inner bacterial membrane, ignoring lipids common to host membranes, and surrounds them—just as dish soap surrounds the grease on pots and pans. This binding breaks the membrane apart, destroying the bacterial cells.
“They find a very interesting detergent-like property that’s dependent on the lipid composition of the membrane that the protein interacts with,” says Jayne Raper, a microbiologist affiliated with Hunter College and New York University School of Medicine who did not participate in the study. Raper has worked on APOL1 and trypanosomes for years and explains that she and others suspected that the other apolipoprotein family members were involved in innate immunity. “The only one that we had any data for was APOL1, but the family is indeed rapidly evolving. And when you see that happen in biology, you always rationalize it must be having a molecular arms race with microbes, because they’re the ones that evolve fast.”
The authors are “among the first to discover an antibacterial function for an apolipoprotein,” Lora Hooper, an immunologist at UT Southwestern Medical Center in Dallas who was not involved in the work, writes in an email to The Scientist. “Normally we think of these proteins as having a dietary function: ferrying lipids throughout the body to be used as energy or cellular building blocks,” she continues. “But the location of APOL3 inside of a cell didn’t exactly jibe with a nutritional function, and so it makes sense that it is doing something completely different, such as defending cells against invading bacteria.”
The next step, according to MacMicking, is to explore other aspects of that defense, in particular, what else APOL3 and others in the family are doing. They “may well have activities that are not just antibacterial, but could be antiviral or antiparasitic,” he says. “We’re in the process of trying to examine the sort of the breadth of antimicrobial activity that these proteins may have.”