Researchers at the Howard Hughes Medical Institute at Yale University School of Medicine, have discovered how a protein called APOL3 that is made throughout much of the body can wipe out invading bacteria by acting like a detergent attacking an oily mark. Tests against Salmonella and other bacteria found that this killer cleaner protein thwarts infections by dissolving bacterial membranes. And while scientists already knew that cells could attack bacterial membranes, the new study uncovers what appears to be the first example of a protective intracellular protein with detergent-like action.

The work offers new insight into a defense mechanism known as cell-autonomous immunity, which human cells use to fight off infection, and could point to the development of new anti-infective approaches. Research lead John MacMicking, PhD, an immunologist at Yale University, said, “This is a case where humans make their own antibiotic in the form a protein that acts like a detergent. We can learn from that.”

MacMicking and colleagues reported on their findings in Science, in a paper titled, “A human apolipoprotein L with detergent-like activity kills intracellular pathogens.”

When it comes to defending the human body, the specialized cells of the immune system act as a crew of cellular bodyguards. But in what the researchers call “the arms race between pathogen and host,” infecting microbes may escape extracellular defense mechanisms and make it to the intracellular environment, where they replicate. Cell-autonomous immunity is a mechanism that has evolved to defend against such intracellular pathogens.

When microbes like Salmonella (green) infect human cells, they overrun the cells’ liquid interiors. Researchers have discovered how cells fight back: with a detergent-like molecule, called APOL3, that kills the bacteria by breaking up their membranes. [R. Gaudet et al./Science 2021]
“This ancient form of host defense protects against intracellular pathogens through direct and indirect effector mechanisms,” the authors explained. In vertebrates, these effector mechanisms are triggered by a type II cytokine, interferon-gamma (IFN-γ), which regulates the transcription of hundreds of IFN-stimulated genes (ISGs) to help combat bacteria, viruses, parasites, and fungi in a wide variety of cell types.

While IFN-γ triggers gene expression in multiple different cell types, including non-immune cells, scientists still know little about the nature of the proteins that are induced by the cytokine, or how such proteins help cells fight pathogens. “The mechanisms and protein machineries involved in this nonclassical or ‘structural’ arm of immunity remain poorly understood,” they commented. “… few ISGs with direct pathogen-neutralizing activity have been characterized … This is especially true within human mucosal or stromal cell lineages that are historically viewed as separate from the classical immune system.”

For their newly reported studies, the researchers infected non-immune human cells with a strain of Salmonella. “We searched for new antimicrobial ISGs in human epithelial (HeLa CCL2) cells, using the virulent Gram-negative bacteria Salmonella enterica serovar Typhimurium (Stm) as an initial infection model,” they reported.

These bacteria belong to a class of microorganism that is bounded by two membranes. The outer bacterial membrane acts like armor, protecting the inner bacterial membrane from threats like antibiotics. “The double membranes surrounding Gram-negative bacteria make them exceptionally difficult to kill,” the team acknowledged.

The researchers’ studies first confirmed that the IFN-γ alarm signal triggered by infection could prevent Salmonella from taking over the human cells, but it still wasn’t known which proteins were involved in that antibacterial attack. To try and identify which genes may be involved, the investigators used CRISPR-Cas9 technology to screen more than 19,000 of the human cells’ genes, looking for IFN-γ-induced genes that might encode protective proteins.

Before killing Salmonella, the detergent-like protein APOL3 (green) must get through the bacteria’s protective outer membrane (red), as shown in the cross sections above. [R. Gaudet et al./Science 2021]
Their results highlighted the apolipoprotein L3 (APOL3) gene, as an ISG that could kill the cytosol-invasive bacteria. APOL3 is one of a family of six genes, but apart from APOL1, which is a secreted extracellular protein found in human serum, the function of the intracellular APOL family members has remained known, the investigators noted.

Their newly reported studies showed that APOL3 destroys the inner bacterial membrane, but to do so receives assistance from a second molecule, GBP1, and probably others. Using high-resolution microscopy and other techniques, the team pieced together the antibacterial mechanism. Their results showed that GBP1 damages the bacterium’s outer membrane, allowing APOL3 through so that it can then break apart the inner membrane—the “coup de grace” that kills the bacterium, MacMicking says.

Like a laundry detergent, APOL3 possesses parts attracted to water and parts drawn to fats or grease. But rather than removing dirt from fabric, these components remove chunks of the bacterial inner membrane, which is composed of lipids. “APOL3 synergizes with other host ISGs in a multipronged attack against the double membrane of Gram-negative bacteria—a formidable barrier that imparts resistance to many classes of antibiotics,” the scientists wrote in a separate summary of their paper. “This study reveals that antibacterial agents that dismantle this barrier during infection naturally exist inside human cells. That these agents are encoded within the IFN-γ–inducible defense program reinforces the importance of this powerful antimicrobial network for cell-autonomous immunity in humans.”

This process of bacterial membrane attack must also be highly selective, MacMicking pointed out, since APOL3 needs to avoid attacking the human host cell’s own membranes. And in fact, the researchers found that APOL3 avoids cholesterol, a major constituent of cell membranes, and instead targets distinctive lipids favored by bacteria.

APOL3 appears likely to be in the toolbox of many cells. MacMicking’s team showed it defends cells within the blood vessels and gut. And because APOL3 appears in a variety of body tissues, the scientists believe it offers wide protection. And while researchers are still a long way from being able to apply this discovery to therapies for infections, deciphering the body’s defenses could provide new tools against bacteria that are increasingly evolving ways to thwart conventional antibiotics. Dialing up cellular detergents and other devices the body uses to kill bacteria, for instance, could help supplement the natural immune response, MacMicking concluded.