Like spies sneaking across enemy lines, the bacteria that cause Lyme disease have a world-class disguise: an invisibility cloak of lipoproteins. Lyme bacteria (Borrelia burgdorferi  sensu lato) interact with their hosts — whether ticks, humans, or other animals — via lipoproteins on their cell surfaces. But because B. burgdorferi are difficult to genetically manipulate and are evolutionarily distant from other better-studied bacteria, scientists still don’t understand how the bacteria use each of these lipoproteins.

Using a cell line of B. burgdorferi  that completely lacks lipoproteins, scientists systematically added back one surface lipoprotein at a time and identified two proteins that helped the bacteria evade detection by the human complement system, the first line of defense for the immune system. These findings will inform the development of new therapeutics and vaccine targets for Lyme disease (1).

The complement system consists of proteins in blood serum that recognize invading microbes and set off a cascade of steps to neutralize them. Because B. burgdorferi travel between their tick and mammalian hosts via a bloodmeal, their first threat to survival is from the complement system.

“The universe of complement evasion proteins is probably relatively large,” said John Leong, a microbiologist at Tufts University and senior author of the study. “We probably haven’t identified all of them, so our goal was to be able to screen the genome for interesting complement evasion proteins.”

Over the years, scientists noticed that many B. burgdorferi  surface lipoproteins bind to components of their host’s complement system. Scientists hypothesize that B. burgdorferi  express so many different lipoproteins on their cell surface because there are multiple ways to activate the complement system and because complement proteins differ slightly among the different animal hosts that B. burgdorferi  infect.

“Lyme disease really results from the Borrelia  moving from the site of infection where the tick bites, through the blood, [and] down into other tissues,” said Mollie Jewett, a Lyme disease researcher at the University of Central Florida who was not involved in the study. “Borrelia  is able to overcome the host barriers to dissemination, and one of those barriers is the complement system. If we can understand how Borrelia  overcomes these barriers, we can try to cut it off at the knees.”

Leong and his team partnered with Brandon Garcia, a structural biologist and expert in the complement system at East Carolina University, to identify B. burgdorferi complement evasion lipoproteins. Many B. burgdorferi  lipoproteins, however, have redundant functions. If the bacteria lose one, another will likely compensate for it.

To characterize the function of individual lipoproteins, Leong and Garcia teamed up with Wolfram Zückert at the University of Kansas Medical Center. Zückert’s team had created a library of B. burgdorferi  that individually express each lipoprotein in the genome. Using this library, Zückert and his team demonstrated that of B. burgdorferi’s 127 putative lipoproteins, approximately 80 of them are located on the bacterial cell surface (2).

Garcia, Leong, and their teams exposed this B. burgdorferi  library to human C1, a protein complex that activates one pathway of the complement system (3). They identified two surface lipoproteins, ElpB and ElpQ, which bound tightly to human C1.

“It immediately seemed like this was a bona fide hit,” said Leong. “The fact that ElpB and ElpQ are two non-identical but highly homologous proteins [that] each had this activity told us from the start that this was a likely, at least, biochemical activity.”

Leong and his team demonstrated that ElpB and ElpQ bound parts of the C1 protein complex that activate the next steps in the complement pathway.

“The final piece, which was not trivial, was developing an assay to really demonstrate that these proteins protected [B. burgdorferi]  from complement killing,” Leong said. His graduate student and coauthor on the study, Michael Pereira, pored over 40-year-old papers to determine the best way to test how well ElpB and ElpQ protected the bacteria from the human complement system in vitro.

With their new assay in hand, Leong and his team reported that B. burgdorferi  expressing ElpQ had a 321-fold higher survival rate than control bacteria that lacked ElpQ. While higher numbers of ElpB-expressing bacteria survived compared to control bacteria, the increase was not statistically significant.

“It’s just a very comprehensive and well thought out study,” said Jewett. “It’s just one step closer to … understanding what key Borrelia  proteins for host pathogen interactions.”

For Leong, determining whether ElpQ and ElpB can protect B. burgdorferi  from the complement system in a live animal host is the next step. To do these follow up experiments, the researchers will need to engineer B. burgdorferi  strains lacking both ElpB and ElpQ. Genetic manipulation is difficult in these bacteria, but new advances in CRISPR for B. burgdorferi  will help move these experiments forward (4).

By understanding how B. burgdorferi uses different lipoproteins to subvert the host complement system, “we can make those barriers stronger somehow or make Borrelia weaker,” said Jewett. “Then the barriers can do their job, and Borrelia  can’t make it to the places where it likes to set up shop. That’s what causes Lyme disease.”

References

1. Pereira, M.J. et al. Lipoproteome screening of the Lyme disease agent identifies inhibitors of antibody-mediated complement killing. Proc Natl Acad Sci USA  119, e2117770119 (2022).
2. Dowdell, A.S. et al. Comprehensive Spatial Analysis of the Borrelia burgdorferi Lipoproteome Reveals a Compartmentalization Bias toward the Bacterial Surface. J Bacteriol  199, e00658-16 (2017).
3. Janeway, C.A. Jr, Travers, P., Walport, M. et al. Immunobiology: The Immune System in Health and Disease. 5^th edition. The complement system and innate immunity. New York: Garland Science (2001). Available from: https://www.ncbi.nlm.nih.gov/books/NBK27100/
4. Takacs, C.N. et al. A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi. Appl Environ Microbiol  87, e02519-e02520 (2020)