Researchers at the VIB-VUB Center for Structural Biology report that removing the armor of the bacterium that causes anthrax slows its growth and negatively affects its ability to cause disease. The study (“Structure of S-layer protein Sap reveals a mechanism for therapeutic intervention in anthrax”), which will appear in Nature Microbiology, may lead to new, effective ways of fighting anthrax and various other diseases, according to the scientists.
Anthrax is a deadly and highly resilient disease, caused by the spore-forming bacterium Bacillus anthracis. The toughness of the spores and the lethality of an anthrax infection via inhalation spurred its development as a biological weapon in the mid-twentieth century. Although the development and stockpiling of anthrax as a bioweapon has been banned by the international community, these regulations are violated at times. Because treatment options are limited and not effective in most cases, this means anthrax remains a potential bioterrorism threat.
As part of its strategy to evade the weapons of the immune system, the anthrax bacterium cloaks itself with a complex, dynamic armor. A poorly understood component of this armor is the Sap S-layer, a single layer of protein that forms a shell around the bacterium.
In this study, researchers successfully applied Nanobodies® (small antibody fragments) to control the assembly of the bacterial armor and study its structure. The Nanobodies were not only effective in preventing the armor from forming, but also proved highly efficient in breaking down existing S-layers. When applied to live bacteria, breaking down the armor slowed bacterial growth and led to drastic changes in the surface of the bacterial cell.
“At present, anthrax mostly affects wildlife and livestock, although it remains a concern for human public health, primarily for people who handle contaminated animal products and as a bioterrorism threat due to the high resilience of spores, a high fatality rate of cases and the lack of a civilian vaccination program. The cell surface of B. anthracis is covered by a protective paracrystalline monolayer, known as surface layer or S-layer, that is composed of the S-layer proteins Sap or EA1. Here, we generate Nanobodies to inhibit the self-assembly of Sap, determine the structure of the Sap S-layer assembly domain (SapAD) and show that the disintegration of the S-layer attenuates the growth of B. anthracis and the pathology of anthrax in vivo,” write the investigators.
“SapAD comprises six β-sandwich domains that fold and support the formation of S-layers independently of calcium. Sap-inhibitory nanobodies prevented the assembly of Sap and depolymerized existing Sap S-layers in vitro. In vivo, nanobody-mediated disruption of the Sap S-layer resulted in severe morphological defects and attenuated bacterial growth. Subcutaneous delivery of Sap inhibitory Nanobodies cleared B. anthracis infection and prevented lethality in a mouse model of anthrax disease. These findings highlight disruption of S-layer integrity as a mechanism that has therapeutic potential in S-layer-carrying pathogens.”
Antonella Fioravanti, PhD, who led the research, said that “I created these Nanobodies as a tool to study the Sap S-layer, but that they would also inhibit bacterial growth was an unexpected bonus.”
The effects were so striking, she added, that the Nanobodies were tested as a treatment in mice infected with B. anthracis. “The results were amazing, all treated mice recovered from lethal anthrax within days,” said Filip Van Hauwermeiren, PhD, who performed the infection studies. “We had been studying ways to stop the lethality of anthrax but had never seen such striking effects as with these Nanobodies,” added his supervisor Mohamed Lamkanfi, PhD, (previously at the VIB-UGhent Center for Inflammation Research, now at Janssen Pharmaceutica and Ghent University).
Therapeutics derived from the Nanobodies discovered in this study may one day fill the currently existing treatment gap, say the scientists. Moreover, targeting the S-layer with nanobodies may be successful in the fight against other bacteria with an S-layer armor. For example, the lab is currently exploring S-layer targeting Nanobodies in Clostridium difficile which causes life-threatening colitis.
The success of the experiments in this study have motivated the researchers to look for other vulnerable targets on bacterial cell surfaces.
“Proteins on the surface of bacteria are interesting antibacterial targets because they are directly accessible. Targeting these proteins means that we have to worry less about that various ways that bacteria are preventing drugs from getting into the cell,” pointed out Han Remaut, PhD, from VIB-VUB.