Synthetic Spores Give Insight into the Real Thing and Reveal Functional Applications


Artificial bacterial spore-like particles. In this image, silica beads that are two micrometers in diameter are coated with a membrane, atop which the basement layer of the Bacillus subtilis spore coat is assembled. The structural component of the coat is covalently linked to a small molecule, a fluorescent dye (shown in red), thereby demonstrating the drug display potential of these particles.

Spores from bacteria, such as Bacillus subtilis, are produced to allow the bacterium’s genetic material to survive harsh environments. When the bacterium senses nutrient depletion, it divides asymmetrically into a forespore and a mother cell. The mother cell engulfs the forespore, and coat proteins synthesized by the mother cell localize to the surface of the forespore. The mother cell eventually ruptures, releasing the mature spore, which is surrounded by a thick shell of approximately 70 different proteins. This protein coat is one of the most durable static biological structures, but, because of its complexity, detailed studies of how the coat forms have been lacking. Kumaran Ramamurthi, Ph.D., of CCR’s Laboratory of Molecular Biology, and his colleagues including postdoctoral fellow and lead author of the study I-Lin Wu, Ph.D., decided to investigate the assembly of the basement layer of the spore coat by decorating spherical membranes supported by silica beads with SpoIVA and SpoVM, proteins which are known to be required for coat assembly.

In the current model of basement layer assembly, SpoVM spontaneously inserts into the convex membrane of the forespore to mark it for coat assembly and to recruit and anchor SpoIVA. In support of this model, the researchers observed a uniform distribution of SpoIVA around the forespore but only in mother cells that expressed SpoVM. Without SpoVM, SpoIVA clumped together in one spot on the forespore. The investigators saw similar results when they incubated phospholipid bilayer-coated silica beads (also called spherical supported lipid bilayers or SSLBs) with purified SpoIVA with or without synthesized SpoVM peptides, respectively. These results indicate that SpoIVA has some natural affinity for membranes but that uniform distribution around a spherical membrane requires SpoVM.

SpoIVA is also known to bind and hydrolyze ATP to generate a conformational change that allows it to polymerize. To see whether ATP is important for the association of SpoIVA with the forespore, the scientists expressed in bacteria a SpoIVA mutant, K30A, which disrupts ATP binding. They found that the K30A mutant fails to coat the forespore surface as well as the wild type protein. Because they also observed more of the mutant in the cytosol, the researchers wondered whether the SpoIVA interaction with the forespore was reversible in the absence of polymerization. Using the SSLB system, they varied the amount of ATP and saw similar adsorption of SpoIVA at all concentrations. However, a competition assay with unlabeled SpoIVA revealed that pre-bound, labeled SpoIVA was eliminated rapidly from the surface of the SSLBs in the absence of ATP whereas with ATP a majority of the labeled SpoIVA remained associated even three days later. These results suggest that there is a dynamic exchange between cytosolic and forespore-bound SpoIVA and that stable association of SpoIVA with the membrane requires ATP hydrolysis.

Because relatively little is known about the structural features of the spore basement layer and their in vitrosystem mimicked the behavior of basement layer proteins in vivo, the investigators used scanning electron microscopy to examine the surface of SSLBs without and with associated proteins. Plain SSLBs were largely smooth, and adding SpoVM did not change the surface. In contrast, the addition of SpoVM and SpoIVA in the presence of ATP caused the surface to become roughened with non-uniform protrusions that were unevenly spaced. This structure resembled previously-reported surfaces of mutant spores that fail to assemble outer layers of the spore coat. SSLBs incubated with SpoVM and SpoIVA without ATP or with SpoVM, ATP, and SpoIVA K30A, however, retained smooth surfaces suggesting the static polymerized protein shell has a distinct structure from one with only adsorbed proteins.

Bacterial spores can be modified with a variety of proteins and have been proposed as delivery systems for vaccines or drugs. However, their use would likely require genetically modified organisms, and the spores themselves may contain additional factors that interfere with the modification of interest. Since a similar synthetic system could eliminate both of these issues, the scientists asked whether their basement layer-encased SSLBs could be modified. Using copper-free click chemistry, they were able to link small molecules, such as trans-cyclooctene and azide, individually or together, to SpoIVA. Likewise, they could attach green fluorescent protein. Thus, these modified synthetic particles may have clinical applications as display platforms for drugs and vaccines or even environmental applications by carrying enzymes that can neutralize pollutants.

Summary Posted: 05/2015

Reference

Wu IL, Narayan K, Castaing JP, Tian F, Subramaniam S, and Ramamurthi KS. A versatile nano display platform from bacterial spore coat proteins. Nature Communications. April 9, 2015. PubMed Link