Pathogenic bacteria such as Yersinia pestis, causative agent of the plague, have a genetic armoury of proteins they use to defend themselves against the immune system when invading a host. Upon invasion, Y. pestis bacteria deploy a molecular cloaking device, made of a protein called Caf1, which allows them to avoid being eaten by a host’s macrophage cells. Caf1 has several interesting structural properties that allow it to carry out this role, such as its ‘non-stick’, bioinert nature. This provides us with a blank canvas for protein engineering, where we can insert different bioactive signals into the protein structure, allowing us to instruct cells in a defined way, e.g., providing them with attachment sites or behavioural cues. We can also exploit Caf1’s unusual properties to use it as a molecular Lego kit, mixing and matching different bioactive Caf1 modules to make multifunctional biomaterials. We aim to use engineered Caf1 proteins to solve problems in the industrial scale production of cells for technologies such as cell therapy and cultivated meat. For example, by mixing adhesive and growth factor signals in a single material, and displaying multiple copies of each signal at once, we can reduce the number of expensive reagents needed. More generally, Caf1 is an excellent example of how bacterial armaments and defences can be re-engineered and adapted to benefit society, rather than cause disease.

The year is 1349 and Europe is in the midst of the worst pandemic it has ever experienced, which will lead to the deaths of 30–50% of the population, in turn causing large societal changes. In this way, the course of history was altered as an estimated 50 million people died – all because of a microscopic bacterium, Yersinia pestis.

Why was this bacterium suddenly so deadly? It was not always the case, as we know from studies of skeletons from the Bronze Age. In these, it appears the bacterium had the capacity to cause disease but lacked the protein which enables its transmission by fleas, hence reducing their ability to cause large-scale pandemics.

Differences in pathogenicity such as this are dependent on the bacterial genetic ‘armoury’, the various attack and defence systems it uses to infect a host. Like most pathogens, Y. pestis has evolved a whole suite of different tools, termed virulence factors, it can use to win the war against the immune system and propagate itself at the expense of the host.

Taking a look inside this genetic arsenal, we find various protein-based weapons. For example, the Yop proteins can be injected into host immune cells and effectively throw a spanner into the works of the machinery they use for phagocytosis (i.e., the engulfment and destruction of invading bacterial cells). Looking further, we find another protein, Caf1, which can act as a cloaking device for the invading bacteria, preventing immune cells from either recognizing or attaching to the bacteria.

How does this work? Caf1 is an unusual protein, in that its structure is incomplete. It adopts what is called an ‘immunoglobulin-like fold’, similar to structures found in both antibodies and proteins of the extracellular matrix, which supports cells in tissues. This fold requires seven β-strands in order to be stable, but Caf1 only has six. It solves this problem in an intriguing way – each Caf1 protein donates a ‘spare’ β-strand to another, forming a linkage and completing the seven-strand fold (Figure 1). As a result of this, the Caf1 monomers can form long, flexible and non-covalent polymers of 250 units or more, measuring in excess of 1.5 μm!

Figure 1

Overview of the Caf1 system. (a) Bacteria containing the caf1 gene express the subunits (green circles), which are targeted to the periplasm (dark brown) by secretion proteins (red). The subunits are then bound by a chaperone and delivered to an outer membrane usher protein (blue), which simultaneously assembles the subunits into polymers and exports them from the cell, where they form a protective gel-like coat around the bacteria (light blue). (b) Close-up of a Caf1 polymer, with the ribbon structure of two individual Caf1 subunits highlighted in green and orange. (c) Close-up of a Caf1 subunit, with the donor strand and acceptor clefts highlighted. (d) The donor strand of one subunit (orange) inserts into the acceptor cleft of another (green) subunit, completing the Caf1 molecular fold and forming a strong, non-covalent linkage. This is the basis for Caf1 polymer formation.

Figure 1

Overview of the Caf1 system. (a) Bacteria containing the caf1 gene express the subunits (green circles), which are targeted to the periplasm (dark brown) by secretion proteins (red). The subunits are then bound by a chaperone and delivered to an outer membrane usher protein (blue), which simultaneously assembles the subunits into polymers and exports them from the cell, where they form a protective gel-like coat around the bacteria (light blue). (b) Close-up of a Caf1 polymer, with the ribbon structure of two individual Caf1 subunits highlighted in green and orange. (c) Close-up of a Caf1 subunit, with the donor strand and acceptor clefts highlighted. (d) The donor strand of one subunit (orange) inserts into the acceptor cleft of another (green) subunit, completing the Caf1 molecular fold and forming a strong, non-covalent linkage. This is the basis for Caf1 polymer formation.

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The bacteria secrete these polymers, which form a gel-like protective coat around the bacterium. The Caf1 coat then ‘hides’ the bacteria from defensive cells such as macrophages by three methods (Figure 2). First, the Caf1 coat acts like a hydrated brush, which hinders contact with the macrophage surface. Next, it lacks any high-affinity sites for the macrophage to latch on to, similar to a rock climber trying to scale a smooth cliff face. Finally, if a macrophage is lucky enough to get a purchase and begins pulling, the Caf1 polymers are immensely strong (for a protein!) and will not break. This prevents exposure of the underlying bacterial surface, which is ripe with targets that the immune system can sense and respond to. This mechanical strength appears to have evolved to be only just higher than the forces that macrophages can exert on the protein, as a single mutation that causes just a 20% drop in breaking strength is enough to allow immune cells to recognize and destroy bacteria, ripping apart the weakened Caf1 in the process.

Figure 2

Effect of mutations on Caf1’s protective ability. Wild-type Caf1 (green) surrounds the bacterium and prevents attachment of macrophage receptors (purple) through a combination of polymer brush effects and lack of high-affinity binding sites, allowing the bacterium to escape unscathed. When an adhesive motif from fibronectin (red dots) is inserted, it provides a high-affinity attachment point for the macrophage receptors, allowing the bacteria to be engulfed and destroyed by phagocytosis. A single-point mutation made in the wild-type Caf1 causes its mechanical stability to drop by about 20%. When bacteria express this version of Caf1 (purple), macrophages can exert a force upon the coat and cause it to rupture, leading to exposure of the bacterial surface. This is readily recognized by macrophage receptors, and so the bacterium is engulfed and destroyed. Therefore, Caf1 uses three factors to successfully defend bacteria against macrophage attack – lack of cell interaction due to hydrated brush effects, lack of high-affinity binding sites and high strength to resist macrophage forces. Figure adapted from Peters et. al., PLOS Pathogens. 2022 Mar 31;18(3):e1010447.

Figure 2

Effect of mutations on Caf1’s protective ability. Wild-type Caf1 (green) surrounds the bacterium and prevents attachment of macrophage receptors (purple) through a combination of polymer brush effects and lack of high-affinity binding sites, allowing the bacterium to escape unscathed. When an adhesive motif from fibronectin (red dots) is inserted, it provides a high-affinity attachment point for the macrophage receptors, allowing the bacteria to be engulfed and destroyed by phagocytosis. A single-point mutation made in the wild-type Caf1 causes its mechanical stability to drop by about 20%. When bacteria express this version of Caf1 (purple), macrophages can exert a force upon the coat and cause it to rupture, leading to exposure of the bacterial surface. This is readily recognized by macrophage receptors, and so the bacterium is engulfed and destroyed. Therefore, Caf1 uses three factors to successfully defend bacteria against macrophage attack – lack of cell interaction due to hydrated brush effects, lack of high-affinity binding sites and high strength to resist macrophage forces. Figure adapted from Peters et. al., PLOS Pathogens. 2022 Mar 31;18(3):e1010447.

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As a result of its presence on the cell surface, Caf1 was a key target for anti-plague vaccine efforts. Indeed, people who have survived infections with Y. pestis often develop antibodies that target Caf1. Bound antibodies act as homing beacons for immune cells and so are able to overcome Caf1’s protective effect by providing them with something easy to latch on to as they engulf and digest the bacteria – in effect providing easy footholds for the rock climber described earlier. After many years of research, an anti-plague vaccine consisting of Caf1 and another protein, called V-antigen, was developed.

As part of these vaccine development efforts, Caf1’s structure was intensively researched as scientists sought better ways of producing it safely in other (non-plague-causing!) bacteria and better ways of presenting it to the immune system. During one of these projects, it was noticed that Caf1 had a very similar structure to a human protein, called fibronectin, which is part of the extracellular matrix (ECM) and provides attachment sites for the cells within tissues. These sites are small, specific regions on ECM proteins and in fibronectin consist of a sequence of just four amino acid residues: arginine, glycine, aspartate and serine (RGDS). As Caf1 can be safely produced by inserting Y. pestis genes into laboratory strains of Escherichia coli, it was a small step to modify the gene sequence to produce engineered versions of Caf1, in this case containing the adhesive RGDS motif from fibronectin (Figure 3). The results were very clear – while the native Caf1 provided a Teflon-like surface that cells could not stick to, the engineered version allowed cells to stick just as if they had been presented with fibronectin itself.

Figure 3

Engineered Caf1 subunits can be combined to form mosaic polymers. The wild-type, unmodified Caf1 subunit (a) can be genetically engineered to contain bioactive motifs (b) such as those from vitronectin (purple), fibronectin (red) and TGF-β (orange). The subunits can be combined to form a multifunctional mosaic polymer (c). The wild-type protein is bioinert and non-adhesive for mammalian cells, causing them to form a rounded phenotype and have poor viability (d), whereas cells grown on the engineered polymers adhere and interact with the inserted motifs (e).

Figure 3

Engineered Caf1 subunits can be combined to form mosaic polymers. The wild-type, unmodified Caf1 subunit (a) can be genetically engineered to contain bioactive motifs (b) such as those from vitronectin (purple), fibronectin (red) and TGF-β (orange). The subunits can be combined to form a multifunctional mosaic polymer (c). The wild-type protein is bioinert and non-adhesive for mammalian cells, causing them to form a rounded phenotype and have poor viability (d), whereas cells grown on the engineered polymers adhere and interact with the inserted motifs (e).

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This result revealed the potential of using Caf1 as a biomaterial – the inert wild-type protein provides a blank slate for engineering in any bioactive motif, allowing us to choose exactly what messages it will convey to cells. Since then, we have been able to generate versions of Caf1 that effectively mimic many ECM proteins, including vitronectin and laminin, as well as growth factors such as BMP2 and VEGF that tell stem cells to develop as bone or endothelial cells to migrate and form new vasculature. Only small parts of these proteins need to be added to Caf1 for transmission of the signal to the cells. In this way, we have made versions of Caf1 that trigger the differentiation of stem cells into bone-producing cells or guide endothelial cells on the first steps of blood vessel formation.

Moreover, as the polymer is formed from many modular Caf1 subunits, it is possible to create ‘mosaic’ Caf1 polymers that convey more than one signal e.g., combining both adhesive and differentiation signals at the same time. There are two ways to do this: genetically engineering the bacteria to express multiple subunits at once, which they then incorporate and secrete as a mosaic polymer, or by exploiting Caf1’s non-covalent method of linking the subunits together. In the latter case, Caf1 polymers that are thermally denatured into monomers are able to spontaneously refold and self-assemble back into oligomers. Thus, different bioactive modules of Caf1 can be mixed and combined in vitro to produce complex mosaic polymers. In this way, Caf1 subunits can be thought of as individual Lego bricks that can be combined and constructed in a multitude of ways. Considering the polymers can also be used to form 3D materials, it really does provide us with a molecular construction set.

The ability to culture cells in the laboratory is the basis of many new technologies. For instance, the development of cell therapies, such as chimeric antigen receptor T cells (CAR-T), is already proving to be remarkably effective at hunting down and destroying difficult to treat cancers, including those that were previously thought untreatable. Another emerging technology is cultivated meat, which aims to replace traditionally farmed meat and seafood with cells grown in a bioreactor. This bypasses the need to grow or kill animals, potentially reducing carbon emissions and preventing marine biodiversity loss.

However, both these technologies present a great technical challenge; cells must be grown in industrial bioreactors on a scale that has never before been reached for animal cells. Part of the challenge is how to mimic the natural environment of the cells while in a system that is not only wholly unlike the body, but is also thousands of times bigger! Biological cues and signals can be delivered by adding natural growth factors into the culture media, but getting the right proportion is challenging. Additionally, they are also incredibly expensive, as their production is non-trivial.

By exploiting the unusual properties of Caf1, we can provide a new way of supplying these biological signals at huge scale. As Caf1 polymers contain many modules, each bearing the bioactive motif, they can transmit these signals more effectively to cells. The Caf1 also attaches to surfaces, so it doesn’t need to be removed every time the media is changed to provide fresh nutrients to cells. The modules can be combined to make more complex signals in a single material. Finally, as the bacteria are so effective at producing Caf1, it can be produced for a much lower cost than other, more challenging proteins.

To summarize, through the study of the mechanisms of deadly bacterial pathogenesis, we can redesign and re-engineer the weapons and defences of a killer in order to enable novel technologies and hopefully benefit society.

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Dr Daniel Peters is a Research Associate at Newcastle University in the Lakey Group, as well as a co-founder and director of MarraBio, established in 2022 to exploit the Caf1 technology. His background is in structural biology and major research interests include the design, engineering and characterization of proteins for synthetic biology applications. Email: daniel.peters@newcastle.ac.uk. Twitter: @DrDanPeters1

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Jeremy Lakey is Professor of Structural Biochemistry at Newcastle University, as well as a co-founder and director of MarraBio. He has worked on proteins of Gram-negative bacteria for many years using biophysical methods to understand their functions and interactions. This led to many industrial collaborations in protein analysis and projects which re-engineered bacterial proteins for new uses. Email: jeremy.lakey@newcastle.ac.uk.

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