To most researchers, the bits of phage-like DNA found in some bacterial genomes are only viral DNA. But through a few serendipitous discoveries, scientists recently realized that what looked like remnants of phage genetic sequences actually encoded tiny, bacterial syringe-like structures with protein payloads sequestered inside. Unlike many other bacterial injection systems, nanosyringes are not bound to the bacterial cell membrane. Instead, bacteria release them into the environment to find and inject their protein cargos into eukaryotic target cells.

Now, scientists are co-opting these bacterial nanosyringes to express therapeutic proteins for human diseases, designing a combined drug and delivery mechanism. These engineered nanosyringes will allow for more efficient drug delivery of protein and peptide-based drugs directly into specific human cells.

“Having a system that can do something as complicated and sophisticated as delivering biologics across membranes — that is entirely genetically encoded — is a synthetic biology dream,” said Joseph Healey, the CEO and co-founder of NanoSyrinx, a nanosyringe biotechnology company. “It's such a captivating solution to what is such a difficult problem.”

A wax moth and tubeworm discovery

The discovery of bacterial nanosyringes has a few unlucky insect larvae to thank. Nick Waterfield, now a microbiologist at the University of Warwick and co-founder of NanoSyrinx, wanted to identify virulence genes in Photorhabdus bacteria. These bacteria release toxins that kill insect larvae over the course of a day. As Waterfield and his team screened virulence gene clusters from four different groups of Photorhabdus bacteria, they discovered that one cluster was so potent that it killed wax moth larvae within 15 minutes (1).

“I was just fascinated to understand what these things were,” said Waterfield. The genetic sequence of the cluster looked like it encoded a structure similar to the tail of a phage, so the team purified the protein and used electron microscopy to take a closer look.

“When we got our first look at them down the electron microscope, it was a bit of a ‘Wow!’ These really do look like syringes because we saw them in an extended and contracted form,” said Waterfield. “We later did some work labeling with an immunogold antibody to show that the toxins were able to be squirted out the end, so that sort of nailed it.”

Since Waterfield’s nanosyringe discovery, scientists have used bioinformatic analyses to identify nanosyringe sequences in many different bacterial species from archaebacteria to both gram-negative and gram-positive bacteria (2).

In fact, a team of scientists recently identified one such nanosyringe system produced by the marine bacteria Pseudoalteromonas luteoviolacea, which associate with tubeworms, microscopic animals that often line the hulls of ships (3). Lead author of the study, Nicholas Shikuma, then a postdoctoral fellow at the California Institute of Technology and now a microbiologist at San Diego State University, found that P. luteoviolacea nanosyringes induced metamorphosis of the tubeworm from its larval to adult form.

Shikuma and his team also observed that only a small subset of the P. luteoviolacea bacteria in a population make and release nanosyringes. Waterfield and his team noticed this feature in Photorhabdus bacteria populations as well (4).

“[The nanosyringes] are produced in a manner that's quite similar to phage in that the bacterium turns on the genes in a subpopulation of the cells and then fills up with the phage tails and then lyses to release them,” said Shikuma. “It's kind of an intriguing phenomenon because it's a form of altruism almost.”

He and his team, including Charles Ericson who is now a PhD student at ETH Zurich, investigated which bacterial genes were required for the nanosyringes to induce tubeworm metamorphosis.

“We found this gene cluster that encodes these contractile injection systems in the syringe-looking things,” said Ericson. Unlike the nanosyringes produced by the Photorhabdus bacteria, P. luteoviolacea nanosyringes form a cluster of approximately 100 syringes. “They look like a spike ball,” Ericson added.

Ericson and Shikuma discovered that P. luteoviolacea encodes both the spikey syringe structure and the proteins it carries that cause tubeworm metamorphosis (5). Because the bacteria encode both the syringe and its protein cargo, Ericson wondered, “If this has something loaded already in it, can we change what's loaded in it? Because that would have really good biotech or medical applications way later on down the line.”

Protein payload swap

Because both the nanosyringe and its protein cargo are genetically encoded by the bacteria cell, scientists realized that by genetically engineering the nanosyringe to express a different protein, they could potentially swap the bacterial protein cargos with therapeutic ones. Additionally, because bacteria release nanosyringes into their surrounding environments, the drug-carrying structures can be easily isolated from bacteria and purified for therapeutic use.

At Waterfield and Healey’s biotech company, NanoSyrinx, the team worked out how to engineer standard laboratory E. coli to express Photorhabdus nanosyringes and to load specific protein payloads inside.

“You essentially do all of the hard work at the cloning bench. You design the chassis you want. You design the payload you want,” said Healey. Then “the magic happens, and the payloads are loaded into the nanosyringes. The nanosyringe is built… They sit there quite happily as loaded nanosyringes full of payload until you crack the E. coli open and then do the protein purification.”

As the NanoSyrinx team optimized this process, one of the biggest challenges they faced was quantifying how much cargo each nanosyringe carried. Unlike the structurally similar phages, which carry DNA or RNA that can be easily measured using techniques such as quantitative PCR, the nanosyringes are proteins carrying additional protein cargo.

“We're having to devise and calibrate whole new suites of analytics that we trust to give us accurate measures,” said Healey.

The NanoSyrinx team have since loaded all kinds of different proteins into their nanosyringes, including human ones. For example, they expressed proapoptotic proteins into their nanosyringes and found that when they mixed them with human white blood cells, the proteins from the nanosyringes induced apoptosis.

“We've not found anything they can't package at least, and they seem to be able to deliver a large variety,” said Waterfield.

A new drug delivery tool in the toolbox

When it comes to diseases that these nanosyringes can treat, the possibilities seem endless. At NanoSyrinx, researchers investigate how to use the nanosyringes to deliver protein-based drugs for oncology, immunology, inflammation, and for gene therapy applications. But nanosyringes will be useful for delivering all sorts of medicine for many different applications.

“When whoever invented the hypodermic syringe said, ‘What disease are you going to solve with it?’ It's a case of well, any disease in which a hypodermic syringe would be useful. It's like that but at a nano scale,” said Waterfield.

Shikuma is also interested in the delivery aspects of these nanosyringes, and he recently started a biotechnology company called Metamorphotech based on P. luteoviolacea’s nanosyringe technology. Beyond drug delivery applications, Shikuma and his team recently discovered that certain Bacteroidales bacterial species in the human gut microbiome also encode nanosyringes, suggesting that these structures may already play a role in human health (6).

“It could be that everybody in the world is walking around with these injection systems in their guts,” said Shikuma. “We just have no idea right now what they're doing, so I find that really intriguing.”

The next hurdle for translating bacterial nanosyringes for treating human disease will be targeting these structures to specific human cells. For P. luteoviolacea, Ericson hypothesized, “We have these little tail fibers at the bottom, kind of towards the needle you can imagine, and we think those are responsible for the specificity of it.”

Whether they help deliver drugs to human cells or induce changes in other eukaryotic cells, nanosyringes efficiently deliver proteins across cell membranes. With increasing numbers of research teams investigating nanosyringes produced by diverse bacteria, the understanding of these structures will only grow.

“There's a huge potential here,” said Ericson. “In a pie in the sky sort of way, I would like to see these things through to where we can bind them to different cells, and we can inject whatever protein we want. That would be grand.”

References

1. Yang, G. et al. Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth. J Bacteriol  188, 2254-61 (2006).
2. Chen, L. et al. Genome-wide Identification and Characterization of a Superfamily of Bacterial Extracellular Contractile Injection Systems. Cell Rep  29, 511-521.e2 (2019).
3. Shikuma, N.J. et al. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science  343, 529-33 (2014).
4. Vlisidou, I. et al. The Photorhabdus asymbiotica virulence cassettes deliver protein effectors directly into target eukaryotic cells eLife  8, e46259 (2019).
5. Ericson, C.F. et al. A contractile injection system stimulates tubeworm metamorphosis by translocating a proteinaceous effector. eLife  8, e46845 (2019).
6. Rojas, M.I. et al. A distinct contractile injection system gene cluster found in a majority of healthy adult human microbiomes. mSystems  5, e00648-20 (2020).