Zooming in on the tools: TS33 Secretion System

Salmonella is one bacterium everyone’s heard of. It’s the scourge of meatpacking plants and involved in spinach recalls every year, causing unwelcome intestinal unrest and dangerous disease when encountered in high enough numbers.

William Picking wants to better understand how to combat this and other food and water system hazards. The Bond Life Sciences Center scientist reached down to take a piece of paper to quickly jot down a drawing of a nifty way some bacteria have evolved to avoid the defenses of the animal cells it infects. One of the focuses of his research is to determine how the bacterial type 3 secretion works. Understanding this secretion apparatus may help scientists create better, more targeted drugs to fight pesky bacterial infections and outbreaks.

His drawing showed the type 3 secretion apparatus (T3SS), the bacterial structure that injects specialized proteins into cells to avoid being detected and attacked by those cells. In some cases, the apparatus actually invades those cells so the bacteria can grow inside them. Scientists like Picking aim to use proteins from this needle-like structure to develop new strategies to prevent disease

This “injectosome” is basically a syringe.  Composed of a base with a channel through it connected to a needle, this apparatus injects into and through the membrane of its host cell to get its proteins inside.

Scientists have a general idea of what the T3SS secretion apparatus looks like, but Picking’s current research focuses on understanding exactly how the injectosome works as a microscopic machine at a deeper level.

Picking and his team zoom in on the specific way that it selects the proteins to be secreted and energizes that process so they can deliver their specialized proteins.

This process must be coordinated so that the proteins are properly injected into the cell, so they invade those cells or, in some cases, kill the host cells trying to harm the bacteria.

The power behind this process comes from the ability of bacteria to recognize the surface of the organism to form a hole in the host cell membrane. The needle then passes those proteins through the hole.

The parts of the injectosome that are inside the bacteria are involved in determining what proteins are secreted and in what order.  This overall internal segment is called the sorting platform and it also provides the actual power that drives the secretion process, causing the secreted protein to be unfolded and then forcing it through the needle and ultimately into the host cell. It makes the injection of this process easier and speeds it up.

“We have a general idea of what the sorting platform looks like but our research focuses on improving our understanding of this structure to get at the mechanisms guiding secretion,” Picking said. “We even want to understand the movements within this structure that are part of the secretion process. “

Picking’s main focuses are bacteria like Salmonella and Shigella, but other bacteria like Pseudomonas aeruginosa can shut down the communication some immune system cells use to combat an attacking microbe.

“By understanding this apparatus at a high resolution, at a detailed structural level, we can potentially design drugs to inhibit the process of infection.” Picking said.

The trick is to reverse engineer a drug that operates like an antibiotic from the knowledge of T33s. That small drug could block the process of infection, and the body’s immune system can kill off the invader.

The problem that arises for scientists is the antibiotic often kills the bacteria. This leads to antibiotic resistance, a process called selective pressure.  That’s not good news. The drug engineered from reverse T33s shuts off the bacteria instead of killing it.  But, by shutting off the bacteria, your immune system will naturally recognize the pathogen or foreign invader and then destroy it itself. This is an ingenious idea, gaining popularity with bacterial scientists meant to avoid the often-inevitable bacterial resistance.

Bacterial resistance is a chronic problem in our world where antibiotics become less effective as the bacteria evolve around its protection. This causes problems the problems you hear in hospital systems where MRSA bacteria threaten life after antibiotics fail to fight them off or even how low levels of antibacterial agents in hand soap create more persistent colonies on surfaces.

Zooming in on the T3SS injectosome may give clues to better fight infection and reduce the number of people affected by dysentery and other common bacterial infections.

“It’s a great goal, not a goal our laboratory will accomplish in the immediate future, but we hope to provide the information for someone else to create these drugs to get around the problems of antibiotic resistance.” Picking said.

By Beni Adelstein | Bond LSC

Research skills aren’t built in a day, but Cynthia Tang’s diligence brought those skills to bear as she recently received a National Institutes of Health fellowship to further her budding career in science.

“Receiving the F30 fellowship means that the NIH sees value in my research proposal, in my training environment at the University of Missouri, and in my potential to become an independent physician-scientist,” said Tang, who works in Henry Wan’s lab at Bond Life Sciences Center. ­

This F30 predoctoral fellowship supports the research of students pursuing M.D.-Ph.D.’s. These awards help lighten the financial burden of a degree path that takes the better part of a decade for Tang and others pursuing their passions in research.

Tang came to the University of Missouri in 2018, started her journey as an M.D. student, then took a break to pursue her Ph.D. In the dual M.D.-Ph.D., the first two years are spent in pre-clinical studies followed by a full-time Ph.D. for three to five years and then students finish up with clinical training.

So, what project captured NIH’s attention?

Tang focused on how Covid-19 spreads and who gets it in rural areas. The aim is to identify people who have a higher chance of getting sick or being hospitalized so preventive measures can be offered earlier.

Lots of data must be gathered to accomplish this. Scientists sequence the genome of coronavirus variants­ to get a detailed profile and compare how that genetic makeup evolves over time. They do this by testing nasopharyngeal swabs—the Q-tips that are commonly used for covid tests—of people suspected of coronavirus infection. They use electronic health records to see who is getting sick, and add demographic information on age and location to the clues researchers analyze.

Tang focuses on rural populations in the U.S., especially in Missouri, because there is a gap in data from those communities. Once those at highest risk are identified, scientists can prioritize early prevention of COVID-19.

“Our biggest motivation for studying rural populations is so that we can better understand the way the virus changes and how to better serve those communities,” she said.

The process of planning for the grant itself enabled Tang to finetune the quality of her work.

“I feel like it helped speed up my Ph.D.,” she said. “We had to break down the research study into all the little pieces of what needs to be done and what to do when things go wrong. Everything must be so well thought out to put the grant together.”

After Tang’s initial fellowship application was rejected, she challenged herself to put together a completely new study design in under two months for her second proposal.

“It was the best thing that ever happened” said Tang, smiling. “I am extremely grateful to Dr. Wan and my thesis committee.”

This fellowship has provided many opportunities to Tang. She traveled overseas to present her findings at international conferences, which connected Bond LSC research with an international science community. Last year she was in Belfast, Ireland, and this month she will be in Valencia, Spain.

Where is Tang now?

She’s working remotely as she wraps up her Ph.D. and will graduate from Mizzou this upcoming December. Once she secures that Ph.D., she will head out to the University of North Carolina at Chapel Hill in January 2023 to finish her clinical training. Then she will enter the homestretch of her dual degree journey, finishing her M.D. in May of 2026.

Cynthia Tang currently serves as president-elect of the American Physician Scientists Association and will continue in this role next spring.

Tang was recently first author on “SARS-CoV-2 and influenza co-infection: A cross-sectional study in central Missouri during the 2021-2022 influenza season.” in the Journal of Virology in 2022.

By Sarah Kiefer

The spikes that protrude from SARS-CoV-2 present a topography of peaks that drive one MU researcher to ask more questions.

To Kamal Singh, a principal investigator at Bond Life Sciences Center, assistant professor in the MU College of Veterinary Medicine, and the director of the Molecular Interactions Core those spikes are a changing map with every new variant of coronavirus, and they lead his lab to study its constant evolution of mutations and proteins.

The Singh lab recently found an unexpectedly high number of APOBEC-mediated mutations among a patient cohort from South India, who were vaccinated and have taken remdesivir  to treat COVID-19. Using data from patients from South India, Saathvik Kannan, a senior at Hickman High School and a computer programmer and researcher for the Singh lab, tracked changes to the virus’ proteins.

“Viruses are known to evolve under pressure, so what’s happening in this case is that antibodies recognize the virus in a cell, and the virus sees that, ‘okay, there is someone to stop me,’ so it makes mutations,” Sing said.

The virus’ genome often alters, by mutating, inserting, or deleting the building blocks within RNA and DNA known as nucleotides, depending upon the pressure that viruses experience. Only some of these sugar-containing nucleotides are mutated, but these small changes can form a new virus variant.

“A few months ago, we didn’t know the reason behind all of these different mutations in patients because we did not look for APOBEC-mediated changes in viral genome,” Singh said.

Kannan found that between October 2022 to January 2023, the mutations among the patient cohort varied between 114 to 83. With that information, he identified 50% of patients who have diabetes and that when they take insulin the APOBEC protein is also induced. This new discovery leads to more questions about those with underlying conditions like diabetes or hypertension, and how they are affected differently by the drug.

“Ideally if a virus were to evolve you would see a linear progression, but you don’t see that here,” Kannan said. “This shows us that APOBEC proteins are likely one of the causes of this evolution in the virus and the makers of these variants.”

When the number of mutations vary significantly, like in the cohort, it re-affirms that the COVID-19 variants evolve independently.

Viruses don’t stop to wait for researchers to find the root of the problem. They constantly change, and in cases where the patients are vaccinated and take remdesivir, the virus evolves by taking advantage of the APOBEC protein.

Singh started down this path of research early on in the pandemic. Through PCR tests — which converts tiny amounts of RNA into DNA then copies it to a measurable level — he learned more about each mutation by comparing new samples to the original viral strain. Until this point, there had only been around 40 identified mutations in the spike protein in the different XBB sub-variant, but Singh’s lab has identified two more. The details have been recently published in The Lancet Journal. The two mutations found in this process, A27S and T747I, were unique to this cohort.

“For the first time we’re able to get more clues on what is actually happening because we were able to compare it to patient samples in the XBB subvariant and we had a wealth of data to work with,” Kannan said.

The Omicron variant yields new mutations constantly — currently at 43 and counting — and presents plenty of opportunity to look for differences. Singh directs his team to comb through variant sequences to identify differences in their genetic code. Soon they begin to identify trends from one month to another.

“The main difference here was that we have high quality patient data this time, from a patient cohort in Southern India, as opposed to getting it from a database,” Singh said. “The patient cohort tells us that the mutations are more than what was reported before.”

From January to June of 2020, Kannan found mutations that were present in one hundred percent of the sequences. When the Alpha Beta variant arrived in November 2020, they immediately knew what the mutated changes were. Their findings were published in the paper, ‘Clinical characteristics and novel mutations of omicron subvariant XBB in Tamil Nadu, India – a cohort study,’ in April 2023 in the Lancet Regional Health – Southeast Asia Journal.

After new variants — such as the XBB sub-variant of Omicron — materialized, Singh’s lab identified existing drugs and new drug compounds that match structural weaknesses presented by mutations.

One way Singh’s lab keeps abreast of new variants is by utilizing the tools they have available to their advantage with in-house programs either in Python or in R programming languages. Their lab can construct non-infectious, virus-like particles that contain all the same characteristics as the virus minus its genome, or its genetic material makeup.

A collaboration between University of Missouri and University of Nebraska Medical Center helped identify the new drug compound, MU-UNMC-2 in addition to the MU-UNMC-1, previously found in late 2021, as potential coronavirus treatments.

Remdesivir, ribavirin, favipiravir, and molnupiravir were among existing drugs that can be used to treat COVID-19 under certain conditions. Remdesivir — previously ineffective as an Ebola virus treatment — initially proved somewhat useful for COVID-19. Molnupavir only showed moderate promise, and the two drugs were approved for emergency use by the FDA for the treatment of COVID-19. Remdesivir now has full FDA approval.

“To address the problem, you have to find the problem. Where is the virus? What can we address? And then we go after it,” Singh said. “My lab was the first one in the world who suggested when the pandemic came that these are a few drugs that can be used and remdesivir was one of them. I’m very proud that I could contribute to human health in that way.”

With knowledge of this new protein in hand, Singh’s lab now is moving forward to focus most of their attention on the APOBEC protein mediated mutations and how underlying health conditions can affect an individual’s reaction to certain drug compounds.

“The virus has been outsmarting us, outfoxing us, until now. As soon as we get something, it changes itself,” Singh said. “That keeps us motivated as we chase it.”

The Singh lab’s research on the XBB sub-variant mutations was published in the paper ‘Omicron SARS-CoV-2 variant: Unique features and their impact on pre-existing anitbodies’ in the “Computational and Structural Biotechnology Journal” in June 2021 and their updated findings can be found in the paper ‘Clinical characteristics and novel mutations of omicron subvariant XBB in Tamil Nadu, India – a cohort study,’ published in “The Lancet Regional Health – Southeast Asia Journal” in April 2023.

Funding provided by: Bond LSC, Swedish research Council, American Lung Association, National Institute of Dental and Craniofacial Research in collaboration with Prof. Gary Weisman of the Bond Life Sciences center.