Anna Waldmann and John Brumell

10 November 2025

By Aideen Teeling

Researchers at The Hospital for Sick Children (SickKids) have discovered that a host protein linked to early onset Parkinson’s disease promotes the spread of Salmonella bacteria between cells.

Upon infecting a host cell, Salmonella immediately hide away from cellular defenses in a specialized compartment called the Salmonella-containing vacuole (SCV). These subcellular bags allow bacteria to replicate in an ideal environment.

“Bacteria are little cell biologists,” says Anna Waldmann, a sixth year PhD student in the department of molecular genetics in the lab of John Brumell.

Explaining her recent publication in PLOS Pathogens, Waldmann describes how bacteria within the SCV secrete proteins, called effector proteins, into the cell via a microscopic needle to modify the environment to be more conducive for replication, effectively using the host cell “to make a living”, she remarks.

However, out of the approximately 40 known proteins secreted by Salmonella in the SCV, scientists have only characterized a few of the interactions occurring between bacterial and host proteins. These dynamic pathogen-host interactions are important for the bacteria to establish an infection and cause disease, and drove the central questions of Waldmann’s work – what host proteins do Salmonella effector proteins interact with and why?

One approach to answering this question is to “tag” all the cellular proteins near the SCV because they are likely to be some of the key interaction partners for bacterial effector proteins.

To do this, the researchers used an adapted protein labeling technique called proximity-dependent biotin identification (BioID). Waldmann says she spent nearly three years getting this system to work properly, testing up to 30 different strategies to ensure the bacteria could secrete the tagging machinery from inside the SCV into the cell effectively. She credits fellow PhD candidate Dustin Ammendolia for helping her troubleshoot.

After months of little success, she began to wonder if she should change her PhD project entirely, until one day in the darkened microscope room when it all worked.

“I looked down the eye piece and saw it was all red,” recalls Waldmann, who received the EPIC Doctoral Award in 2023. The tagged proteins were finally visible and surrounding the SCV just as intended.

With the BioID system working, Waldmann teamed up with collaborators at the Princess Margaret Cancer Centre at the University Health Network to identify the newly labelled proteins, producing a list of host proteins that were likely interacting with, and possibly regulating, the SCV.

One protein called VPS13C, which has been linked to Parkinson’s disease, stood out because it represented a large portion of tagged proteins.

“It was the biggest hit,” says Waldmann, who is also the co-chair of EPIC’s trainee advisory committee.

To explore the role of VPS13C in Salmonella infection, the researchers created “knock-out” cells which did not express the VPS13C protein.

When laboratory project coordinator and “electron microscopy wizard” Ren Li looked at the Salmonella-infected VPS13C knock-out cell lines, they observed something striking. The morphology of the SCV completely changed in the absence of VPS13C.

In cells missing VPS13C, the researchers observed that SCVs were significantly larger and contained more than one bacterium per vacuole, which is atypical during normal infection. It became clear that VPS13C is an essential factor that maintains the shape and size of the SCV.

Through subsequent experiments, Waldmann and her colleagues demonstrated that VPS13C does not affect Salmonella’s ability to replicate in a host cell. However, the loss of VPS13C significantly reduced the number of bacteria that went on to infect neighbouring cells, which could hinder the bacteria’s ability to establish systemic infection.

Mutations in VPS13C have been associated with early onset Parkinson’s disease, which prompted the researchers to investigate the effect of genetic changes on Salmonella infection. They found that, in cells with mutated VPS13C, the SCV did not form and function normally as it did in cells with unmutated VPS13C protein.

Waldmann says these findings underscore the importance of studying the shared pathways that are used by bacterial invaders and neurodegenerative diseases, an area of growing interest that could uncover new insights about how to prevent and manage these debilitating conditions.

This study was supported by the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. Waldmann is also funded by the University of Toronto’s Open Fellowship and the Emerging & Pandemic Infections Consortium.