Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth

Many clinically relevant Gram-negative pathogens like Yersinia, Vibrio, Escherichia, Pseudomonas, and Shigella encode the type III secretion system (T3SS) to inject effector proteins into host cells¹. In many bacterial species, the T3SS is under strict regulatory control². For example, translocation of Yersinia T3SS effector proteins into target host cells is critical to subvert host defense mechanisms and enable bacterial colonization of host tissues. However, Yersinia T3SS activity is metabolically burdensome and can trigger recognition by host immune receptors³. Accordingly, regulators that sense specific environmental cues control the expression of T3SS genes in many bacterial species. As pathogens such as Yersinia experience environmental changes during their infection cycle that impact the expression of critical virulence factors, it is important to develop laboratory conditions that mimic salient features of host niches occupied by bacterial pathogens. Specifically, oxygen tension and iron availability differ among various tissue sites in a spatiotemporal manner and impact the expression of virulence genes such as the T3SS^4,5,6. Therefore, the goal of this method is to assess how oxygen and iron impact the expression of the Yersinia T3SS. This will provide insight into the dynamics of the host-pathogen interaction.

The method described here details how to culture Yersinia pseudotuberculosis aerobically and anaerobically, as well as how to deplete Yersinia iron stores during aerobic or anaerobic growth. There are a few important considerations highlighted here regarding successfully culturing bacteria under these variable conditions. First, anaerobic culturing requires additional glucose supplementation, a modification that is noted in the media recipe. Second, since Y. pseudotuberculosis employs siderophores and other iron uptake systems that can robustly scavenge iron from the environment, special attention is devoted to ensuring the culture media and laboratory glassware are as free of iron as possible⁷. Previous studies have used iron chelators such as dipyridyl to deplete iron from rich-media bacterial cultures to mimic iron starvation^8,9. However, depleting Yersinia iron stores to induce iron starvation requires the removal of residual iron in glassware and media as well as prolonged growth in the absence of iron. This protocol details how to acid wash glassware and chelate media to remove residual iron, in addition to culturing the bacteria for several generations to ensure thorough iron starvation. Iron starvation can be ensured by measuring relative transcript levels of well-characterized iron-responsive genes across conditions, as demonstrated here with yfeA, and bfd.

The culmination of this protocol demonstrates how to precipitate secreted T3SS effector proteins from each of these conditions by treating the culture supernatant with trichloroacetic acid (TCA) and visualizing secreted proteins through SDS-PAGE. Finally, relative T3SS activity is assessed by visualizing secreted proteins via silver staining and quantifying relative levels of T3SS effector proteins, referred to as Yersinia outer proteins (Yops)¹⁰.

T3SS activity assays generally utilize specific antibodies to detect T3SS effector protein levels in the culture supernatant. However, western blotting antibodies for T3SS effector proteins are often not commercially available. Therefore, special attention has been taken to ensure that the final visualization of T3SS activity in this method does not require specific antibodies, and instead can leverage silver staining, which allows for the visualization of all secreted proteins. While this method is specifically tailored and optimized for Y. pseudotuberculosis, it can be adapted to other bacterial species, though the exact media conditions and incubation times will vary.