On the Trail of Salmonella

Understanding the function of Salmonella’s needle complex could prove crucial to more effective prevention of infectious outbreaks. “The secretion system is essential for virulence—consequently, reducing or inhibiting its function might be a good strategy for therapeutic intervention,” said biochemist and biophysicist Thomas Marlovits, PhD, a group leader at the Vienna Institutes IMP (Research Institute of Molecular Pathology)-IMBA (Institute of Molecular Biotechnology) in Austria.

Dr. Marlovits and his team have recently developed the most detailed three-dimensional images ever created of Salmonella’s needle complex, using an advanced technique called cryo-electron microscopy (cryoEM). “CryoEM allows the direct visualization of large molecules (such as the needle complex) in a near native-like environment,” he explained. “The sample preparation involves rapid freezing in a buffer of choice, which renders a fully hydrated, near native-like state of the needle complex particles, and is in sharp contrast to conventional preparation methods that usually include staining procedures of the molecules. We are then able to observe the sample material under this fully hydrated state, thanks to the special capabilities of the microscope, such as keeping the sample at -180 degrees Celsius and using low-dose conditions, which means that very few electrons interact with the molecule of interest. All together, these procedures keep the material intact in its finest details.”

Conventional electron microscopy, on the other hand, would use staining, which can easily destroy the fine details; high-vacuum conditions, which present the sample in conditions far from its native state; and a high dose of electrons, which would destroy the sample in fractions of a second without recovering high-resolution information.

The need to use low-dose conditions, however, presents scientists with a new visualization challenge: low contrast. It’s almost like trying to make out a person’s face in very dim light. Dr. Marlovits and his team have used specialized software to overcome this problem. “It allows us to use statistical methods to improve the signal-to-noise ratio in the images by ‘finding’ all the particles that are observed in a specific orientation relative to the electron beam,” he said. “Once found, we put all these particles in one basket and can generate an ‘average’ image, which now has a good signal-to-noise ratio.”