Deciphering the motility apparatus of bacteria
HZI scientists elucidate how bacteria assemble flagella outside the cell
Many bacteria move by rotating long, thin filaments called flagella. Flagella are made of several tens of thousands building blocks outside the bacterial cell and grow up to ten times longer than the bacterial cell body. They allow bacteria to swim towards a nutrient source or to approach cells of the human mucosa in order to infect them. This means that flagella are also tools in infection processes and might be suitable as potential targets for new agents against pathogenic bacteria. The details of how flagella are assembled and how this process might be inhibited remained elusive. Scientists of the Helmholtz Centre for Infection Research (HZI) in Braunschweig now elucidated this mechanism using real-time observations of growing flagella. The researchers published their results in the freely accessible journal, eLife.
Even for bacteria it is not satisfactory to just drift through life: Because every day is full of situations, in which a bacterium needs to move actively in its environment – for example when it searches for food or a suitable host. Under these circumstances, the bacterium forms flagella – i.e long, rotating filaments that can be used like a propeller to propel the bacterium. Bacteria use certain sensors to measure chemical signals in their environment and then use these signals to control the direction of rotation of the flagella. For example salmonellae form flagella when they detect copious amounts of nutrients: They interpret this as evidence that they are inside the intestine of their host. Then they use their flagellar propulsion system to swim through the mucous layer of the intestinal mucosa to reach the cells of the intestinal wall and then penetrate them, which ultimately leads to an infection. Bacteria of the Escherichia coli genus show the opposite response: They form flagella only if they measure too little nutrients in order to be able to swim to a new nutrient source.
Flagella can be up to 20 µm in length, which is one 50th of a millimetre. The bacterial cell body is only 2 µm in length and the bacteria initiate the production of flagella by building a protein pump in their membrane, which simultaneously serves as an anchor for the flagellum. To build the long, external filament the pump excretes a large number of building blocks of a single protein called flagellin into the tube-like structure. The flagellin units travel through the tube structure until they reach the tip of the filament, where they self-assemble outside the cell and thereby elongate the flagellum. "The pump uses energy derived from an ion and charge difference across the inner membrane for the secretion of flagellin building blocks," says Dr Marc Erhardt, who is the head of the "Infection Biology of Salmonella" junior research group at the Helmholtz Centre for Infection Research (HZI). "But, until now, it was not known how this highly complex structure assembles outside the cell from thousands of simple components and which energy source drives the secretion process."
To solve this puzzle, Marc Erhardt's team observed the growth of individual flagella. In one of the experiments, they labelled flagellin components of salmonellae with fluorescent antibodies. They induced the bacteria to form flagella, then they excited the dye to emit light and watching through the microscope recorded the growth of the flagella. This allowed them to detect that the growth rate of flagella decreased the longer the filaments got. "A previously accepted model described a mechanism, in which the flagella would always grow at the same rate regardless of their length," says Erhardt. "However, our observations in real-time showed that the growth rate decreases with the length of the filament and that there must be a different underlying mechanism."
This result was confirmed by another experiment: As before, the scientists made salmonellae produce flagella and then dyed the flagellin components with various fluorescent dyes while the flagellum was growing. They always changed the dye after a predetermined period of time – for example every 30 minutes. Ultimately, they were able to determine that the flagellar sections formed in the first time interval were the longest and that the sections formed in subsequent intervals kept getting shorter. "We explain these results to mean that the pump – i.e. the export system – permanently pumps flagellin from the bacterial cell into the tube of the flagellum and therefore the filament can grow rapidly when short," says Marc Erhardt. "With the filament getting longer, the flagellin units need more time to get from the pump to the end and accordingly, the growth slows down." Since there is no transport system for these components, they diffuse through the flagellum – which means they kind of drift along. With increasing distance to cover, this takes longer and longer which provides a simple explanation why flagella do not grow indefinitely.
This mechanism is also supported by a mathematic model based on the measured values of the researchers from the microscopy experiments for input. "The flagellar protein pump is related to other secretory systems of bacteria, which transport toxins into host cells during an infection," says Erhardt. "Our results also provide explanations for the protein export by these molecular syringes." The researchers of the HZI aim to specifically eliminate such virulence factors, which are needed by many bacterial pathogens to infect host cells. For this purpose, they are looking for suitable agents that interfere with the formation or function of these nanomachines to disarm pathogenic bacteria.
Thibaud T. Renault, Anthony O. Abraham, Tobias Bergmiller, Guillaume Paradis, Simon Rainville, Emmanuelle Charpentier, Călin C. Guet, Yuhai Tu, Keiichi Namba, James P. Keener, Tohru Minamino, and Marc Erhardt: Bacterial flagella grow through an injection-diffusion mechanism. eLife 2017; DOI: 10.7554/eLife.23136