Many Gram-negative bacteria use a type III secretion system (T3SS) to manipulate eukaryotic cells. The injection of virulence proteins into the target cells is a precisely timed process that prevents unnecessary waste of these toxins and avoids alerting the immune system. A gatekeeper complex blocks premature secretion and is essential for a successful infection. However, little is known about how this complex regulates protein secretion by the T3SS.

Using live cell microscopy, we found that in Yersinia enterocolitica, all gatekeeper proteins localize in the bacterial cytosol. Applying and adapting optogenetic methods, we succeeded to control individual gatekeepers with high spatial and temporal resolution in a reversible manner. Sequestration of the gatekeppers led to uncontrolled secretion, indicating that they are prevented from carrying out their regulatory function. Interestingly, this control required the sequestration of gatekeepers to a liquid-liquid pase separation compartment, while sequestration to the membrane did not impede the function of the gatekeeper proteins.

Our observations support a model in which the gatekeepers perform their regulatory role in the cytosol by a yet unexplained mechanism. This data on the localization of the complex leads to a better understanding of T3SS-regulation and host cell sensing in Yersinia enterocolitica infection, paving the way for possible applications, such as the cotrolled application of the T3SS or targeting the gatekeeper proteins to prevent disease.