Genetic Dissection of the Signaling Cascade that Controls Activation of the Shigella Type III Secretion System from the Needle Tip

Many Gram-negative bacterial pathogens use type III secretion systems (T3SSs) for virulence. The Shigella T3SS consists of a hollow needle, made of MxiH and protruding from the bacterial surface, anchored in both bacterial membranes by multimeric protein rings. Atop the needle lies the tip complex (TC), formed by IpaD and IpaB. Upon physical contact with eukaryotic host cells, T3S is initiated leading to formation of a pore in the eukaryotic cell membrane, which is made of IpaB and IpaC. Through the needle and pore channels, further bacterial proteins are translocated inside the host cell to meditate its invasion. IpaD and the needle are implicated in transduction of the host cell-sensing signal to the T3S apparatus. Furthermore, the sensing-competent TC seems formed of 4 IpaDs topped by 1 IpaB. However, nothing further is known about the activation process. To investigate IpaB's role during T3SS activation, we isolated secretion-deregulated IpaB mutants using random mutagenesis and a genetic screen. We found ipaB point mutations in leading to defects in secretion activation, which sometimes diminished pore insertion and host cell invasion. We also demonstrated IpaB communicates intramolecularly and intermolecularly with IpaD and MxiH within the TC because mutations affecting these interactions impair signal transduction.