Type III Secretion Systems (T3SS) are multiprotein, syringe-shaped nanomachines that deliver virulence factors, known as effector proteins, from bacteria into host cells. T3SS are common in Gram-negative bacteria and are key for the progression of many infectious diseases, including bubonic plague, dysentery, and food poisoning. The essential nature of the T3SS in disease progression, and its high degree of structural conservation among bacteria make it an attractive target for the development of anti-virulence compounds. We propose to use cryo-electron tomography and subtomogram averaging to elucidate the in-situ architecture of Enteropathogenic E. coli and Salmonella typhimurium T3SS. Furthermore, we aim to perform single particle analysis on purified T3SS components in reconstituted environments. The structural insights obtained from these studies will help guide the rational design of anti-virulence compounds that can disrupt protein secretion and impair pathogenesis, without exerting selective pressures that drive antibiotic resistance.