Structural and Functional Characterization of the Shigella flexneri Type Three Secretion System (T3SS) ATPase Spa47

Shigella flexneri is a bacterial pathogen that causes shigellosis, a severe form of dysentery hallmarked by massive fluid loss and hemorrhaging of the intestines. Though treatable with certain antibiotics, shigellosis presents a worldwide health concern with an estimated 90 million infections and greater than 100,000 deaths annually. Shigella relies on a specialized transport system, called the type three secretion system (T3SS), to directly inject bacterial proteins into human host cells and cause infection. The complex protein machinery of the T3SS is highly conserved among related bacteria including Salmonella (food poisoning), Pseudomonas (lung infections), and Yersinia (plague). The apparatus includes a hollow needle-like structure through which specialized effector proteins are secreted and a sorting platform that resides at the base. We have recently characterized the Shigella sorting platform protein, Spa47, as an ATPase which likely provides the necessary energy for the formation of the T3SS needle and subsequent secretion of bacterial proteins into host cells. In the work presented here, we solved several high-resolution crystal structures of Spa47 which we use to model an activated Spa47 complex and guide the design of key Spa47 mutations, providing the first insight into both the structure and function of what we believe represents the “powerhouse protein” supporting Shigella infection. These findings add to our understanding of how an important class of bacteria cause infection and provide a strong platform for follow-up studies evaluating the regulation of Spa47 oligomerization in vivo as a much needed means of treating and potentially preventing shigellosis and related diseases

Utah State University Flutes Solo Recital

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Development and characterization of a highly soluble construct of the Shigella flexneri translocator protein IpaC

Shigella flexneri is a Gram-negative pathogen that causes Shigellosis, a severe form of dysentery hallmarked by massive fluid loss and hemorrhaging of the intestines. Though treatable with antibiotics, shigellosis results in more than 125 million reported cases and 14,000 deaths annually. S. flexneri relies on a type three secretion system (T3SS) as its primary virulence factor. The type three secretion apparatus (T3SA) resembles a needle-like structure through which the effector proteins are injected in the cytoplasm of the eukaryotic host. The distal end of the needle is the tip complex which is a sophisticated hetero-complex responsible for interaction with the host cell membrane. This system relies on the presence of dedicated chaperone proteins that bind to effectors within the cytoplasm of the pathogen and release it upon secretion through the apparatus. The S. flexneri tip complex includes a pentamer of the hydrophilic protein IpaD which regulates secretion through the apparatus as well as the hydrophobic translocator proteins IpaB and IpaC. Both IpaB and IpaC are required for proper pore formation in the host cell membrane, though little is known about their specific roles in this interaction. The hydrophobic character of IpaC requires either a chaperone protein, IpgC, or a detergent to maintain solubility in solution. Here we identify a stable construct of IpaC, which requires neither detergent nor its chaperone to maintain solubility and perform a series of biophysical characterization experiments on both the IpaC construct alone and as a part of the chaperone-bound heterocomplex