MxiN Differentially Regulates Monomeric and Oligomeric Species of the <i>Shigella</i> Type Three Secretion System ATPase Spa47

<i>Shigella</i> rely entirely on the action of a single type three secretion system (T3SS) to support cellular invasion of colonic epithelial cells and to circumvent host immune responses. The ATPase Spa47 resides at the base of the <i>Shigella</i> needle-like type three secretion apparatus (T3SA), supporting protein secretion through the apparatus and providing a likely means for native virulence regulation by <i>Shigella</i> and a much needed target for non-antibiotic therapeutics to treat <i>Shigella</i> infections. Here, we show that MxiN is a differential regulator of Spa47 and that its regulatory impact is determined by the oligomeric state of the Spa47 ATPase, with which it interacts. <i>In vitro</i> and <i>in vivo</i> characterization shows that interaction of MxiN with Spa47 requires the six N-terminal residues of Spa47 that are also necessary for stable Spa47 oligomer formation and activation. This interaction with MxiN negatively influences the activity of Spa47 oligomers while upregulating the ATPase activity of monomeric Spa47. Detailed kinetic analyses of monomeric and oligomeric Spa47 in the presence and absence of MxiN uncover additional mechanistic insights into the regulation of Spa47 by MxiN, suggesting that the MxiN/Spa47 species resulting from interaction with monomeric and oligomeric Spa47 are functionally distinct and that both could be involved in <i>Shigella</i> T3SS regulation. Uncovering regulation of Spa47 by MxiN addresses an important gap in the current understanding of how <i>Shigella</i> controls T3SA activity and provides the first description of differential T3SS ATPase regulation by a native T3SS protein

Deoxycholate-Enhanced <i>Shigella</i> Virulence Is Regulated by a Rare π‑Helix in the Type Three Secretion System Tip Protein IpaD

Type three secretion systems (T3SS) are specialized nanomachines that support infection by injecting bacterial proteins directly into host cells. The <i>Shigella</i> T3SS has uniquely evolved to sense environmental levels of the bile salt deoxycholate (DOC) and upregulate virulence in response to DOC. In this study, we describe a rare <i>i</i> + 5 hydrogen bonding secondary structure element (π-helix) within the type three secretion system tip protein IpaD that plays a critical role in DOC-enhanced virulence. Specifically, engineered mutations within the π-helix altered the pathogen’s response to DOC, with one mutant construct in particular exhibiting an unprecedented reduction in virulence following DOC exposure. Fluorescence polarization binding assays showed that these altered DOC responses are not the result of differences in affinity between IpaD and DOC, but rather differences in the DOC-dependent T3SS tip maturation resulting from binding of IpaD to translocator/effector protein IpaB. Together, these findings begin to uncover the complex mechanism of DOC-enhanced <i>Shigella</i> virulence while identifying an uncommon structural element that may provide a much needed target for non-antibiotic treatment of <i>Shigella</i> infection

Visible-Light-Driven Valorization of Biomass Intermediates Integrated with H₂ Production Catalyzed by Ultrathin Ni/CdS Nanosheets

Photocatalytic upgrading of crucial biomass-derived intermediate chemicals (i.e., furfural alcohol, 5-hydroxy­methyl­furfural (HMF)) to value-added products (aldehydes and acids) was carried out on ultrathin CdS nanosheets (thickness ∼1 nm) decorated with nickel (Ni/CdS). More importantly, simultaneous H₂ production was realized upon visible light irradiation under ambient conditions utilizing these biomass intermediates as proton sources. The remarkable difference in the rates of transformation of furfural alcohol and HMF to their corresponding aldehydes in neutral water was observed and investigated. Aided by theoretical computation, it was rationalized that the slightly stronger binding affinity of the aldehyde group in HMF to Ni/CdS resulted in the lower transformation of HMF to 2,5-diformyl­furan compared to that of furfural alcohol to furfural. Nevertheless, photo­catalytic oxidation of furfural alcohol and HMF under alkaline conditions led to complete transformation to the respective carboxylates with concomitant production of H₂

Binding Affects the Tertiary and Quaternary Structures of the <i>Shigella</i> Translocator Protein IpaB and Its Chaperone IpgC

<i>Shigella flexneri</i> uses its type III secretion system (T3SS) to promote invasion of human intestinal epithelial cells as the first step in causing shigellosis, a life-threatening form of dysentery. The <i>Shigella</i> type III secretion apparatus (T3SA) consists of a basal body that spans the bacterial envelope and an exposed needle that injects effector proteins into target cells. The nascent <i>Shigella</i> T3SA needle is topped with a pentamer of the needle tip protein invasion plasmid antigen D (IpaD). Bile salts trigger recruitment of the first hydrophobic translocator protein, IpaB, to the tip complex where it senses contact with a host membrane. In the bacterial cytoplasm, IpaB exists in a complex with its chaperone IpgC. Several structures of IpgC have been determined, and we recently reported the 2.1 Å crystal structure of the N-terminal domain (IpaB^74.224) of IpaB. Like IpgC, the IpaB N-terminal domain exists as a homodimer in solution. We now report that when the two are mixed, these homodimers dissociate and form heterodimers having a nanomolar dissociation constant. This is consistent with the equivalent complexes copurified after they had been co-expressed in <i>Escherichia coli</i>. Fluorescence data presented here also indicate that the N-terminal domain of IpaB possesses two regions that appear to contribute additively to chaperone binding. It is also likely that the N-terminus of IpaB adopts an alternative conformation as a result of chaperone binding. The importance of these findings within the functional context of these proteins is discussed

Galdieria_in_Richmond_Mine_FISH

Micrographs of two G. sulphuraria cells detected using Cya1208 rRNA probe in an A-drift biofilm from the Richmond Min

208_Genomes

List of 208 sequenced genomes used to generate protein families and phylogenetic tree