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

Bernard, Abram; Jessop, T. Carson; Kumar, Prashant; Dickenson, Nicholas E.

doi:10.1021/acs.biochem.7b00836

Cited by 16 publications

(14 citation statements)

References 58 publications

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“… 2011 ; Bernard et al. 2017 ), leading to an injection-enhancing effect by supporting the recruitment of hydrophobic translocator proteins to the maturing tip complex in preparation of host membrane contact. Even though the tip proteins of the T3SS of Shigella and Salmonella SPI-1 are structurally very similar, deoxycholate binding does not have an enhancing effect on secretion in Salmonella (Wang et al.…”

Section: Host-cell Sensing and Injectionmentioning

confidence: 99%

Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells

Wagner

Grin

Malmsheimer

et al. 2018

FEMS Microbiology Letters

150

134

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Virulence-associated type III secretion systems (T3SS) serve the injection of bacterial effector proteins into eukaryotic host cells. They are able to secrete a great diversity of substrate proteins in order to modulate host cell function, and have evolved to sense host cell contact and to inject their substrates through a translocon pore in the host cell membrane. T3SS substrates contain an N-terminal signal sequence and often a chaperone-binding domain for cognate T3SS chaperones. These signals guide the substrates to the machine where substrates are unfolded and handed over to the secretion channel formed by the transmembrane domains of the export apparatus components and by the needle filament. Secretion itself is driven by the proton motive force across the bacterial inner membrane. The needle filament measures 20–150 nm in length and is crowned by a needle tip that mediates host-cell sensing. Secretion through T3SS is a highly regulated process with early, intermediate and late substrates. A strict secretion hierarchy is required to build an injectisome capable of reaching, sensing and penetrating the host cell membrane, before host cell-acting effector proteins are deployed. Here, we review the recent progress on elucidating the assembly, structure and function of T3SS injectisomes.

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Section: Host-cell Sensing and Injectionmentioning

confidence: 99%

Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells

Wagner

Grin

Malmsheimer

et al. 2018

FEMS Microbiology Letters

150

134

View full text Add to dashboard Cite

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“…Secreted IpaC levels were detected and compared using a Bio-Rad ChemiDoc imaging system and the associated Image Lab analysis software. As validated previously [37], a monoclonal antibody against the cytoplasmic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a cytoplasmic control in the western blot to ensure that the IpaC detected in the supernatant was secreted through the injectisome and was not the result of cell lysis. A complete set of whole cell extract GAPDH controls is included in S3 Fig.…”

Section: Quantitation Of S Flexneri T3ss Translocator Secretionmentioning

confidence: 99%

Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence

et al. 2020

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Type three secretion systems (T3SS) are complex nano-machines that evolved to inject bacterial effector proteins directly into the cytoplasm of eukaryotic cells. Many high-priority human pathogens rely on one or more T3SSs to cause disease and evade host immune responses, underscoring the need to better understand the mechanisms through which T3SSs function and their role(s) in supporting pathogen virulence. We recently identified the Shigella protein Spa47 as an oligomerization-activated T3SS ATPase that fuels the T3SS and supports overall Shigella virulence. Here, we provide both in vitro and in vivo characterization of Spa47 oligomerization and activation in the presence and absence of engineered ATPase-inactive Spa47 mutants. The findings describe mechanistic details of Spa47-catalyzed ATP hydrolysis and uncover critical distinctions between oligomerization mechanisms capable of supporting ATP hydrolysis in vitro and those that support T3SS function in vivo. Concentration-dependent ATPase kinetics and experiments combining wild-type and engineered ATPase inactive Spa47 mutants found that monomeric Spa47 species isolated from recombinant preparations exhibit low-level ATPase activity by forming short-lived oligomers with active site contributions from at least two protomers. In contrast, isolated Spa47 oligomers exhibit enhanced ATP hydrolysis rates that likely result from multiple preformed active sites within the oligomeric complex, as is predicted to occur within the context of the type three secretion system injectisome. High-resolution fluorescence microscopy, T3SS activity, and virulence phenotype analyses of Shigella strains co-expressing wild-type Spa47 and the ATPase inactive Spa47 mutants demonstrate that the N-terminus of Spa47, not ATPase activity, is responsible for incorporation into the injectisome where the mutant strains exhibit a dominant negative effect on T3SS function and Shigella virulence. Together, the findings presented here help to close a significant gap in our understanding of how T3SS ATPases are activated and define restraints with respect to how ATP hydrolysis is ultimately coupled to T3SS function in vivo.

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“…A π-helix also plays a role in infection caused by Shigella passing through the human small intestine. In response to high concentrations of the bile salt deoxycholate (DOC), which binds to invasion plasmid antigen D (IpaD) of the type three secretion system of Shigella, a π-helical shift within IpaD leads to association with invasion plasmid antigen B (IpaB) and eventually invasion of host cells [38]. Based on a sequence homology of middle domains from EcDosC and 78 DGC-containing GCS homologues, the π-helical kink of EcDosC (H223, K224) is highly conserved [24] and both these residues and the π-helical region are predicted in BpeGReg (native residues numbering H225, K226) and PccGCS (native residues numbering H237, K238) ( Figure 2).…”

Section: Discussionmentioning

confidence: 99%

π-Helix controls activity of oxygen-sensing diguanylate cyclases

Walker

Potter

et al. 2020

Bioscience Reports

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The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O 2 levels, including modulating synthesis of cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates biofilm formation. While GCS proteins have been demonstrated to regulate O 2 -dependent pathways, the mechanism by which the O 2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O 2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O 2 -dependent GCS activity.identified in numerous bacteria with a variety of output domains that are regulated by the O 2 -binding signal, including methyl accepting chemotaxis protein (MCP), kinase, and diguanylate cyclase (DGC) [12,13]. In Bacillus subtilis, the MCP-containing GCS regulates aerotaxis, allowing the organism to move to its preferred location within an O 2 gradient [14]. In contrast, the DGC-containing GCS from the plant pathogen Pectobacterium carotovorum (PccGCS) regulates O 2 -dependent motility, virulence factor production, and rotting within a plant host, highlighting the importance of O 2 sensing and GCS proteins in controlling bacterial phenotypes [11].Within the GCS protein family, the most common effect of O 2 binding to the heme of the globin domain is an increase in the activity of the output domain [13]. This increase in GCS enzymatic activity often requires oligomerization of GCS monomers to yield activity of the output domains; GCS proteins with diguanylate cyclase, histidine kinase, and adenylate cylcase output domains have been shown to function as homodimers and higher order oligomers [15][16][17][18][19][20]. Because of the multi-domain organization and oligomerization of GCS proteins, questions have arisen regarding the overall structure and path of signal transduction within the protein family. ...

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

Cited by 16 publications

References 58 publications

Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells

Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells

Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence

π-Helix controls activity of oxygen-sensing diguanylate cyclases

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