Steps for Shigella Gatekeeper Protein MxiC Function in Hierarchical Type III Secretion Regulation

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions

Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation

Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram-negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaD's C-terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation

The Extreme C Terminus of Shigella flexneri IpaB Is Required for Regulation of Type III Secretion, Needle Tip Composition, and Binding▿

Type III secretion systems (T3SSs) are widely distributed virulence determinants of Gram-negative bacteria. They translocate bacterial proteins into host cells to manipulate them during infection. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region, and a hollow needle protruding from the bacterial surface. The distal tip of mature, quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. However, its mode of needle association is unknown. Here, we show that deletion of 3 or 9 residues at the C terminus of IpaB leads to fast constitutive secretion of late effectors, as observed in a ΔipaB strain. Like the ΔipaB mutant, mutants with C-terminal mutations also display hyperadhesion. However, unlike the ΔipaB mutant, they are still invasive and able to lyse the internalization vacuole with nearly wild-type efficiency. Finally, the mutant proteins show decreased association with needles and increased recruitment of IpaC. Taken together, these data support the notion that the state of the tip complex regulates secretion. We propose a model where the quiescent needle tip has an “off” conformation that turns “on” upon host cell contact. Our mutants may adopt a partially “on” conformation that activates secretion and is capable of recruiting some IpaC to insert pores into host cell membranes and allow invasion

Isolation of Salmonella Mutants Resistant to the Inhibitory Effect of Salicylidene acylhydrazides on Flagella-Mediated Motility

Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB ((P28T)) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB ((P28T)) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the F(O)F(1)-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification

Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the T3SS needle tip complex

Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies

Comparative schematic representation of the bacterial flagellar export apparatus and F_O/F₁ ATPase.

<p>The bacterial cytoplasmic membrane (<i>CM</i>) is shown in dark gray blocks. <i>Peri</i> stands for periplasmic face and <i>Cyto</i> for cytoplasmic face. The names components that are functionally and/or structurally related between the two complexes (as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052179#s4" target="_blank">Discussion</a>) are outlined in black and share similar colours and/or shapes.</p

Hypermotility and flagellar numbers for isolated mutants.

<p><b>A</b>) Quantitative motility assays of each mutant relative to Δ<i>fliHI/flhB</i>* in the presence of DMSO. Dots represent individual colonies. Lines represent means. Kruskal-Wallis test indicated a difference between groups (p = 0.0001) and a Dunn's post-test indicated that both mutant strains were significantly more motile than Δ<i>fliHI/flhB</i>* (p<0.001). <b>B</b>) Number of flagella for Δ<i>fliHI/flhB</i>*, HM15 and DI15. The display purposes, the data from two independent experiments were pooled. Lines represent means. Using Poisson regression analysis of unpooled data, HM18 had an overall significantly increased number of flagella/cell (p<0.001).</p

Effect of INP0404 and INP0405 on motility of weakly motile <i>Salmonella</i> Typhimurium strains lacking subunits of the cytoplasmic portion of the flagellar export apparatus.

<p>Motility assays were performed for the indicated strains in the position displayed on the circle above the plate photographs and using DMSO alone or the compounds indicated on the left. An experiment representative of two independent ones is shown. When the motility of Δ<i>fliHI/flhB*</i> and Δ<i>fliHIJ/flhB*</i> was expressed as a ratio of their motility in either drug relative to DMSO, the average values obtained were 0.69 and 0.73, respectively, for INP0404 and 0.65 and 0.61, respectively, for INP0405.</p

Influence of the DI15 and HM18 backgrounds on the function of flagellar and virulence-related T3SSs.

<p>Strain Δ<i>fliHI/flhB</i>*, DI15 and HM18 were grown to late exponential phase in the presence of DMSO, or 50 µM INP0404 or INP0405 and normalised for OD₆₀₀. Cultures were separated into cell <i>pellet</i> and culture <i>supernatant</i> by centrifugation, followed by SDS-PAGE and Western blotting with anti-FliC (flagellin, <i>top</i>) and anti-SipC (SPI1 T3SS effector, <i>bottom</i>) antibodies. A representative experiment is shown.</p