Internalization of Pseudomonas aeruginosaStrain PAO1 into epithelial cells is promoted by interaction of a T6SS effector with the microtubule network

Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction

The target cell genus does not matter.

International audienceTwo type VI secreted phospholipases D of Pseudomonas aeruginosa were identified as trans-kingdom virulence effectors, targeting both prokaryotic and eukaryotic host cells. Each of them triggers killing bacterial competitors and internalization into non-phagocytic cells. These type VI lipolytic enzymes are widely distributed among pathogens and may constitute a conserved strategy

The target cell genus does not matter.

International audienceTwo type VI secreted phospholipases D of Pseudomonas aeruginosa were identified as trans-kingdom virulence effectors, targeting both prokaryotic and eukaryotic host cells. Each of them triggers killing bacterial competitors and internalization into non-phagocytic cells. These type VI lipolytic enzymes are widely distributed among pathogens and may constitute a conserved strategy

The T6SSs of Pseudomonas aeruginosa Strain PAO1 and Their Effectors: Beyond Bacterial-Cell Targeting.

International audiencePseudomonas aeruginosa is an opportunistic pathogen responsible for many diseases such as chronic lung colonization in cystic fibrosis patients and acute infections in hospitals. The capacity of P. aeruginosa to be pathogenic toward several hosts is notably due to different secretion systems. Amongst them, P. aeruginosa encodes three Type Six Secretion Systems (T6SS), named H1- to H3-T6SS, that act against either prokaryotes and/or eukaryotic cells. They are independent from each other and inject diverse toxins that interact with different components in the host cell. Here we summarize the roles of these T6SSs in the PAO1 strain, as well as the toxins injected and their targets. While H1-T6SS is only involved in antiprokaryotic activity through at least seven different toxins, H2-T6SS and H3-T6SS are also able to target prokaryotic as well as eukaryotic cells. Moreover, recent studies proposed that H2- and H3-T6SS have a role in epithelial cells invasion by injecting at least three different toxins. The diversity of T6SS effectors is astounding and other effectors still remain to be discovered. In this review, we present a table with other putative P. aeruginosa strain PAO1 T6SS-dependent effectors. Altogether, the T6SSs of P. aeruginosa are important systems that help fight other bacteria for their ecological niche, and are important in the pathogenicity process

Gene transfer: conjugation.

International audienceConjugation is a gene transfer process in which a recipient bacterium receives DNA from a donor bacterium by cell-to-cell contact through conjugative pili. Conjugation is mediated by certain plasmids or transposons. Here, we describe plasmid conjugation

Divergent control of two type VI secretion systems by RpoN in Pseudomonas aeruginosa

International audienceThree Type VI Secretion System (T6SS) loci called H1- to H3-T6SS coexist in Pseudomonas aeruginosa. H1-T6SS targets prokaryotic cells whereas H2-T6SS mediates interactions with both eukaryotic and prokaryotic host cells. Little is known about the third system, except that it may be connected to H2-T6SS during the host infection. Here we show that H3-T6SS is required for P. aeruginosa PAO1 virulence in the worm model. We demonstrate that the two putative H3-T6SS operons, called "left" and "right", are coregulated with H2-T6SS by the Las and Rhl Quorum Sensing systems. Interestingly, the RpoN σ54 factor has divergent effects on the three operons. As for many T6SSs, RpoN activates the expression of H3-T6SS left. However, RpoN unexpectedly represses the expression of H3-T6SS right and also H2-T6SS. Sfa2 and Sfa3 are putative enhancer binding proteins encoded on H2-T6SS and H3-T6SS left. In other T6SSs EBPs can act as σ54 activators to promote T6SS transcription. Strikingly, we found that the RpoN effects of H3-T6SS are Sfa-independent while the RpoN mediated repression of H2-T6SS is Sfa2-dependent. This is the first example of RpoN repression of a T6SS being mediated by a T6SS-encoded EBP

Engineering probiotic bacteria to fight enteric pathogens?

<p>Commensal bacteria (in blue) could be genetically engineered (depicted with a syringe) to be resistant to a pathogen’s T6SS attack by providing them with genes encoding an antitoxin (depicted as a light blue shield) or by providing them with their own T6SS (shown as a light blue crossbow) that specifically targets the pathogen (in yellow).</p

The T6SS-mediated bacterial warfare in the host gut.

<p><b>(A)</b> Commensal <i>Bacteriodes fragilis</i> bacteria (in blue) target and kill enterotoxigenic <i>B</i>. <i>fragilis</i> (in yellow) in a T6SS-dependent manner (shown as crossbows), providing colonization resistance to the host. <b>(B)</b> <i>Salmonella</i> Typhimurium (in yellow) uses its T6SS crossbow to kill <i>Klebsiella oxytoca</i> (in blue), a potential nutritional competitor, allowing <i>Salmonella</i> to expand in the host gut. Dead bacteria are represented in grey, the gut epithelium is represented in light blue, and green cubes represent similar sugars metabolized by <i>Salmonella and Klebsiella</i>.</p

Sfa2 negatively controls <i>H2-T6SS</i> expression while <i>H3-T6SS</i> expression is Sfa-independent.

<p>The expression of <i>H3-T6SS</i> left <b>(A)</b>, <i>H3-T6SS</i> right <b>(B)</b>, and <i>H2-T6SS </i><b>(C)</b> after 6 hours of growth after 9 hours <b>(A & B)</b> or 7 hours <b>(C)</b> at 37°C in the WT strain (blue bars), in a PAO1<i>sfa2</i> mutant (red bars), and in a PAO1<i>sfa3</i> mutant (green bars). Expression is given in Miller units. Each experiment was done in triplicate and independently repeated three times; error bars indicate the standard deviation.</p