Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells.
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ABSTRACT:
SUBMITTER: Lommel S
PROVIDER: S-EPMC1084051 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are diarrheagenic pathogens that colonize the gut mucosa via attaching-and-effacing lesion formation. EPEC and EHEC utilize a type III secretion system (T3SS) to translocate effector proteins that subvert host cell signalling to sustain colonization and multiplication. EspH, a T3SS effector that modulates actin dynamics, was implicated in the elongation of the EHEC actin pedestals. In this study we found that EspH is necessary for both efficient pedestal formation and pedestal elongation during EPEC infection. We report that EspH induces actin polymerization at the bacterial attachment sites independently of the Tir tyrosine residues Y474 and Y454, which are implicated in binding Nck and IRSp53/ITRKS respectively. Moreover, EspH promotes recruitment of neural Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex to the bacterial attachment site, in a mechanism involving the C-terminus of Tir and the WH1 domain of N-WASP. Dominant negative of WASP-interacting protein (WIP), which binds the N-WASP WH1 domain, diminished EspH-mediated actin polymerization. This study implicates WIP in EPEC-mediated actin polymerization and pedestal elongation and represents the first instance whereby N-WASP is efficiently recruited to the EPEC attachment sites independently of the Tir:Nck and Tir:IRTKS/IRSp53 pathways. Our study reveals the intricacies of Tir and EspH-mediated actin signalling pathways that comprise of distinct, convergent and synergistic signalling cascades.
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Project description:Shigella flexneri is an intracellular bacterium that hijacks the host actin cytoskeleton to invade and disseminate within the colonic epithelium. Shigella's virulence factors induce actin polymerization, leading to bacterial uptake, actin tail formation, actin-mediated motility, and cell-to-cell spreading. Many host factors involved in the Shigella-prompted actin rearrangements remain elusive. Here, we studied the role of a host protein receptor for activated C kinase 1 (RACK1) in actin cytoskeleton dynamics and Shigella infection. We used time-lapse imaging to demonstrate that RACK1 facilitates Shigella-induced actin cytoskeleton remodeling at multiple levels during infection of epithelial cells. Silencing RACK1 expression impaired Shigella-induced rapid polymerizing structures, reducing host cell invasion, bacterial motility, and cell-to-cell spreading. In uninfected cells, RACK1 silencing reduced jasplakinolide-mediated filamentous actin aggregate formation and negatively affected actin turnover in fast polymerizing structures, such as membrane ruffles. Our findings provide a role of RACK1 in actin cytoskeleton dynamics and Shigella infection.
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Project description:Enteropathogenic Escherichia coli, Shigella flexneri, and Listeria monocytogenes induce localized actin polymerization at the cytoplasmic face of the plasma membrane or within the host cytoplasm, creating unique actin-rich structures termed pedestals or actin tails. The process is known to be mediated by the actin-related protein 2 and 3 (Arp2/3) complex, which in these cases acts downstream of neural Wiskott-Aldrich syndrome protein (N-WASP) or of a listerial functional homolog of WASP family proteins. Here, we show that zonula occludens-1 (ZO-1), a protein in the tight junctions of polarized epithelial cells, is recruited to actin tails and pedestals. Immunocytochemical analysis revealed that ZO-1 was stained most in the distal part of the actin-rich structures, and the incorporation was mediated by the proline-rich region of the ZO-1 molecule. The direct clustering of membrane-targeted Nck, which is known to activate the N-WASP-Arp2/3 pathway, triggered the formation of the ZO-1-associated actin tails. The results suggest that the activation of the Arp2/3 complex downstream of N-WASP or a WASP-related molecule is a key to the formation of the particular actin-rich structures that bind with ZO-1. We propose that an analysis of the recruitment on a molecular basis will lead to an understanding of how ZO-1 recognizes a distinctive actin-rich structure under pathophysiological conditions.
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Project description:Shigella flexneri, which replicates in the cytoplasm of intestinal epithelial cells, can use the Embden-Meyerhof-Parnas, Entner-Doudoroff, or pentose phosphate pathway for glycolytic carbon metabolism. To determine which of these pathways is used by intracellular S. flexneri, mutants were constructed and tested in a plaque assay for the ability to invade, replicate intracellularly, and spread to adjacent epithelial cells. Mutants blocked in the Embden-Meyerhof-Parnas pathway (pfkAB and pykAF mutants) invaded the cells but formed very small plaques. Loss of the Entner-Doudoroff pathway gene eda resulted in small plaques, but the double eda edd mutant formed normal-size plaques. This suggested that the plaque defect of the eda mutant was due to buildup of the toxic intermediate 2-keto-3-deoxy-6-phosphogluconic acid rather than a specific requirement for this pathway. Loss of the pentose phosphate pathway had no effect on plaque formation, indicating that it is not critical for intracellular S. flexneri. Supplementation of the epithelial cell culture medium with pyruvate allowed the glycolysis mutants to form larger plaques than those observed with unsupplemented medium, consistent with data from phenotypic microarrays (Biolog) indicating that pyruvate metabolism was not disrupted in these mutants. Interestingly, the wild-type S. flexneri also formed larger plaques in the presence of supplemental pyruvate or glucose, with pyruvate yielding the largest plaques. Analysis of the metabolites in the cultured cells showed increased intracellular levels of the added compound. Pyruvate increased the growth rate of S. flexneri in vitro, suggesting that it may be a preferred carbon source inside host cells.
| S-EPMC4097621 | biostudies-literature
Project description:Shigella flexneri two-component regulatory systems (TCRS) are responsible for sensing changes in environmental conditions and regulating gene expression accordingly. We examined 12 TCRS that were previously uncharacterized for potential roles in S. flexneri growth within the eukaryotic intracellular environment. We demonstrate that the TCRS EvgSA, NtrBC, and RstBA systems are required for wild-type plaque formation in cultured epithelial cells. The phenotype of the NtrBC mutant depended in part on the Nac transcriptional regulator. Microarray analysis was performed to identify S. flexneri genes differentially regulated by the NtrBC system or Nac in the intracellular environment. This study contributes to our understanding of the transcriptional regulation necessary for Shigella to effectively adapt to the mammalian host cell.
| S-EPMC4097594 | biostudies-literature
Project description:Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.
| S-EPMC3837999 | biostudies-literature
Project description:Antibiotic treatment plays an essential role in preventing Shigella infection. However, incidences of global rise in antibiotic resistance create a major challenge to treat bacterial infection. In this context, there is an urgent need for newer approaches to reduce S. flexneri burden. This study largely focuses on the role of the herbal compound capsaicin (Caps) in inhibiting S. flexneri growth and evaluating the molecular mechanism behind bacterial clearance. Here, we show for the first time that Caps inhibits intracellular S. flexneri growth by inducing autophagy. Activation of autophagy by Caps is mediated through transcription factor TFEB, a master regulator of autophagosome biogenesis. Caps induced the nuclear localization of TFEB. Activation of TFEB further induces the gene transcription of autophagosomal genes. Our findings revealed that the inhibition of autophagy by silencing TFEB and Atg5 induces bacterial growth. Hence, Caps-induced autophagy is one of the key factors responsible for bacterial clearance. Moreover, Caps restricted the intracellular proliferation of S. flexneri-resistant strain. The efficacy of Caps in reducing S. flexneri growth was confirmed by an animal model. This study showed for the first time that S. flexneri infection can be inhibited by inducing autophagy. Overall observations suggest that Caps activates TFEB to induce autophagy and thereby combat S. flexneri infection.
| S-EPMC9298657 | biostudies-literature
Project description:Upon infection of mammalian cells, enterohemorrhagic E. coli (EHEC) O157:H7 utilizes a type III secretion system to translocate the effectors Tir and EspF(U) (aka TccP) that trigger the formation of F-actin-rich 'pedestals' beneath bound bacteria. EspF(U) is localized to the plasma membrane by Tir and binds the nucleation-promoting factor N-WASP, which in turn activates the Arp2/3 actin assembly complex. Although N-WASP has been shown to be required for EHEC pedestal formation, the precise steps in the process that it influences have not been determined. We found that N-WASP and actin assembly promote EHEC-mediated translocation of Tir and EspF(U) into mammalian host cells. When we utilized the related pathogen enteropathogenic E. coli to enhance type III translocation of EHEC Tir and EspF(U), we found surprisingly that actin pedestals were generated on N-WASP-deficient cells. Similar to pedestal formation on wild type cells, Tir and EspF(U) were the only bacterial effectors required for pedestal formation, and the EspF(U) sequences required to interact with N-WASP were found to also be essential to stimulate this alternate actin assembly pathway. In the absence of N-WASP, the Arp2/3 complex was both recruited to sites of bacterial attachment and required for actin assembly. Our results indicate that actin assembly facilitates type III translocation, and reveal that EspF(U), presumably by recruiting an alternate host factor that can signal to the Arp2/3 complex, exhibits remarkable versatility in its strategies for stimulating actin polymerization.
| S-EPMC2924363 | biostudies-literature
Project description:BACKGROUND: Shigella bacteria cause dysentery, which remains a significant threat to public health. Shigella flexneri is the most common species in both developing and developed countries. Five Shigella genomes have been sequenced, revealing dynamic and diverse features. To investigate the intra-species diversity of S. flexneri genomes further, we have sequenced the complete genome of S. flexneri 5b strain 8401 (abbreviated Sf8401) and compared it with S. flexneri 2a (Sf301). RESULTS: The Sf8401 chromosome is 4.5-Mb in size, a little smaller than that of Sf301, mainly because the former lacks the SHI-1 pathogenicity island (PAI). Compared with Sf301, there are 6 inversions and one translocation in Sf8401, which are probably mediated by insertion sequences (IS). There are clear differences in the known PAIs between these two genomes. The bacteriophage SfV segment remaining in SHI-O of Sf8401 is clearly larger than the remnants of bacteriophage SfII in Sf301. SHI-1 is absent from Sf8401 but a specific related protein is found next to the pheV locus. SHI-2 is involved in one intra-replichore inversion near the origin of replication, which may change the expression of iut/iuc genes. Moreover, genes related to the glycine-betaine biosynthesis pathway are present only in Sf8401 among the known Shigella genomes. CONCLUSION: Our data show that the two S. flexneri genomes are very similar, which suggests a high level of structural and functional conservation between the two serotypes. The differences reflect different selection pressures during evolution. The ancestor of S. flexneri probably acquired SHI-1 and SHI-2 before SHI-O was integrated and the serotypes diverged. SHI-1 was subsequently deleted from the S. flexneri 5b genome by recombination, but stabilized in the S. flexneri 2a genome. These events may have contributed to the differences in pathogenicity and epidemicity between the two serotypes of S. flexneri.
| S-EPMC1550401 | biostudies-literature
Project description: Not available
| S-EPMC2828053 | biostudies-literature