Pseudomonas aeruginosa survives in epithelia by ExoS-mediated inhibition of autophagy and mTOR.
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ABSTRACT:
SUBMITTER: Rao L
PROVIDER: S-EPMC7857434 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:One major factor that contributes to the virulence of Pseudomonas aeruginosa is its ability to reside and replicate unchallenged inside airway epithelial cells. The mechanism by which P. aeruginosa escapes destruction by intracellular host defense mechanisms, such as autophagy, is not known. Here, we show that the type III secretion system effector protein ExoS facilitates P. aeruginosa survival in airway epithelial cells by inhibiting autophagy in host cells. Autophagy inhibition is independent of mTOR activity, as the latter is also inhibited by ExoS, albeit by a different mechanism. Deficiency of the critical autophagy gene Atg7 in airway epithelial cells, both in vitro and in mouse models, greatly enhances the survival of ExoS-deficient P. aeruginosa but does not affect the survival of ExoS-containing bacteria. The inhibitory effect of ExoS on autophagy and mTOR depends on the activity of its ADP-ribosyltransferase domain. Inhibition of mTOR is caused by ExoS-mediated ADP-ribosylation of RAS, whereas autophagy inhibition is due to the suppression of autophagic Vps34 kinase activity.
| S-SCDT-EMBOR-2020-50613-T | biostudies-other
Project description:Type III secretion is used by many gram-negative bacterial pathogens to directly deliver protein toxins (effectors) into targeted host cells. In all cases, secretion of effectors is triggered by host cell contact, although the mechanism is unclear. In Pseudomonas aeruginosa, expression of all type III secretion-related genes is up-regulated when secretion is triggered. We were able to visualize this process using a green fluorescent protein reporter system and to use it to monitor the ability of bacteria to trigger effector secretion on cell contact. Surprisingly, the action of one of the major type III secreted effectors, ExoS, prevented triggering of type III secretion by bacteria that subsequently attached to cells, suggesting that triggering of secretion is feedback regulated. Evidence is presented that translocation (secretion of effectors across the host cell plasma membrane) of ExoS is indeed self-regulated and that this inhibition of translocation can be achieved by either of its two enzymatic activities. The translocator proteins PopB, PopD, and PcrV are secreted via the type III secretion system and are required for pore formation and translocation of effectors across the host cell plasma membrane. Here we present data that secretion of translocators is in fact not controlled by calcium, implying that triggering of effector secretion on cell contact represents a switch in secretion specificity, rather than a triggering of secretion per se. The requirement for a host cell cofactor to control effector secretion may help explain the recently observed phenomenon of target cell specificity in both the Yersinia and P. aeruginosa type III secretion systems.
| S-EPMC2293250 | biostudies-literature
Project description:Otitis media (OM) is a broad term describing a group of infectious and inflammatory disorders of the middle ear. Despite antibiotic therapy, acute OM can progress to chronic suppurative otitis media (CSOM) characterized by ear drum perforation and purulent discharge. Pseudomonas aeruginosa is the most common pathogen associated with CSOM. Although, macrophages play an important role in innate immune responses but their role in the pathogenesis of P. aeruginosa-induced CSOM is not known. The objective of this study is to examine the interaction of P. aeruginosa with primary macrophages. We observed that P. aeruginosa enters and multiplies inside human and mouse primary macrophages. This bacterial entry in macrophages requires both microtubule and actin dependent processes. Transmission electron microscopy demonstrated that P. aeruginosa was present in membrane bound vesicles inside macrophages. Interestingly, deletion of oprF expression in P. aeruginosa abrogates its ability to survive inside macrophages. Our results suggest that otopathogenic P. aeruginosa entry and survival inside macrophages is OprF-dependent. The survival of bacteria inside macrophages will lead to evasion of killing and this lack of pathogen clearance by phagocytes contributes to the persistence of infection in CSOM. Understanding host-pathogen interaction will provide novel avenues to design effective treatment modalities against OM.
| S-EPMC5114284 | biostudies-literature
Project description:Neutrophils are the first line of defense against bacterial infections, and the generation of reactive oxygen species is a key part of their arsenal. Pathogens use detoxification systems to avoid the bactericidal effects of reactive oxygen species. Here we demonstrate that the Gram-negative pathogen Pseudomonas aeruginosa is susceptible to reactive oxygen species but actively blocks the reactive oxygen species burst using two type III secreted effector proteins, ExoS and ExoT. ExoS ADP-ribosylates Ras and prevents it from interacting with and activating phosphoinositol-3-kinase (PI3K), which is required to stimulate the phagocytic NADPH-oxidase that generates reactive oxygen species. ExoT also affects PI3K signaling via its ADP-ribosyltransferase activity but does not act directly on Ras. A non-ribosylatable version of Ras restores reactive oxygen species production and results in increased bacterial killing. These findings demonstrate that subversion of the host innate immune response requires ExoS-mediated ADP-ribosylation of Ras in neutrophils.
| S-EPMC5478421 | biostudies-literature
Project description:Pseudomonas aeruginosa uses its type III secretion system to inject the effector proteins ExoS and ExoU into eukaryotic cells, which subverts these cells to the bacterium's advantage and contributes to severe infections. We studied the environmental reservoirs of exoS+ and exoU+ strains of P. aeruginosa by collecting water, soil, moist substrates and plant samples from environments in the Chicago region and neighbouring states. Whole-genome sequencing was used to determine the phylogeny and type III secretion system genotypes of 120 environmental isolates. No correlation existed between geographic separation of isolates and their genetic relatedness, which confirmed previous findings of both high genetic diversity within a single site and the widespread distribution of P. aeruginosa clonal complexes. After excluding clonal isolates cultured from the same samples, 74 exoS+ isolates and 16 exoU+ isolates remained. Of the exoS+ isolates, 41 (55%) were from natural environmental sites and 33 (45%) were from man-made sites. Of the exoU+ isolates, only 3 (19%) were from natural environmental sites and 13 (81%) were from man-made sites (p < 0.05). These findings suggest that man-made water systems may be a reservoir from which patients acquire exoU+ P. aeruginosa strains.
| S-EPMC6108916 | biostudies-literature
Project description:Type III cytotoxins contribute to the ability of bacterial pathogens to subvert the host innate immune system. ExoS (453 amino acids) is a bifunctional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96 to 232 comprise a Rho GTPase activating protein domain, while residues 233 to 453 comprise a 14-3-3-dependent ADP-ribosyltransferase domain. An N-terminal domain (termed the membrane localization domain [MLD]) targets ExoS to the Golgi-endoplasmic reticulum (Golgi-ER) of mammalian cells. This study identifies an amino acid motif that is responsible for the membrane binding properties of the MLD. Deletion mapping showed that the MLD included a symmetrical leucine-rich motif within residues 51 to 77 of ExoS. The terminal dileucines and internal leucines and an isoleucine within the MLD, but not charged or other hydrophobic residues, targeted a reporter protein to the Golgi-ER region of HeLa cells. Mutations of the leucines within the MLD did not affect type III secretion or translocation into HeLa cells but limited the ability of ExoS to ADP-ribosylate Ras GTPases. Mutations of charged residues within the MLD did not affect type III secretion, delivery into HeLa cells, or the ability of ExoS to ADP-ribosylate Ras GTPases. The organization of the leucines within the MLD of ExoS is different from that of previously described leucine-rich motifs but is present in several other bacterial proteins. This implies a role for intracellular targeting in the efficient targeting of mammalian cells by type III cytotoxins.
| S-EPMC1307044 | biostudies-literature
Project description:Hospital-acquired pneumonia is associated with high rates of morbidity and mortality, and dissemination to the bloodstream is a recognized risk factor for particularly poor outcomes. Yet the mechanism by which bacteria in the lungs gain access to the bloodstream remains poorly understood. In this study, we used a mouse model of Pseudomonas aeruginosa pneumonia to examine this mechanism. P. aeruginosa uses a type III secretion system to deliver effector proteins such as ExoS directly into the cytosol of eukaryotic cells. ExoS, a bi-functional GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT), inhibits phagocytosis during pneumonia but has also been linked to a higher incidence of dissemination to the bloodstream. We used a novel imaging methodology to identify ExoS intoxicated cells during pneumonia and found that ExoS is injected into not only leukocytes but also epithelial cells. Phagocytic cells, primarily neutrophils, were targeted for injection with ExoS early during infection, but type I pneumocytes became increasingly injected at later time points. Interestingly, injection of these pneumocytes did not occur randomly but rather in discrete regions, which we designate ""fields of cell injection" (FOCI). These FOCI increased in size as the infection progressed and contained dead type I pneumocytes. Both of these phenotypes were attenuated in infections caused by bacteria secreting ADPRT-deficient ExoS, indicating that FOCI growth and type I pneumocyte death were dependent on the ADPRT activity of ExoS. During the course of infection, increased FOCI size was associated with enhanced disruption of the pulmonary-vascular barrier and increased bacterial dissemination into the blood, both of which were also dependent on the ADPRT activity of ExoS. We conclude that the ADPRT activity of ExoS acts upon type I pneumocytes to disrupt the pulmonary-vascular barrier during P. aeruginosa pneumonia, leading to bacterial dissemination.
| S-EPMC4474835 | biostudies-literature
Project description:BackgroundIn nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood.ResultsIn this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after 14 years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K+ was elicited by high concentration of Na+ in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that 78 genes had differential expression between the parental strain and its derivative clones. Sixty-one transcripts were up-regulated, while 17 were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions.ConclusionsThe individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress.
| S-EPMC6591848 | biostudies-literature
Project description:Earlier studies reported that a cell membrane protein, Annexin A2 (AnxA2), plays multiple roles in the development, invasion, and metastasis of cancer. Recent studies demonstrated that AnxA2 also functions in immunity against infection, but the underlying mechanism remains largely elusive. Using a mouse infection model, we reveal a crucial role for AnxA2 in host defense against Pseudomonas aeruginosa, as anxa2(-/-) mice manifested severe lung injury, systemic dissemination, and increased mortality compared with wild-type littermates. In addition, anxa2(-/-) mice exhibited elevated inflammatory cytokines (TNF-?, IL-6, IL-1?, and IFN-?), decreased bacterial clearance by macrophages, and increased superoxide release in the lung. We further identified an unexpected molecular interaction between AnxA2 and Fam13A, which activated Rho GTPase. P. aeruginosa infection induced autophagosome formation by inhibiting Akt1 and mTOR. Our results indicate that AnxA2 regulates autophagy, thereby contributing to host immunity against bacteria through the Akt1-mTOR-ULK1/2 signaling pathway.
| S-EPMC4592832 | biostudies-literature
Project description:The intestinal tract is considered the most important reservoir of Pseudomonas aeruginosa in intensive care units (ICUs). Gut colonization by P. aeruginosa underlies the development of invasive infections such as gut-derived sepsis. Intestinal colonization by P. aeruginosa is associated with higher ICU mortality rates. The translocation of endogenous P. aeruginosa from the colonized intestinal tract is an important pathogenic phenomenon. Here we identify bacterial and host proteins associated with bacterial penetration through the intestinal epithelial barrier. We first show by comparative genomic hybridization analysis that the exoS gene, encoding the type III effector protein, ExoS, was specifically detected in a clinical isolate that showed higher virulence in silkworms following midgut injection. We further show using a silkworm oral infection model that exoS is required both for virulence and for bacterial translocation from the midgut to the hemolymph. Using a bacterial two-hybrid screen, we show that the mammalian factor FXYD3, which colocalizes with and regulates the function of Na,K-ATPase, directly binds ExoS. A pulldown assay revealed that ExoS binds to the transmembrane domain of FXYD3, which also interacts with Na,K-ATPase. Na,K-ATPase controls the structure and barrier function of tight junctions in epithelial cells. Collectively, our results suggest that ExoS facilitates P. aeruginosa penetration through the intestinal epithelial barrier by binding to FXYD3 and thereby impairing the defense function of tight junctions against bacterial penetration.
| S-EPMC2976341 | biostudies-literature