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1.
Elife ; 62017 12 12.
Article in English | MEDLINE | ID: mdl-29231810

ABSTRACT

Disruption of the sumoylation/desumoylation equilibrium is associated with several disease states such as cancer and infections, however the mechanisms regulating the global SUMO balance remain poorly defined. Here, we show that infection by Shigella flexneri, the causative agent of human bacillary dysentery, switches off host sumoylation during epithelial cell infection in vitro and in vivo and that this effect is mainly mediated by a calcium/calpain-induced cleavage of the SUMO E1 enzyme SAE2, thus leading to sumoylation inhibition. Furthermore, we describe a mechanism by which Shigella promotes its own invasion by altering the sumoylation state of RhoGDIα, a master negative regulator of RhoGTPase activity and actin polymerization. Together, our data suggest that SUMO modification is essential to restrain pathogenic bacterial entry by limiting cytoskeletal rearrangement induced by bacterial effectors. Moreover, these findings identify calcium-activated calpains as powerful modulators of cellular sumoylation levels with potentially broad implications in several physiological and pathological situations.


Subject(s)
Calcium/metabolism , Calpain/metabolism , Dysentery, Bacillary/microbiology , Host-Pathogen Interactions , Shigella flexneri/pathogenicity , Ubiquitin-Activating Enzymes/metabolism , Dysentery, Bacillary/metabolism , Dysentery, Bacillary/pathology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Epithelial Cells/pathology , HeLa Cells , Humans , Proteolysis , Signal Transduction , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , rho Guanine Nucleotide Dissociation Inhibitor alpha/metabolism
2.
Cell Host Microbe ; 17(2): 164-77, 2015 Feb 11.
Article in English | MEDLINE | ID: mdl-25600187

ABSTRACT

Salmonella invasion of intestinal epithelial cells requires extensive, though transient, actin modifications at the site of bacterial entry. The actin-modifying protein villin is present in the brush border where it participates in the constitution of microvilli and in epithelial restitution after damage through its actin-severing activity. We investigated a possible role for villin in Salmonella invasion. The absence of villin, which is normally located at the bacterial entry site, leads to a decrease in Salmonella invasion. Villin is necessary for early membrane-associated processes and for optimal ruffle assembly by balancing the steady-state level of actin. The severing activity of villin is important for Salmonella invasion in vivo. The bacterial phosphatase SptP tightly regulates villin phosphorylation, while the actin-binding effector SipA protects F-actin and counterbalances villin-severing activity. Thus, villin plays an important role in establishing the balance between actin polymerization and actin severing to facilitate the initial steps of Salmonella entry.


Subject(s)
Actin Cytoskeleton/metabolism , Endocytosis , Epithelial Cells/physiology , Host-Pathogen Interactions , Microfilament Proteins/metabolism , Microvilli/physiology , Salmonella typhimurium/physiology , Bacterial Proteins/metabolism , Cell Line , Epithelial Cells/microbiology , Humans , Intestinal Mucosa/microbiology , Intestinal Mucosa/physiology , Microvilli/microbiology , Protein Tyrosine Phosphatases/metabolism
3.
EMBO Rep ; 15(9): 965-72, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25097252

ABSTRACT

Shigella flexneri, the etiological agent of bacillary dysentery, invades the human colonic epithelium and causes its massive inflammatory destruction. Little is known about the post-translational modifications implicated in regulating the host defense pathway against Shigella. Here, we show that SUMO-2 impairs Shigella invasion of epithelial cells in vitro. Using mice haploinsufficient for the SUMO E2 enzyme, we found that sumoylation regulates intestinal permeability and is required to restrict epithelial invasion and control mucosal inflammation. Quantitative proteomics reveals that Shigella infection alters the sumoylation status of a restricted set of transcriptional regulators involved in intestinal functions and inflammation. Consistent with this, sumoylation restricts the pro-inflammatory transcriptional response of Shigella-infected guts. Altogether, our results show that the SUMO pathway is an essential component of host innate protection, as it reduces the efficiency of two key steps of shigellosis: invasion and inflammatory destruction of the intestinal epithelium.


Subject(s)
Dysentery, Bacillary/metabolism , Intestines/microbiology , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation/genetics , Animals , Dysentery, Bacillary/genetics , Dysentery, Bacillary/microbiology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Epithelial Cells/pathology , Haploinsufficiency/genetics , Host-Pathogen Interactions/genetics , Humans , Inflammation/metabolism , Inflammation/microbiology , Inflammation/pathology , Intestines/pathology , Mice , Protein Processing, Post-Translational/genetics , Shigella flexneri/metabolism , Shigella flexneri/pathogenicity , Small Ubiquitin-Related Modifier Proteins/genetics
4.
Dev Biol ; 323(2): 189-96, 2008 Nov 15.
Article in English | MEDLINE | ID: mdl-18801354

ABSTRACT

In insects the enzyme phenoloxidase (PO) catalyzes melanin deposition at the wound site and around parasitoid eggs. Its proenzyme prophenoloxidase (proPO) is proteolytically cleaved to active phenoloxidase by a cascade consisting of serine proteases and inhibited by serpins. The Drosophila genome encodes 29 serpins, of which only two, Serpin-27A (Spn27A) and Necrotic, have been analyzed in detail. Using a genetic approach, we demonstrate that the so far uncharacterized Serpin-28D (Spn28D, CG7219) regulates the proPO cascade in both hemolymph and tracheal compartments. spn28D is the serpin gene most strongly induced upon injury. Inactivation of spn28D causes pupal lethality and a deregulated developmental PO activation leading to extensive melanization of tissues in contact with air and pigmentation defects of the adult cuticle. Our data also show that Spn28D regulates hemolymph PO activity in both larvae and adults at a different level than Spn27A. Our data support a model in which Spn28D confines PO availability by controlling its initial release, while Spn27A is rather limiting the melanization reaction to the wound site. This study further highlights the complexity of the proPO cascade that can be differentially regulated in different tissues during development.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Hemolymph/enzymology , Monophenol Monooxygenase/metabolism , Pigmentation , Serpins/metabolism , Animals , Catechol Oxidase/metabolism , Enzyme Precursors/metabolism , Larva/enzymology , Melanins/metabolism , Mutation/genetics , Phenotype , Pupa/enzymology , RNA Interference , Trachea/abnormalities
5.
Cell Host Microbe ; 4(2): 147-58, 2008 Aug 14.
Article in English | MEDLINE | ID: mdl-18692774

ABSTRACT

Metazoans tolerate commensal-gut microbiota by suppressing immune activation while maintaining the ability to launch rapid and balanced immune reactions to pathogenic bacteria. Little is known about the mechanisms underlying the establishment of this threshold. We report that a recently identified Drosophila immune regulator, which we call PGRP-LC-interacting inhibitor of Imd signaling (PIMS), is required to suppress the Imd innate immune signaling pathway in response to commensal bacteria. pims expression is Imd (immune deficiency) dependent, and its basal expression relies on the presence of commensal flora. In the absence of PIMS, resident bacteria trigger constitutive expression of antimicrobial peptide genes (AMPs). Moreover, pims mutants hyperactivate AMPs upon infection with Gram-negative bacteria. PIMS interacts with the peptidoglycan recognition protein (PGRP-LC), causing its depletion from the plasma membrane and shutdown of Imd signaling. Therefore, PIMS is required to establish immune tolerance to commensal bacteria and to maintain a balanced Imd response following exposure to bacterial infections.


Subject(s)
Down-Regulation , Drosophila Proteins/metabolism , Drosophila/immunology , Drosophila/microbiology , Immune Tolerance , Signal Transduction , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cells, Cultured , Drosophila Proteins/genetics , Escherichia coli/immunology , Escherichia coli/physiology , Female , Gene Expression , Intestines/immunology , Intestines/microbiology , Intestines/physiology , Male , Transcription Factors/genetics , Transcription Factors/metabolism
6.
J Immunol ; 177(11): 7880-8, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-17114459

ABSTRACT

Although many cytokine receptors generate their signals via the STAT3 pathway, the IL-10R appears unique in promoting a potent anti-inflammatory response (AIR) via STAT3 to antagonize proinflammatory signals that activate the innate immune response. We found that heterologous cytokine receptor systems that activate STAT3 but are naturally refractory (the IL-22R), or engineered to be refractory (the IL-6, leptin, and erythropoietin receptors), to suppressor of cytokine signaling-3-mediated inhibition activate an AIR indistinguishable from IL-10. We conclude that the AIR is a generic cytokine signaling pathway dependent on STAT3 but not unique to the IL-10R.


Subject(s)
Gene Expression , Inflammation , Interleukin-10/metabolism , Interleukin-6/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/immunology , Animals , Enzyme-Linked Immunosorbent Assay , Immunoblotting , Mice , Oligonucleotide Array Sequence Analysis , Receptors, Interleukin-10/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/metabolism
7.
Mol Cell Biol ; 26(21): 7821-31, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16894030

ABSTRACT

The founding member of the inhibitor of apoptosis protein (IAP) family was originally identified as a cell death inhibitor. However, recent evidence suggests that IAPs are multifunctional signaling devices that influence diverse biological processes. To investigate the in vivo function of Drosophila melanogaster IAP2, we have generated diap2 null alleles. diap2 mutant animals develop normally and are fully viable, suggesting that diap2 is dispensable for proper development. However, these animals were acutely sensitive to infection by gram-negative bacteria. In Drosophila, infection by gram-negative bacteria triggers the innate immune response by activating the immune deficiency (imd) signaling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the pathway of mammalian tumor necrosis factor receptor 1 (TNFR1). diap2 mutant flies failed to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumbed to bacterial infection. Our genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1, and Relish. Therefore, DIAP2 functions in the host immune response to gram-negative bacteria. In contrast, we find that the Drosophila TNFR-associated factor (Traf) family member Traf2 is dispensable in resistance to gram-negative bacterial infection. Taken together, our genetic data identify DIAP2 as an essential component of the Imd signaling cascade, protecting the organism from infiltrating microbes.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/physiology , Gram-Negative Bacteria/metabolism , Gram-Negative Bacterial Infections/metabolism , Immunity, Innate/physiology , Inhibitor of Apoptosis Proteins/metabolism , NF-kappa B/metabolism , Signal Transduction/physiology , Animals , Animals, Genetically Modified , Anti-Infective Agents/metabolism , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Drosophila Proteins/genetics , Epistasis, Genetic , Inhibitor of Apoptosis Proteins/genetics , Male , Survival Rate , TNF Receptor-Associated Factor 2/genetics , TNF Receptor-Associated Factor 2/metabolism
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