scholarly journals SseL Deubiquitinates RPS3 to Inhibit Its Nuclear Translocation

Pathogens ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 86 ◽  
Author(s):  
Miaomiao Wu ◽  
Samir El Qaidi ◽  
Philip Hardwidge
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Translocation ◽  
Host Cells ◽  
Secretion Systems ◽  
Virulence Proteins ◽  
E Coli ◽  
Active Site Cysteine ◽  
T3ss Effector ◽  
Type Iii Secretion Systems ◽  
Important Host

Many Gram-negative bacterial pathogens use type III secretion systems to deliver virulence proteins (effectors) into host cells to counteract innate immunity. The ribosomal protein S3 (RPS3) guides NF-κB subunits to specific κB sites and plays an important role in the innate response to bacterial infection. Two E. coli effectors inhibit RPS3 nuclear translocation. NleH1 inhibits RPS3 phosphorylation by IKK-β, an essential aspect of the RPS3 nuclear translocation process. NleC proteolysis of p65 generates an N-terminal p65 fragment that competes for full-length p65 binding to RPS3, thus also inhibiting RPS3 nuclear translocation. Thus, E. coli has multiple mechanisms by which to block RPS3-mediated transcriptional activation. With this in mind, we considered whether other enteric pathogens also encode T3SS effectors that impact this important host regulatory pathway. Here we report that the Salmonella Secreted Effector L (SseL), which was previously shown to function as a deubiquitinase and inhibit NF-κB signaling, also inhibits RPS3 nuclear translocation by deubiquitinating this important host transcriptional co-factor. RPS3 deubiquitination by SseL was restricted to K63-linkages and mutating the active-site cysteine of SseL abolished its ability to deubiquitinate and subsequently inhibit RPS3 nuclear translocation. Thus, Salmonella also encodes at least one T3SS effector that alters RPS3 activities in the host nucleus.

scholarly journals Cooperative Immune Suppression by Escherichia coli and Shigella Effector Proteins

2018 ◽  
Vol 86 (4) ◽  
Author(s):  
Maarten F. de Jong ◽  
Neal M. Alto
Keyword(s):  
Escherichia Coli ◽  
Defense Mechanisms ◽  
Immune Defense ◽  
Effector Proteins ◽  
Innate Immune ◽  
Host Cells ◽  
Secretion Systems ◽  
Content Type ◽  
E Coli ◽  
Type Iii Secretion Systems

ABSTRACT The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/ Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.

scholarly journals Effect of the O-Antigen Length of Lipopolysaccharide on the Functions of Type III Secretion Systems in Salmonella enterica

2009 ◽  
Vol 77 (12) ◽  
pp. 5458-5470 ◽  
Author(s):  
Stefanie U. Hölzer ◽  
Markus C. Schlumberger ◽  
Daniela Jäckel ◽  
Michael Hensel
Keyword(s):  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Defense Mechanisms ◽  
Host Cells ◽  
Effector Protein ◽  
Secretion Systems ◽  
Type Iii ◽  
O Antigen ◽  
T3ss Effector ◽  
Type Iii Secretion Systems

ABSTRACT The virulence of Salmonella enterica critically depends on the functions of two type III secretion systems (T3SS), with the Salmonella pathogenicity island 1 (SPI1)-encoded T3SS required for host cell invasion and the SPI2-T3SS enabling Salmonella to proliferate within host cells. A further T3SS is required for the assembly of the flagella. Most serovars of Salmonella also possess a lipopolysaccharide with a complex O-antigen (OAg) structure. The number of OAg units attached to the core polysaccharide varies between 16 and more than 100 repeats, with a trimodal distribution. This work investigated the correlation of the OAg length with the functions of the SPI1-T3SS and the SPI2-T3SS. We observed that the number of repeats of OAg units had no effect on bacterial motility. The interaction of Salmonella with epithelial cells was altered if the OAg structure was changed by mutations in regulators of OAg. Strains defective in synthesis of very long or long and very long OAg species showed increased translocation of a SPI1-T3SS effector protein and increased invasion. Invasion of a strain entirely lacking OAg was increased, but this mutant strain also showed increased adhesion. In contrast, translocation of a SPI2-T3SS effector protein and intracellular replication were not affected by modification of the OAg length. Mutant strains lacking the entire OAg or long and very long OAg were highly susceptible to complement killing. These observations indicate that the architecture of the outer membrane of Salmonella is balanced to permit sufficient T3SS function but also to confer optimal protection against antimicrobial defense mechanisms.

scholarly journals Role ofSalmonellaPathogenicity Island 1 Protein IacP inSalmonella entericaSerovar Typhimurium Pathogenesis

2011 ◽  
Vol 79 (4) ◽  
pp. 1440-1450 ◽  
Author(s):  
Jin Seok Kim ◽  
Jeong Seon Eom ◽  
Jung Im Jang ◽  
Hyeon Guk Kim ◽  
Doo Won Seo ◽  
...  
Keyword(s):  
Effector Proteins ◽  
Infection Model ◽  
Secretion Systems ◽  
Virulence Proteins ◽  
Type Iii ◽  
Mouse Infection Model ◽  
T3ss Effector ◽  
Cytoplasmic Proteins ◽  
Type Iii Secretion Systems ◽  
Serovar Typhimurium

ABSTRACTGram-negative bacteria, includingSalmonella entericaserovar Typhimurium, exploit type III secretion systems (T3SSs) through which virulence proteins are delivered into the host cytosol to reinforce invasive and replicative niches in their host. Although many secreted effector proteins and membrane-bound structural proteins in the T3SS have been characterized, the functions of many cytoplasmic proteins still remain unknown. In this study, we found that IacP, encoded bySalmonellapathogenicity island 1, was important for nonphagocytic cell invasion and bacterial virulence. When theiacPgene was deleted from severalSalmonellaserovar Typhimurium strains, the invasion into INT-407 epithelial cells was significantly decreased compared to that of their parental strains, and retarded rearrangements of actin fibers were observed for theiacPmutant-infected cells. Although IacP had no effect on the secretion of type III translocon proteins, the levels of secretion of the effector proteins SopB, SopA, and SopD into the culture medium were decreased in theiacPmutant. In a mouse infection model, mice infected with theiacPmutant exhibited alleviated pathological signs in the intestine and survived longer than did wild-type-infected mice. Taken together, IacP plays a key role inSalmonellavirulence by regulating the translocation of T3SS effector proteins.

scholarly journals The T3SS Effector Protease NleC Is Active within Citrobacter rodentium

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 589
Author(s):  
Md Kamrul Hasan ◽  
Samir El Qaidi ◽  
Philip R. Hardwidge
Keyword(s):  
Type Iii Secretion ◽  
Effector Proteins ◽  
Host Cells ◽  
Citrobacter Rodentium ◽  
Secretion Systems ◽  
Gram Negative ◽  
Virulence Proteins ◽  
T3ss Effector ◽  
Enhanced Resistance ◽  
Cleavage Motif

Whether type III secretion system (T3SS) effector proteins encoded by Gram-negative bacterial pathogens have intra-bacterial activities is an important and emerging area of investigation. Gram-negative bacteria interact with their mammalian hosts by using secretion systems to inject virulence proteins directly into infected host cells. Many of these injected protein effectors are enzymes that modify the structure and inhibit the function of mammalian proteins. The underlying dogma is that T3SS effectors are inactive until they are injected into host cells, where they then fold into their active conformations. We previously observed that the T3SS effectors NleB and SseK1 glycosylate Citrobacter rodentium and Salmonella enterica proteins, respectively, leading to enhanced resistance to environmental stress. Here, we sought to extend these studies to determine whether the T3SS effector protease NleC is also active within C. rodentium. To do this, we expressed the best-characterized mammalian substrate of NleC, the NF-κB p65 subunit in C. rodentium and monitored its proteolytic cleavage as a function of NleC activity. Intra-bacterial p65 cleavage was strictly dependent upon NleC. A p65 mutant lacking the known CE cleavage motif was resistant to NleC. Thus, we conclude that, in addition to NleB, NleC is also enzymatically active within C. rodentium.

scholarly journals The S. Typhi effector StoD is an E3/E4 ubiquitin ligase which binds K48- and K63-linked diubiquitin

2019 ◽  
Vol 2 (3) ◽  
pp. e201800272
Author(s):  
Melanie A McDowell ◽  
Alexander MP Byrne ◽  
Elli Mylona ◽  
Rebecca Johnson ◽  
Agnes Sagfors ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Type Iii Secretion ◽  
Intracellular Signaling ◽  
Binding Assays ◽  
Secretion Systems ◽  
E Coli ◽  
T3ss Effector ◽  
C Terminus ◽  
Type Iii Secretion Systems ◽  
Β Sheet

Salmonella enterica (e.g., serovars Typhi and Typhimurium) relies on translocation of effectors via type III secretion systems (T3SS). Specialization of typhoidal serovars is thought to be mediated via pseudogenesis. Here, we show that the Salmonella Typhi STY1076/t1865 protein, named StoD, a homologue of the enteropathogenic Escherichia coli/enterohemorrhagic E. coli/Citrobacter rodentium NleG, is a T3SS effector. The StoD C terminus (StoD-C) is a U-box E3 ubiquitin ligase, capable of autoubiquitination in the presence of multiple E2s. The crystal structure of the StoD N terminus (StoD-N) at 2.5 Å resolution revealed a ubiquitin-like fold. In HeLa cells expressing StoD, ubiquitin is redistributed into puncta that colocalize with StoD. Binding assays showed that StoD-N and StoD-C bind the same exposed surface of the β-sheet of ubiquitin, suggesting that StoD could simultaneously interact with two ubiquitin molecules. Consistently, StoD interacted with both K63- (KD = 5.6 ± 1 μM) and K48-linked diubiquitin (KD = 15 ± 4 μM). Accordingly, we report the first S. Typhi–specific T3SS effector. We suggest that StoD recognizes and ubiquitinates pre-ubiquitinated targets, thus subverting intracellular signaling by functioning as an E4 enzyme.

scholarly journals Role for Myosin II in Regulating Positioning of Salmonella-Containing Vacuoles and Intracellular Replication

2008 ◽  
Vol 76 (6) ◽  
pp. 2722-2735 ◽  
Author(s):  
Julie A. Wasylnka ◽  
Malina A. Bakowski ◽  
Jason Szeto ◽  
Maikke B. Ohlson ◽  
William S. Trimble ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Distinct Type ◽  
Host Cells ◽  
Secretion Systems ◽  
Virulence Proteins ◽  
Type Iii Secretion Systems ◽  
Serovar Typhimurium ◽  
Microtubule Motor

ABSTRACT Salmonella enterica serovar Typhimurium grows within host cells in a permissive compartment termed the Salmonella-containing vacuole (SCV). These bacteria use two distinct type III secretion systems (T3SS) to deliver virulence proteins (effectors) into cells. Effectors secreted by the Salmonella pathogenicity island 1 (SPI-1)-encoded T3SS mediate invasion and early SCV maturation steps, while those secreted by the SPI-2 T3SS affect the SCV at later stages postinfection. Some SPI-2 effectors modulate microtubule motor activity on the SCV. Here, we show that the actin-based motor myosin II also affects SCV dynamics during infection. Following invasion, myosin II is required for SCV positioning near the nucleus of host cells. Later, myosin II counteracts the activities of the SPI-2 effectors PipB2 and SseJ to maintain SCV positioning and stability, respectively. Myosin II activity was required for maximal bacterial growth in macrophages. Rho kinase activity was required for SCV positioning. The effector SopB, a known activator of Rho GTPases, was found to be required for SCV positioning, and transfection of cells with SopB was sufficient to induce myosin II phosphorylation. These studies reveal a novel role for myosin II in controlling SCV dynamics during infection and suggest that SopB activates myosin II.

scholarly journals Small-Molecule Type III Secretion System Inhibitors Block Assembly of the Shigella Type III Secreton

2008 ◽  
Vol 191 (2) ◽  
pp. 563-570 ◽  
Author(s):  
Andreas K. J. Veenendaal ◽  
Charlotta Sundin ◽  
Ariel J. Blocker
Keyword(s):  
Type Iii Secretion ◽  
Bacterial Infections ◽  
Shigella Flexneri ◽  
Effector Proteins ◽  
Host Cells ◽  
Incomplete Penetrance ◽  
Secretion Systems ◽  
Bacterial Surface ◽  
Type Iii ◽  
Type Iii Secretion Systems

ABSTRACT Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

scholarly journals A Salmonella type III effector, PipA, works in a different manner than the PipA family effectors GogA and GtgA

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248975
Author(s):  
Momo Takemura ◽  
Takeshi Haneda ◽  
Hikari Idei ◽  
Tsuyoshi Miki ◽  
Nobuhiko Okada
Keyword(s):  
Hela Cells ◽  
Critical Role ◽  
Effector Proteins ◽  
Host Cells ◽  
Microbial Pathogens ◽  
Secretion Systems ◽  
Type Iii ◽  
Zinc Metalloprotease ◽  
Type Iii Secretion Systems ◽  
Serovar Typhimurium

Nuclear factor-kappa B (NF-κB) plays a critical role in the host defense against microbial pathogens. Many pathogens modulate NF-κB signaling to establish infection in their host. Salmonella enterica serovar Typhimurium (S. Typhimurium) possesses two type III secretion systems (T3SS-1 and T3SS-2) and directly injects many effector proteins into host cells. It has been reported that some effectors block NF-κB signaling, but the molecular mechanism of the inactivation of NF-κB signaling in S. Typhimurium is poorly understood. Here, we identified seven type III effectors—GogA, GtgA, PipA, SseK1, SseK2, SseK3, and SteE—that inhibited NF-κB activation in HeLa cells stimulated with TNF-α. We also determined that only GogA and GtgA are involved in regulation of the activation of NF-κB in HeLa cells infected with S. Typhimurium. GogA, GtgA, and PipA are highly homologous to one another and have the consensus zinc metalloprotease HEXXH motif. Our experiments demonstrated that GogA, GtgA, and PipA each directly cleaved NF-κB p65, whereas GogA and GtgA, but not PipA, inhibited the NF-κB activation in HeLa cells infected with S. Typhimurium. Further, expressions of the gogA or gtgA gene were induced under the SPI-1-and SPI-2-inducing conditions, but expression of the pipA gene was induced only under the SPI-2-inducing condition. We also showed that PipA was secreted into RAW264.7 cells through T3SS-2. Finally, we indicated that PipA elicits bacterial dissemination in the systemic stage of infection of S. Typhimurium via a T3SS-1-independent mechanism. Collectively, our results suggest that PipA, GogA and GtgA contribute to S. Typhimurium pathogenesis in different ways.

scholarly journals A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems

2019 ◽  
Author(s):  
Sibel Westerhausen ◽  
Melanie Nowak ◽  
Claudia Torres-Vargas ◽  
Ursula Bilitewski ◽  
Erwin Bohn ◽  
...  
Keyword(s):  
Protein Secretion ◽  
High Throughput ◽  
Type Iii Secretion ◽  
Molecular Mechanisms ◽  
Host Cells ◽  
Target Cells ◽  
Secretion Systems ◽  
Type Iii ◽  
Nanoluc Luciferase ◽  
Type Iii Secretion Systems

AbstractThe elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a lack of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess secretion and injection through the type III secretion system encoded by Salmonella pathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type III secretion-dependent injection of effector proteins into host cells.ImportanceThe ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or even into target cells is one of the foundations of interbacterial as well as pathogen-host interaction. While great progress has been made in elucidating assembly and structure of secretion systems, our understanding of their secretion mechanism often lags behind, not last because of the challenge to quantitatively assess secretion function. Here, we developed a luciferase-based assay to enable the simple, quick, quantitative, and high throughput-compatible assessment of secretion and injection through virulence-associated type III secretion systems. The assay allows detection of minute amounts of secreted substrate proteins either in the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an enabling technology to elucidate the mechanisms of secretion and injection of type III secretion systems and is likely adaptable to assay secretion through other bacterial secretion systems.

scholarly journals An Amino-Terminal Secretion Signal Is Required for YplA Export by the Ysa, Ysc, and Flagellar Type III Secretion Systems of Yersinia enterocolitica Biovar 1B

2005 ◽  
Vol 187 (17) ◽  
pp. 6075-6083 ◽  
Author(s):  
Sasha M. Warren ◽  
Glenn M. Young
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Distinct Type ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Secretion Systems ◽  
Type Iii ◽  
Secretion Signal ◽  
Amino Terminal ◽  
Type Iii Secretion Systems

ABSTRACT Yersinia enterocolitica biovar 1B maintains three distinct type III secretion (TTS) systems, which independently operate to target proteins to extracellular sites. The Ysa and Ysc systems are prototypical contact-dependent TTS systems that translocate toxic effectors to the cytosols of targeted eukaryotic host cells during infection. The flagellar TTS system is utilized during the assembly of the flagellum and is required for secretion of the virulence-associated phospholipase YplA to the bacterial milieu. When ectopically produced, YplA is also a secretion substrate for the Ysa and Ysc TTS systems. In this study, we define elements that allow YplA recognition and export by the Ysa, Ysc, and flagellar TTS systems. Fusion of various amino-terminal regions of YplA to Escherichia coli alkaline phosphatase (PhoA) lacking its native secretion signal demonstrated that the first 20 amino acids or corresponding mRNA codons of YplA were sufficient for export of YplA-PhoA chimeras by each TTS system. Export of native YplA by each of the three TTS systems was also found to depend on the integrity of its amino terminus. Introduction of a frameshift mutation or deletion of yplA sequences encoding the amino-terminal 20 residues negatively impacted YplA secretion. Deletion of other yplA regions was tolerated, including that resulting in the removal of amino acid residues 30 through 40 of the polypeptide and removal of the 5′ untranslated region of the mRNA. This work supports a model in which independent and distantly related TTS systems of Y. enterocolitica recognize protein substrates by a similar mechanism.

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