scholarly journals Dual perspective proteomics infectome profiling discovers Salmonella type III secretion system effector functions in macrophages

2021 ◽  
Author(s):  
Jennifer Geddes-McAlister ◽  
A. Sukumaran ◽  
S. L. Vogt ◽  
J. L. Roland ◽  
S. E. Woodward ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Bacterial Pathogens ◽  
Host Cells ◽  
Secretion Systems ◽  
Specific Expression ◽  
Type Iii ◽  
Cell Functions ◽  
Serovar Typhimurium ◽  
Dual Perspective

Intracellular bacterial pathogens have evolved sophisticated infection strategies, including the release and secretion of virulence factors to interfere with host cell functions and to perturb immune responses. For Salmonella enterica serovar Typhimurium (S. Typhimurium), the type III secretion systems encoded on Salmonella pathogenicity islands (SPI) 1 and 2 mediates invasion of the bacterium into innate immune cells and regulates bacterial replication and survival within the hostile environment of the host, respectively. Here, we explore the temporal and strain-specific dual perspective response of both the host and pathogen during cellular infection via quantitative proteomics. We report time- and pathogenicity island-specific expression and secretion of infection-associated proteins (i.e., SL1344_1263, SL1344_3112, SL1344_1563, and YnhG) and regulated immune response proteins in macrophage, including Cd86, Cd40, Casp4, C3, IL-1?, and Cd69). Through intracellular macrophage and in vivo murine models of infection, we reveal a role in virulence for three of the bacterial proteins (SL1344_1263, SL1344_1563, and YnhG), defining their importance as novel T3SS effectors. We characterize the temporal intra- and extracellular production of the effectors and identify their interaction networks in host cells, representing inhibitory and stimulatory pathways mounted by invading bacterial pathogens.

scholarly journals The First 45 Amino Acids of SopA Are Necessary for InvB Binding and SPI-1 Secretion

2006 ◽  
Vol 188 (7) ◽  
pp. 2411-2420 ◽  
Author(s):  
Wendy Higashide ◽  
Daoguo Zhou
Keyword(s):  
Type Iii Secretion ◽  
Catalytic Domain ◽  
Effector Proteins ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Secretion Systems ◽  
Reporter System ◽  
Type Iii ◽  
Serovar Typhimurium ◽  
Type Iii Protein Secretion

ABSTRACT Salmonella enterica serovar Typhimurium encodes two type III secretion systems (TTSSs) within pathogenicity island 1 (SPI-1) and island 2 (SPI-2). These type III protein secretion and translocation systems transport a panel of bacterial effector proteins across both the bacterial and the host cell membranes to promote bacterial entry and subsequent survival inside host cells. Effector proteins contain secretion and translocation signals that are often located at their N termini. We have developed a ruffling-based translocation reporter system that uses the secretion- and translocation-deficient catalytic domain of SopE, SopE78-240, as a reporter. Using this assay, we determined that the N-terminal 45 amino acid residues of Salmonella SopA are necessary and sufficient for directing its secretion and translocation through the SPI-1 TTSS. SopA1-45, but not SopA1-44, is also able to bind to its chaperone, InvB, indicating that SPI-1 type III secretion and translocation of SopA require its chaperone.

scholarly journals Optimization of the Delivery of Heterologous Proteins by the Salmonella enterica Serovar Typhimurium Type III Secretion System for Vaccine Development

2006 ◽  
Vol 74 (10) ◽  
pp. 5826-5833 ◽  
Author(s):  
Li-Mei Chen ◽  
Gabriel Briones ◽  
Ruben O. Donis ◽  
Jorge E. Galán
Keyword(s):  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Vaccine Development ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Antigen Delivery ◽  
Secretion Systems ◽  
Type Iii ◽  
Serovar Typhimurium

ABSTRACT Type III protein secretion systems, which are organelles with the capacity to deliver bacterial proteins into host cells, have been adapted to deliver heterologous antigens for vaccine development. A limitation of these antigen delivery systems is that some proteins are not amenable to secretion through this pathway. We show here that proteins from the simian and human immunodeficiency viruses that are not permissive for secretion through a Salmonella enterica serovar Typhimurium type III secretion system can be modified to travel this secretion pathway by introduction of discrete mutations. Proteins optimized for secretion were presented more efficiently via the major histocompatibility complex class I pathway and were able to induce a better immune response.

scholarly journals Identification of New Secreted Effectors in Salmonella enterica Serovar Typhimurium

2005 ◽  
Vol 73 (10) ◽  
pp. 6260-6271 ◽  
Author(s):  
Kaoru Geddes ◽  
Micah Worley ◽  
George Niemann ◽  
Fred Heffron
Keyword(s):  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Bacterial Species ◽  
Effector Proteins ◽  
Host Cells ◽  
Secretion Systems ◽  
Cell Cytoplasm ◽  
Type Iii ◽  
Serovar Typhimurium ◽  
Infected Cells

ABSTRACTA common theme in bacterial pathogenesis is the secretion of bacterial products that modify cellular functions to overcome host defenses. Gram-negative bacterial pathogens use type III secretion systems (TTSSs) to inject effector proteins into host cells. The genes encoding the structural components of the type III secretion apparatus are conserved among bacterial species and can be identified by sequence homology. In contrast, the sequences of secreted effector proteins are less conserved and are therefore difficult to identify. A strategy was developed to identify virulence factors secreted bySalmonella entericaserovar Typhimurium into the host cell cytoplasm. We constructed a transposon, which we refer to as mini-Tn5-cycler, to generate translational fusions betweenSalmonellachromosomal genes and a fragment of the calmodulin-dependent adenylate cyclase gene derived fromBordetella pertussis(cyaA′). In-frame fusions to bacterial proteins that are secreted into the eukaryotic cell cytoplasm were identified by high levels of cyclic AMP in infected cells. The assay was sufficiently sensitive that a single secreted fusion could be identified among several hundred that were not secreted. This approach identified three new effectors as well as seven that have been previously characterized. A deletion of one of the new effectors,steA(Salmonellatranslocated effector A), attenuated virulence. In addition, SteA localizes to thetrans-Golgi network in both transfected and infected cells. This approach has identified new secreted effector proteins inSalmonellaand will likely be useful for other organisms, even those in which genetic manipulation is more difficult.

scholarly journals High-Throughput Screening of Type III Secretion Determinants Reveals a Major Chaperone-Independent Pathway

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Nadja Heinz Ernst ◽  
Analise Z. Reeves ◽  
Julia E. Ramseyer ◽  
Cammie F. Lesser
Keyword(s):  
High Throughput ◽  
Type Iii Secretion ◽  
Bacterial Pathogens ◽  
Host Cells ◽  
Review Of The Literature ◽  
Secretion Systems ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Effector Secretion

ABSTRACTNumerous Gram-negative bacterial pathogens utilize type III secretion systems (T3SSs) to inject tens of effector proteins directly into the cytosol of host cells. Through interactions with cognate chaperones, type III effectors are defined and recruited to the sorting platform, a cytoplasmic component of these membrane-embedded nanomachines. However, notably, a comprehensive review of the literature reveals that the secretion of most type III effectors has not yet been linked to a chaperone, raising questions regarding the existence of unknown chaperones as well as the universality of chaperones in effector secretion. Here, we describe the development of the first high-throughput type III secretion (T3S) assay, a semiautomated solid-plate-based assay, which enables the side-by-side comparison of secretion of over 20Shigellaeffectors under a multitude of conditions. Strikingly, we found that the majority ofShigellaeffectors are secreted at equivalent levels by wild-type and variants ofShigellathat no longer encode one or all knownShigellaT3S effector chaperones. In addition, we found thatShigellaeffectors are efficiently secreted from a laboratory strain ofEscherichia coliexpressing the coreShigellatype III secretion apparatus (T3SA) but no otherShigella-specific proteins. Furthermore, we observed that the sequences necessary and sufficient to define chaperone-dependent and -independent effectors are fundamentally different. Together, these findings support the existence of a major, previously unrecognized, noncanonical chaperone-independent secretion pathway that is likely common to many T3SSs.IMPORTANCEMany bacterial pathogens use specialized nanomachines, including type III secretion systems, to directly inject virulence proteins (effectors) into host cells. Here, we present the first extensive analysis of chaperone dependence in the process of type III effector secretion, providing strong evidence for the existence of a previously unrecognized chaperone-independent pathway. This noncanonical pathway is likely common to many bacteria, as an extensive review of the literature reveals that the secretion of multiple type III effectors has not yet been knowingly linked to a chaperone. While additional studies will be required to discern the molecular details of this pathway, its prevalence suggests that it can likely serve as a new target for the development of antimicrobial agents.

scholarly journals Inhibition of Type III Secretion in Salmonella enterica Serovar Typhimurium by Small-Molecule Inhibitors

2007 ◽  
Vol 51 (7) ◽  
pp. 2631-2635 ◽  
Author(s):  
Debra L. Hudson ◽  
Abigail N. Layton ◽  
Terry R. Field ◽  
Alison J. Bowen ◽  
Hans Wolf-Watz ◽  
...  
Keyword(s):  
Small Molecules ◽  
Type Iii Secretion ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Gram Negative Bacteria ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Cultured Epithelial Cells ◽  
Serovar Typhimurium

ABSTRACT Type III secretion systems (T3SS) are conserved in many pathogenic gram-negative bacteria. Small molecules that specifically target T3SS in Yersinia and Chlamydia spp. have recently been identified. Here we show that two such compounds inhibit Salmonella T3SS-1, preventing secretion of T3SS-1 effectors, invasion of cultured epithelial cells, and enteritis in vivo.

scholarly journals Tandem Translation Generates a Chaperone for the Salmonella Type III Secretion System Protein SsaQ

2011 ◽  
Vol 286 (41) ◽  
pp. 36098-36107 ◽  
Author(s):  
Xiu-Jun Yu ◽  
Mei Liu ◽  
Steve Matthews ◽  
David W. Holden
Keyword(s):  
Type Iii Secretion ◽  
Ex Vivo ◽  
Eukaryotic Cell ◽  
Effector Proteins ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Function Loss ◽  
Bacterial Cytoplasm

Type III secretion systems (T3SSs) of bacterial pathogens involve the assembly of a surface-localized needle complex, through which translocon proteins are secreted to form a pore in the eukaryotic cell membrane. This enables the transfer of effector proteins from the bacterial cytoplasm to the host cell. A structure known as the C-ring is thought to have a crucial role in secretion by acting as a cytoplasmic sorting platform at the base of the T3SS. Here, we studied SsaQ, an FliN-like putative C-ring protein of the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS. ssaQ produces two proteins by tandem translation: a long form (SsaQL) composed of 322 amino acids and a shorter protein (SsaQS) comprising the C-terminal 106 residues of SsaQL. SsaQL is essential for SPI-2 T3SS function. Loss of SsaQS impairs the function of the T3SS both ex vivo and in vivo. SsaQS binds to its corresponding region within SsaQL and stabilizes the larger protein. Therefore, SsaQL function is optimized by a novel chaperone-like protein, produced by tandem translation from its own mRNA species.

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.

Sign in / Sign up

Export Citation Format

Share Document