Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m111.278663 ◽

2011 ◽

Vol 286 (41) ◽

pp. 36098-36107 ◽

Cited By ~ 28

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.

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

PLoS ONE ◽

10.1371/journal.pone.0248975 ◽

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.

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

10.1101/745471 ◽

2019 ◽

Cited By ~ 1

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.

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Bridging the Gap: Type III Secretion Systems in Plant-Beneficial Bacteria

Microorganisms ◽

10.3390/microorganisms10010187 ◽

2022 ◽

Vol 10 (1) ◽

pp. 187

Author(s):

Antoine Zboralski ◽

Adrien Biessy ◽

Martin Filion

Keyword(s):

Type Iii Secretion ◽

Plant Pathogens ◽

Plant Immunity ◽

Effector Proteins ◽

Secretion Systems ◽

Beneficial Bacteria ◽

Living Plant ◽

Pseudomonas Spp ◽

Type Iii ◽

Type Iii Secretion Systems

Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines translocating effector proteins into the cytoplasm of eukaryotic cells. They have been intensively studied for their important roles in animal and plant bacterial diseases. Over the past two decades, genome sequencing has unveiled their ubiquitous distribution in many taxa of Gram-negative bacteria, including plant-beneficial ones. Here, we discuss the distribution and functions of the T3SS in two agronomically important bacterial groups: the symbiotic nodule-forming nitrogen-fixing rhizobia and the free-living plant-beneficial Pseudomonas spp. In legume-rhizobia symbiosis, T3SSs and their cognate effectors play important roles, including the modulation of the plant immune response and the initiation of the nodulation process in some cases. In plant-beneficial Pseudomonas spp., the roles of T3SSs are not fully understood, but pertain to plant immunity suppression, biocontrol against eukaryotic plant pathogens, mycorrhization facilitation, and possibly resistance against protist predation. The diversity of T3SSs in plant-beneficial bacteria points to their important roles in multifarious interkingdom interactions in the rhizosphere. We argue that the gap in research on T3SSs in plant-beneficial bacteria must be bridged to better understand bacteria/eukaryotes rhizosphere interactions and to support the development of efficient plant-growth promoting microbial inoculants.

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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

Journal of Bacteriology ◽

10.1128/jb.187.17.6075-6083.2005 ◽

2005 ◽

Vol 187 (17) ◽

pp. 6075-6083 ◽

Cited By ~ 15

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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Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity

Microorganisms ◽

10.3390/microorganisms8030357 ◽

2020 ◽

Vol 8 (3) ◽

pp. 357 ◽

Cited By ~ 4

Author(s):

Juan Luis Araujo-Garrido ◽

Joaquín Bernal-Bayard ◽

Francisco Ramos-Morales

Keyword(s):

Type Iii Secretion ◽

Enterohemorrhagic Escherichia Coli ◽

Host Cells ◽

Secretion Systems ◽

Type Iii ◽

Cellular Processes ◽

Host Cell Death ◽

Pathway Activation ◽

Type Iii Secretion Systems ◽

Arginine Residues

Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targets.

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Evidence for Targeting of Yop Effectors by the Chromosomally Encoded Ysa Type III Secretion System of Yersinia enterocolitica

Journal of Bacteriology ◽

10.1128/jb.184.20.5563-5571.2002 ◽

2002 ◽

Vol 184 (20) ◽

pp. 5563-5571 ◽

Cited By ~ 32

Author(s):

Briana M. Young ◽

Glenn M. Young

Keyword(s):

Yersinia Enterocolitica ◽

Type Iii Secretion ◽

Immunoblot Analysis ◽

Effector Proteins ◽

Secretion Systems ◽

Necrosis Factor Alpha ◽

Type Iii ◽

Type Iii Secretion Systems ◽

Molecular Masses ◽

Factor Alpha

ABSTRACT Yersinia enterocolitica O:8 has two contact-dependent type III secretion systems (TTSSs). The Ysa TTSS is encoded by a set of genes located on the chromosome and exports Ysp proteins. The Ysc TTSS and the Yop effector proteins it exports are encoded by genes located on plasmid pYVe8081. In this study, secretion of YspG, YspH, and YspJ by the Ysa TTSS was shown to require pYVe8081. Furthermore, mutations that blocked the function of the Ysc TTSS did not affect YspG, YspH, and YspJ production. This indicated that YspG, YspH, and YspJ are encoded by genes located on pYVe8081 and that they may correspond to Yops. A comparison of Ysps with Yop effectors secreted by Y. enterocolitica indicated that YspG, YspH, and YspJ have apparent molecular masses similar to those of YopN, YopP, and YopE, respectively. Immunoblot analysis demonstrated that antibodies directed against YopN, YopP, and YopE recognized YspG, YspH, and YspJ. Furthermore, mutations in yopN, yopP, and yopE specifically blocked YopN, YopP, and YopE secretion by the Ysc TTSS and YspG, YspH, and YspJ secretion by the Ysa TTSS. These results indicate YspG, YspH, and YspJ are actually YopN, YopP, and YopE. Additional analysis demonstrated that YopP and YspH secretion was restored to yopP mutants by complementation in trans with a wild-type copy of the yopP gene. Examination of Y. enterocolitica-infected J774A.1 macrophages revealed that both the Ysc and Ysa TTSSs contribute to YopP-dependent suppression of tumor necrosis factor alpha production. This indicates that both the Ysa and Ysc TTSSs are capable of targeting YopP and that they influence Y. enterocolitica interactions with macrophages. Taken together, these results suggest that the Ysa and Ysc TTSSs contribute to Y. enterocolitica virulence by exporting both unique and common subsets of effectors.

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