Salmonella Pathogenicity Island 2-Mediated Overexpression of Chimeric SspH2 Proteins for Simultaneous Induction of Antigen-Specific CD4 and CD8 T Cells

ABSTRACT Salmonella enterica serovar Typhimurium employs two different type III secretion systems (TTSS) encoded within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) for targeting of effector proteins into the cytosol of eukaryotic cells during different stages of the infection cycle. The SPI1 TTSS translocates virulence factors across the plasma membrane when the bacterium initially contacts the host cell. In contrast, the SPI2 TTSS functions to translocate proteins across the membrane of the Salmonella-containing vacuole and promotes intracellular survival and replication. The aim of the present study was to directly compare the potentials of SPI1 and SPI2 type III effector proteins to act as carrier molecules for a heterologous antigen. The p60 protein of Listeria monocytogenes was used as a model antigen to construct chimeric SopE2 (SPI1), SifA (SPI2), and SspH2 (SPI2) proteins. SPI1- and SPI2-dependent up- and down-regulation of hybrid gene expression led to sequential translocation of p60 fusion proteins into the cytosol of Salmonella-infected macrophages. Mice orally immunized with recombinant Salmonella strains expressing these hybrid proteins revealed comparable numbers of p60-specific CD8 T cells. However, only overexpression of translocated SspH2/p60 from a medium-copy-number vector induced simultaneous antigen-specific CD4 and CD8 T-cell responses, suggesting that SspH2 is an attractive carrier molecule for foreign-protein delivery.

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Superior Protective Immunity against Murine Listeriosis by Combined Vaccination with CpG DNA and Recombinant Salmonella enterica Serovar Typhimurium

Infection and Immunity ◽

10.1128/iai.00700-09 ◽

2009 ◽

Vol 77 (12) ◽

pp. 5501-5508 ◽

Cited By ~ 10

Author(s):

Christina Berchtold ◽

Klaus Panthel ◽

Stefan Jellbauer ◽

Brigitte Köhn ◽

Elisabeth Roider ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Salmonella Enterica ◽

Protective Immunity ◽

Cd8 T Cell ◽

Cpg Dna ◽

Memory Cd8 T Cells ◽

Type Iii ◽

Serovar Typhimurium

ABSTRACT Preexisting antivector immunity can severely compromise the ability of Salmonella enterica serovar Typhimurium live vaccines to induce protective CD8 T-cell frequencies after type III secretion system-mediated heterologous protein translocation in orally immunized mice. To circumvent this problem, we injected CpG DNA admixed to the immunodominant p60217-225 peptide from Listeria monocytogenes subcutaneously into BALB/c mice and coadministered a p60-translocating Salmonella strain by the orogastric route. The distribution of tetramer-positive p60217-225-specific effector and memory CD8 T cells was analyzed by costaining of lymphocytes with CD62L and CD127. In contrast to the single oral application of recombinant Salmonella or single immunization with CpG and p60, in the spleens from mice immunized with a combination of both vaccine types a significantly higher level of p60-specific CD8 T cells with a predominance of the effector memory T-cell subset was detected. In vivo protection studies revealed that this CD8 T-cell population conferred sterile protective immunity against a lethal infection with L. monocytogenes. However, p60-specific central memory CD8 T cells induced by single vaccination with CpG and p60 were not able confer effective protection against rapidly replicating intracellular Listeria. In conclusion, we provide compelling evidence that the combination of Salmonella type III-mediated antigen delivery and CpG immunization is an attractive novel vaccination strategy to modulate CD8 differentiation patterns toward distinct antigen-specific T-cell subsets with favorable protective capacities.

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

Journal of Bacteriology ◽

10.1128/jb.188.7.2411-2420.2006 ◽

2006 ◽

Vol 188 (7) ◽

pp. 2411-2420 ◽

Cited By ~ 12

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.

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Role ofSalmonellaPathogenicity Island 1 Protein IacP inSalmonella entericaSerovar Typhimurium Pathogenesis

Infection and Immunity ◽

10.1128/iai.01231-10 ◽

2011 ◽

Vol 79 (4) ◽

pp. 1440-1450 ◽

Cited By ~ 15

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.

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Salicylidene Acylhydrazides That Affect Type III Protein Secretion in Salmonella enterica Serovar Typhimurium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00223-07 ◽

2007 ◽

Vol 51 (8) ◽

pp. 2867-2876 ◽

Cited By ~ 77

Author(s):

Aurel Negrea ◽

Eva Bjur ◽

Sofia Eriksson Ygberg ◽

Mikael Elofsson ◽

Hans Wolf-Watz ◽

...

Keyword(s):

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Effector Proteins ◽

Secretion Systems ◽

Type Iii ◽

Type Iii Secretion Systems ◽

Serovar Typhimurium ◽

Type Iii Protein Secretion ◽

Bacterial Replication

ABSTRACT A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector β-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1. Salicylidene acylhydrazides also affected intracellular bacterial replication in murine macrophage-like cells and blocked the transport of an epitope-tagged SPI2 effector protein. Two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We conclude that salicylidene acylhydrazides affect bacterial T3SS activity in S. enterica and hence could be used as lead substances when designing specific inhibitors of bacterial T3SSs in order to pharmaceutically intervene with bacterial virulence.

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Identification of New Secreted Effectors in Salmonella enterica Serovar Typhimurium

Infection and Immunity ◽

10.1128/iai.73.10.6260-6271.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6260-6271 ◽

Cited By ~ 76

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.

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Discovery of Novel Secreted Virulence Factors fromSalmonella entericaSerovar Typhimurium by Proteomic Analysis of Culture Supernatants

Infection and Immunity ◽

10.1128/iai.00771-10 ◽

2010 ◽

Vol 79 (1) ◽

pp. 33-43 ◽

Cited By ~ 79

Author(s):

George S. Niemann ◽

Roslyn N. Brown ◽

Jean K. Gustin ◽

Afke Stufkens ◽

Afshan S. Shaikh-Kidwai ◽

...

Keyword(s):

Virulence Factors ◽

Type Iii Secretion ◽

Learning Algorithm ◽

Effector Proteins ◽

Secretion Systems ◽

Type Iii Effectors ◽

Type Iii ◽

Type Iii Secretion Systems ◽

Serovar Typhimurium ◽

Proteomic Study

ABSTRACTSalmonella entericaserovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded inSalmonellapathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (ΔssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ΔssaLmutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE,SVM-basedidentification andevaluation ofvirulenceeffectors) and selected 12 candidate proteins for further characterization. Using CyaA′ reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for fullSalmonellavirulence.

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Interactions of the Transmembrane Polymeric Rings of the Salmonella enterica Serovar Typhimurium Type III Secretion System

mBio ◽

10.1128/mbio.00158-10 ◽

2010 ◽

Vol 1 (3) ◽

Cited By ~ 28

Author(s):

Sarah Sanowar ◽

Pragya Singh ◽

Richard A. Pfuetzner ◽

Ingemar André ◽

Hongjin Zheng ◽

...

Keyword(s):

Type Iii Secretion ◽

Effector Proteins ◽

Carboxyl Terminus ◽

Gram Negative Bacteria ◽

Secretion Systems ◽

Gram Negative ◽

Type Iii ◽

Serovar Typhimurium ◽

Cross Links ◽

And Function

ABSTRACT The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function. IMPORTANCE Many biological macromolecular complexes are composed of symmetrical homomers, which assemble into larger structures. Some complexes, such as secretion systems, span biological membranes, forming hydrophilic domains to move substrates across lipid bilayers. Type III secretion systems (T3SSs) deliver bacterial effector proteins directly to the host cell cytoplasm and allow for critical interactions between many Gram-negative pathogenic bacterial species and their hosts. Progress has been made towards the goal of determining the three-dimensional structure of the secretion apparatus by determination of high-resolution crystal structures of individual protein subunits and low-resolution models of the assembly, using reconstructions of cryoelectron microscopy images. However, a more refined picture of the localization of periplasmic ring structures within the assembly and their interactions has only recently begun to emerge. This work localizes T3SS transmembrane rings and identifies structural elements that affect substrate switching and are essential to the assembly of components that are inserted into host cell membranes.

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Role of Autocleavage in the Function of a Type III Secretion Specificity Switch Protein in Salmonella enterica Serovar Typhimurium

mBio ◽

10.1128/mbio.01459-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 31

Author(s):

Julia V. Monjarás Feria ◽

Matthew D. Lefebre ◽

York-Dieter Stierhof ◽

Jorge E. Galán ◽

Samuel Wagner

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Effector Proteins ◽

Switching Function ◽

Gram Negative Bacteria ◽

Wild Type ◽

Gram Negative ◽

Type Iii ◽

Autocatalytic Cleavage ◽

Serovar Typhimurium

ABSTRACTType III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the “switch protein,” a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of theSalmonella entericaserovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage eventper sedoes not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function.IMPORTANCEBacterial interaction with eukaryotic hosts often involves complex molecular machines for targeted delivery of bacterial effector proteins. One such machine, the type III secretion system of some Gram-negative bacteria, serves to inject a multitude of structurally diverse bacterial proteins into the host cell. Critical to the function of these systems is their ability to secrete proteins in a strict hierarchical order, but it is unclear how the mechanism of switching works. Central to the switching mechanism is a highly conserved inner membrane protease that undergoes autocatalytic cleavage. Although it has been suggested previously that the autocleavage event is the trigger for substrate switching, we show here that this is not the case. Rather, our results show that cleavage allows the proper conformation of the protein to render it competent for its switching function. These findings may help develop inhibitors of type III secretion machines that offer novel therapeutic avenues to treat various infectious diseases.

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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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