scholarly journals Identification of Proteins Secreted via Vibrio parahaemolyticus Type III Secretion System 1

2006 ◽  
Vol 74 (2) ◽  
pp. 1032-1042 ◽  
Author(s):  
Takahiro Ono ◽  
Kwon-Sam Park ◽  
Mayumi Ueta ◽  
Tetsuya Iida ◽  
Takeshi Honda
Keyword(s):  
Hela Cells ◽  
Type Iii Secretion ◽  
Vibrio Parahaemolyticus ◽  
Type Iii Secretion System ◽  
Gene Clusters ◽  
Secretion System ◽  
Secreted Proteins ◽  
Large Chromosome ◽  
Two Dimensional Gel Electrophoresis ◽  
Type Iii

ABSTRACT Vibrio parahaemolyticus, a gram-negative marine bacterium, is an important pathogen causing food-borne gastroenteritis or septicemia. Recent genome sequencing of the RIMD2210633 strain (a Kanagawa phenomenon-positive clinical isolate of serotype O3:K6) revealed that the strain has two sets of gene clusters that encode the type III secretion system (TTSS) apparatus. The first cluster, TTSS1, is located on the large chromosome, and the second, TTSS2, is on the small chromosome. Previously, we reported that TTSS1 is involved in the cytotoxicity of the RIMD2210633 strain against HeLa cells. Here, we analyzed proteins secreted via the TTSS apparatus encoded by TTSS1 by using two-dimensional gel electrophoresis and identified the proteins encoded by genes VP1680, VP1686, and VPA450. To investigate the roles of those secreted proteins, we constructed and analyzed a series of deletion mutants. Flow cytometry analysis using fluorescence-activated cell sorting with fluorescein isothiocyanate-labeled annexin V demonstrated that the TTSS1-dependent cell death was by apoptosis. The cytotoxicity to HeLa cells was related to one of the newly identified secreted proteins encoded by VP1680. Adenylate cyclase fusion protein studies proved that the newly identified secreted proteins were translocated into HeLa cells. Thus, these appear to be the TTSS effector proteins in V. parahaemolyticus.

scholarly journals A Degenerate Type III Secretion System from Septicemic Escherichia coli Contributes to Pathogenesis

2005 ◽  
Vol 187 (23) ◽  
pp. 8164-8171 ◽  
Author(s):  
Diana Ideses ◽  
Uri Gophna ◽  
Yossi Paitan ◽  
Roy R. Chaudhuri ◽  
Mark J. Pallen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Gene Clusters ◽  
Secretion System ◽  
Effector Proteins ◽  
E Coli ◽  
Type Iii

ABSTRACT The type III secretion system (T3SS) is an important virulence factor used by several gram-negative bacteria to deliver effector proteins which subvert host cellular processes. Enterohemorrhagic Escherichia coli O157 has a well-defined T3SS involved in attachment and effacement (ETT1) and critical for virulence. A gene cluster potentially encoding an additional T3SS (ETT2), which resembles the SPI-1 system in Salmonella enterica, was found in its genome sequence. The ETT2 gene cluster has since been found in many E. coli strains, but its in vivo role is not known. Many of the ETT2 gene clusters carry mutations and deletions, raising the possibility that they are not functional. Here we show the existence in septicemic E. coli strains of an ETT2 gene cluster, ETT2sepsis, which, although degenerate, contributes to pathogenesis. ETT2sepsis has several premature stop codons and a large (5 kb) deletion, which is conserved in 11 E. coli strains from cases of septicemia and newborn meningitis. A null mutant constructed to remove genes coding for the putative inner membrane ring of the secretion complex exhibited significantly reduced virulence. These results are the first demonstration of the importance of ETT2 for pathogenesis.

scholarly journals A Type III Secretion System Is Required for Aeromonas hydrophila AH-1 Pathogenesis

2004 ◽  
Vol 72 (3) ◽  
pp. 1248-1256 ◽  
Author(s):  
H. B. Yu ◽  
P. S. Srinivasa Rao ◽  
H. C. Lee ◽  
S. Vilches ◽  
S. Merino ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Gene Clusters ◽  
Opportunistic Pathogen ◽  
Secretion System ◽  
Virulence Determinants ◽  
Degenerate Primers ◽  
Type Iii ◽  
Insertional Inactivation

ABSTRACT Aeromonas hydrophila is a gram-negative opportunistic pathogen in fish and humans. Many bacterial pathogens of animals and plants have been shown to inject anti-host virulence determinants into the hosts via a type III secretion system (TTSS). Degenerate primers based on lcrD family genes that are present in every known TTSS allowed us to locate the TTSS gene cluster in A. hydrophila AH-1. A series of genome walking steps helped in the identification of 25 open reading frames that encode proteins homologous to those in TTSSs in other bacteria. PCR-based analysis showed the presence of lcrD homologs (ascV) in all of the 33 strains of A. hydrophila isolated from various sources. Insertional inactivation of two of the TTSS genes (aopB and aopD) led to decreased cytotoxicity in carp epithelial cells, increased phagocytosis, and reduced virulence in blue gourami. These results show that a TTSS is required for A. hydrophila pathogenesis. This is the first report of sequencing and characterization of TTSS gene clusters from A. hydrophila. The TTSS identified here may help in developing suitable vaccines as well as in further understanding of the pathogenesis of A. hydrophila.

scholarly journals SepZ/EspZ Is Secreted and Translocated into HeLa Cells by the Enteropathogenic Escherichia coli Type III Secretion System

2005 ◽  
Vol 73 (7) ◽  
pp. 4327-4337 ◽  
Author(s):  
Kristen J. Kanack ◽  
J. Adam Crawford ◽  
Ichiro Tatsuno ◽  
Mohamed A. Karmali ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Bacterial Attachment ◽  
Host Cells ◽  
Dependent Manner ◽  
Cell Infection ◽  
Type Iii

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a major bacterial cause of infantile diarrhea in developing countries and is the prototype for a group of gastrointestinal pathogens causing characteristic attaching and effacing (A/E) histopathology on intestinal epithelia. A/E pathogens utilize a type III secretion system (TTSS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to deliver effector proteins into host cells. Here, we investigate sequence divergence of the LEE-encoded SepZ protein and identify it as a TTSS-secreted and -translocated molecule. SepZ is hypervariable among A/E pathogens, with sequences sharing between 60 to 81% amino acid identity with SepZ of EPEC. A SepZ-CyaA fusion was secreted and translocated into HeLa cells in a TTSS-dependent manner. Additionally, we determined that the first 20 amino acids of SepZ were sufficient to direct its translocation. In contrast to previous studies suggesting a role in invasion and the structure and/or regulation of the TTSS, we found that SepZ does not mediate uptake of EPEC into host cells or affect translocation and tyrosine phosphorylation of the translocated intimin receptor. Immunohistochemistry reveals that, after an extended HeLa cell infection, accumulated SepZ can be detected beneath the site of bacterial attachment in a subset of pedestal regions. To indicate its newly identified status as a translocated effector protein, we propose to rename SepZ as EspZ.

scholarly journals Transcriptional Downregulation of a Type III Secretion System under Reducing Conditions in Bordetella pertussis

2020 ◽  
Vol 202 (21) ◽  
Author(s):  
Masataka Goto ◽  
Tomoko Hanawa ◽  
Akio Abe ◽  
Asaomi Kuwae
Keyword(s):  
Bordetella Pertussis ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Culture Conditions ◽  
Secretion System ◽  
Host Cells ◽  
Secreted Proteins ◽  
Content Type ◽  
Type Iii ◽  
Reducing Conditions

ABSTRACT Bordetella pertussis uses a type III secretion system (T3SS) to inject virulence proteins into host cells. Although the B. pertussis T3SS was presumed to be involved in host colonization, efficient secretion of type III secreted proteins from B. pertussis has not been observed. To investigate the roles of type III secreted proteins during infection, we attempted to optimize culture conditions for the production and secretion of a type III secreted protein, BteA, in B. pertussis. We observed that B. pertussis efficiently secretes BteA in ascorbic acid-depleted (AsA−) medium. When L2 cells, a rat lung epithelial cell line, were infected with B. pertussis cultured in the AsA− medium, BteA-dependent cytotoxicity was observed. We also performed an immunofluorescence assay of L2 cells infected with B. pertussis. Clear fluorescence signals of Bsp22, a needle structure of T3SS, were detected on the bacterial surface of B. pertussis cultured in the AsA− medium. Since ascorbic acid is known as a reducing agent, we cultured B. pertussis in liquid medium containing other reducing agents such as 2-mercaptoethanol and dithioerythritol. Under these reducing conditions, the production of type III secreted proteins was repressed. These results suggest that in B. pertussis, the production and secretion of type III secreted proteins are downregulated under reducing conditions. IMPORTANCE The type III secretion system (T3SS) of Bordetella pertussis forms a needlelike structure that protrudes from the bacterial cell surface. B. pertussis uses a T3SS to translocate virulence proteins called effectors into host cells. The culture conditions for effector production in B. pertussis have not been investigated. We attempted to optimize culture medium compositions for producing and secreting type III secreted proteins. We found that B. pertussis secretes type III secreted proteins in reducing agent-deprived liquid medium and that BteA-secreting B. pertussis provokes cytotoxicity against cultured mammalian cells. These results suggest that redox signaling is involved in the regulation of B. pertussis T3SS.

scholarly journals Vibrio parahaemolyticus Inhibition of Rho Family GTPase Activation Requires a Functional Chromosome I Type III Secretion System

2008 ◽  
Vol 76 (5) ◽  
pp. 2202-2211 ◽  
Author(s):  
Timothy Casselli ◽  
Tarah Lynch ◽  
Carolyn M. Southward ◽  
Bryan W. Jones ◽  
Rebekah DeVinney
Keyword(s):  
Virulence Factors ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Vibrio Parahaemolyticus ◽  
Secretion System ◽  
Type Iii ◽  
Rho Family Gtpases ◽  
Rho Family ◽  
Gtpase Activation ◽  
Chromosome I

ABSTRACT Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis; however, its virulence mechanisms are not well understood. The identification of type III secreted proteins has provided candidate virulence factors whose functions are still being elucidated. Genotypic strain variability contributes a level of complexity to understanding the role of different virulence factors. The ability of V. parahaemolyticus to inhibit Rho family GTPases and cause cytoskeletal disruption was examined with HeLa cells. After HeLa cells were infected, intracellular Rho activation was inhibited in response to external stimuli. In vitro activation of Rho, Rac, and Cdc42 isolated from infected HeLa cell lysates was also inhibited, indicating that the bacteria were specifically targeting GTPase activation. The inhibition of Rho family GTPase activation was retained for clinical and environmental isolates of V. parahaemolyticus and was dependent on a functional chromosome I type III secretion system (CI-T3SS). GTPase inhibition was independent of hemolytic toxin genotype and the chromasome II (CII)-T3SS. Rho inhibition was accompanied by a shift in the total actin pool to its monomeric form. These phenotypes were abrogated in a mutant strain lacking the CI-T3S effector Vp1686, suggesting that the inhibiting actin polymerization may be a downstream effect of Vp1686-dependent GTPase inhibition. Although Vp1686 has been previously characterized as a potential virulence factor in macrophages, our findings reveal an effect on cultured HeLa cells. The ability to inhibit Rho family GTPases independently of the CII-T3SS and the hemolytic toxins may provide insight into the mechanisms of virulence used by strains lacking these virulence factors.

scholarly journals Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length

2002 ◽  
Vol 184 (5) ◽  
pp. 1244-1252 ◽  
Author(s):  
Koichi Tamano ◽  
Eisaku Katayama ◽  
Takahito Toyotome ◽  
Chihiro Sasakawa
Keyword(s):  
Hela Cells ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretory Protein ◽  
Secretion System ◽  
Host Cells ◽  
Functional Type ◽  
Wild Type ◽  
Needle Length ◽  
Type Iii

ABSTRACT The Shigella type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion. The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ. In S. flexneri, the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated. Here we show that Spa32, encoded by one of the spa genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity. When the spa32 gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm. Upon introduction of a cloned spa32 into the spa32 mutant, the bacteria produced needles of wild-type length. The spa32 mutant overexpressing MxiH produced extremely long (>5 μm) needles. Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system. The spa32 mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells. Upon introduction of Salmonella invJ, encoding InvJ, which has 15.4% amino acid identity with Spa32, into the spa32 mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells. These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in Shigella spp. and is involved in the control of needle length. Furthermore, its function is interchangeable with that of Salmonella InvJ.

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