scholarly journals Evidence for HrpXo-Dependent Expression of Type II Secretory Proteins in Xanthomonas oryzae pv. oryzae

2004 ◽  
Vol 186 (5) ◽  
pp. 1374-1380 ◽  
Author(s):  
Ayako Furutani ◽  
Seiji Tsuge ◽  
Kouhei Ohnishi ◽  
Yasufumi Hikichi ◽  
Takashi Oku ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Gene ◽  
Secretion System ◽  
Xanthomonas Oryzae ◽  
Nucleotide Sequence Analysis ◽  
Secretory Proteins ◽  
Type Ii ◽  
Wild Type ◽  
Type Iii

ABSTRACT Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by hrp genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that cysP2 has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a cysP2::gus fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the hrp regulatory gene hrpXo is also involved in the expression of not only hrp genes and type III secretory proteins but also some type II secretory proteins.

scholarly journals Evidence for a Type III Secretion System in Aeromonas salmonicida subsp. salmonicida

2002 ◽  
Vol 184 (21) ◽  
pp. 5966-5970 ◽  
Author(s):  
Sarah E. Burr ◽  
Katja Stuber ◽  
Thomas Wahli ◽  
Joachim Frey
Keyword(s):  
Type Iii Secretion ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Aeromonas Salmonicida ◽  
Broad Host Range ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iii

ABSTRACT Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis, is an important fish pathogen. We have screened this bacterium with a broad-host-range probe directed against yscV, the gene that encodes the archetype of a highly conserved family of inner membrane proteins found in every known type III secretion system. This has led to the identification of seven open reading frames that encode homologues to proteins functioning within the type III secretion systems of Yersinia species. Six of these proteins are encoded by genes comprising a virA operon. The A. salmonicida subsp. salmonicida yscV homologue, ascV, was inactivated by marker replacement mutagenesis and used to generate an isogenic ascV mutant. Comparison of the extracellular protein profiles from the ascV mutant and the wild-type strain indicates that A. salmonicida subsp. salmonicida secretes proteins via a type III secretion system. The recently identified ADP-ribosylating toxin AexT was identified as one such protein. Finally, we have compared the toxicities of the wild-type A. salmonicida subsp. salmonicida strain and the ascV mutant against RTG-2 rainbow trout gonad cells. While infection with the wild-type strain results in significant morphological changes, including cell rounding, infection with the ascV mutant has no toxic effect, indicating that the type III secretion system we have identified plays an important role in the virulence of this pathogen.

scholarly journals Salmonella enterica subspecies enterica serovar Enteritidis Salmonella pathogenicity island 2 type III secretion system: role in intestinal colonization of chickens and systemic spread

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2770-2781 ◽  
Author(s):  
Amanda L. S. Wisner ◽  
Taseen S. Desin ◽  
Birgit Koch ◽  
Po-King S. Lam ◽  
Emil M. Berberov ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Wild Type ◽  
Type Iii ◽  
Systemic Spread

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. For the purposes of our studies we used a chicken isolate of S. Enteritidis (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen-free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type strain Sal18: Sal18 attTn7 : : tet and Sal18 attTn7 : : cat, while the other two groups received the wild-type strain (Sal18 attTn7 : : tet) and one of two mutant strains. From this study, we concluded that S. Enteritidis strains deficient in the SPI-1 and SPI-2 systems were outcompeted by the wild-type strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains: the wild-type strain, and three other strains lacking either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge, we observed a reduced systemic spread of the SPI-2 mutants, but by day 3, the systemic distribution levels of the mutants matched that of the wild-type strain. Based on these two studies, we conclude that the S. Enteritidis SPI-2 T3SS facilitates invasion and systemic spread in chickens, although alternative mechanisms for these processes appear to exist.

scholarly journals The Edwardsiella piscicida Type III Translocon Protein EseC Inhibits Biofilm Formation by Sequestering EseE

2019 ◽  
Vol 85 (8) ◽  
Author(s):  
Ying Li Liu ◽  
Tian Tian He ◽  
Lu Yi Liu ◽  
Jia Yi ◽  
Pin Nie ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Wild Type ◽  
Content Type ◽  
Type Iii ◽  
Edwardsiella Piscicida

ABSTRACT The type III secretion system (T3SS) is one of the most important virulence factors of the fish pathogen Edwardsiella piscicida. It contains three translocon proteins, EseB, EseC, and EseD, required for translocation of effector proteins into host cells. We have previously shown that EseB forms filamentous appendages on the surface of E. piscicida, and these filamentous structures mediate bacterial cell-cell interactions promoting autoaggregation and biofilm formation. In the present study, we show that EseC, but not EseD, inhibits the autoaggregation and biofilm formation of E. piscicida. At 18 h postsubculture, a ΔeseC strain developed strong autoaggregation and mature biofilm formation, accompanied by enhanced formation of EseB filamentous appendages. This is in contrast to the weak autoaggregation and immature biofilm formation seen in the E. piscicida wild-type strain. EseE, a protein that directly binds to EseC and also positively regulates the transcription of the escC-eseE operon, was liberated and showed increased levels in the absence of EseC. This led to augmented transcription of the escC-eseE operon, thereby increasing the steady-state protein levels of intracellular EseB, EseD, and EseE, as well as biofilm formation. Notably, the levels of intracellular EseB and EseD produced by the ΔeseE and ΔeseC ΔeseE strains were similar but remarkably lower than those produced by the wild-type strain at 18 h postsubculture. Taken together, we have shown that the translocon protein EseC inhibits biofilm formation through sequestering EseE, a positive regulator of the escC-eseE operon. IMPORTANCE Edwardsiella piscicida, previously known as Edwardsiella tarda, is a Gram-negative intracellular pathogen that mainly infects fish. The type III secretion system (T3SS) plays a pivotal role in its pathogenesis. The T3SS translocon protein EseB is required for the assembly of filamentous appendages on the surface of E. piscicida. The interactions between the appendages facilitate autoaggregation and biofilm formation. In this study, we explored the role of the other two translocon proteins, EseC and EseD, in biofilm formation. We have demonstrated that EseC, but not EseD, inhibits the autoaggregation and biofilm formation of E. piscicida, providing new insights into the regulatory mechanism involved in E. piscicida biofilm formation.

scholarly journals icmT Is Essential for Pore Formation-Mediated Egress of Legionella pneumophila from Mammalian and Protozoan Cells

2002 ◽  
Vol 70 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Maelle Molmeret ◽  
O. A. Terry Alli ◽  
Steven Zink ◽  
Antje Flieger ◽  
Nicholas P. Cianciotto ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Type Strain ◽  
Pore Formation ◽  
Wild Type Strain ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Type Ii Secretion System

ABSTRACT The final step of the intracellular life cycle of Legionella pneumophila and other intracellular pathogens is their egress from the host cell after termination of intracellular replication. We have previously isolated five spontaneous mutants of L. pneumophila that replicate intracellularly similar to the wild-type strain but are defective in pore formation-mediated cytolysis and egress from mammalian and protozoan cells, and the mutants have been designated rib (release of intracellular bacteria). Here, we show that the rib mutants are not defective in the activity of enzymes secreted through the type II secretion system, including phospholipase A, lysophospholipase A, and monoacylglycerol lipase, although they are potential candidates for factors that lyse host cell membranes. In addition, the pilD and lspG mutants, which are defective in the type II secretion system, are not defective in the pore-forming toxin. We show that all five rib mutants have an identical point mutation (deletion) following a stretch of poly(T) in the icmT gene. Spontaneous revertants of the rib mutants, due to an insertion of a nucleotide following the poly(T) stretch in icmT, have been isolated and shown to have regained the wild-type phenotype. We constructed an icmT insertion mutant (AA100kmT) in the chromosome of the wild-type strain by allelic exchange. The AA100kmT mutant was as defective as the rib mutant in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. Both the rib mutant and the AA100kmT mutant were complemented by the icmT gene for their phenotypic defect. rtxA, a gene that is thought to have a minor role in pore formation, was not involved in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. We conclude that the icmT gene is essential for pore formation-mediated lysis of mammalian and protozoan cells and the subsequent bacterial egress.

scholarly journals Campylobacter jejuni FlpA Binds Fibronectin and Is Required for Maximal Host Cell Adherence

2009 ◽  
Vol 192 (1) ◽  
pp. 68-76 ◽  
Author(s):  
Michael E. Konkel ◽  
Charles L. Larson ◽  
Rebecca C. Flanagan
Keyword(s):  
Amino Acids ◽  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Type Strain ◽  
Wild Type Strain ◽  
Major Constituent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Wild Type ◽  
Type Iii

ABSTRACT Campylobacter jejuni is one of the most frequent bacterial causes of food-borne gastrointestinal disease in developed countries. Previous work indicates that the binding of C. jejuni to human intestinal cells is crucial for host colonization and disease. Fibronectin (Fn), a major constituent of the extracellular matrix, is a ∼250-kDa glycoprotein present at regions of cell-to-cell contact in the intestinal epithelium. Fn is composed of three types of repeating units: type I (∼45 amino acids), type II (∼60 amino acids), and type III (∼90 amino acids). The deduced amino acid sequence of C. jejuni flpA (Cj1279c) contains at least three Fn type III domains. Based on the presence of the Fn type III domains, we hypothesized that FlpA contributes to the binding of C. jejuni to human INT 407 epithelial cells and Fn. We assessed the contribution of FlpA in C. jejuni binding to host cells by in vitro adherence assays with a C. jejuni wild-type strain and a C. jejuni flpA mutant and binding of purified FlpA protein to Fn by enzyme-linked immunosorbent assay (ELISA). Adherence assays revealed the binding of the C. jejuni flpA mutant to INT 407 epithelial cells was significantly reduced compared with that for a wild-type strain. In addition, rabbit polyclonal serum generated against FlpA blocked C. jejuni adherence to INT 407 cells in a concentration-dependent manner. Binding of FlpA to Fn was found to be dose dependent and saturable by ELISA, demonstrating the specificity of the interaction. Based on these data, we conclude that FlpA mediates C. jejuni attachment to host epithelial cells via Fn binding.

scholarly journals Involvement of the XpsN Protein in Formation of the XpsL-XpsM Complex in Xanthomonas campestris pv. campestris Type II Secretion Apparatus

2001 ◽  
Vol 183 (2) ◽  
pp. 528-535 ◽  
Author(s):  
Hsien-Ming Lee ◽  
Shiaw-Wei Tyan ◽  
Wei-Ming Leu ◽  
Ling-Yun Chen ◽  
David Chanhen Chen ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Mutant Strain ◽  
Type Strain ◽  
Xanthomonas Campestris ◽  
Wild Type Strain ◽  
Type Ii ◽  
Wild Type ◽  
In The Wild ◽  
Steady State Levels ◽  
Type Gene

ABSTRACT The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestrispv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from thexpsM mutant strain, and vice versa. Introduction of the wild-type xpsM gene carried on a plasmid into thexpsM mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the xpsN mutant strain. They were recovered either by reintroducing the wild-type xpsNgene or by introducing extra copies of wild-type xpsL orxpsM individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of X. campestris pv. campestris caused inhibition of secretion. Complementation of an xpsL orxpsM mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the xpsN gene on the plasmid along with thexpsL and the xpsM genes caused more severe inhibition in both cases. Furthermore, complementation of thexpsN mutant strain was also inhibited. In both the wild-type strain and a strain with the xps gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.

scholarly journals Spa32 Regulates a Switch in Substrate Specificity of the Type III Secreton of Shigella flexneri from Needle Components to Ipa Proteins

2002 ◽  
Vol 184 (13) ◽  
pp. 3433-3441 ◽  
Author(s):  
Juana Magdalena ◽  
Abderrahman Hachani ◽  
Mustapha Chamekh ◽  
Noureddine Jouihri ◽  
Pierre Gounon ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Congo Red ◽  
Type Strain ◽  
Wild Type Strain ◽  
Physical Contact ◽  
Target Cells ◽  
Virulence Plasmid ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iii

ABSTRACT Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a Shigella spa32 mutant and studied its phenotype. The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK. The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.

scholarly journals The Type III Secretion Chaperone HpaB Controls the Translocation of Effector and Noneffector Proteins From Xanthomonas campestris pv. vesicatoria

2018 ◽  
Vol 31 (1) ◽  
pp. 61-74 ◽  
Author(s):  
Felix Scheibner ◽  
Nadine Hartmann ◽  
Jens Hausner ◽  
Christian Lorenz ◽  
Anne-Katrin Hoffmeister ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Type Strain ◽  
Xanthomonas Campestris ◽  
Molecular Mechanisms ◽  
Wild Type Strain ◽  
Effector Proteins ◽  
Wild Type ◽  
Type Iii ◽  
In The Wild ◽  
Secretion Chaperone

Pathogenicity of the gram-negative bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which translocates effector proteins into plant cells. Effector proteins contain N-terminal T3S and translocation signals and interact with the T3S chaperone HpaB, which presumably escorts effectors to the secretion apparatus. The molecular mechanisms underlying the recognition of effectors by the T3S system are not yet understood. In the present study, we analyzed T3S and translocation signals in the type III effectors XopE2 and XopJ from X. campestris pv. vesicatoria. Both effectors contain minimal translocation signals, which are only recognized in the absence of HpaB. Additional N-terminal signals promote translocation of XopE2 and XopJ in the wild-type strain. The results of translocation and interaction studies revealed that the interaction of XopE2 and XopJ with HpaB and a predicted cytoplasmic substrate docking site of the T3S system is not sufficient for translocation. In agreement with this finding, we show that the presence of an artificial HpaB-binding site does not promote translocation of the noneffector XopA in the wild-type strain. Our data, therefore, suggest that the T3S chaperone HpaB not only acts as an escort protein but also controls the recognition of translocation signals.

scholarly journals Two-Component Sensor RhpS Promotes Induction of Pseudomonas syringae Type III Secretion System by Repressing Negative Regulator RhpR

2007 ◽  
Vol 20 (3) ◽  
pp. 223-234 ◽  
Author(s):  
Yanmei Xiao ◽  
Lefu Lan ◽  
Chuntao Yin ◽  
Xin Deng ◽  
Douglas Baker ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Deletion Strain ◽  
Wild Type ◽  
Two Component ◽  
In The Wild

The Pseudomonas syringae type III secretion system (T3SS) is induced during interaction with the plant or culture in minimal medium (MM). How the bacterium senses these environments to activate the T3SS is poorly understood. Here, we report the identification of a novel two-component system (TCS), RhpRS, that regulates the induction of P. syringae T3SS genes. The rhpR and rhpS genes are organized in an operon with rhpR encoding a putative TCS response regulator and rhpS encoding a putative biphasic sensor kinase. Transposon insertion in rhpS severely reduced the induction of P. syringae T3SS genes in the plant as well as in MM and significantly compromised the pathogenicity on host plants and hypersensitive response-inducing activity on nonhost plants. However, deletion of the rhpRS locus allowed the induction of T3SS genes to the same level as in the wild-type strain and the recovery of pathogenicity upon infiltration into plants. Overexpression of RhpR in the ΔrhpRS deletion strain abolished the induction of T3SS genes. However, overexpression of RhpR in the wild-type strain or overexpression of RhpR(D70A), a mutant of the predicted phosphorylation site of RhpR, in the ΔrhpRS deletion strain only slightly reduced the induction of T3SS genes. Based on these results, we propose that the phosphorylated RhpR represses the induction of T3SS genes and that RhpS reverses phosphorylation of RhpR under the T3SS-inducing conditions. Epistasis analysis indicated that rhpS and rhpR act upstream of hrpR to regulate T3SS genes.

scholarly journals Xanthomonas oryzae pv. Oryzae Avirulence Genes Contribute Differently and Specifically to Pathogen Aggressiveness

2000 ◽  
Vol 13 (12) ◽  
pp. 1322-1329 ◽  
Author(s):  
Jianfa Bai ◽  
Seong-Ho Choi ◽  
Grisel Ponciano ◽  
Hei Leung ◽  
Jan E. Leach
Keyword(s):  
Gene Family ◽  
Type Strain ◽  
Wild Type Strain ◽  
Family Members ◽  
Xanthomonas Oryzae ◽  
Wild Type ◽  
Homologous Genes ◽  
Avr Gene ◽  
Avr Genes ◽  
Pathogen Aggressiveness

Genomic copies of three Xanthomonas oryzae pv. oryzae avirulence (avr) genes, avrXa7, avrXa10, and avrxa5, and four homologous genes, aB3.5, aB3.6, aB4.3, and aB4.5, were mutagenized individually or in combination to study the roles of avr genes in one component of pathogen fitness, i.e., aggressiveness or the amount of disease X. oryzae pv. oryzae causes in susceptible rice lines. These X. oryzae pv. oryzae genes are members of the highly related Xanthomonas avrBs3 gene family. Compared to the wild-type strain, X. oryzae pv. oryzae strains with mutations in avrXa7, avrxa5, and the four homologous genes caused shorter lesions on rice line IR24, which contains no resistance genes relevant to the wild-type strain. The contribution of each gene to lesion length varied, with avrXa7 contributing the most and avrXa10 showing no measurable effect on aggressiveness. The functional, plasmidborne copies of avrXa7, aB4.5, and avrxa5 restored aggressiveness only to strains with mutations in avrXa7, aB4.5, and avrxa5, respectively. Mutations in avrXa7 were not complemented by plasmids carrying any other avr gene family members. These data indicate that some, but not all, avr family members contribute to pathogen aggressiveness and that the contributions are quantitatively different. Furthermore, despite their sequence similarity, the aggressiveness functions of these gene family members are not interchangeable. The results suggest that selection and pyramiding resistance genes can be guided by the degree of fitness penalty that is empirically determined in avr gene mutations.

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