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PubMed2002

Evidence for targeting of Yop effectors by the chromosomally encoded Ysa type III secretion system of Yersinia enterocolitica.

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.

Animals↗

PubMed2002

A new type IV secretion system promotes conjugal transfer in Agrobacterium tumefaciens.

Two DNA transfer systems encoded by the tumor-inducing (Ti) plasmid have been previously identified in Agrobacterium tumefaciens. The virB operon is required for the transfer of transferred DNA to the plant host, and the trb system encodes functions required for the conjugal transfer of the Ti plasmid between cells of Agrobacterium. Recent availability of the genome sequence of Agrobacterium allowed us to identify a third system that is most similar to the VirB type IV secretion system of Bartonella henselae. We have designated this system avhB for Agrobacterium virulence homologue virB. The avhB loci reside on pAtC58 and encode at least 10 proteins (AvhB2 through AvhB11), 7 of which display significant similarity to the corresponding virulence-associated VirB proteins of the Ti plasmid. However, the AvhB system is not required for tumor formation; rather, it mediates the conjugal transfer of the pAtC58 cryptic plasmid between cells of Agrobacterium. This transfer occurs in the absence of the Ti plasmid-encoded VirB and Trb systems. Like the VirB system, AvhB products promote the conjugal transfer of the IncQ plasmid RSF1010, suggesting that these products comprise a mating-pair formation system. The presence of plasmid TiC58 or plasmid RSF1010 reduces the conjugal transfer efficiency of pAtC58 10- or 1,000-fold, respectively. These data suggest that complex substrate interactions exist among the three DNA transfer systems of Agrobacterium.

Agrobacterium tumefaciens↗

PubMed2006

Functional evaluation of a novel constitutive promoter F1 of Bacillus pumilus, as a rice epiphytic strain, and construction of an efficient expression and secretion system under the control of F1.

To establish a constitutive, high-efficiency expression and secretion system for Bacillus pumilus, the function of a promoter and the abilities of three signal peptides in B. pumilus DX01 were tested. F1, cloned from the rice epiphyte B. pumilus strain DX01, had strong transcription activity and was a vegetative-phase constitutive promoter. The signal sequences of Bacillus subtilis levansucrase (sacB) and subtilisin, as well as B. pumilus DX01 RNase signal sequence could drive the secretion of E. coli beta-lactamase from B. pumilus DX01 efficiently, among which the signal sequence of B. subtilis sacB was the most effective. Likewise, they could also direct the secretion of green fluorescence protein (GFP) from DX01.

Bacillus↗

PubMed2002

SseF and SseG are translocated effectors of the type III secretion system of Salmonella pathogenicity island 2 that modulate aggregation of endosomal compartments.

The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI 2) is important for intracellular proliferation in infected host cells. Intracellular Salmonella use this system to translocate a set of effector proteins into the host cell. We studied the role of SseF and SseG, two SPI 2-encoded proteins. SseF and SseG are not required for translocation of effector proteins such as SseJ, encoded by genes outside of SPI 2. Rather, both proteins are translocated and interact with phagosomal membranes after translocation. In infected epithelial cells the formation of Salmonella-induced filaments, endosomal aggregates rich in lysosomal glycoproteins, is dependent on the function of SPI 2. We observed that, in mutant strains deficient for sseF or sseG, the formation of aggregated endosomes can take place, but the composition of the structures is different from those observed in cells infected with Salmonella wild type. These observations indicate that SseF and SseG modulate the aggregation of host endosomes.

Animals↗

PubMed1996

Evidence for a ninth gene, ptlI, in the locus encoding the pertussis toxin secretion system of Bordetella pertussis and formation of a PtlI-PtlF complex.

The pertussis toxin secretion system of Bordetella pertussis initially was thought to comprise eight proteins, PtlA-PtlH. We have investigated the existence of another protein, PtlI, encoded by a putative gene located between ptlD and ptlE. A B. pertussis strain expressing a ptlI::phoA translational fusion possessed alkaline phosphatase activity, suggesting that ptlI encodes a protein. In B. pertussis, a protein with an apparent molecular weight of approximately 5,200 (similar to that predicted by the ptlI sequence) was immunoreactive with an antibody raised to a PtlI-maltose-binding protein fusion protein. PtlE expression in a mutant sustaining an in-frame deletion in ptlI indicated that ptlE starts further downstream than initially predicted. PtlF, not detected in the ptlI deletion mutant, was restored partially by expressing ptlI in trans. A 36-kDa species, consistent with a PtlI-PtlF complex, was immunoreactive with antibodies to PtlI and PtlF in nonreduced cell extracts of a Bordetella bronchiseptica strain which overexpresses the Ptl proteins. Upon dithiothreitol treatment, the 36-kDa species was diminished greatly or undetectable. In B. pertussis, PtlI and PtlF co-precipitated with antibody to PtlF. These findings demonstrate the existence of PtlI and a PtlI-PtlF complex, providing the first description of an interaction between Ptl proteins.

Bacterial Proteins↗

PubMed2006

Evolutionary links between FliH/YscL-like proteins from bacterial type III secretion systems and second-stalk components of the FoF1 and vacuolar ATPases.

Bacterial type III secretion drives flagellar biosynthesis and mediates bacterial-eukaryotic interactions. Type III secretion is driven by an ATPase that is homologous to the catalytic subunits of proton-translocating ATPases, such as the F(o)F(1) ATPase. Here we use PSI-BLAST searches to show that some noncalatytic components are also conserved between type III secretion systems and proton-translocating ATPases. In particular, we show that the FliH/YscL-like proteins and the E subunits of vacuolar ATPases represent fusions of domains homologous to second-stalk components of the F(o)F(1) ATPase (the b and delta subunits).

Amino Acid Sequence↗

PubMed1997

Effects of pleiotrophic mutations, degUh and spoOA, on the production of foreign proteins using the heterologous secretion system of Bacillus subtilis.

To elevate the production of foreign target proteins using a heterologous protein secretion system of Bacillus subtilis, two different pleiotrophic mutations, degUh for increase of transcriptional level of target genes and spoOA for reduction of extracellular protease activity of a host strain were introduced, respectively. The productivities of three differently originated enzymes, beta-lactamase, streptokinase and human pancreatic trypsin inhibitor (hPSTI) were examined under the each mutation background. By the degUh mutation, the activities of all three enzymes secreted in the culture were increased, although the increased levels were different from 1.2-fold (streptokinase) to 1.8-fold (beta-lactamase). The lower productivity of streptokinase compared to other enzymes under the degUh background was caused by the higher susceptibility to proteolytic degradation. The increased transcriptional level of the beta-lactamase gene by degUh mutation resulted in the accumulation of unprocessed precursor protein in the cytoplasm and cytoplasmic membrane. In the case of the spoOA mutation background, the differences in the levels of the secreted target proteins were not significant and observed only after a stationary phase of the growth.

Bacillus subtilis↗

PubMed1999

The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner.

We present here data showing that the Avr proteins HrmA and AvrPto are secreted in culture via the native Hrp pathways from Pseudomonas syringae pathovars that produce these proteins. Moreover, their secretion is strongly affected by the temperature and pH of the culture medium. Both HrmA and AvrPto were secreted at their highest amounts when the temperature was between 18 and 22 degrees C and when the culture medium was pH 6.0. In contrast, temperature did not affect the secretion of HrpZ. pH did affect HrpZ secretion, but not as strongly as it affected the secretion of HrmA. This finding suggests that there are at least two classes of proteins that travel the P. syringae pathway: putative secretion system accessory proteins, such as HrpZ, which are readily secreted in culture; and effector proteins, such as HrmA and AvrPto, which apparently are delivered inside plant cells and are detected in lower amounts in culture supernatants under the appropriate conditions. Because HrmA was shown to be a Hrp-secreted protein, we have changed the name of hrmA to hopPsyA to reflect that it encodes a Hrp outer protein from P. syringae pv. syringae. The functional P. syringae Hrp cluster encoded by cosmid pHIR11 conferred upon P. fluorescens but not Escherichia coli the ability to secrete HopPsyA in culture. The use of these optimized conditions should facilitate the identification of additional proteins traveling the Hrp pathway and the signals that regulate this protein traffic.

Bacterial Outer Membrane Proteins↗

PubMed2003

Protein binding between PcrG-PcrV and PcrH-PopB/PopD encoded by the pcrGVH-popBD operon of the Pseudomonas aeruginosa type III secretion system.

Of the proteins encoded by the pcrGVH-popBD operon of the Pseudomonas aeruginosa type III secretion system, PcrG bound to PcrV and PcrH bound to PopB/PopD. In addition, Yersinia LcrG bound to PcrV, and Yersinia LcrH bound to PopD. The results imply a highly functional conservation of type III secretion between P. aeruginosa and Yersinia species.

Animals↗

PubMed2001

Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure.

Enteropathogenic Escherichia coli (EPEC) secretes several Esp proteins via the type III secretion system (secreton). EspA, EspB, and EspD are required for translocation of the effector proteins into host cells, in which EspB and EspD are thought to form a pore in the host membrane. Recent study has shown that EspA forms a filamentous structure that assembles as a physical bridge between bacteria and host cell surfaces, which then functions as a conduit for the translocation of bacterial effectors into host cells. To investigate the supermolecular structure of the type III secreton in EPEC, we partially purified it from the bacteria membrane and observed it via transmission electron microscopy. The EPEC type III secreton was composed of a basal body and a needle part and was similar to those of Salmonella and Shigella, except for a sheath-like structure at the tip of the needle. The length of sheath-like structures varied; it extended more than 600 nm and was 10 times longer than the Shigella needle part. The putative major needle component, EscF, was required for both secretion of Esp proteins and needle complex formation. Interestingly, elongation of the sheath-like structure was observed under constitutive expression of EspA but not of EscF. Furthermore, the transmission electron microscopy view with immunogold labeled anti-EspA antibodies clearly showed that EspA is a component of the sheath-like structure. This study revealed, to our knowledge for the first time, the supermolecular structure of the EPEC type III secreton and its direct association with the EspA-sheath-like structure.

Amino Acid Sequence↗

PubMed1998

Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival.

Salmonella pathogenicity island 2 (SPI-2) encodes a putative type III secretion system necessary for systemic infection in animals. We have investigated the transcriptional organization and regulation of SPI-2 by creating gfp fusions throughout the entire gene cluster. These gfp fusions demonstrated that SPI-2 genes encoding structural, regulatory and previously uncharacterized putative secreted proteins are preferentially expressed in the intracellular environment of the host macrophage. Furthermore, the transcription of these genes within host cells was dependent on the two-component regulatory system SsrA/SsrB and an acidic phagosomal environment. Most SPI-2 mutants failed to replicate to the same level as wild-type strains in murine macrophages and human epithelial cells. In orally infected mice, SPI-2 mutants colonized the Peyer's patches but did not progress to the mesenteric lymph nodes. We conclude that SPI-2 genes are specifically expressed upon entry into mammalian cells and are required for intracellular growth in host cells in vivo and in vitro.

Animals↗

PubMed2004

Two periplasmic disulfide oxidoreductases, DsbA and SrgA, target outer membrane protein SpiA, a component of the Salmonella pathogenicity island 2 type III secretion system.

The formation of disulfide is essential for the folding, activity, and stability of many proteins secreted by Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into proteins exported from the cytoplasm to periplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we examined the role of the Dsb enzymes in Salmonella pathogenesis, and we demonstrated that DsbA, but not DsbC, is required for the full expression of virulence in a mouse infection model of Salmonella enterica serovar Typhimurium. Salmonella strains carrying a dsbA mutation showed reduced function mediated by type III secretion systems (TTSSs) encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). To obtain a more detailed understanding of the contribution of DsbA to both SPI-1 and SPI-2 TTSS function, we identified a protein component of the SPI-2 TTSS apparatus affected by DsbA. Although we found no substrate protein for DsbA in the SPI-1 TTSS apparatus, we identified SpiA (SsaC), an outer membrane protein of SPI-2 TTSS, as a DsbA substrate. Site-directed mutagenesis of the two cysteine residues present in the SpiA protein resulted in the loss of SPI-2 function in vitro and in vivo. Furthermore, we provided evidence that a second disulfide oxidoreductase, SrgA, also oxidizes SpiA. Analysis of in vivo mixed infections demonstrated that a Salmonella dsbA srgA double mutant strain was more attenuated than either single mutant, suggesting that DsbA acts in concert with SrgA in vivo.

Animals↗

PubMed2002

Salmonella enterica serovar Pullorum requires the Salmonella pathogenicity island 2 type III secretion system for virulence and carriage in the chicken.

Functional mutations were made in the type III secretion systems encoded by Salmonella pathogenicity island 1 (SPI 1) and Salmonella pathogenicity island 2 (SPI 2) of Salmonella enterica serovar Pullorum, the cause of pullorum disease in poultry. Their role in cell invasion in vitro, and in virulence in vivo was determined. The SPI 1 mutant showed decreased invasiveness for chicken cells but was capable of causing disease in orally infected 1-day-old chicks, although it showed some reduction in virulence. The SPI 2 mutant showed no reduction in invasiveness, but was fully attenuated for virulence in 1-day-old chicks, and was not detected following oral infection in 1-week-old chickens. Following intravenous infection, the SPI 2 mutant was also attenuated and cleared more rapidly than the parent strain. This indicates that S. Pullorum requires SPI 2 for virulence and persistence but SPI 1 appears to contribute to, but is not essential for, the virulence of S. Pullorum.

Animals↗

PubMed2006

Conservation of type III secretion system genes in Bradyrhizobium isolated from soybean.

The distribution of rhcRST genes encoding the type III secretion system (T3SS) in a collection of Bradyrhizobium strains was characterized by PCR and Southern blot hybridization. The polymorphism of the corresponding sequences amplified by PCR was characterized by RFLP and sequencing together with those available in the databank. Genomic group I is characterized by the presence of Bradyrhizobium elkanii strains and group II by the presence of B. japonicum and B. liaoningense strains. Highly conserved T3SS-like genes were detected by PCR in all Bradyrhizobium strains isolated from soybean belonging to genomic group II, and in none of the strains belonging to genomic group I. These data were confirmed by Southern blot hybridization that further indicated the presence of sequences showing similarity to the rhcRST sequence in B. elkanii strains. The high level of conservation of rhcRST among Bradyrhizobia of genomic group II and sharing the same host-plant suggests that T3SS-like genes might have undergone horizontal genetic transfer within this genomic group. When considering the three Rhizobiaceae genera, a clear congruence was recorded between the rhcRST, rRNA gene and ITS sequences in bacteria harbouring sequences encoding T3SS, suggesting a relatively ancient emergence of the T3SS in these genera.

Base Sequence↗

PubMed2026

Genome sequences of pirAB+ and pirAB- Vibrio campbellii strains isolated from shrimp ponds with mortality outbreaks carry type VI secretion systems.

Vibrio campbellii strains PH1401 and PH1409 were isolated from shrimp ponds with documented mortality outbreaks in the Philippines. PH1401 and PH1409 share identity with V. campbellii strain BoB-53. Whole-genome analysis reveals full-length pirAB binary toxin genes in PH1401. Both strains carry three type VI secretion systems.

Vibrio campbellii↗

PubMed2004

Variation in the effectors of the type III secretion system among Photorhabdus species as revealed by genomic analysis.

Entomopathogenic bacteria of the genus Photorhabdus harbor a type III secretion system. This system was probably acquired prior to the separation of the species within this genus. Furthermore, the core components of the secretion machinery are highly conserved but the predicted effectors differ between Photorhabdus luminescens and P. asymbiotica, two highly related species with different hosts.

Base Sequence↗

PubMed2026

Systematic analysis of the type VII secretion system in Streptococcus gallolyticus subsp. gallolyticus reveals genomic diversity and functional associations.

Streptococcus gallolyticus subsp. gallolyticus (Sgg) is an opportunistic pathobiont associated with bacteremia, infective endocarditis, and colorectal cancer. However, the genomic diversity of this subspecies and the distribution of key virulence determinants, particularly the type VII secretion system (T7SS), remain poorly characterized. Here, we performed genomic analyses of 76 Sgg strains from diverse geographic and host origins. Core- and pan-genome analyses, multi locus sequence typing, and phylogenetic reconstruction revealed dominant sequence types (STs) that correlate with geographic origin or source of isolation. Furthermore, systematic characterization of the T7SS locus identified five new T7SS subtypes and demonstrated a strong association between T7SS subtype and ST. We further expanded the known repertoire of T7SS LXG domain-containing polymorphic toxins (LXG toxins) in Sgg substantially through genome-wide searches. Distinct distribution patterns were observed for the LXG toxins across the strains. Lastly, our data indicated that T7SS subtype was significantly associated with biofilm formation capacity of Sgg strains. Together, these findings advance our understanding of Sgg genomic diversity, reveal substantial lineage-associated variation in T7SS architecture and effector repertoires, and suggest a previously unrecognized connection between T7SS and biofilm formation in Sgg.

LXG toxins↗

PubMed2003

Extracellular proteins involved in soybean cultivar-specific nodulation are associated with pilus-like surface appendages and exported by a type III protein secretion system in Sinorhizobium fredii USDA257.

Several gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes proteins called Nops (nodulation outer proteins) into the extracellular environment upon flavonoid induction. Mutation analysis and the nucleotide sequence of a 31.2-kb symbiosis (sym) plasmid DNA region of USDA257 revealed the existence of a TTSS locus in this symbiotic bacterium. This locus includes rhc (rhizobia conserved) genes that encode components of a TTSS and proteins that are secreted into the environment (Nops). The genomic organization of the TTSS locus of USDA257 is remarkably similar to that of another broad-host range symbiont, Rhizobium sp. strain NGR234. Flavonoids that activate the transcription of the nod genes of USDA257 also stimulate the production of novel filamentous appendages known as pili. Electron microscope examination of isolated pili reveals needle-like filaments of 6 to 8 nm in diameter. The production of the pili is dependent on a functional nodD1 and the presence of a nod gene-inducing compound. Mutations in several of the TTSS genes negate the ability of USDA257 to elaborate pili. Western blot analysis using antibodies raised against purified NopX, Nop38, and Nop7 reveals that these proteins were associated with the pili. Mutations in rhcN, rhcJ, rhcC, and ttsI alter the ability of USDA257 to form nodules on Glycine max and Macroptilium atropurpureum.

Bacterial Proteins↗