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PubMed2004

Inverted pathogenicity: the use of pathogen-specific molecular mechanisms for prevention or therapy of disease.

The term "inverted pathogenicity" stands for the exploitation of microbial toxins, virulence factors and cellular mechanisms for preventive or therapeutic purposes. This mini-review will focus on the major pathogenicity concept of Salmonella and Yersinia and how to use its underlying molecular principle for the development of a novel vaccination strategy. Both bacterial species employ a type III secretion system which mediates secretion and direct delivery (translocation) of antihost factors into the cytosol of eukaryotic cells. One of the best studied type III effector proteins is the 25-kDa Yersinia outer protein E (YopE). During the interaction of Yersinia with professional phagocytes, YopE translocation disturbs eukaryotic cytoskeleton dynamics and inhibits phagocytosis. YopE is a GTPase-activating protein that is active towards G proteins from the Rho family. Fusion of the N-terminal 138 amino acids of YopE comprising the translocation domain of the type III molecule to listeriolysin O (LLO) or p60 of Listeria monocytogenes results in hybrid proteins that are engaged and translocated by both Yersinia and Salmonella type III secretion systems. Oral immunization of mice with attenuated Yersinia or Salmonella vaccine strains expressing translocated chimeric YopE leads to pronounced LLO- or p60-peptide-specific CD8 T-cell responses that confer protective immunity. Surprisingly, cytosolic delivery of YopE/LLO by Yersinia also results in LLO-specific CD4 T-cell priming.

Animals↗
PubMed2006

Anti-tumor immunotherapy via antigen delivery from a live attenuated genetically engineered Pseudomonas aeruginosa type III secretion system-based vector.

Immunotherapy requiring an efficient T lymphocyte response is initiated by antigen delivery to antigen-presenting cells. Several studies have assessed the efficiency of various antigen loading procedures, including microbial vectors. Here a live strain of Pseudomonas aeruginosa was engineered to translocate a recombinant antigenic protein into mammalian cells via the type III secretion system, a bacterial device translocating effector proteins into host cells. Optimization of the vector included virulence attenuation and determination of the N-terminal sequence allowing translocation of fused antigens into cells. In vitro delivery of an ovalbumin fragment by the bacterial vector into dendritic cells induced the activation of ovalbumin-specific CD8(+) T lymphocytes. Mice injected with the ovalbumin-delivering vector developed ovalbumin-specific CD8(+) T lymphocytes and were resistant to a subsequent challenge with an ovalbumin-expressing melanoma. Moreover, in a curative assay, injection of the vaccine vector 5 and 12 days after tumor implantation led to a complete cure in five of six animals. These results highlight the utility of type III secretion system-based vectors for anti-tumor immunotherapy.

Animals↗
PubMed2007

Genome sequence and analysis of the soil cellulolytic actinomycete Thermobifida fusca YX.

Thermobifida fusca is a moderately thermophilic soil bacterium that belongs to Actinobacteria. It is a major degrader of plant cell walls and has been used as a model organism for the study of secreted, thermostable cellulases. The complete genome sequence showed that T. fusca has a single circular chromosome of 3,642,249 bp predicted to encode 3,117 proteins and 65 RNA species with a coding density of 85%. Genome analysis revealed the existence of 29 putative glycoside hydrolases in addition to the previously identified cellulases and xylanases. The glycosyl hydrolases include enzymes predicted to exhibit mainly dextran/starch- and xylan-degrading functions. T. fusca possesses two protein secretion systems: the sec general secretion system and the twin-arginine translocation system. Several of the secreted cellulases have sequence signatures indicating their secretion may be mediated by the twin-arginine translocation system. T. fusca has extensive transport systems for import of carbohydrates coupled to transcriptional regulators controlling the expression of the transporters and glycosylhydrolases. In addition to providing an overview of the physiology of a soil actinomycete, this study presents insights on the transcriptional regulation and secretion of cellulases which may facilitate the industrial exploitation of these systems.

Actinomycetales↗
PubMed2004

A Brevibacillus choshinensis system that secretes cytoplasmic proteins.

Brevibacillus choshinensis has previously been shown to be a useful strain for the secretion of heterologous proteins via the Sec secretory pathway. This pathway involves the secretion of proteins prior to folding, whereas the alternative TAT (twin-arginine translocation) pathway enables pre-folded proteins to be secreted. We have modified the signal peptide of the Brevibacillus expression vector pNCMO2 to accommodate a Sec avoidance signal as well as the twin arginines required for secretion via the TAT system. Use of this modified signal peptide with the phosphotriesterase OpdA enabled B. choshinensis transformants to express and secrete the enzyme in an active and substantially pure form. The system was also used successfully to secrete two cytoplasmic proteins, the phosphotriesterase HocA from Pseudomonas monteilii and the phenylcarbamate-degrading enzyme, PCD, from Arthrobacter oxydans. The inhibitors carbonyl cyanide m-chlorophenyl hydrazine and sodium azide were used to confirm that secretion was occurring via the TAT secretion pathway. The modified B. choshinensis system we have developed may have general utility in secreting a wide range of heterologous proteins in active and conveniently processed form.

Bacterial Proteins↗
PubMed1997

Secreted protein acidic and rich in cysteine expression after subtotal nephrectomy and blockade of the renin-angiotensin system.

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular matrix-associated protein with antiadhesive, antiproliferative, and matrix remodeling properties. SPARC gene and protein expression were investigated after subtotal nephrectomy (STNx), a model of noninflammatory progressive renal injury. In addition, the effect of blockade of the renin-angiotensin system by the angiotensin-converting enzyme inhibitor ramipril or by the angiotensin II receptor antagonist valsartan was examined. The STNx rats developed hypertension, proteinuria, and renal impairment. These changes were associated with a 2.4-fold increase in SPARC gene expression in STNx compared with SHAM kidneys (P < 0.001). In situ hybridization revealed increased SPARC mRNA in glomeruli and interstitial cells, as well as de novo expression by tubular epithelial cells at sites of renal injury. Immunofluorescence studies confirmed concordant changes in SPARC protein. Both ramipril and valsartan ameliorated renal injury and significantly reduced SPARC overexpression in the STNx animals. The findings of the present study suggest that SPARC, in the context of its known biological actions, may influence some of the pathological features associated with significant renal mass reduction.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1997

Improvement of the production of foreign proteins using a heterologous secretion vector system in Bacillus subtilis: effects of resistance to glucose-mediated catabolite repression.

To improve protein production, a heterologous secretion vector system was constructed with the aid of the amyR2 region. The operator sequence (amyO) of the amyR2 region on the secretion vector was changed through site-directed mutagenesis to eliminate carbon-source-mediated catabolite repression. Three substitutional (AG, G5, G10), one deletional (delta HH), and one insertional (AGHF) mutant promoters were obtained. The expression level and the degree of catabolite repression of amyR2 and the mutant promoters were examined with a single copy system using an integrational promoter probe vector, pDH32. Under glucose-free culture conditions, expression levels from all mutant promoters except HH were 1.4 to 1.5 fold higher than that from amyR2. While the expression of the amyR2 promoter was repressed by 90% in the presence of 2% glucose, expression levels of the mutant promoters were repressed by only 1% to 50%. To evaluate the advantage of the mutant promoters in production of foreign proteins by the heterologous secretion system, beta-lactamase and human pancreatic secretory trypsin inhibitor (hPSTI) were expressed by the mutant promoters. When B. subtilis LKS87 was used as a host strain, the production of the target proteins using the respective mutant promoter was increased by about 1.5 fold under glucose-free culture conditions. Under the high glucose culture conditions, secretion of target proteins produced from the mutant promoters increased 1.5 to 2 fold, whereas those by the amyR2 promoter were reduced to between 50% and 60%. The additive effect of degUh mutation on protein production was not observed under high glucose culture conditions. In addition, such culture conditions inhibited proteolytic degradation of secreted target proteins after the stationary growth phase even in B. subtilis LKS88 (degUh mutant). Thus, our results indicated that the mutant promoters, which are resistant to glucose-mediated catabolite repression, are very useful for over-production of foreign proteins under the high glucose culture conditions using the heterologous expression-secretion system in B. subtilis.

Bacillus subtilis↗
PubMed2005

The DotL protein, a member of the TraG-coupling protein family, is essential for Viability of Legionella pneumophila strain Lp02.

Legionella pneumophila is able to survive inside phagocytic cells by an internalization route that bypasses fusion of the nascent phagosome with the endocytic pathway to allow formation of a replicative phagosome. The dot/icm genes, a major virulence system of L. pneumophila, encode a type IVB secretion system that is required for intracellular growth. One Dot protein, DotL, has sequence similarity to type IV secretion system coupling proteins (T4CPs). In other systems, coupling proteins are not required for viability of the organism. Here we report the first example of a strain, L. pneumophila Lp02, in which a putative T4CP is essential for viability of the organism on bacteriological media. This result is particularly surprising since the majority of the dot/icm genes in Lp02 are dispensable for growth outside of a host cell, a condition that does not require a functional Dot/Icm secretion complex. We were able to isolate suppressors of the Delta dotL lethality and found that many contained mutations in other components of the Dot/Icm secretion system. A systematic analysis of dot/icm deletion mutants revealed that the majority of them (20 of 26) suppressed the lethality phenotype, indicating a partially assembled secretion system may be the source of Delta dotL toxicity in the wild-type strain. These results are consistent with a model in which the DotL protein plays a role in regulating the activity of the L. pneumophila type IV secretion apparatus.

Bacterial Proteins↗
PubMed2006

Cre reporter system to monitor the translocation of type III secreted proteins into host cells.

Central to the study of type III secretion systems is the availability of reporter systems to monitor bacterial protein translocation into host cells. We report here the development of a bacteriophage P1 Cre recombinase-based system to monitor the translocation of bacterial proteins into mammalian cells. Bacteriophage P1 Cre recombinase fused to the secretion and translocation signals of Salmonella enterica serovar Typhimurium of the type III secreted protein SopE was secreted in a type III secretion system-dependent fashion. More importantly, the SopE-Cre chimera was translocated into host cells via the type III secretion system and activated the expression of luciferase and green fluorescent protein reporters of Cre recombinase activity.

Animals↗
PubMed2007

A type IV-secretion-like system is required for conjugative DNA transport of broad-host-range plasmid pIP501 in gram-positive bacteria.

Plasmid pIP501 has a very broad host range for conjugative transfer among a wide variety of gram-positive bacteria and gram-negative Escherichia coli. Functionality of the pIP501 transfer (tra) genes in E. coli was proven by pIP501 retrotransfer to Enterococcus faecalis (B. Kurenbach, C. Bohn, J. Prabhu, M. Abudukerim, U. Szewzyk, and E. Grohmann, Plasmid 50:86-93, 2003). The 15 pIP501 tra genes are organized in a single operon (B. Kurenbach, J. Kopeć, M. Mägdefrau, K. Andreas, W. Keller, C. Bohn, M. Y. Abajy, and E. Grohmann, Microbiology 152:637-645, 2006). The pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA. Three of the 15 pIP501-encoded Tra proteins show significant sequence similarity to the Agrobacterium type IV secretion system proteins VirB1, VirB4, and VirD4. Here we report a comprehensive protein-protein interaction map of all of the pIP501-encoded Tra proteins determined by the yeast two-hybrid assay. Most of the interactions were verified in vitro by isolation of the protein complexes with pull-down assays. In conjunction with known or postulated functions of the pIP501-encoded Tra proteins and computer-assisted prediction of their cellular location, we propose a model for the first type IV-secretion-like system encoded by a conjugative plasmid from gram-positive bacteria.

Bacterial Proteins↗
PubMed2001

Expression of ExsA in trans confers type III secretion system-dependent cytotoxicity on noncytotoxic Pseudomonas aeruginosa cystic fibrosis isolates.

Twelve Pseudomonas aeruginosa cystic fibrosis isolates that are not able to exert a type III secretion system (TTSS)-dependent cytotoxicity towards phagocytes have been further studied. The strains, although possessing TTSS genes and exsA, which encodes a positive regulator of the TTSS regulon, showed no transcriptional activation of the exsCBA regulatory operon. The expression of exsA in trans restored the in vitro secretion of TTSS proteins and ex vivo cytotoxicity.

Bacterial Proteins↗
PubMed2000

OmpR regulates the two-component system SsrA-ssrB in Salmonella pathogenicity island 2.

Salmonella pathogenicity island 2 (SPI-2) encodes a putative, two-component regulatory system, SsrA-SsrB, which regulates a type III secretion system needed for replication inside macrophages and systemic infection in mice. The sensor and regulator homologs, ssrAB (spiR), and genes within the secretion system, including the structural gene ssaH, are transcribed after Salmonella enters host cells. We have studied the transcriptional regulation of ssrAB and the secretion system by using gfp fusions to the ssrA and ssaH promoters. We found that early transcription of ssrA, after entry into macrophages, is most efficient in the presence of OmpR. An ompR mutant strain does not exhibit replication within cultured macrophages. Furthermore, footprint analysis shows that purified OmpR protein binds directly to the ssrA promoter region. We also show that minimal medium, pH 4.5, induces SPI-2 gene expression in wild-type but not ompR mutant strains. We conclude that the type III secretion system of SPI-2 is regulated by OmpR, which activates expression of ssrA soon after Salmonella enters the macrophage.

Bacterial Proteins↗
PubMed2025

WhiB6 Transduces Contact-Dependent Signaling in Mycobacterium smegmatis and Coordinately Induces Both ESX-1 and ESX-4.

Bacteria have evolved complex conditional pathways that respond to environmental stresses and signals. We use conjugation in Mycobacterium smegmatis to identify contact-recognition and response pathways that mediate interactions between donor and recipient cells. Contact with a compatible donor cell initiates a response in the recipient that requires the ESX-1 secretion system and subsequently activates the dormant ESX-4 secretion system. The links of this signal transduction pathway, the mechanism of coordination and dependency between ESX-1 and ESX-4 secretion systems, are unknown. Previous studies identified SigM as a cell-contact responsive sigma factor dedicated to activating ESX-4. WhiB proteins are iron-sulfur-binding stress-response transcription factors exclusively found in Actinobacteria. WhiB6 has been shown to regulate ESX-1 associated gene expression in other mycobacteria. Here, we show that WhiB6 is required both for conjugation and for transducing cell-contact dependent signaling in the recipient cell. Our RNA-seq, ChIP-seq, and proteomic profiling data define a WhiB6 regulon that supports conjugative cell-cell interaction. The WhiB6 regulon includes genes encoding ESX-1, ESX-4, SigM, as well as dispersed operons that likely support ESX secretion. Our data demonstrate that WhiB6 is epistatic to SigM and ESX-4 in this signal transduction pathway. This work shows that WhiB6 functions as a signal transduction node in recipient cells: it coordinates the expression of two ESX systems and it also induces uncharacterized proteins that collectively constitute a complete secretion response to recipient contact with a donor cell.

Mycobacterium smegmatis↗
PubMed1997

Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2.

We have investigated the structure and transcriptional organization of 13 genes of Salmonella Pathogenicity Island 2 (SPI2) that encode components of the second type III secretion apparatus of Salmonella typhimurium. ssaK, L, M, V, N, O, P, Q, R, S, T, U constitute one operon of 10 kb. ssaJ lles upstream of ssaK and is the terminal gene of another operon. The deduced products of ssaJ, ssaK, ssaV, ssaN, ssaO, ssaQ, ssaR, ssaS, ssaT, and ssaU show greatest similarity to the Yersinia spp. genes yscJ, yscL, lcrD, yscN, yscO, yscQ, yscR, yscS, yscT, and yscU, respectively. The products of the ssaL, ssaM and ssaP genes do not have significant similarity to products of other type III secretion systems, and might be important for the specific function of the SPI2 type III secretion system. Bacterial strains carrying different ssa mutations display minor alterations in terms of serum sensitivity when compared with the wild-type strain, but none are defective in replication within macrophage-like RAW 264.7 cells. However, some of the ssa mutant strains invade HEp2 cells less efficiently and are less cytotoxic to RAW 264.7 macrophages than the wild-type strain. We show that the invasion defect is correlated with a lack of SipC in culture supernatants of these mutant strains. SipC is a product of the SPI1 type III secretion system of S. typhimurium, and is important for epithelial cell invasion. Therefore, mutations in SPI2 can affect the SPI1 secretion system, which raises the possibility of an interaction between the two type III secretion systems.

Amino Acid Sequence↗
PubMed2003

The Hrp pilus: learning from flagella.

Plant pathogenic bacteria deliver avirulence and virulence effector proteins into plant cells via the hrp-gene-encoded type III secretion system. A key component of this secretion system is a surface appendage called the Hrp pilus. Recent results suggest that the Hrp pilus serves as a conduit for type III protein secretion and that it is assembled in a manner similar to the flagellum. The Hrp pilus is likely to be the functional equivalent of the needle extension, assembled by type III secretion systems of mammalian pathogenic bacteria.

Animals↗
PubMed2003

Detection of type III secretion genes as a general indicator of bacterial virulence.

Type III secretion systems of Gram-negative bacteria are specific export machineries for virulence factors which allow their translocation to eukaryotic cells. Since they correlate with bacterial pathogenicity, their presence is used as a general indicator of bacterial virulence. By comparing the genetic relationship of the major type III secretion systems we found the family of genes encoding the inner-membrane channel proteins represented by the Yersinia enterocolitica lcrD (synonym yscV) and its homologous genes from other species an ideal component for establishing a general detection approach for type III secretion systems. Based on the genes of the lcrD family we developed gene probes for Gram-negative human, animal and plant pathogens. The probes comprise lcrD from Y. enterocolitica, sepA from enteropathogenic Escherichia coli, invA from Salmonella typhimurium, mxiA from Shigella sonnei, as well as hrcV from Erwinia amylovora. In addition we included as a control probe the flhA gene from E. coli K-12 to validate our approach. FlhA is part of the flagellar export apparatus which shows a high degree of similarity with type III secretions systems, but is not involved in pathogenicity. The probes were evaluated by screening a series of pathogenic as well as non-pathogenic bacteria. The probes detected type III secretion in pathogens where such systems were either known or were expected to be present, whereas no positive hybridization signals could be found in non-pathogenic Gram-negative bacteria. Gram-positive bacteria were devoid of known type III secretion systems. No interference due to the genetic similarity between the type III secretion system and the flagellar export apparatus was observed. However, potential type III secretion systems could be detected in bacteria where no such systems have been described yet. The presented approach provides therefore a useful tool for the assessment of the virulence potential of bacterial isolates of human, animal and plant origin. Moreover, it is a powerful means for a first safety assessment of poorly characterized strains intended to be used in biotechnological applications.

Animals↗
PubMed2006

Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta via Ipaf.

Macrophages respond to Salmonella typhimurium infection via Ipaf, a NACHT-leucine-rich repeat family member that activates caspase-1 and secretion of interleukin 1beta. However, the specific microbial salmonella-derived agonist responsible for activating Ipaf is unknown. We show here that cytosolic bacterial flagellin activated caspase-1 through Ipaf but was independent of Toll-like receptor 5, a known flagellin sensor. Stimulation of the Ipaf pathway in macrophages after infection required a functional salmonella pathogenicity island 1 type III secretion system but not the flagellar type III secretion system; furthermore, Ipaf activation could be recapitulated by the introduction of purified flagellin directly into the cytoplasm. These observations raise the possibility that the salmonella pathogenicity island 1 type III secretion system cannot completely exclude 'promiscuous' secretion of flagellin and that the host capitalizes on this 'error' by activating a potent host-defense pathway.

Animals↗
PubMed2004

An adenylate cyclase-controlled signaling network regulates Pseudomonas aeruginosa virulence in a mouse model of acute pneumonia.

Infections caused by the opportunistic pathogen Pseudomonas aeruginosa involve the interplay of several bacterial virulence factors. It has recently been established that the delivery of toxic effector proteins by the type III secretion system is an important virulence mechanism in several animal models. Furthermore, the expression of the type III secretion system and its effectors has been correlated with a poor clinical outcome during human infections. A novel cyclic AMP (cAMP) regulatory network that controls the expression of virulence factors, including the type III secretion system, was examined to determine its contribution to P. aeruginosa colonization and dissemination in a mouse pneumonia model. Mutants lacking the two genome-encoded adenylate cyclases, CyaA and CyaB, and the cAMP-dependent regulator Vfr were examined. Based on the enumeration of bacteria in lungs, livers, and spleens, as well as the assessment of mouse lung pathology, mutations in the cyaB and vfr genes resulted in a more significantly attenuated phenotype than mutations in cyaA. Moreover, in this model, expression of the type III secretion system was essential for lung colonization and pathology. Strains with mutations in the exsA gene, which encodes a type III regulatory protein, or pscC, which encodes an essential component of the secretion apparatus, were also significantly attenuated. Finally, we demonstrate that virulence can be restored in an adenylate cyclase mutant by the overexpression of exsA, which specifically restores expression of the type III secretion system in the absence of a functional cAMP-dependent regulatory network.

Adenylyl Cyclases↗