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PubMed2025

Sucrose and malic acid in the tobacco plant induce hrp regulon in a phytopathogen Ralstonia pseudosolanacearum.

Genes encoding a type III secretion system in Ralstonia pseudosolanacearum are regulated by HrpB as an hrp regulon and induced only in plants. This study aimed to identify the plant signals that induce the hrp regulon and confirm the signal recognition mechanism. Signaling molecules that induce hrpB expression were screened using resting cells of the hrpB-lacZ reporter strain. Only the soluble fraction of smashed tobacco seedlings induced hrpB expression. The heated soluble fraction retained its hrpB-inducing activity, indicating that the signaling molecules were not proteins. When the soluble fraction was fractionated into acidic, neutral, and basic components, both the acidic and neutral fractions induced hrpB expression. As neutral compounds, sucrose, glucose, and fructose have been found to induce hrpB expression. Sucrose-induced hrpB expression was greatly reduced in the prhA mutant, indicating that the TonB-dependent receptor PrhA perceives sugars. Among the organic acids found in the acidic fractions, malic acid most efficiently induced hrpB expression, which was reduced by the mutation of a hybrid histidine kinase gene of a two-component system, rsc1598, indicating that Rsc1598 may sense malic acid. We demonstrated direct binding of Rsc1598 to malic acid using isothermal titration calorimetry.IMPORTANCESimilar to other Gram-negative plant pathogens, the type III secretion system (T3SS) is the most important virulence factor in Ralstonia pseudosolanacearum. The genes for the T3SS are regulated as an hrp regulon, activated only when the pathogen encounters the plants, indicating that the pathogen must sense plant signals. For the first time, we identified two signaling compounds, sucrose and malic acid, that are abundantly found in tobacco roots. The hrp operon was induced even in non-host plants, possibly because sucrose and malic acid are common in plants. We also found that R. pseudosolanacearum membrane proteins received sucrose and malic acid independently. As a next step, antagonists of signaling molecules can be screened.

Malates↗

PubMed1992

Expression of a gene encoding a scorpion insectotoxin peptide in yeast, bacteria and plants.

The nucleotide sequence encoding the scorpion insectotoxin I5A was chemically synthesized and expressed in yeast, bacteria and tobacco. The I5A peptides produced in these organisms were purified using an immunoaffinity chromatography procedure. I5A produced using the bacterial secretion system was efficiently secreted and released into the culture medium. In contrast, only a trace amount of I5A was detected in bacterial cytosols when expressed from a direct expression vector, suggesting that I5A was unstable in bacterial cells. I5A secreted from yeast using an alpha-factor signal sequence was shown to have an N-terminal (Glu-Ala)2 extension, indicating incomplete processing of the secreted peptide by dipeptidyl aminopeptidase A. In tobacco, a nonsecreted form of the protein was produced. No measurable insect toxicity was observed when insect larvae were assayed, regardless of whether I5A was produced in yeast, bacteria or tobacco. The lack of toxicity is almost certainly the result of improper folding due to incorrect disulfide bond formation. The inability to produce a biologically active peptide must be overcome before scorpion toxins might be used for the genetic engineering of plants for insect resistance. The yeast and bacterial expression systems described here may be useful for further studies on the problem of expressing a biologically active peptide.

Amino Acid Sequence↗

PubMed2004

The secretome of the plant pathogenic bacterium Erwinia chrysanthemi.

Erwinia chrysanthemi causes soft-rot diseases of many plants by secreting a battery of enzymes which degrade the plant cell walls. We initiated a proteomic analysis to create a reference map of the E. chrysanthemi secretome. Extracellular proteins were isolated from E. chrysanthemi culture supernatants and resolved by two-dimensional electrophoresis. By analysis of mutants, Western blotting, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) 55 spots representing 25 unique proteins were identified. In uninduced conditions, we identified spots corresponding to the cellulase Cel5, the proteases PrtA, PrtB, and PrtC, the flagellin FliC, and some intracellular proteins whose presence probably resulted from spontaneous cell lysis. We identified another secreted protein, AvrL, homologous to an avirulence protein of Xanthomonas campestris. After culture in conditions inducing pectinase production, i.e., in the presence of galacturonate and plant extract, we identified spots corresponding to the endopectate lyases PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ, the pectin acetylesterases PaeX and PaeY, the pectin methylesterase PemA, and the polygalacturonase PehX. In the presence of other inducing compounds, we detected the rhamnogalacturonate lyase RhiE and the esterase FaeD. Analysis of mutants, altered for one type of secretion system, was performed to determine the targets of each system. The type I system Prt was necessary for the secretion of three proteases. No proteins secreted by the type III Hrp system could be detected in E. chrysanthemi supernatants. In addition to the already known substrates (eleven pectinases and one cellulase), this analysis revealed that the type II Out system mediates secretion of the esterase FaeD and of the Avr-like protein AvrL.

Bacterial Proteins↗

PubMed2002

Type IV secretion and Brucella virulence.

The type IV secretion system, encoded by the virB region, is a key virulence factor for Brucella. The 12 genes of the region form an operon that is specifically induced by phagosome acidification in cells after phagocytosis. We speculate that the system serves to secrete unknown effector molecules, which allow Brucella to pervert the host cell endosomal pathways and to create a novel intracellular compartment in which it can replicate.

Animals↗

PubMed2002

Bacterial secrets of secretion: EuroConference on the biology of type IV secretion processes.

Type IV secretion systems (TFSS) mediate secretion or direct cell-to-cell transfer of virulence factors (proteins or protein-DNA complexes) from many Gram-negative animal, human and plant pathogens, such as Agrobacterium tumefaciens, Bartonella tribocorum, Bordetella pertussis, Brucella suis, Helicobacter pylori, Legionella pneumophila and Rickettsia prowazekii, into eukaryotic cells. Bacterial conjugation is also classified as a TFSS-like process mediating the spread of broad-host-range plasmids between Gram-negative bacteria such as RP4 and R388, which carry antibiotic resistance genes. Genetic, biochemical, cell biological and structural biology experiments led to significant progress in the understanding of several aspects of TFSS processes. X-ray crystallography revealed that homologues of the A. tumefaciens inner membrane-associated proteins VirB11 and VirD4 from H. pylori and R388, respectively, may form channels for substrate translocation or assembly of the transmembrane TFSS machinery. Biochemical and cell biological experiments revealed interactions between components of the periplasmic core components VirB8, VirB9 and VirB10, which may form the translocation channel. Analysis of A. tumefaciens virulence proteins VirE2 and VirF suggested that the periplasmic translocation route of the pertussis toxin from B. pertussis may be more generally valid than previously anticipated. Secretion and modification of toxins from H. pylori and L. pneumophila profoundly affect host cell metabolism, thus entering the discipline of cellular microbiology. Finally, results from genome sequencing projects revealed the presence of up to three TFSS in a single organism, and the analysis of their interplay and adaptation to different functions will be a future challenge. TFSS-carrying plasmids were discovered in different ecosystems, suggesting that genetic exchange may speed up their evolution and adaptation to different cell-cell interactions.

Animals↗

PubMed2005

Crystal structure of the protease-resistant core domain of Yersinia pestis virulence factor YopR.

Yersinia pestis, the causative agent of the plague, employs a type III secretion system (T3SS) to secrete and translocate virulence factors into to the cytoplasm of mammalian host cells. One of the secreted virulence factors is YopR. Little is known about the function of YopR other than that it is secreted into the extracellular milieu during the early stages of infection and that it contributes to virulence. Hoping to gain some insight into the function of YopR, we determined the crystal structure of its protease-resistant core domain, which consists of residues 38-149 out of 165 amino acids. The core domain is composed of five alpha-helices that display unexpected structural similarity with one domain of YopN, a central regulator of type III secretion in Y. pestis. This finding raises the possibility that YopR may play a role in the regulation of type III secretion.

Amino Acid Sequence↗

PubMed2005

Regulation of type III secretion hierarchy of translocators and effectors in attaching and effacing bacterial pathogens.

Human enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and the mouse pathogen Citrobacter rodentium (CR) belong to the family of attaching and effacing (A/E) bacterial pathogens. They possess the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system. These pathogens secrete a number of proteins into culture media, including type III effector proteins and translocators that are required for the translocation of effectors into host cells. Preliminary evidence indicated that the LEE-encoded SepL and Rorf6/SepD may form a molecular switch that controls the secretion of translocators and effectors in CR. Here, we show that SepL and SepD indeed perform this function in A/E pathogens such as EHEC and EPEC. Their sepL and sepD mutants do not secrete translocators but exhibit enhanced secretion of effectors. We demonstrate that SepL and SepD interact with each other and that both SepL and SepD are localized to the bacterial membranes. Furthermore, we demonstrate that culture media influence the type III secretion profile of EHEC, EPEC, and CR and that low-calcium concentrations inhibit secretion of translocators but promote the secretion of effectors, similar to effects on type III secretion by mutations in sepL and sepD. However, the secretion profile of the sepD and sepL mutants is not affected by these culture conditions. Collectively, our results suggest that SepL and SepD not only are necessary for efficient translocator secretion in A/E pathogens but also control a switch from translocator to effector secretion by sensing certain environmental signals such as low calcium.

Bacterial Outer Membrane Proteins↗

PubMed2003

Inter- and intraclonal diversity of the Pseudomonas aeruginosa proteome manifests within the secretome.

The proteomes of cultured Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis (CF) lungs were compared by using genetically divergent clones and isogenic morphotypes of one strain. Cellular extracts gave very similar protein patterns in two-dimensional gels, suggesting that the conserved species-specific core genome encodes proteins that are expressed under standard culture conditions in vitro. In contrast, the protein profiles of extracts of culture supernatants were dependent on the growth phase, and there were significant differences between clones. The profiles also varied within clonally related morphotypes from one CF patient, including a hyperpiliated small-colony variant. Mass spectrometry revealed that this variant overexpressed proteins secreted by the type I secretion system (including proteins involved in iron acquisition) and by the type III secretion system. Furthermore, the proteins in the supernatant extracts from the small-colony variant which were recognized by sera from different CF patients varied greatly. We concluded that the secretome expression is a sensitive measure of P. aeruginosa strain variation.

Antibodies, Bacterial↗

PubMed2004

Selective decontamination of the digestive tract attenuated the myocardial inflammation and dysfunction that occur with burn injury.

This study examined the effects of oral antibiotics to selectively decontaminate the digestive tract (SDD) on postburn myocardial signaling, inflammation, and function. We hypothesized that antibiotic therapy to eliminate pathogens from the gastrointestinal (GI) tract would reduce myocardial inflammatory responses and improve postburn myocardial performance. Sprague-Dawley rats received polymyxin E (15 mg), tobramycin (6 mg), and 5-flucytosin (100 mg) by oral gavage twice daily for 3 days preburn and 24 h postburn. Experimental groups included 1) sham burn given vehicle (3 ml water), 2) sham plus SDD, 3) burn over 40% total body surface area (TBSA) plus SDD, and 4) burn over 40% TBSA given vehicle. All burns received lactated Ringer solution (4 mg.kg(-1).%burn(-1)); myocardial signaling (PKCepsilon/p38 MAPK/NF-kappaB) was studied 2, 4, and 24 h postburn; and cytokine secretion (systemic and myocyte secreted cytokines, ELISA) and cardiac function were examined 24 h postburn. Vehicle-treated burn injury increased myocardial PKCepsilon/p38 MAPK expression, promoted NF-kappaB nuclear translocation, promoted TNF-alpha, IL-1beta, IL-6, and IL-10 secretion, and impaired myocardial function. SDD attenuated burn-related proinflammatory myocardial signaling, cytokine secretion, and myocardial contractile defects. Our data suggest that burn-related loss of GI barrier function and translocation of microbial products serve as upstream mediators of postburn myocardial inflammatory signaling and dysfunction.

Animals↗

PubMed2005

ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa.

Type III secretion systems are toxin delivery systems that are present in a large number of pathogens. A hallmark of all type III secretion systems studied to date is that expression of one or more of their components is induced upon cell contact. It has been proposed that this induction is controlled by a negative regulator that is itself secreted by means of the type III secretion machinery. Although candidate proteins for this negative regulator have been proposed in a number of systems, for the most part, a direct demonstration of their role in regulation is lacking. Here, we report the discovery of ExsE, a negative regulator of type III secretion gene expression in Pseudomonas aeruginosa. Deletion of exsE deregulates expression of the type III secretion genes. We provide evidence that ExsE is itself secreted by means of the type III secretion machinery and physically interacts with ExsC, a positive regulator of the type III secretion regulon. Taken together, these data demonstrate that ExsE is the secreted negative regulator that couples triggering of the type III secretion machinery to induction of the type III secretion genes.

Bacterial Proteins↗

PubMed1996

Modulation of virulence factor expression by pathogen target cell contact.

Upon contact with the eukaryotic cell, Yersinia pseudotuberculosis increased the rate of transcription of virulence genes (yop), as determined by in situ monitoring of light emission from individual bacteria expressing luciferase under the control of the yopE promoter. The microbe-host interaction triggered export of LcrQ, a negative regulator of Yop expression, via the Yop-type III secretion system. The intracellular concentration of LcrQ was thereby lowered, resulting in increased expression of Yops. These results suggest a key role for the type III secretion system of pathogenic bacteria to coordinate secretion with expression of virulence factors after physical contact with the target cell.

Bacterial Adhesion↗

PubMed1997

Folding and stability of the Z and S(iiyama) genetic variants of human alpha1-antitrypsin.

Z (Glu342 --> Lys) and S(iiyama) (Ser53 --> Phe) genetic variations of human alpha1-antitrypsin (alpha1-AT) cause a secretion blockage in the hepatocytes, leading to alpha1-AT deficiency in the plasma. Using in vitro folding analysis, we have shown previously that these mutations interfere with the proper folding of polypeptides. To understand the fundamental cause for the secretion defect of the Z and S(iiyama) variants of alpha1-AT, we investigated in vivo folding and stability of these variant alpha1-AT using the secretion system of yeast Saccharomyces cerevisiae. Various thermostable mutations suppressing the folding block of the Z variant in vitro corrected the secretion defect as well as the intracellular degradation in the yeast secretion system. Significantly, the extent of suppression in the secretion defect of Z protein was proportional to the extent of suppression in the folding defect, assuring that the in vivo defect associated with the Z variant is primarily derived from the folding block. In contrast, the folding and secretion efficiency of S(iiyama) was not much improved by the same mutations. In addition, none of the rarely secreted S(iiyama) alpha1-AT carrying the stabilizing mutations for the wild type and Z variant were active. It appears that the major defect in S(iiyama) variant is the loss of stability in contrast to the kinetic block of folding in the Z variant.

Hot Temperature↗

PubMed2005

Bacteriophage-encoded type III effectors in Salmonella enterica subspecies 1 serovar Typhimurium.

Salmonella spp. are Gram-negative bacteria which cause infections ranging from mild, self-limiting enterocolitis to systemic (typhoid) disease. Recent work has established that the genetic makeup varies considerably between different Salmonella strains. Phages play an important role in this diversity. In fact, Salmonella has emerged as a prime example for the involvement of virulence factor encoding phages in the emergence of new epidemic strains. Among other virulence factors, Salmonella enterica utilizes two specialized protein secretion systems termed type III secretion systems (TTSS) to deliver effector proteins into host cells which manipulate host cell signaling cascades. These two TTSS and several effectors are encoded within Salmonella pathogenicity islands 1 and 2. Some effectors including SopE, SspH1, SseI and SopE2 are encoded by phages or phage remnants. These phage-encoded effectors seem to be transferred between different Salmonella strains. They have attracted much interest because they might contribute to the evolution of Salmonella spp. Here we will focus on SopEPhi which encodes the SPI-1 effector SopE. It provides an excellent example to illustrate how horizontally transferred effector proteins are integrated into the complex regulatory network of a TTSS in a recipient bacterium. Additional data supporting the hypothesis are presented. This is a prerequisite to allow optimization of the bacterium host cell interaction by reassortment of the phage-encoded effector protein repertoire.

Bacterial Proteins↗

PubMed1990

Genetic analysis of an MDR-like export system: the secretion of colicin V.

The extracellular secretion of the antibacterial toxin colicin V is mediated via a signal sequence independent process which requires the products of two linked genes: cvaA and cvaB. The nucleotide sequence of cvaB reveals that its product is a member of a subfamily of proteins, involved in the export of diverse molecules, found in both eukaryotes and prokaryotes. This group of proteins, here referred to as the 'MDR-like' subfamily, is characterized by the presence of a hydrophobic region followed by a highly conserved ATP binding fold. By constructing fusions between the structural gene for colicin V, cvaC, and a gene for alkaline phosphatase, phoA, lacking its signal sequence, it was determined that 39 codons in the N-terminus of cvaC contained the structural information to allow CvaC-PhoA fusion proteins to be efficiently translocated across the plasma membrane of Escherichia coli in a CvaA/CvaB dependent fashion. This result is consistent with the location of point mutations in the cvaC gene which yielded export deficient colicin V. The presence of the export signal at the N-terminus of CvaC contrasts with the observed C-terminal location of the export signal for hemolysin, which also utilizes an MDR-like protein for its secretion. It was also found that the CvaA component of the colicin V export system shows amino acid sequence similarities with another component involved in hemolysin export, HlyD. The role of the second component in these systems and the possibility that other members of the MDR-like subfamily will also have corresponding second components are discussed. A third component used in both colicin V and hemolysin extracellular secretion is the E. coli host outer membrane protein, TolC.

Alkaline Phosphatase↗

PubMed2006

Pseudomonas aeruginosa type III-secreted toxin ExoT inhibits host-cell division by targeting cytokinesis at multiple steps.

Pseudomonas aeruginosa is an opportunistic pathogen that requires preexisiting epithelial injury to cause acute infections. We report that P. aeruginosa inhibits mammalian cytokinesis in a type III secretion system and exotoxin T (ExoT)-dependent manner. ExoT is a bifunctional type III secretion system effector protein that contains an N-terminal GTPase-activating protein domain and a C-terminal ADP-ribosyl transferase domain. Each of its domains inhibits cytokinesis in a kinetically, morphologically, and mechanistically distinct manner. The GTPase-activating protein-mediated inhibition of cytokinesis occurs early, likely as a consequence of its inhibitory effect on RhoA. The ADP-ribosyl transferase domain inhibits late steps of cytokinesis by blocking syntaxin-2 localization to the midbody, an event essential for completion of cytokinesis. These findings provide an example of a bacterial pathogen targeting cytokinesis.

ADP Ribose Transferases↗

PubMed2004

The type III secreted protein BopD in Bordetella bronchiseptica is complexed with BopB for pore formation on the host plasma membrane.

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although BopB, BopN, BopD, and Bsp22 have been identified as type III secreted proteins, these proteins remain to be characterized. In this study, in order to clarify the function of BopD during Bordetella infection, a BopD mutant was generated. Although secretion of BopD into the culture supernatant was completely abolished by the bopD mutation, the secretion of other type III secreted proteins was not affected by this mutation. It has been reported that severe cytotoxicity, including cell detachment from the substrata, and release of lactate dehydrogenase (LDH) into the supernatant are induced in L2 cells by wild-type B. bronchiseptica infection, and these phenotypes are dependent on the type III secretion system. In contrast, neither cell detachment nor LDH release was induced in L2 cells infected with the BopD mutant. Furthermore, the hemolytic activity of the BopD mutant was greatly impaired compared with that of the wild-type strain. On the basis of the results of coimmunoprecipitation assays with anti-BopB antibodies, we conclude that BopD has the ability to associate with BopB. Finally, we show that the BopD-BopB complex is responsible for the pore formation in the host plasma membrane that functions as the conduit for the transition of effector proteins into host cells.

Animals↗

PubMed2004

Type V protein secretion: simplicity gone awry?

Since its discovery in the late 1980's, the family of secreted proteins termed the autotransporters has been expanding continuously to become the largest group of secreted proteins in Gram-negative bacteria. The type V secretion pathway, which includes the autotransporters (type Va) together with the two-partner secretion system (type Vb) and the Oca family (type Vc), can be defined by secreted proteins that are (i) translocated across the outer membrane via a transmembrane pore formed by a beta-barrel and (ii) contain all the information required for translocation through the cell envelope. In the light of new discoveries and controversies in this research field, the secretion process of autotransporters, or the type Va secretion system, will be discussed here and placed in the context of the more general field of bacterial protein translocation.

Amino Acid Sequence↗

PubMed2004

Structural biology of bacterial pathogenesis.

Recent years have seen a rapid increase in structural information on proteins implicated in bacterial pathogenesis. The different modes by which bacteria establish contact with their host tissues are exemplified by the structures of bacterial adhesins in complex with their cognate host receptor. A more detailed structural understanding of the various Gram-negative secretion systems has emerged with the determination of the structures of type I and type IV secretion system components, and with the elucidation of the mechanism of fibre formation in the chaperone-usher pathway of pilus biogenesis. Finally, the structures of complexes of secreted virulence factors bound to their host targets have unravelled the mechanisms by which bacterial pathogens exploit cellular processes to their advantage.

Adhesins, Bacterial↗