Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of beta-lactamase when the latter was fused to a C-terminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the beta-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.

Pathogenic bacteria of the genus Photorhabdus are naturally found in symbiotic association with soil entomopathogenic nematodes, and are of increasing economic interest in view of their potential for the development of novel biopesticides. This bipartite natural system is currently used for the biological control of crop pests in several countries. However, an increasing number of Photorhabdus strains have recently been isolated from human clinical specimens in both the United States and Australia, associated with locally invasive soft tissue infections and disseminated bacteraemia. In view of their growing use in biological control, which increases the potential rate of exposure of humans to these pathogens, we decided to undertake a comparative study of the genomic differences between insect and human pathogenic strains of Photorhabdus, in an attempt to understand the genetic mechanisms involved in the apparent change of host specificity, presumably responsible for their recently acquired capacity to infect humans. The data presented here demonstrates that major genomic differences exist between strains of Photorhabdus exhibiting virulence against insects or humans. Several individual genes, coding for virulence factors, were isolated and shown to be specific to the Photorhabdus asymbiotica human pathogens. One of these genes, sopB, encoding a host cell invasion factor translocated via the type III secretion system, has been cloned and the comparison of its genomic context in different pathogens strongly indicates that horizontal gene transfer is implicated in the acquisition of these virulence factors specific to the human pathogens. The precise role of this and other virulence factors identified here in the pathogenicity of P. asymbiotica towards humans is currently under investigation.

Photorhabdus temperata strain K122 exhibited oral toxicity against Prays oleae with an LC50 of 58.1×106cellsml−1. Recombinant P. temperata strains expressing the cry1Aa and/or cry1Ia genes of Bacillus thuringiensis have been constructed. The two cry genes, encoding δ-endotoxins, were placed under the control of the lac promoter and IPTG dependent expression in P. temperata was demonstrated. The presence of the cry genes in K122 resulted in a clear improvement of oral toxicity. This improvement was of 6.2-, 6.6-, and 14.6-fold for the strains K122(pBCcry1Aa), K122(pBScry1Ia), and K122(pBCcry1Aa+pBScry1Ia), respectively. Furthermore, determination of the Synergistic Factor between Cry1Aa and Cry1Ia showed that they act synergistically. This work demonstrates that the heterologous expression of B. thuringiensis cry genes in P. temperata can be used to improve and broaden its host range for insect control.

Some bacterial phenotypes measured in vitro can be used to access bacterial virulence, on the premise that they are positively correlated with data from in vivo experiments. We show here that in vitro assessment of bacterial phenotypes, such as adherence and cytotoxicity, are positively correlated with data from in vivo experiments in Drosophila and can be used to assess bacterial virulence in vivo. Manipulation of environmental parameters, such as iron availability, induced changes in the phenotypes measured in vitro that correlated with changes in vivo virulence of all strains tested. Applying these assays, we demonstrate the pathogenic potential of a Pseudomonas fluorescens strain, initially isolated as a non-pathogenic milk contaminant. This strain displayed adherence and cytotoxicity comparable to those of the Pseudomonas aeruginosa pathogenic strain PAK, and colonized the infected flies as rapidly as the PAK strain. These results indicate that this “a priori” non-pathogenic bacterium is capable of escaping the host immune response, supporting the use of in vitro tests for screening of potential pathogens.

Pathogens have developed multiple strategies that allow them to exploit host resources and resist the immune response. To study how Drosophila flies deal with infectious diseases in a natural context, we investigated the interactions between Drosophila and a newly identified entomopathogen, Pseudomonas entomophila. Flies orally infected with P. entomophila rapidly succumb despite the induction of both local and systemic immune responses, indicating that this bacterium has developed specific strategies to escape the fly immune response. Using a combined genetic approach on both host and pathogen, we showed that P. entomophila virulence is multi-factorial with a clear differentiation between factors that trigger the immune response and those that promote pathogenicity. We demonstrate that AprA, an abundant secreted metalloprotease produced by P. entomophila, is an important virulence factor. Inactivation of aprA attenuated both the capacity to persist in the host and pathogenicity. Interestingly, aprA mutants were able to survive to wild-type levels in immune-deficient Relish flies, indicating that the protease plays an important role in protection against the Drosophila immune response. Our study also reveals that the major contribution to the fly defense against P. entomophila is provided by the local, rather than the systemic immune response. More precisely, our data points to an important role for the antimicrobial peptide Diptericin against orally infectious Gram-negative bacteria, emphasizing the critical role of local antimicrobial peptide expression against food-borne pathogens.

By enriching a random transposon insertion bank of Pseudomonas fluorescens for mutants affected in their adherence to the human extracellular matrix protein fibronectin, we isolated 23 adherence minus mutants. Mutants showed a defect in their ability to develop a biofilm on an abiotic surface and were impaired for virulence when tested in an in vivo virulence model in the fruit fly, Drosophila melanogaster. Molecular characterisation of these mutants showed that the transposon insertions localised to two distinct chromosomal locations, which were subsequently cloned and characterised from two mutants. A search in the databanks identified two loci in the Pseudomonas aeruginosa PAO1 genome with significant homology to the genes interrupted by the transposon insertions. Mutant IVC6 shows homology to gmd, coding for the enzyme GDP-mannose dehydratase, involved in the synthesis of A-band- O-antigen-containing lipopolysaccharide (LPS). Mutant IVG7 is significantly similar to a probable outer membrane protein of strain PAO1, with no specific function attributed thus far, yet with significant homology to Escherichia coli FadL, involved in long-chain fatty acid transport. We propose that this protein, together with LPS, is involved in the first steps of P. fluorescens adherence leading to host colonisation. Results presented here also demonstrate the pathogenic potential of P. fluorescens, assessed in an in vivo Drosophila model system, correlated with its ability to adhere to the human extracellular matrix protein, fibronectin. Correlation between the mutant phenotypes with identified virulence factors and their actual role in the virulence of P. fluorescens is discussed.

By enriching a random transposon insertion bank of Pseudomonas fluorescens for mutants affected in their adherence to the human extracellular matrix protein fibronectin, we isolated 23 adherence minus mutants. Mutants showed a defect in their ability to develop a biofilm on an abiotic surface and were impaired for virulence when tested in an in vivo virulence model in the fruit fly, Drosophila melanogaster. Molecular characterisation of these mutants showed that the transposon insertions localised to two distinct chromosomal locations, which were subsequently cloned and characterised from two mutants. A search in the databanks identified two loci in the Pseudomonas aeruginosa PAO1 genome with significant homology to the genes interrupted by the transposon insertions. Mutant IVC6 shows homology to gmd, coding for the enzyme GDP-mannose dehydratase, involved in the synthesis of A-band- O-antigen-containing lipopolysaccharide (LPS). Mutant IVG7 is significantly similar to a probable outer membrane protein of strain PAO1, with no specific function attributed thus far, yet with significant homology to Escherichia coli FadL, involved in long-chain fatty acid transport. We propose that this protein, together with LPS, is involved in the first steps of P. fluorescens adherence leading to host colonisation. Results presented here also demonstrate the pathogenic potential of P. fluorescens, assessed in an in vivo Drosophila model system, correlated with its ability to adhere to the human extracellular matrix protein, fibronectin. Correlation between the mutant phenotypes with identified virulence factors and their actual role in the virulence of P. fluorescens is discussed.

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C. Aagard Y. Abu Kwaik M. Achtman J. Adams S. Ades B. Adler N. M. Agabian D. Agranoff B. Ahmer H. Aiba J. R. Aist S. Aizawa B. Akerley K. Aktories J. F. Alderete E. Allan J.-C. Alonso K. Altendorf C. AlvarezDominguez S. Ambudkar R. Amils O. Amster-Choder L. Anderson M. S. Anderson B. Andre N. Andrews Y. Anraku M. A. Apicella H. Arai Y. Arakawa J. P. Armitage P. Arnold C. M. Arraiano H. N. Arst S. Arvidson T. Atlung Y. E. Auffray J. Ayala S. Aymerich P. Babitzke M. Bagdasarian T. Baker M. Balasubramanian M. Bally L. M. Banta F. Banuett C. BarbeÁs J. T. Barbieri A. G. Barbour G. J. Barcak J. Bardwell M.-A. Barny F. Barras C. E. Barry S. Bartnicki-Garcia B. L. Bassler S. Baumberg A. J. BaÈumler P. Bavoil K. W. Bayles H. Bayley T. Beals T. Beatty J. M. Becker J. Beckwith D. M. Bedwell S. V. Beer K. Begg P. Beguin J.-P. Belaich J. G. Belasco M. Belfort P. Belhumeur D. Belin S. Bell R. Belland R. A. Bender H. BeÂneÂdetti M. Benedik T. Beppu P. Berche D. E. Berg H. C. Berg O. Berg L. W. Bergman B. Bergman B. Berks L. E. Bermudez H. D. Bernstein M. J. Berridge P. Bertin D. E. Bessen M. Bessman S. M. Beverley J. K. Bhattacharjee M. J. Bibb T. A. Bickle A. Binns A. P. Bird L. Birnbaumer W. R. Bishai L. F. Bisson W. Bitter M. Black J. S. Blanchard U. BlaÈsi M. R. Blatt A. S. Bleiweis M. A. Blight J. B. Bliska A. Blocker I. C. Blom®eld B. R. Bloom K. S. Bloom P. Blount A. BoÈck A. L. Bognar G. A. Bohach U. Bonas K. J. Boor W. Boos I. R. Booth J. C. Boothroyd P. Boquet T. B. BoreÂn P. Borgia M. Bott E. C. BoÈ ttger J.-P. Bouche C. Boucher P. Bouloc D. H. Boxer E. Boye E. Boy-Marcotte H. Brade D. Bramhill S. Brantl V. Braun G. H. Braus D. Bray E. Bremer P. J. Brennan C. Breton E. I. Breukink P. Brick B. A. Bridges S. Bron J. Broome-Smith A. J. P. Brown E. J. Brown N. Brown C. Bruand R. BruÈckner J. A. Bruenn Y. V. Brun M. Brunner H. Buc M. Buck R. Buckingham M. Buckle T. J. Buckley B. Bukau C. E. Bulawa L. A. Bulla R. A. Burne D. L. Burns C. M. Burns C. Burucoa S. J. W. Busby H. Bussey M. J. Buttner B. R. Byers R. L. Calendar J. M. Calvo D. A. Campbell J. Campbell J. G. Cannon R. A. Capaldi M. Caparon A. Caprioli E. Carafoli N. H. Carbonetti M. Carlson E. Carniel M. Carrington M. Cashel G. Cecchini D. W. Celander A. M. Chakrabarty T. Chakraborty M. Chal®e R. Chalmers A. Chambers M. Chandler N. W. Charon K. Chater A. K. Chatterjee D. K. Chattoraj C. W. Chen J. Chernoff G. S. Chhatwal A. Chopin M. C. Chopin P. J. Christie G. Christiensen N. P. Cianciotto C. G. Clark D. P. Clark A. J. Clarke A. K. Clarke J.-P. Claverys P. Cleary D. Clemens J. M. CleÂment D. B. Clewell R. T. Clubb A. J. Clutterbuck A. Cochran S. N. Cohen J. A. Cole S. C. T. Cole D. C. Coleman F. Collard J. Collier R. J. Collier C. M. Collins A. Collmer B. Coloma M. J. Colston G. Condemine J. Content A. M. Cook D. A. Cooksey B. T. Cookson T. G. Cooper D. P. Corey G. R. Cornelis P. Cornelis P. Cossart P. A. Cotter J. W. Coulton D. L. Court M. Couturier A. Covacci T. Cover A. F. Cowman G. Cox M. M. Cox R. A. Cox J. Coyette N. Craig W. Cramer J. E. Cronan E. Crooke J. H. Crosa G. A. M. Cross E. Cubib J. Cullum E. Cundliffe Molecular Microbiology (1999) 33(2), 443±447

Molecular MicrobiologyVolume 19, Issue 3 p. 643-645 The Gram-negative cell envelope ‘springs’ to life: coiled-coil trans-envelope proteins Andréa Pimenta, Andréa Pimenta Institut de Génétique et Microbiologie, URA 1354, CNRS–Bâtiment 409, Université de Paris XI, 91405 Orsay Cedex, FranceSearch for more papers by this authorMark Blight, Mark Blight Institut de Génétique et Microbiologie, URA 1354, CNRS–Bâtiment 409, Université de Paris XI, 91405 Orsay Cedex, FranceSearch for more papers by this authorDavid Clarke, David Clarke Institut de Génétique et Microbiologie, URA 1354, CNRS–Bâtiment 409, Université de Paris XI, 91405 Orsay Cedex, FranceSearch for more papers by this authorI. Barry Holland, I. Barry Holland Institut de Génétique et Microbiologie, URA 1354, CNRS–Bâtiment 409, Université de Paris XI, 91405 Orsay Cedex, FranceSearch for more papers by this author Andréa Pimenta, Andréa Pimenta Institut de Génétique et Microbiologie, URA 1354, CNRS–Bâtiment 409, Université de Paris XI, 91405 Orsay Cedex, FranceSearch for more papers by this authorMark Blight, Mark Blight Institut de Génétique et Microbiologie, URA 1354, CNRS–Bâtiment 409, Université de Paris XI, 91405 Orsay Cedex, FranceSearch for more papers by this authorDavid Clarke, David Clarke Institut de Génétique et Microbiologie, URA 1354, CNRS–Bâtiment 409, Université de Paris XI, 91405 Orsay Cedex, FranceSearch for more papers by this authorI. Barry Holland, I. Barry Holland Institut de Génétique et Microbiologie, URA 1354, CNRS–Bâtiment 409, Université de Paris XI, 91405 Orsay Cedex, FranceSearch for more papers by this author First published: February 1996 https://doi.org/10.1046/j.1365-2958.1996.t01-3-442924.xCitations: 16 I. Barry Holland E-mail Holland@igmors.u-psud.fr; Tel. (1) 69 41 66 99; Fax (1) 69 41 78 08 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume19, Issue3February 1996Pages 643-645 RelatedInformation

Heterologous proteins synthesized in the Gram-negative bacterium Escherichia coli in bioreactor culture may accumulate in one of three 'compartments': the cytoplasm, the periplasm, or the extracellular medium. Many overexpressed proteins from various origins have been purified from each of these locations. However, to date, each system has required specific tailoring to meet the stringent requirements for each protein product to ensure correct folding, activity and appropriate yield. The E. coli haemolysin secretion system appears to provide a flexible mechanism with which to secrete a wide variety of heterologous fusion proteins into the extracellular medium.

Haemolysin B (HlyB) is essential for secretion of the 107 × 103 Mr haemolysin A protein from Escherichia coli and is a member of a family of highly conserved, apparently ATP-dependent surface proteins in many organisms. We have shown in this study that both HlyB and HlyD fractionate primarily with the cytoplasmic membrane of E. coli and are accessible to proteases after removal of the outer membrane. We have measured experimentally the topological organization of HlyB within the membrane by construction of fusions to β-lactamase as a reporter. The predicted folding of HlyB, with a minimum of six transmembrane segments, does not always coincide with regions of highest average hydrophobicity. This suggests that HlyB may have a novel organization within the bilayer. From our data and comparative sequence analysis, we have been able to predict very similar topological models for the other members of the HlyB family.

Conference Article| April 01 1991 Bacterial haemolysin and mammallan P-glycoprotein I. Barry Holland; I. Barry Holland *Institut de Génétique et Microbiologie, Université Paris-Sud, Bâtiment 409, 91405 Orsay Cedex, France Search for other works by this author on: This Site PubMed Google Scholar Odile Possot; Odile Possot *Institut de Génétique et Microbiologie, Université Paris-Sud, Bâtiment 409, 91405 Orsay Cedex, France Search for other works by this author on: This Site PubMed Google Scholar Mark Blight; Mark Blight †Department of Genetics, University of Leicester, Leicester LE1 7RN, U.K. Search for other works by this author on: This Site PubMed Google Scholar Kevin Yue Kevin Yue †Department of Genetics, University of Leicester, Leicester LE1 7RN, U.K. Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Received: December 18 1990 Online ISSN: 1470-8752 Print ISSN: 0300-5127 © 1991 Biochemical Society1991 Biochem Soc Trans (1991) 19 (2): 252–255. https://doi.org/10.1042/bst0190252 Article history Received: December 18 1990 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation I. Barry Holland, Odile Possot, Mark Blight, Kevin Yue; Bacterial haemolysin and mammallan P-glycoprotein. Biochem Soc Trans 1 April 1991; 19 (2): 252–255. doi: https://doi.org/10.1042/bst0190252 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: CF, cystic fibrosis, MDR, multi-drug resistance This content is only available as a PDF. © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.

In the secretion of polypeptides from Gram-negative bacteria, the outer membrane constitutes a specific barrier which has to be circumvented. In the majority of systems, secretion is two-step process, with initial export to the periplasm involving an N-terminal signal sequence. Transport across the outer membrane then involves a variable number of ancillary polypeptides including both periplasmic and outer membrane. While such ancillary proteins are probably specific for each secreted protein, the mechanism of movement across the outer membrane is unknown. In contrast to these systems, secretion of theE. coli hemolysin (HlyA) has several distinctive features. These include a novel targeting signal located within the last 50 or so C-terminal amino acids, the absence of any periplasmic intermediates in transfer, and a specific membrane-bound translocator, HlyB, with important mammalian homologues such as P-glycoprotein (Mdr) and the cystic fibrosis protein. In this review we discuss the nature of the HlyA targeting signal, the structure and function of HlyB, and the probability that HlyA is secreted directly to the medium through a trans-envelope complex composed of HlyB and HlyD.