Alkaline Phosphatase Reporter Transposon for Identification of Genes Encoding Secreted Proteins in Gram-Positive Microorganisms

Gibson, Carmela M.; Caparon, Michael G.

doi:10.1128/aem.68.02.928-932.2002

Cited by 27 publications

(28 citation statements)

References 40 publications

(39 reference statements)

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“…Statistical significance was determined using a two-tailed paired t test with the StatView statistical analysis software (SAS Institute Inc., Cary, NC). (38), as well as previous reports of Tn4001 in HSC5 (15,38), demonstrated that transposition with Tn4001 results in single random integration of the transposon into the chromosome.…”

Section: Vol 77 2009 Stm In S Pyogenes 1855mentioning

confidence: 93%

“…Direct chromosomal sequencing of the transposon insertion, as opposed to subcloning and sequencing, allowed confirmation of single transposon insertions, whereas if multiple insertions had occurred in the chromosome, getting a single pure sequence off of the transposon would not have been possible. Previous reports have demonstrated the ability of a single copy of the Tn4001 transposon to insert into the HSC5 S. pyogenes genome (15,38). Each sequence was subsequently analyzed by comparison to known sequences in the databases of the National Center for Biotechnology Information.…”

Section: Methodsmentioning

confidence: 99%

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First Streptococcus pyogenes Signature-Tagged Mutagenesis Screen Identifies Novel Virulence Determinants

Kizy

Neely

2009

Infect Immun

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The virulence of bacterial pathogens is a complex process that requires the dynamic expression of many genes for the pathogens to invade and circumvent host defenses, as well as to proliferate in vivo. In this study, we employed a large-scale screen, signature-tagged mutagenesis (STM), to identify Streptococcus pyogenes virulence genes important for pathogenesis within the host. Approximately 1,200 STM mutants were created and screened using the zebrafish infectious disease model. The transposon insertion site was identified for 29 of the 150 mutants that were considered attenuated for virulence. Previously reported streptococcal virulence genes, such as mga, hasA, amrA, smeZ, and two genes in the sil locus, were identified, confirming the utility of the model for revealing genes important for virulence. Multiple genes not previously implicated in virulence were also identified, including genes encoding putative transporters, hypothetical cytosolic proteins, and macrolide efflux pumps. The STM mutant strains display various levels of attenuation, and multiple separate insertions were identified in either the same gene or the same locus, suggesting that these factors are important for this type of acute, invasive infection. We further examined two such genes, silB and silC of a putative quorum-sensing regulon, and determined that they are significant virulence factors in our model of necrotizing fasciitis. sil locus promoter expression was examined under various in vitro conditions, as well as in zebrafish tissues, and was found to be differentially induced. This study was a unique investigation of S. pyogenes factors required for successful invasive infection.Severe Streptococcus pyogenes infections, such as streptococcal toxic shock syndrome and necrotizing fasciitis, result in high mortality rates that range from 25% to 50% (7, 43). The severity and incidence of these invasive S. pyogenes diseases have increased since the 1980s despite what had been considered near eradication of S. pyogenes infections in the previous decades of the 20th century due to the development of antibiotics (13, 43). The reasons for this resurgence remain vague, despite numerous reports indicating the prevalence of particular strains associated with severe S. pyogenes infections (9, 13). Recent studies assessed factors that are present in various serotypes, but they failed to determine specific pathogen and host factors that are common denominators of invasive S. pyogenes disease (34, 55). Moreover, from the host perspective, Kotb et al. eloquently illustrated that certain human leukocyte class II alleles confer a predisposition to severe S. pyogenes disease (29). Undoubtedly, the complexity and versatility of this organism, particularly its remarkable ability to alter its broad spectrum of virulence factors in order to survive in different environments, are the foundation for its success.Although in vitro studies have permitted analysis of virulence determinants, the conditions are selective, as the systems cannot completely mimic ho...

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Section: Vol 77 2009 Stm In S Pyogenes 1855mentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

First Streptococcus pyogenes Signature-Tagged Mutagenesis Screen Identifies Novel Virulence Determinants

Kizy

Neely

2009

Infect Immun

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“…The alkaline phosphatase reporter transposon (TnFuZ) developed by Gibson and Caparon allowed the study of proteins exported from Gram-positive bacteria; in S. pyogenes, two exported proteins without signal peptide have been described. In addition, it is known that some well-characterized proteins of S. pyogenes (a-enolase, glyceraldehyde-3-phosphate dehydrogenase) which are surface-exposed lack a defined export signal (Gibson & Caparon, 2002).…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of a fibronectin-binding protein from Clostridium difficile

et al. 2003

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A 68 kDa fibronectin-binding protein (Fbp68) from Clostridium difficile displaying significant homology to several established or putative Fbps from other bacteria was identified. The one-copy gene is highly conserved in C. difficile isolates. Fbp68 was expressed in Escherichia coli in fusion with glutathione S-transferase; the fusion protein and the native Fbp68 were purified. Immunoblot analysis and cell fractionation experiments revealed that Fbp68 is present on the surface of the bacteria. Far-immuno dot-blotting demonstrated that Fbp68 was capable of fixing fibronectin. Indirect immunofluorescence and ELISA were employed to demonstrate that C. difficile could bind both soluble and immobilized fibronectin. With competitive adherence inhibition assays it was shown that antibodies raised against Fbp68 partially inhibited attachment of C. difficile to fibronectin and Vero cells. Furthermore, Vero cells could fix purified membrane-immobilized Fbp68. Thus Fbp68 appears to be one of the several adhesins identified to date in C. difficile.

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“…This sequence has been shown to contain a functional signal sequence directing either protein export or cell surface localization (Gibson & Caparon, 2002). The deposited Spy2033 sequence has an abnormally long 64 aa signal sequence.…”

Section: Summary Featuresmentioning

confidence: 99%

Pattern searches for the identification of putative lipoprotein genes in Gram-positive bacterial genomes

2002

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INTRODUCTIONThe bacterial cell envelope is of major significance as the focal point for interaction between the bacterium and its environment, notably the host in the case of bacterial pathogens. Bacterial cell envelope proteins thus carry out numerous important functions including roles in adhesion, nutrient acquisition and a variety of interactions with host defences. In Gram-positive bacteria the matrix of peptidoglycan and other wall polymers surrounding the cell is not an efficient barrier to the passage of small macromolecules, due both to its inherent structural permeability and to its turnover during normal cell growth. Indeed, peptidoglycan may be sufficiently permeable as to allow the passage of globular proteins of up to "50 kDa in size (Demchick & Koch, 1996 ;Dijkstra & Keck, 1996). Consequently, in the absence of a retentive outer membrane, Grampositive bacteria have evolved distinct mechanisms for retaining proteins within their cell envelopes, including covalent linkage to the peptidoglycan and non-covalent binding to teichoic acids and other cell envelope polymers (Navarre & Schneewind, 1999 ;Cossart & Jonquieres, 2000 ;Janulczyk & Rasmussen, 2001). Also significant among these mechanisms is membraneanchoring of bacterial lipoproteins (Lpps) by covalent N-terminal lipidation (Braun & Wu, 1994 ; Sutcliffe & Russell, 1995). Lpps in Gram-positive bacteria perform important roles as substrate-binding proteins (SBPs) in ABC transporter systems ; in antibiotic resistance ; in cell signalling ; in protein export and folding ; in sporulation and germination ; in conjugation and various other functions (Sutcliffe & Russell, 1995 valent to those carried out by periplasmic proteins of Gram-negative bacteria.The archetypal bacterial Lpp is the murein lipoprotein of Escherichia coli characterized by Braun and coworkers (reviewed in Braun & Wu, 1994). Structural studies revealed that a diacylglycerol moiety is thioether linked to an N-terminal cysteine of this Lpp and that this lipid group serves to orientate the protein by anchoring it to the inner leaflet of the outer membrane. Chemically identical lipid modifications have since been proposed, primarily on the basis of protein sequence analysis (see below), for a great many proteins in both Gram-positive and Gram-negative bacteria, although relatively few have received extensive biochemical characterization.Biosynthesis of bacterial lipoprotein of the ' Braun ' type proceeds via a well conserved pathway that is apparently unique to prokaryotes (Fig. 1). Following signal-peptidedirected export of the prolipoprotein (proLpp), the enzyme prolipoprotein diacylglycerol transferase (Lgt) uses phospholipid substrates and catalyses the addition of a diacylglycerol lipid unit onto the thiol of a crucial conserved cysteine which is located within a ' lipobox ' motif at the cleavage region of the proLpp signal peptide (Sankaran et al., 1995 ; Qi et al., 1995). Subsequently, the signal peptide is removed by a specific lipoprotein signal peptidase II (Lsp) enzyme which ...

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Alkaline Phosphatase Reporter Transposon for Identification of Genes Encoding Secreted Proteins in Gram-Positive Microorganisms

Cited by 27 publications

References 40 publications

First Streptococcus pyogenes Signature-Tagged Mutagenesis Screen Identifies Novel Virulence Determinants

First Streptococcus pyogenes Signature-Tagged Mutagenesis Screen Identifies Novel Virulence Determinants

Identification and characterization of a fibronectin-binding protein from Clostridium difficile

Pattern searches for the identification of putative lipoprotein genes in Gram-positive bacterial genomes

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