| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109-1078, USA
2 Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110, USA
3 Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, MI 48109-0620, USA
Correspondence
Eric S. Krukonis
ekrukoni{at}umich.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
yapC mutant may reflect the relatively low level of yapC expression in vitro, as assessed by RT-PCR, and/or redundant functions expressed in vitro. These data demonstrate several activities for YapC that may function during Y. pestis infection.
A supplementary table of primers is available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Plague is one of the most deadly infectious diseases in history, killing 
25 % of the population of Western Europe during the years 1347–1351 (Cantor, 2001; Perry & Fetherston, 1997).
To efficiently establish infection, bacteria express a variety of adhesins on their surface. While a number of Yersinia adhesins have been extensively studied, some of the best-characterized adhesins of Y. pseudotuberculosis and Yersinia enterocolitica are not expressed in Y. pestis. These include invasin (Isberg et al., 1987; Rosqvist et al., 1990) and YadA (Rosqvist et al., 1990; Tamm et al., 1993; Yang & Isberg, 1993), which have been mutated by IS100 element insertion and frameshift mutation, respectively (Deng et al., 2002; Parkhill et al., 2001).
Two known adhesins that are expressed by Y. pestis are pH 6 antigen and plasminogen activator. pH 6 antigen (encoded by psaA) was first described as a surface component of Y. pestis induced at temperatures 
35 °C and pH 
6.7 (Ben-Efraim et al., 1961) that forms 4 nm thick fibrils on the bacterial surface (Lindler & Tall, 1993). In addition, pH 6 antigen acts as an adhesin for cultured cells and agglutinates red blood cells from several animal species (Bichowsky-Slomnicki & Ben-Efraim, 1963; Yang et al., 1996). Plasminogen activator (Pla) also mediates Y. pestis adhesion to eukaryotic cells and extracellular matrix (Kienle et al., 1992; Lahteenmaki et al., 1998). Pla is localized on the outer membrane of Y. pestis and cleaves and activates plasminogen to facilitate bacterial dissemination from peripheral tissues to other organs (Beesley et al., 1967; Sodeinde et al., 1992; Welkos et al., 1997). It has also been reported to be an invasin for Y. pestis (Cowan et al., 2000; Lahteenmaki et al., 2001).
One of the hallmarks of Y. pestis infection is the delivery of cytotoxic Yop proteins from the bacterium to the host cell cytoplasm via a type III secretion system (T3SS; Cornelis et al., 1998), a process that requires adhesion to the host cell (Rosqvist et al., 1990). The fact that adhesins in addition to the T3SS translocators, YopB and YopD of Yersinia species, are required for Yop delivery was demonstrated using an uncharacterized non-adherent derivative of Y. pestis strain EV76, in which Yop-mediated cytotoxicity was restored upon expression of the adhesins invasin or YadA of Y. pseudotuberculosis (Rosqvist et al., 1990). However, Y. pestis strains defective for pH 6 antigen (Lindler et al., 1990) or plasminogen activator (Brubaker et al., 1965) can still mediate Yop delivery to host cells, as measured by maintenance of virulence. Thus, additional adhesins of Y. pestis are predicted to mediate the cell adhesion required for Yop delivery and other virulence-associated events.
To identify additional Y. pestis adhesins that are potentially important for virulence, we investigated the role of the annotated Y. pestis autotransporter protein YapC, a homologue (33.7 % identity) of TibA, an adhesin/invasin of enterotoxigenic Escherichia coli (ETEC) (Lindenthal & Elsinghorst, 2001).
Autotransporters are characterized by the presence of an N-terminal ‘passenger’ domain, which can possess an array of functions, and a C-terminal translocation domain that forms a β-barrel allowing secretion of the passenger domain to the cell surface (Henderson et al., 2004). Some autotransporters remain intact on the cell surface, while others can be cleaved by a variety of mechanisms, including cleavage by an endogenous protease activity in the passenger domain (Hendrixson et al., 1997), cleavage by a different protease in the bacterial cell (Egile et al., 1997; Shere et al., 1997) or a recently characterized intramembrane self-cleavage mechanism within the β-barrel structure (Dautin et al., 2007). Neither TibA nor YapC appear to direct their own cleavage via a passenger-domain-mediated protease activity (Elsinghorst & Weitz, 1994; Lindenthal & Elsinghorst, 2001; Yen et al., 2007), and residues required for the newly discovered intramembrane self-cleavage mechanism are lacking in both TibA and YapC (Dautin et al., 2007). YapC is also predicted to belong to the AT-1 family of monomeric autotransporters (Yen et al., 2007).
TibA of E. coli is a glycosylated surface protein and glycosylation is required for its cell-binding activity (Sherlock et al., 2005). In the case of TibA, the glycosylating enzyme, TibC, is encoded immediately upstream of tibA within the same operon. In the case of YapC, genome analysis revealed no neighbouring glycosylating enzyme homologue, although this does not exclude the possibility that YapC is glycosylated.
In addition to mediating adhesion and invasion of epithelial cells, TibA enhances biofilm formation and autoaggregation in ETEC (Sherlock et al., 2005). We demonstrate here that YapC of Y. pestis mediates adhesion of recombinant E. coli to murine macrophages and human epithelial cells. YapC also facilitates E. coli autoaggregation and formation of biofilm-like deposits. Recent studies also found that YapC was surface localized when expressed in E. coli and that it could mediate autoaggregation (Yen et al., 2007). These activities may be important for colonization, survival or transmission during Y. pestis infections.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Gene deletions in Y. pestis KIM5 were performed using PCR products and the method described by Yu et al. (2000) and Datsenko & Wanner (2000). Briefly, the pKD4 kanamycin-resistance cassette was amplified by PCR with primers having 5' extension sequences from regions flanking the gene(s) targeted for deletion. Input template DNA in the PCR reaction was digested with DpnI, and the DpnI-resistant PCR product was transformed into Y. pestis KIM5 that had been previously transformed with pKD46, encoding the 
-RED recombinase, and induced for 4–5 h with 10 mM arabinose before preparation of electrocompetent cells. Kanamycin-resistant colonies were selected and the deletion(s) confirmed by PCR. The strain was then transformed with pCP20, encoding the FLP recombinase, which resulted in excision of the kanamycin-resistance gene. Plasmids were removed from the strains by growth in heart infusion broth (HIB) without drug selection. All incubations were performed at 28 °C. HEp-2 cells (ATCC CCL-23) were cultured in MEM+10 % fetal calf serum (FCS) and RAW264.7 macrophages (ATCC TIB-71) were cultured in Dulbecco's modified Eagle's medium DMEM (Gibco)+10 % FCS.
|
) were cut by appropriate enzymes, gel purified, and the PCR products were ligated into pMMB207. PCR products were designed to include the natural Shine–Dalgarno ribosome-binding site of the gene of interest, but lack the natural promoter. Expression was induced in E. coli with IPTG. E. coli DH5
was transformed with the ligation products and chloramphenicol-resistant colonies were selected. Clones were confirmed by DNA sequencing. Plasmids were then purified and transformed into the Fim– E. coli strain AAEC185 (Blomfield et al., 1991).
Outer-membrane preparations and SDS-PAGE.
E. coli AAEC185 and derivatives were cultured overnight in LB medium supplemented with 10 µg chloramphenicol ml–1 and 100 µM IPTG at 28 °C or 37 °C and outer-membrane preparations were made by the method described by Hantke (1984). Briefly, 1 ml of a 3 ml overnight culture induced with 100 µM IPTG was pelleted at 4 °C. Cells were resuspended in 1 ml 0.2 M Tris/HCl (pH 8.0) and centrifuged again at 4 °C for 5 min. Cells were resuspended in 50 µl 0.2 M Tris/HCl (pH 8.0) and the following were added in order: 100 µl 0.2 M Tris/HCl (pH 8.0)/1 M sucrose, 10 µl 10 mM EDTA (pH 8.0), 10 µl 2 mg lysozyme ml–1 and 320 µl double-distilled (dd) H2O. Preparations were mixed and incubated at room temperature for 10 min, then 10 µl 1 mg DNase ml–1 was added and mixed, followed by 500 µl 2 % Triton X-100/10 mM MgCl2/50 mM Tris/HCl (pH 8.0). Preparations were mixed well and centrifuged at 4 °C in a microcentrifuge for 30 min at maximum speed. Supernatants were saved (referred to as ‘soluble fraction’ in Fig. 2) and pellets resuspended in 300 µl ddH2O. Preparations were then centrifuged at 4 °C for 15 min, washed twice more with ddH2O and resuspended in 100 µl SDS-PAGE sample buffer and boiled for 5 min. Isolated outer membranes were subjected to SDS-PAGE followed by Coomassie blue staining.
|
-subunit (RNAP) antibodies, cells were grown and processed as described for the outer-membrane preparations. In addition, the extent of shearing of pH 6 antigen (PsaA) filaments from the bacterial surface was assessed by Western blot analysis of culture supernatants. Western blots were probed with a 1 : 1000 dilution of a rabbit anti-PsaA antibody, kindly provided by Dr Susan Straley (Dept of Microbiology, Immunology & Molecular Genetics, University of Kentucky). Anti-RNIAP Western blots were probed with a 1 : 1000 dilution of mouse anti-RNAP antibody generated against E. coli RNAP (Neoclone). Blots were washed and probed with anti-rabbit-horeseradish peroxidase (HRP) or anti-mouse-HRP secondary antibodies, respectively (Zymed) prior to developing with Supersignal West Pico ECL substrate (Pierce).
Cytotoxicity assay.
HEp-2 cells were grown to 80 % confluency in 24-well tissue culture plates (Falcon, Becton Dickinson) in minimal Eagle's medium (MEM) with Earle's salts and L-glutamine (Gibco) supplemented with 10 % FCS, 1 % non-essential amino acids and 1 % sodium pyruvate. RAW 264.7 cells were cultured until 80 % confluency in 24-well plates containing DMEM (high glucose, with L-glutamine and pyridoxine hydrochloride, without sodium pyruvate) supplemented with 10 % FCS.
Y. pestis KIM5, KIM5 pCD1– and different deletion mutants were cultured overnight at 28 °C in HIB (pH 7.0) with shaking. Cultures were diluted to OD620 0.15 in HIB pH 7.0 and incubated for a further 4 h at 28 °C with shaking. The cultures were pelleted and resuspended in tissue culture medium without serum to OD620 1.5 Plates were washed twice with 1 ml serum-free MEM or DMEM and 1 ml medium without serum was added to each well. Then 50 µl of resuspended bacteria was added to achieve an m.o.i. of approximately 100 bacteria per cell. Cell morphology was observed for rounding under a phase-contrast microscope every hour for 4 h. Cell rounding is an indication of Yop-mediated cytotoxicity.
Adhesion assay.
Twenty-four-well cell culture plates were prepared as described above. E. coli AAEC185 containing pMMB207, pMMB207-yapC or pMMB207-psaABC was cultured overnight (16 h) at 28 °C with shaking in Luria–Bertani (LB) medium supplemented with 10 µg chloramphenicol ml–1 and 100 µM IPTG. Bacterial cultures were centrifuged and the bacteria were resuspended in serum-free MEM or DMEM at OD600 0.6. Cells were then diluted 1 : 100 into serum-free MEM or DMEM and 100 µl aliquots were used to infect 24-well plates seeded overnight with HEp-2 or RAW264.7 cells (after washing the cells twice with serum-free MEM or DMEM) in 500 µl MEM or DMEM respectively (an m.o.i. of approximately 1–3 bacteria per cell). RAW264.7 cells were treated with 5 µg cytochalasin D ml–1 in DMEM for 45 min prior to addition of bacteria to inhibit actin-dependent macrophage phagocytosis. Plates were incubated at 37 °C, 5 % CO2 for 2 h. Wells were then washed twice with PBS followed by cell lysis in 0.5 ml ddH2O containing 0.1 % Triton X-100 for 10 min. at room temperature. Wells were washed one more time with PBS and samples were pooled with the Triton X-100 lysis fraction of recovered bacteria. Duplicate wells were used to determine the total number of bacteria per well. For this purpose, all medium and washes were pooled. Tenfold dilutions in PBS were plated on LB agar plates containing 10 µg chloramphenicol ml–1. The plates were incubated at 37 °C overnight and the output c.f.u. were enumerated. Adhesion is expressed as the percentage of adherent bacteria relative to the total number of bacteria in a parallel infected well (total inoculum after 2 h).
Adhesion assays for Y. pestis strains were performed similarly, except that bacteria were grown in the absence of IPTG and chloramphenicol and cells were plated on HIB agar at 28 °C for 48 h prior to quantification.
Autoaggregation assay.
E. coli AAEC185 containing the empty vector pMMB207, pMMB207-yapC or pMMB207-psaABC were grown overnight in LB with 10 µg chloramphenicol ml–1 and 100 µM IPTG. Cells were pelleted and resuspended at an OD600 of 1.5 in 2 ml DMEM in glass test tubes. Tubes were incubated in a 37 °C water bath and OD600 was read every 15 min, 30 min or 60 min as the assay progressed. As bacteria aggregated, they settled out of the DMEM solution, resulting in a decrease in OD600.
Biofilm formation assay.
Crystal violet staining was used to detect cells attached to polystyrene as described by O'Toole et al. (1999). Briefly, overnight bacterial cultures were diluted 1 : 100 into LB plus 10 µg chloramphenicol ml–1 and 100 µM IPTG in flat-bottomed polystyrene culture plates (Costar Corning) and incubated 24 h at 37 °C or 28 °C without shaking. The optical density of the cultures was read in a microplate reader (Perkin Elmer Lambda Reader) at 595 nm. Plates were washed twice with PBS, and 0.01 % crystal violet was added and incubated for 15 min at room temperature. Wells were washed three times with distilled water, and stained bacteria were solubilized with 80 % ethanol and 20 % acetone mixture. Absorbance of the mixture was measured by the microplate reader at 595 nm. Results were normalized for bacterial culture density. Y. pestis assays were performed similarly except that no IPTG or chloramphenicol was added and cells were grown in a defined medium, PMH2 (Gong et al., 2001; Staggs & Perry, 1991).
RT-PCR.
Expression of yapC under various in vitro growth conditions was assessed using reverse transcription (RT)-PCR. KIM5 or KIM5 yapC was grown under three different conditions in HIB: 28 °C pH 7, 37 °C pH 6 and 37 °C pH 7. Bacterial cells were prepared for RNA extraction with Trizol according to the manufacturer's recommendations (Invitrogen). RNA samples were treated with DNase I for 10 min at 37 °C to degrade contaminating DNA followed by inactivation of DNase I with 2 mM EDTA and heating to 65 °C for 10 min. RNA was then precipitated with sodium acetate and ethanol and washed with 70 % ethanol prior to performing the RT-PCRs. RNA samples of 500 ng were used for reverse transcription, using random hexamer primers and Superscript II reverse transcriptase as described by the manufacturer (Invitrogen). PCR amplification was performed using the yapC or 16S rRNA primer pairs listed in Supplementary Table S1 (available with the online version of this paper). Thirty cycles of amplification were performed using an annealing temperature of 47 °C. Products were then run on a 2 % agarose gel, stained with ethidium bromide and imaged for visualization of appropriately sized PCR products. Negative control samples were processed in parallel, but no reverse transcriptase was added. A positive control PCR reaction was carried out using KIM5 chromosomal DNA.
Detecting YapC surface expression.
YapC was detected on the surface of Y. pestis using an anti-YapC antibody generated in the laboratory of Dr Virginia Miller (Department of Molecular Microbiology, Washington University School of Medicine). Y. pestis KIM5 or the KIM5 yapC mutant was grown overnight in HIB at 28 °C pH 7 (starting pH). Overnight cultures were diluted 1 : 50 into HIB and grown under three conditions: 28 °C pH 7, 37 °C pH 7 and 37 °C pH 6. Cells were grown to mid-exponential phase (4 h) and then cells were pelleted, washed once with 1 ml PBS and resuspended at an equivalent OD620 concentration (OD620 0.424) in an Eppendorf tube. Cells were then fixed for 20 min at room temperature with 500 µl 4 % paraformaldehyde (with rolling), washed once with 1 ml PBS and blocked for 3 h at room temperature with 1 ml PBS+1 % BSA (with rolling). Cells were then pelleted and resuspended in 500 µl PBS+0.2 % BSA+0.05 % Tween-20+a 1 : 500 dilution of the rabbit anti-YapC antibody. Bacteria and antibody were co-incubated overnight at 4 °C with rolling. Cells were then pelleted and washed twice with 1 ml PBS (with resuspension and pelleting), after which 500 µl PBS+0.2 % BSA+0.05 % Tween-20+1 : 1000 goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (Zymed) was incubated with the bacteria for 45 min at room temperature. Cells were then washed three times with 1 ml PBS and once with 1 ml 100 mM Tris pH 8.0, and reactions for detection of antibody binding were performed in 500 µl 100 mM Tris pH 8.0+4 mg ml–1 of the alkaline phosphatase substrate PNPP (Fluka). Reactions were developed for between 19 and 27 min prior to spinning out the bacteria and transferring the supernatants to a 96-well plate for reading in an ELISA plate reader at 405 nm. Results were normalized to YapC surface expression of KIM5 grown overnight in HIB at 28 °C. A405 readings were corrected for the time of PNPP development. E. coli samples were processed identically, except that they were induced with 100 µM IPTG during the 4 h growth in LB at 37 °C and exposed to PNPP for 8 min.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) to murine-derived macrophage-like cells (RAW264.7) or human-derived epithelial-like cells (HEp-2). Adhesion of E. coli expressing YapC to RAW264.7 macrophages and HEp-2 cells was analysed by a c.f.u. assay (see Methods). For these studies, the known Y. pestis adhesin pH 6 antigen (encoded by the psaABC locus) served as a positive control for efficient cell binding. E. coli expressing YapC showed a twofold increase in adhesion to RAW264.7 cells (28 %, corresponding to 1 bacterium per cell using our m.o.i. of 3) when compared to E. coli carrying the empty expression vector pMMB207 (Fig. 1a). The positive control, pH 6 antigen, provided 59 % adhesion (2 bacteria per cell) to RAW264.7 macrophages. On HEp-2 cells (where background binding by E. coli AAEC185 was lower), YapC showed a 3.3-fold increase (up to 2.1 % adhesion) in cell adhesion compared to E. coli with plasmid pMMB207 (0.65 % adhesion, Fig. 1b). pH 6 antigen conferred upon E. coli AAEC185 23.9 % adhesion for HEp-2 cells, demonstrating its strong adhesive ability. Giemsa staining of adhesion assays verified these findings, as E. coli expressing either YapC or pH 6 antigen showed higher numbers of bacteria bound per cell than E. coli carrying empty vector (data not shown).
|
yapC mutation on Y. pestis adhesion. A KIM5 yapC mutant had no defect in adhesion to either RAW264.7 macrophages or HEp-2 cells when pregrown at 28 °C or 37 °C (data not shown). Since the Caf1 capsule of Y. pestis (expressed at 37 °C) has been shown to interfere with the ability of some Y. pestis proteins to bind mammalian cells (Liu et al., 2006) and capsule production interferes with the activity of the E. coli TibA protein (Sherlock et al., 2005), we also constructed a caf1yapC mutant of KIM5. The caf1yapC mutant also had no cell-binding defect for RAW264.7 or HEp-2 cells (data not shown). The yapC mutant also had no defect in delivery of cytotoxic Yop proteins (a process which requires adhesins; Rosqvist et al., 1990) as measured by pCD1-dependent cell rounding (data not shown). The lack of an effect on cell binding in the yapC mutant suggests that Y. pestis expresses multiple adhesins capable of host cell binding or that expression of YapC in vitro is low.
Expression of YapC in the outer membrane
To confirm the expression of YapC in the outer membrane of E. coli, we cultured strains carrying the pMMB207-yapC plasmid overnight at 28 °C or 37 °C in Luria–Bertani (LB) medium supplemented with 100 µM IPTG to induce yapC expression and outer-membrane proteins were prepared according to the Triton X-100 extraction method of Hantke (1984). YapC was clearly visible as a predicted 65 kDa band (consistent with signal peptide processing) present in E. coli outer membranes (Fig. 2a
, lanes 2 and 5), and its identity was confirmed by mass spectrometry (data not shown). Although pH 6 antigen was expressed, as evidenced by its ability to mediate cell adhesion (Fig. 1), it was poorly detected in our outer membrane preparations, when stained with Coomassie blue (Fig. 2a, lanes 3 and 6; PsaA is a predicted 15 kDa protein). Since fimbria-like structures are often shed or sheared from the surface of bacterial cells during processing (Hoschutzky et al., 1989), we looked at the culture supernatants after spinning overnight cultures of bacteria at 7000 r.p.m. in a laboratory microcentrifuge. Anti-PsaA (the structural subunit of pH 6 antigen) Western blots performed on bacterial cell extracts or supernatants that were normalized to represent the amounts produced by similar numbers of bacteria demonstrated that the vast majority of PsaA filaments are sheared from AAEC185 during this initial bacterial pelleting step (Fig. 2b, lanes 12 and 24). PsaA was present in the culture supernatants of both 28 °C and 37 °C preparations of AAEC185/pMMMB207-psaABC (Fig. 2b). We were even able to visualize various polymerized forms (presumably incompletely denatured in our SDS-PAGE system) of pH 6 antigen (monomer to tetramer; Fig. 2b, lanes 12 and 24). It appears that only a small proportion of PsaA remains membrane-associated during our outer membrane preparation protocol, yet clearly PsaA-expressing E. coli cells have pH 6 antigen-mediated adhesive activity (leading to 24–59 % adhesion, depending on the cell line). Comparisons of RNA polymerase contamination from the cytosolic/inner-membrane Triton X-100-soluble fraction demonstrated minimal contamination when whole-cell lysates were compared with outer-membrane preparations (Fig. 2c).
There is an E. coli protein (denoted by an asterisk in Fig. 2b) that is cross-reactive with anti-PsaA antisera. Based on its fractionation, this protein is from either the cytoplasm or the inner membrane (Triton X-100-soluble fraction, Fig. 2b). The fact that we see some of this cross-reactive protein in supernatants from YapC-expressing E. coli suggests that YapC expression may be somewhat toxic to E. coli, causing some cells to lyse and release intracellular contents into the supernatant.
YapC surface expression in E. coli was also demonstrated using a recently generated anti-YapC antibody. AAEC185 carrying pMMB207 (empty vector) or pMMB207-yapC was grown for 4 h in LB with 100 µM IPTG induction. Cells were washed, fixed and processed for anti-YapC surface labelling as described in Methods. Expression of YapC in E. coli gave a more than fourfold increase in anti-YapC antibody recognition (Fig. 2d).
YapC mediates E. coli autoaggregation in tissue culture media
Another phenotype associated with TibA (the YapC homologue) in ETEC is autoaggregation (Sherlock et al., 2005). Autoaggregation in tissue culture medium is also associated with virulence in Yersinia species (Laird & Cavanaugh, 1980). Therefore, we investigated the ability of YapC to direct E. coli autoaggregation in DMEM tissue culture medium at 37 °C. These studies were performed in the Fim– E. coli derivative AAEC185 since the E. coli Fim system also mediates autoaggregation in tissue culture media (S. Felek & E. S. Krukonis, unpublished observations). Bacteria were induced for YapC or pH 6 antigen expression overnight at 28 °C with 100 µM IPTG, pelleted, resuspended in DMEM at 37 °C at an OD600 of 1.5 and monitored for aggregation by OD600 measurements over time. Expression of YapC led to aggregation of E. coli AAEC185 within 15–30 min and a 50 % reduction in OD600 occurred by approximately 45 min after resuspension in DMEM (Fig. 3). AAEC185 expressing the empty plasmid pMMB207 or pH 6 antigen showed no autoaggregation for up to 6 h (360 min) post-transfer to DMEM (Fig. 3).
|
and Methods).
A recent report by Yen et al. (2007) also demonstrated that expression of YapC in E. coli leads to autoaggregation, although in this case bacterial aggregation was tested in LB bacterial culture medium, not tissue culture medium.
Y. pestis also undergoes autoaggregation when grown at 28 °C. This phenotype is blocked by expression of the Caf1 protein capsule at 37 °C (S. Felek & E. S. Krukonis, unpublished observations). We tested our KIM5 yapC mutant for clumping in several media, including HIB, LB, PBS and DMEM. There was no difference in autoaggregation in any media between KIM5 and KIM5 yapC (data not shown). Subsequent investigations have revealed that a major determinant of KIM5 autoaggregation is a metabolic gene that is likely involved in LPS modifications (S. Felek & E. S. Krukonis, unpublished observations). Deletion of this locus results in a dramatic decrease in autoaggregation of KIM5.
YapC promotes biofilm-like deposits in E. coli
Since autoaggregation of bacteria is a critical step in biofilm formation, we assessed the ability of YapC to mediate formation of biofilm-like deposits in E. coli. Furthermore, the ETEC YapC homologue, TibA, has been shown to mediate biofilm formation in ETEC.
Biofilm assays were performed by incubating E. coli AAEC185 containing vector pMMB207 or pMMB207-yapC in LB medium in sterile 96-well polystyrene plates. YapC expression led to a 5.3-fold increase in AAEC185 biofilm formation (as measured by crystal violet staining) when the bacteria were grown at 37 °C and an 8-fold increase when grown at 28 °C (Fig. 4). Thus, YapC has the potential to play a role in Y. pestis biofilm formation in both the human host and the flea vector.
|
yapC mutant were grown overnight in PMH2 medium (Gong et al., 2001; Staggs & Perry, 1991) at 28 °C or 37 °C in sterile 96-well polystyrene plates. We did not detect a defect in biofilm-like deposits in the yapC mutant at either temperature (data not shown).
Given that at 37 °C YapC may be masked by the F1 capsule of KIM5, we also tested an isogenic caf1 mutant for biofilm formation in the presence or absence of YapC. Again, we saw no difference in biofilm-like deposits in a KIM5 caf1 mutant compared to the caf1yapC mutant (data not shown). It should be noted that KIM5 lacks the hms locus responsible for biofilm-dependent flea blockage by Y. pestis (Hinnebusch et al., 1996). Thus, biofilm formation in the KIM5 assays is via a different mechanism and is considered hms-independent.
Expression profile of yapC under various growth conditions
Using RT-PCR, Yen et al. (2007) found that yapC of Y. pestis KIM10+ is expressed in HIB at 28 °C. Since the three activities of YapC – cell adhesion, autoaggregation and biofilm formation – may be important in either the human host or the flea vector, we assessed yapC expression in Y. pestis under various in vitro growth conditions.
Parental KIM5 cells or a yapC mutant were grown overnight at 28 °C pH 7, diluted to OD620 0.2 and grown for 4 h at 37 °C pH 7, at 37 °C pH 6 (conditions which induce pH 6 antigen expression) or at 28 °C pH 7. RNA was isolated using a Trizol extraction protocol and RT-PCR was performed as described in Methods. Primers within the yapC-coding region led to amplification of a 113 bp product under all conditions and this product was absent in the yapC deletion mutant (Fig. 5a). This amplification product was also dependent upon reverse transcriptase (only present in ‘+’ lanes in Fig. 5). Thus, it was not due to DNA contamination. We also amplified the 16S rRNA transcript by RT-PCR and showed it was amplified equally well in both KIM5 and the yapC derivative (resulting in a 125 bp band, Fig. 5b), indicating that the yapC samples were competent for productive RT-PCR. Although these results indicate that yapC is expressed in Y. pestis under all three of the growth conditions we routinely use in the laboratory, such end-point (30 cycle) PCR reactions are non-quantitative. By comparing the yapC and 16S rRNA reaction products, it appears that yapC is expressed less well (Fig. 5a, b). Upon further investigation, by examining PCR products after 50 rounds of amplification, using several different dilutions of the template cDNA, it was determined that yapC expression levels are about 1000-fold lower than that of 16S rRNA (data not shown).
|
yapC mutant were grown overnight at 28 °C (starting pH 7) and then diluted 1 : 50 for 4 h growth under various conditions. YapC expression on the surface of KIM5 was assessed using an anti-YapC antibody (Methods). The only condition where we observed an increase in YapC surface expression compared to the yapC isogenic control was when KIM5 was grown overnight to stationary phase at 28 °C (Fig. 5c; P=0.1). Thus, YapC appears to be poorly expressed in vitro (possibly induced in stationary phase), but may be expressed during infection. The fact that YapC is poorly expressed in vitro likely contributes to our inability to observe a strong yapC mutant phenotype in our various assays. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Ofek et al., 2003). Many bacterial pathogens maintain multiple adhesins to enable binding to various cell types. In some bacteria, more than ten adhesins have been described (Ofek et al., 2003).
TibA is an ETEC glycoprotein that has been shown to mediate glycosylation-dependent binding to and invasion of several cell types (Elsinghorst & Kopecko, 1992; Elsinghorst & Weitz, 1994; Lindenthal & Elsinghorst, 2001). In this study, we found that YapC, a Y. pestis TibA homologue, can function as an adhesin. It promotes adhesion of E. coli AAEC185 to both RAW264.7 macrophages and HEp-2 cells (Fig. 1). We also confirmed the strong adhesive ability of the known Y. pestis adhesin, pH 6 antigen (Fig. 1; Yang et al., 1996). A recent study by Yen et al. (2007) found that YapC expressed in E. coli was unable to mediate haemagglutination of sheep red blood cells. In those experiments, the starting dilution of bacterial cells was 1 : 10. We could detect weak haemagglutination activity in E. coli expressing YapC to a bacterial dilution of 1 : 4 (data not shown). It is also possible that RAW264.7 macrophages and HEp-2 cells express a YapC receptor that is poorly expressed on erythrocytes. It should be noted that in our HEp-2 and RAW264.7 adhesion studies, some component of the increased adhesion might be due to the autoaggregative activity of YapC in E. coli (Fig. 3). YapC expressed in E. coli also mediated a two- to fourfold increase in adhesion to two other human-derived cell lines: THP-1 monocyte-like cells and A549 cells derived from lung epithelium (data not shown).
Another important bacterial process dependent on surface structures is biofilm formation. Biofilms are often important for bacterial virulence, facilitating colonization and protecting bacteria from host immune defence and antimicrobial treatments (Fux et al., 2005; Stewart & William Costerton, 2001). Attachment of bacteria to a surface is the first step in biofilm formation, followed by replication to form microcolonies to produce a mature biofilm (Jefferson, 2004). Biofilm formation blocks the proventriculus of the flea and is important for transmission of bubonic plague (Darby et al., 2002; Jarrett et al., 2004). The HmsT and HmsP proteins have been shown previously to control biofilm production in Y. pestis at 28 °C; these proteins are thought to be critical only for the flea (ambient temperature) stage of the Y. pestis life cycle since HmsT is degraded at 37 °C (Kirillina et al., 2004). We found that YapC mediates biofilm formation at both 37 °C and 28 °C in E. coli (Fig. 4). Thus, YapC may mediate biofilm formation during infection of mammalian hosts and/or the flea vector. YapC-mediated autoaggregation of bacterial cells (Fig. 3) may also contribute to biofilm formation. The ETEC YapC homologue, TibA, also induces biofilm formation in E. coli (Sherlock et al., 2005). When tested in Y. pestis, a yapC mutant of KIM5 had no defect in biofilm formation (data not shown). Autoaggregation in DMEM, perhaps an early step in biofilm formation, was also not affected in the yapC mutant (data not shown). Thus, while we have shown that YapC possesses certain activities, there are apparently redundant proteins with similar activities in Y. pestis. In the case of biofilm formation, we know that expression of several annotated Y. pestis chaperone/usher systems also results in biofilm formation (Felek et al., 2007) and we have recently identified a locus in Y. pestis that controls autoaggregation (S. Felek & E. S. Krukonis, unpublished). In addition, the expression of YapC in vitro appears to be fairly low (Fig. 5). The fact that YapC is poorly expressed in vitro likely contributes to our inability to observe a yapC mutant phenotype in our various assays.
It has been demonstrated that adhesins are required for Yop delivery in Y. pestis (Rosqvist et al., 1990). Yop delivery leads to cytotoxicity in eukaryotic cells. Our results and previous studies by others demonstrate that Y. pestis has at least three adhesins: YapC (Fig. 1), pH 6 antigen (Fig. 1 and Yang et al., 1996) and plasminogen activator (Kienle et al., 1992; Lahteenmaki et al., 1998). We tested various Y. pestis mutant strains for cytotoxicity in cultured cells. A yapC or psaA mutant of Y. pestis KIM5 showed wild-type levels of cytotoxicity for RAW264.7 macrophages and HEp-2 cells (data not shown), as assessed by cell rounding. As expected, a KIM5 pCD1– (Yop-negative control) strain did not cause cytotoxicity (data not shown). In addition, a triple mutant KIM8 (plasminogen activator negative, pla) playapCpsaA strain was also able to deliver Yop proteins to eukaryotic cells with wild-type efficiency when pregrown at 28 °C pH 7 (data not shown). It should be noted that a psaA mutant shows a 1–2 h delay in Yop-mediated cytotoxicity when pregrown at 37 °C pH 6 (data not shown). This pH 6 antigen-dependent reduction in Yop delivery confirms the ability of pH 6 antigen to facilitate delivery of T3SS proteins, as was shown previously in a non-adherent mutant of Pseudomonas aeruginosa, where expression of pH 6 antigen established sufficient contact to allow type III secretion of the toxin ExoS (Sundin et al., 2002).
The results discussed above suggest that there are redundant adhesins in Y. pestis for cell binding and Yop delivery. These could include the recently described adhesive autotransporters YapN and YapK (Yen et al., 2007), or the chaperone/usher-dependent surface structure encoded by Y. pestis KIM locus y0561 (Felek et al., 2007). Alternatively, an uncharacterized adhesin may be specifically required for Yop delivery. It is important to note that the observation of redundant adhesins in vitro does not necessarily mean that the same adhesins are redundant in vivo. Environmental signalling and various forms of regulation may result in the expression of specific adhesins during different stages of Y. pestis infection. It may also be the case that Yop delivery to macrophages and neutrophils may be less dependent on a specific adhesin in addition to the YopB/D translocation complex, due to the phagocytic nature of these cells (Boyd et al., 2000). In the closely related pathogen Y. pseudotuberculosis, at least two adhesins, invasin and YadA, have been shown to be able to mediate Yop delivery (Rosqvist et al., 1990, 1994). This ability to deliver Yop proteins has also been shown to be dependent on adhesin length, with a minimum length required to establish proper contact for type III secretion (Mota et al., 2005). However, neither invasin nor YadA is functional in Y. pestis, due to IS element insertion or frame shift mutation, respectively (Deng et al., 2002; Parkhill et al., 2001).
In summary, we have demonstrated that YapC, an annotated autotransporter of Y. pestis, can mediate cell adhesion to HEp-2 (human) cells and RAW264.7 (mouse) macrophages, can lead to autoaggregation and can facilitate biofilm formation. Each of these activities can play an important role in the efficiency of establishing an infection or overcoming the host innate immune response. The role of TibA in ETEC pathogenesis is not defined, but the various activities of TibA and its Y. pestis homologue, YapC, suggest that these molecules may play important roles in pathogenesis. While other factors of Y. pestis may also possess some of these activities, we hypothesize that YapC may be an important player in the infection process.
|
ACKNOWLEDGEMENTS |
|---|
-RED recombination system in Y. pestis. We thank Dr Susan Straley for providing anti-PsaA antibody. We thank Dr Virginia Miller for providing the anti-YapC antibody. Many thanks go to Dr James Bliska for helpful discussions throughout this work. Finally, we thank Drs Victor DiRita, Michele Swanson and J. Chris Fenno for critical reading of the manuscript. Edited by: P. van der Ley
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Beesley, E. D., Brubaker, R. R., Janssen, W. A. & Surgalla, M. J. (1967). Pesticins. III. Expression of coagulase and mechanism of fibrinolysis. J Bacteriol 94, 19–26.
Ben-Efraim, S., Aronson, M. & Bichowsky-Slomnicki, L. (1961). New antigenic component of Pasteurella pestis formed under specified conditions of pH and temperature. J Bacteriol 81, 704–714.
Bichowsky-Slomnicki, L. & Ben-Efraim, S. (1963). Biological activities in extracts of Pasteurella pestis and their relation to the "pH 6 antigen". J Bacteriol 86, 101–111.
Blomfield, I. C., McClain, M. S. & Eisenstein, B. I. (1991). Type 1 fimbriae mutants of Escherichia coli K12: characterization of recognized afimbriate strains and construction of new fim deletion mutants. Mol Microbiol 5, 1439–1445.[Medline]
Boyd, A. P., Grosdent, N., Totemeyer, S., Geuijen, C., Bleves, S., Iriarte, M., Lambermont, I., Octave, J.-N. & Cornelis, G. R. (2000). Yersinia enterocolitica can deliver Yop proteins into a wide range of cell types: development of a delivery system for heterologous proteins. Eur J Cell Biol 79, 659–671.[CrossRef][Medline]
Brubaker, R. R., Beesley, E. D. & Surgalla, M. J. (1965). Pasteurella pestis: role of pesticin I and iron in experimental plague. Science 149, 422–424.
Cantor, N. (2001). In the Wake of the Plague. New York: Perennial.
Cornelis, G. R., Boland, A., Boyd, A. P., Geuijen, C., Iriarte, M., Neyt, C., Sory, M.-P. & Stainier, I. (1998). The virulence plasmid of Yersinia, an antihost genome. Microbiol Mol Biol Rev 62, 1315–1352.
Cowan, C., Jones, H., Kaya, Y., Perry, R. & Straley, S. (2000). Invasion of epithelial cells by Yersinia pestis: evidence for a Y. pestis-specific invasin. Infect Immun 68, 4523–4530.
Darby, C., Hsu, J. W., Ghori, N. & Falkow, S. (2002). Caenorhabditis elegans: plague bacteria biofilm blocks food intake. Nature 417, 243–244.[CrossRef][Medline]
Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.
Dautin, N., Barnard, T. J., Anderson, D. E. & Bernstein, H. D. (2007). Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism. EMBO J 26, 1942–1952.[CrossRef][Medline]
Deng, W., Burland, V., Plunkett, G., III, Boutin, A., Mayhew, G. F., Liss, P., Perna, N. T., Rose, D. J., Mau, B. & other authors (2002). Genome sequence of Yersinia pestis KIM. J Bacteriol 184, 4601–4611.
Egile, C., d'Hauteville, H., Parsot, C. & Sansonetti, P. J. (1997). SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri. Mol Microbiol 23, 1063–1073.[CrossRef][Medline]
Elsinghorst, E. A. & Kopecko, D. J. (1992). Molecular cloning of epithelial cell invasion determinants from enterotoxigenic Escherichia coli. Infect Immun 60, 2409–2417.
Elsinghorst, E. A. & Weitz, J. A. (1994). Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein. Infect Immun 62, 3463–3471.
Felek, S., Runco, L., Thanassi, D. G. & Krukonis, E. S. (2007). Characterization of six novel chaperone/usher systems in Yersinia pestis. In 9th International Symposium on Yersinia, pp. 97–105. Lexington, KY, USA: Springer.
Fux, C. A., Costerton, J. W., Stewart, P. S. & Stoodley, P. (2005). Survival strategies of infectious biofilms. Trends Microbiol 13, 34–40.[CrossRef][Medline]
Gong, S., Bearden, S. W., Geoffroy, V. A., Fetherston, J. D. & Perry, R. D. (2001). Characterization of the Yersinia pestis Yfu ABC inorganic iron transport system. Infect Immun 69, 2829–2837.
Hantke, K. (1984). Cloning of the repressor protein gene of iron-regulated systems in Escherichia coli K12. Mol Gen Genet 197, 337–341.[CrossRef][Medline]
Henderson, I. R., Navarro-Garcia, F., Desvaux, M., Fernandez, R. C. & Ala'Aldeen, D. (2004). Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68, 692–744.
Hendrixson, D. R., de la Morena, M. L., Stathopoulos, C. & St Geme, J. W., III (1997). Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein. Mol Microbiol 26, 505–518.[CrossRef][Medline]
Hinnebusch, B. J., Perry, R. D. & Schwan, T. G. (1996). Role of the Yersinia pestis hemin storage (hms) locus in the transmission of plague by fleas. Science 273, 367–370.[Abstract]
Hoschutzky, H., Lottspeich, F. & Jann, K. (1989). Isolation and characterization of the -galactosyl-1,4-β-galactosyl-specific adhesin (P adhesin) from fimbriated Escherichia coli. Infect Immun 57, 76–81.
Isberg, R. R., Voorhis, D. L. & Falkow, S. (1987). Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell 50, 769–778.[CrossRef][Medline]
Jarrett, C. O., Deak, E., Isherwood, K. E., Oyston, P. C., Fischer, E. R., Whitney, A. R., Kobayashi, S. D., DeLeo, F. R. & Hinnebusch, B. J. (2004). Transmission of Yersinia pestis from an infectious biofilm in the flea vector. J Infect Dis 190, 783–792.[CrossRef][Medline]
Jefferson, K. K. (2004). What drives bacteria to produce a biofilm? FEMS Microbiol Lett 236, 163–173.[Medline]
Kienle, Z., Emody, L., Svanborg, C. & O'Toole, P. (1992). Adhesive properties conferred by the plasminogen activator of Yersinia pestis. J Gen Microbiol 138, 1679–1687.
Kirillina, O., Fetherston, J. D., Bobrov, A. G., Abney, J. & Perry, R. D. (2004). HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis. Mol Microbiol 54, 75–88.[CrossRef][Medline]
Lahteenmaki, K., Virkola, R., Saren, A., Emody, L. & Korhonen, T. K. (1998). Expression of plasminogen activator Pla of Yersinia pestis enhances bacterial attachment to the mammalian extracellular matrix. Infect Immun 66, 5755–5762.
Lahteenmaki, K., Kukkonen, M. & Korhonen, T. K. (2001). The Pla surface protease/adhesin of Yersinia pestis mediates bacterial invasion into human endothelial cells. FEBS Lett 504, 69–72.[CrossRef][Medline]
Laird, W. J. & Cavanaugh, D. C. (1980). Correlation of autoagglutination and virulence of yersiniae. J Clin Microbiol 11, 430–432.
Lindenthal, C. & Elsinghorst, E. A. (2001). Enterotoxigenic Escherichia coli TibA glycoprotein adheres to human intestine epithelial cells. Infect Immun 69, 52–57.
Lindler, L. E. & Tall, B. D. (1993). Yersinia pestis pH 6 antigen forms fimbriae and is induced by intracellular association with macrophages. Mol Microbiol 8, 311–324.[Medline]
Lindler, L. E., Klempner, M. S. & Straley, S. C. (1990). Yersinia pestis pH 6 antigen: genetic, biochemical, and virulence characterization of a protein involved in the pathogenesis of bubonic plague. Infect Immun 58, 2569–2577.
Liu, F., Chen, H., Galvan, E. M., Lasaro, M. A. & Schifferli, D. M. (2006). Effects of Psa and F1 on the adhesive and invasive interactions of Yersinia pestis with human respiratory tract epithelial cells. Infect Immun 74, 5636–5644.
Morales, V. M., Backman, A. & Bagdasarian, M. (1991). A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97, 39–47.[CrossRef][Medline]
Mota, L. J., Journet, L., Sorg, I., Agrain, C. & Cornelis, G. R. (2005). Bacterial injectisomes: needle length does matter. Science 307, 1278
Niemann, H. H., Schubert, W.-D. & Heinz, D. W. (2004). Adhesins and invasins of pathogenic bacteria: a structural view. Microbes Infect 6, 101–112.[CrossRef][Medline]
O'Toole, G. A., Pratt, L. A., Watnick, P. I., Newman, D. K., Weaver, V. B. & Kolter, R. (1999). Genetic approaches to study of biofilms. Methods Enzymol 310, 91–109.[CrossRef][Medline]
Ofek, I., Hasty, D. L. & Doyle, R. J. (2003). Bacterial Adhesion to Animal Cells and Tissues. Washington, DC: American Society for Microbiology.
Parkhill, J., Wren, B. W., Thomson, N. R., Titball, R. W., Holden, M. T., Prentice, M. B., Sebaihia, M., James, K. D., Churcher, C. & other authors (2001). Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413, 523–527.[CrossRef][Medline]
Perry, R. D. & Fetherston, J. D. (1997). Yersinia pestis – etiologic agent of plague. Clin Microbiol Rev 10, 35–66.[Abstract]
Rosqvist, R., Forsberg, A., Rimpilainen, M., Bergman, T. & Wolf-Watz, H. (1990). The cytotoxic protein YopE of Yersinia obstructs the primary host defence. Mol Microbiol 4, 657–667.[Medline]
Rosqvist, R., Magnusson, K. & Wolf-Watz, H. (1994). Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J 13, 964–972.[Medline]
Shere, K. D., Sallustio, S., Manessis, A., D'Aversa, T. G. & Goldberg, M. B. (1997). Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin-based motility. Mol Microbiol 25, 451–462.[CrossRef][Medline]
Sherlock, O., Vejborg, R. M. & Klemm, P. (2005). The TibA adhesin/invasin from enterotoxigenic Escherichia coli is self recognizing and induces bacterial aggregation and biofilm formation. Infect Immun 73, 1954–1963.
Sodeinde, O. A., Subrahmanyam, Y. V., Stark, K., Quan, T., Bao, Y. & Goguen, J. D. (1992). A surface protease and the invasive character of plague. Science 258, 1004–1007.
Staggs, T. M. & Perry, R. D. (1991). Identification and cloning of a fur regulatory gene in Yersinia pestis. J Bacteriol 173, 417–425.
Stewart, P. S. & William Costerton, J. (2001). Antibiotic resistance of bacteria in biofilms. Lancet 358, 135–138.[CrossRef][Medline]
Sundin, C., Wolfgang, M. C., Lory, S., Forsberg, A. & Frithz-Lindsten, E. (2002). Type IV pili are not specifically required for contact dependent translocation of exoenzymes by Pseudomonas aeruginosa. Microb Pathog 33, 265–277.[CrossRef][Medline]
Tamm, A., Tarkkanen, A.-M., Korhonen, T. K., Kuusela, P., Toivanen, P. & Skurnik, M. (1993). Hydrophobic domains affect collagen-binding specificity and surface polymerization as well as the virulence potential of the YadA protein of Yersinia enterocolitica. Mol Microbiol 10, 995–1011.[Medline]
Welkos, S. L., Friedlander, A. M. & Davis, K. J. (1997). Studies on the role of plasminogen activator in systemic infection by virulent Yersinia pestis strain C092. Microb Pathog 23, 211–223.[CrossRef][Medline]
Yang, Y. & Isberg, R. (1993). Cellular internalization in the absence of invasin expression is promoted by the Yersinia pseudotuberculosis yadA product. Infect Immun 61, 3907–3913.
Yang, Y., Merriam, J., Mueller, J. & Isberg, R. (1996). The psa locus is responsible for thermoinducible binding of Yersinia pseudotuberculosis to cultured cells. Infect Immun 64, 2483–2489.[Abstract]
Yen, Y. T., Karkal, A., Bhattacharya, M., Fernandez, R. C. & Stathopoulos, C. (2007). Identification and characterization of autotransporter proteins of Yersinia pestis KIM. Mol Membr Biol 24, 28–40.[CrossRef][Medline]
Yu, D., Ellis, H. M., Lee, E.-C., Jenkins, N. A., Copeland, N. G. & Court, D. L. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97, 5978–5983.
Received 20 June 2007;
revised 31 January 2008;
accepted 5 March 2008.
This article has been cited by other articles:
|
|
 |
|
  M. Zhu, D. Ajdic, Y. Liu, D. Lynch, J. Merritt, and J. A. Banas Role of the Streptococcus mutans irvA Gene in GbpC-Independent, Dextran-Dependent Aggregation and Biofilm Formation Appl. Envir. Microbiol., November 15, 2009; 75(22): 7037 - 7043. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. D. Crane, S. L. Warner, and C. M. Bosio A Novel Role for Plasmin-Mediated Degradation of Opsonizing Antibody in the Evasion of Host Immunity by Virulent, but Not Attenuated, Francisella tularensis J. Immunol., October 1, 2009; 183(7): 4593 - 4600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-h. Hu, C.-s. Liu, J.-h. Hou, and L. Sun Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain Appl. Envir. Microbiol., July 1, 2009; 75(13): 4333 - 4340. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Zhou and R. Yang Molecular Darwinian Evolution of Virulence in Yersinia pestis Infect. Immun., June 1, 2009; 77(6): 2242 - 2250. [Full Text] [PDF]
|
|
|
|
|
|
S. Felek and E. S. Krukonis The Yersinia pestis Ail Protein Mediates Binding and Yop Delivery to Host Cells Required for Plague Virulence Infect. Immun., February 1, 2009; 77(2): 825 - 836. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Lawrenz, J. D. Lenz, and V. L. Miller A Novel Autotransporter Adhesin Is Required for Efficient Colonization during Bubonic Plague Infect. Immun., January 1, 2009; 77(1): 317 - 326. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
, pMMB207; 
, YapC; 
, PsaABC.
