| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844-3052, USA
Correspondence
Gregory A. Bohach
gbohach{at}uidaho.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
These authors contributed equally to this work.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Today, treatment and prevention are not yet 100 % effective and a better understanding of pathogenesis is needed to enhance protection (McEvedy, 1988; Titball & Williamson, 2004; Zietz & Dunkelberg, 2004). Y. pestis carries a number of pathogenesis genes on the chromosome, but the pCD1 plasmid is essential for virulence. pCD1 encodes a type III secretion system and effector proteins with several functions, including circumventing host innate immunity (Leigh et al., 2005; Lindler et al., 1990).
Y. pestis enters epithelial cells, another mechanism to circumvent the immune response (Cowan et al., 2000). In airborne disease, penetration of epithelial cells may promote development of pulmonary lesions (Liu et al., 2006). Although Pla protease enhances interaction with epithelial cells (Cowan et al., 2000; Lahteenmaki et al., 1998, 2001a), Psa fimbria have also been implicated. Neither is sufficient to confer complete internalization. Thus, Y. pestis adherence and internalization mechanisms are not yet completely elucidated (Liu et al., 2006).
Outer-membrane proteins (Omps) have β-strand structures with membrane-spanning domains, and participate in channelling, antibiotic resistance and signal transduction (Bockmann & Caflisch, 2005). One Omp, OmpX, was first described for Enterobacter cloacae (Stoorvogel et al., 1991), but homologues, including PagC, Lom, Rck and Ail (the attachment–invasion locus protein of Yersinia spp.), were identified in other Gram-negative bacteria (Dupont et al., 2004; Heffernan et al., 1992a; Mecsas et al., 1995). Proteins in this family promote invasion, resistance to complement-mediated killing, survival in macrophages, and internalization in epithelial cells (Cirillo et al., 1996).
In the Y. pestis KIM genomic database, four ORFs for OmpX or Ail variants have been identified: y1324, y1682, y2034 (Caspi et al., 2006; Karp et al., 2005) and y2446 (UniProtKB/TrEMBL database). The y1324 gene encodes a protein which has a predicted molecular mass of 21 569 Da (including its signal sequence), has high sequence identity (99 and 68.5 %) to Ail in Y. pseudotuberculosis and Yersinia enterocolitica, respectively, and is designated OmpX. The other three genes, y1682, y2034 and y2446 encode predicted proteins with 37.5, 46.4 and 45.9 %, amino acid sequence identity, respectively, with Y. enterocolitica Ail.
Although Y. enterocolitica Ail has been extensively studied (Miller et al., 1990, 2001), similar studies have not been reported for its Y. pestis homologues. Two previous studies showed that loss of an unidentified protein in the Y. pestis outer membrane affects autoaggregation (Podladchikova & Rykova, 2006) and adhesion to phagocytes (Kukleva et al., 2000). The present study was conducted to characterize Y. pestis OmpX by engineering an OmpX deletion mutation in Y. pestis KIM6+ and comparing the properties of this mutant strain to the parental and complemented strains.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
Strains and plasmids used in the study are listed in Table 1. A spontaneous Nalr mutant was obtained by plating Y. pestis KIM6+ on LB containing Nal. The pMS20 suicide plasmid, with sacBR encoding levensucrase, and Cmr, was used as described previously (Gay et al., 1983). In some experiments, Y. pestis strains constitutively expressing green fluorescent protein (GFP) were used. These were constructed by electroporating pFVP25.1 (a gift from G. Mallo; Caenorhabdidis Genetics Center, Minneapolis, MN; http://www.cbs.umn.edu/CGC), which encodes Ampr and GFP.
|
) were grown in 6 % CO2 (37 °C) in growth medium (GM) [low glucose Dulbecco's modified Eagle's medium (Gibco) supplemented with 10 % (v/v) fetal bovine serum (FBS) (HighClone) and 1 % (v/v) penicillin/streptomycin solution (Gibco)].
Construction of a Y. pestis KIM6+ Nalr ompX deletion mutant.
The ompX gene was amplified by PCR using primers complementary to the predicted promoter and terminator regions and containing engineered XbaI and EcoRV sites, respectively. The resulting 750 bp PCR product was cloned into pMS20 (Smith, 2000) and the construct (pMHZ1) was transformed into Escherichia coli CC118 
pir. A representative clone was digested with MfeI and NdeI (sites in ompX), generating a 426 bp deletion. A gene conferring Knr (neomycin phosphotransferase; npt), with flanking FRT (flippase recognition target) sites, was amplified by PCR using pKD4 (Datsenko & Wanner, 2000) as a template and, and cloned into pMHZ1. The resulting construct, pMHZ2 carrying Knr, was transformed into E. coli S17-1 pir. A transformant harbouring pMHZ2 was mated with Y. pestis KIM6+ Nalr as described previously (Smith, 2000) and counter-selected on LB agar with Nal and Cm. This merodiploid strain (ompX+/ompX : : npt) was generated by a homologous single-crossover recombination. It was maintained and served as (i) a single copy ompX complementation control and (ii) an isogenic precursor for selecting the ompX : : npt disruption. The latter was isolated on LB agar containing sucrose to select for a second crossover event while maintaining selection for the ompX : : npt disruption. Sucrose-resistant, Cm-sensitive, colonies were tested by PCR for the ompX : : npt disruption (generation of a 1828 bp product). DNA sequencing was used to confirm all constructs.
PCR.
Primers (Table 1
) were purchased from Integrated DNA Technologies and reagents were from Invitrogen. Primers used to amplify Tn5 npt contained engineered MfeI and NdeI restriction sites. In the reverse primer, a mutation was introduced to exclude the NdeI restriction site naturally present in the FRT region. Amplification of this region, including the FRT sequences, enables deletion of the antibiotic resistance gene using the flippase-bearing pCP20 plasmid (Datsenko & Wanner, 2000). PCR products were visualized on agarose gels and sizes were determined using the 1 kb Plus DNA ladder (Fisher Scientific).
Expression of ompX and the downstream (y1325) hcaT gene were measured by real-time PCR. Total RNA was extracted as described previously (Rebeil et al., 2006). Reverse transcription with hexanucleotides (Roche), real-time PCR using the SYBR green I dye master mix and an ABI 7000 thermocycler (Perkin–Elmer Applied Biosystems), and data analysis were as described previously (Seo et al., 2007). Relative quantities of mRNA were normalized to the amount of proS mRNA in the samples. RNA isolation for each strain was done in duplicate on two different days and real-time PCR was performed in triplicate for each RNA sample.
Culture growth measurements.
Growth rate comparisons were done in LB broth (50 ml) at 28 and 37 °C on an orbital shaker (200 r.p.m.). A Beckman Coulter DU530 spectrophotometer (Beckman Instruments) was used to measure OD600 of the cultures hourly, with vigorous vortexing before each reading.
Internalization assays.
Y. pestis cells from 
12 h (OD600 0.55–0.75) cultures grown at 28 °C (200 r.p.m.) in LB medium were washed in PBS (0.01 M sodium phosphate, 0.8% Nacl, pH 7.2) and resuspended in internalization medium (IM; GM lacking FBS and antibiotics). Dilutions were made in PBS containing 0.1 % Triton X-100 and 0.2 % glycerol to determine cell numbers by plate counts. HEp-2 cells (1.5x105 per well) in GM were incubated in 24-well plates (6 % CO2, 37 °C). After 42 h, the cell monolayers were washed three times with IM and 1 ml adjusted Y. pestis suspension was added to each well to produce co-cultures with m.o.i. values between 10 and 20. The plates were centrifuged (5 min, 200 g, 18 °C) and incubated for 1 h as described above. Each well was washed three times with IM and the co-cultures were incubated for 1.5 h in IM with 500 µg gentamicin per well (Gibco) to kill extracellular Y. pestis. The wells were washed three times and trypsin-like enzyme (Tryp-Le Express, Gibco) was added and incubated for 7 min to detach the HEp-2 cells. Triton X-100 (0.025 %) was added to release intracellular bacteria and bacterial numbers were determined from plate counts. Initial experiments, using standard techniques (Shaffer & Goldin, 1974), showed that inactivating ompX did not significantly alter the gentamicin minimal bactericidal concentration (MBC; 3.9–7.8 µg ml–1).
Cell-association assays.
Procedures were conducted as described above except that, after 1 h incubation, the HEp-2/Y. pestis co-cultures were washed nine times to remove unbound bacteria and the gentamicin exposure step was omitted. In some experiments, microscopy was used to assess cell association of Y. pestis strains expressing GFP from plasmid pFVP25.1. HEp-2 cells were incubated for 48 h in a four-chamber coverglass (Nalge Nunc International) starting with an initial inoculum of 5.0x104 bacteria ml–1. The assays were performed as described above except that after the ninth wash the cells were fixed with 3.7 % formaldehyde and stained with 16.5 nM phalloidin conjugated with Alexa Fluor 546 as described by the manufacturer (Invitrogen). Images of nine 0.7 µm-thick slices collected by LSM5 Pa laser scanning microscope (Carl Zeiss MicroImaging) with a Plan-Apochromat 63x/1.4 Oil DIC lens were processed by Zeiss LSM Image Examiner version 3.2.0.70.
Analysis of OmpX expression.
To examine OmpX levels at various growth phases and temperatures, bacteria were grown for 12 h as described above, washed in PBS, and pelleted by centrifugation at 12 000 g at 4 °C for 5 min. Proteins were extracted with a buffer containing 8 M urea (Sigma), 2 % CHAPS (EMD Chemicals), 20 mM DTT (Bio-Rad), bromophenol blue, Protease Inhibitors (Amersham Biosciences), and 100x Nuclease (Amersham Biosciences). Protein samples and the Benchmark protein ladder (Invitrogen) were resolved by SDS-PAGE (Laemmli, 1970) on 12.5 % polyacrylamide gels and visualized by Coomassie blue staining.
Tandem mass spectrometry (MS/MS) analysis.
Protein bands observed in SDS-PAGE gels were excised manually, destained, and subjected to standard protease digestion procedures (Shevchenko et al., 1996). Samples were processed in high-recovery tubes from Axygen. Proteins were digested overnight at 37 °C using trypsin (Worthington) and 0.1 % n-octyl β-glucoside (Fluka) (Katayama et al., 2001). Peptides were recovered with extraction buffer containing 50 % acetonitrile and 5 % trifluoroacetic acid and the resulting sample was concentrated under vacuum and resuspended in 5 % acetonitrile/0.1 % formic acid.
MS/MS analysis was performed using a Waters Nanoacquity ultra performance liquid chromatography (UPLC) unit, as described previously (Lee et al., 2006). Digested peptides (2 µl) were loaded onto a fused silica capillary column (Atlantis dC18, 3 µm, 75 µmx100 mm) in tandem with a trap column (Symmetry C18, 5 µm, 180 µmx20 mm; Waters). The peptide mixture was separated using a gradient of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile at 0.4 µl min–1. The column effluent was delivered directly to a Premiere quadrupole-time-of-flight mass spectrometer (Waters) equipped with a nanospray electrospray ion source. The MS and UPLC were controlled and mass spectra were acquired and analysed using MassLynx 4.0 software. All spectra were obtained in the positive ion mode. The survey scans used a 400–2000 Da mass range in continuum mode and up to three peptides with charges 2, 3 or 4 were sequenced (MS/MS) at a given retention time using 50–2000 Da mass window.
ProteinLynx Global Server 2.2 and Protein Expression Informatics System software Version 1.0 were used for MS/MS spectra analysis, peptide sequencing and protein identification. MS/MS data were compared to protein sequence databases from the University of Wisconsin (http://www.genome.wisc.edu/sequencing/pestis.htm) and the Sanger Institute (http://www.sanger.ac.uk/Projects/Y_pestis/) and results were analysed using Mascot software (Matrix Science).
Serum-resistance assays.
Venous blood was collected from healthy donors and processed as described previously (Vandenbosch et al., 1987) to obtain normal human serum (NHS). Heat-inactivated serum (HIS) was generated at 56 °C for 30 min to inactivate complement. During the exponential phase of growth, bacterial cells (grown as above) were collected, washed twice in PBS, diluted 100-fold, mixed with an equal volume of NHS or HIS, and incubated at 37 °C. Viable bacteria were quantified by plate counts as described above following incubation in sera for 0, 0.5 or 1.0 h.
Statistical analysis.
Data were analysed using either the Mann–Whitney or Student's t tests. These analyses were conducted with SigmaPlot 9.0 (Systat Software) software.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Ail (Sanger Institute; Y. pestis CO92) begins with ATG and encodes a putative 26-residue signal peptide. ORF Finder (NCBI Tools for Bioinformatics Research) predicted the signal peptide sequence in the Y. pestis CO92 database for both genes.
|
). pMHZ2 was introduced into Y. pestis KIM6+ Nalr by conjugation. Y. pestis KIM6+ Nalr Cmr colonies represented pMHZ2 co-integrates with an ompX/ompX : : npt genotype as confirmed by PCR (results not shown). PCR analysis of one isolate generated the predicted 750 bp product representing chromosomal ompX, and the predicted 1829 bp product representing the introduced ompX : : npt fragment. This ompX merodiploid strain (Y. pestis ompX+/ompX : : npt) was used as a positive control for subsequent experiments examining the properties of the Y. pestis ompX : : npt strain. A second crossover, deleting the wild-type chromosomal ompX and the pMHZ2 co-integrate, and conferring Cmr, was selected on LB agar with Kn and sucrose. A sucrose-resistant colony, that was also Knr and Cms, was chosen for further study after confirming the ompX disruption by PCR. PCR analysis of this Y. pestis ompX : : npt strain generated only the predicted 1829 bp fragment (results not shown).
Real-time PCR data indicated that ompX transcription was not significantly different in the parental and complemented strains (mean change in threshold cycle (
CT): –6.550 and –6.285; P=0.182), and expression by the Y. pestis ompX : : npt deletion mutant was undetectable. Furthermore, expression of the downstream y1325 (hcaT) gene was not significantly different in any strain [mean CT: 8.16 (parental), 8.82 (deletion), 8.22 (merodiploid); P=0.279 (parental vs deletion), and P=0.907 (parental vs merodiploid)].
Proteomic analysis
SDS-PAGE analysis of Y. pestis whole-cell lysates (Fig. 2) indicated that the Y. pestis KIM6+ Nalr ompX : : npt strain lacked a protein band at approx. 19 kDa which was present in parental Y. pestis KIM6+ Nalr, control merodiploid Y. pestis KIM6+ Nalr ompX+/ompX : : npt, and Y. pestis KIM6– strains. This protein was expressed at both 28 and 37 °C and during exponential and stationary phases. This protein band from gels containing the parental strain, and corresponding gel slices from the mutants, was excised, digested with trypsin, and sequenced. MS/MS data were used to search protein databases for Y. pestis KIM and Y. pestis CO92. This revealed that the missing protein was identical to the mature form of OmpX (gene tag y1324) and Ail (gene tag ypo2905) in these two databases, respectively. In this report, we used the nomenclature in the Y. pestis KIM database (OmpX rather than Ail) to be consistent with the gene designation used in the database for the strain most closely related to the strain used in our study.
|
; Nielsen & Krogh, 1998) (Fig. 1). This suggests that the signal peptide is cleaved between Ala and Glu (Fig. 1); Y. enterocolitica Ail is cleaved at an analogous site (Miller et al., 1990). The predicted molecular mass of mature OmpX, calculated with ProtParameters (Gasteiger et al., 2005), is 17.47 kDa, slightly smaller than the 19 kDa size estimated by SDS-PAGE.
Disruption of ompX does not impair the growth of Y. pestis in vitro
To ascertain whether the mutation affected bacterial growth rate, growth kinetics were monitored for the parental strain and its mutant derivatives at 28 and 37 °C. The ompX disruption did not impair growth at either temperature. In fact, the Y. pestis KIM6+ Nalr ompX : : npt deletion mutant had a small but reproducible increase in growth rate at 28 °C compared to the parental and merodiploid control strains (doubling times 97, 101 and 107 min respectively; data not shown). This increased growth rate was more prominent at 37 °C; the deletion mutant had a doubling time of 118 min compared to 152 and 154 min, respectively, for the parental and merodiploid control strains. When compared to the parental and control strains, the increase in growth rate was statistically significant (P<0.009) for cultures grown at 37 °C, but not for those grown at 28 °C.
Disruption of ompX results in loss of autoaggregation and pellicle formation
Certain strains of Y. pestis produce characteristic flocculent growth in broth cultures (Bobrov et al., 2002). This phenotype was readily observable for the parental Y. pestis KIM6+ Nalr strain, the merodiploid Y. pestis KIM6+ Nalr ompX+/ompX : : npt control strain, and the pgm-deficient strain Y. pestis KIM6– (Fig. 3a), which autoaggregated when grown at either 28 or 37 °C. Despite being subjected to shaking, the bacterial growth settled to the bottom when grown at 28 °C. Smaller aggregates formed but remained dispersed in cultures grown at 37 °C. In addition, these strains formed pellicles attaching to the sides of the tubes at the air–liquid interface. Loss of these characteristics coincided with disruption of the ompX gene. Y. pestis KIM6+ Nalr ompX : : npt grew as a homogeneous suspension at both 28 and 37 °C, and neither clumping nor pellicle formation was observed at either temperature. Microscopic analysis revealed that the parental and merodiploid strains formed large aggregates, while the Y. pestis KIM6+ Nalr ompX : : npt mutant grew as individual cells or in smaller clusters (Fig. 3b).
|
; Heffernan et al., 1994; Miller & Falkow, 1988). To determine whether Y. pestis OmpX performs similar functions, we examined the effect of inactivating the ompX gene on interactions with cultured HEp-2 human epithelial cells. The overall association of Y. pestis KIM6+ Nalr ompX : : npt with HEp-2 cells was significantly impaired in comparison to Y. pestis KIM6+ Nalr and Y. pestis KIM6+ Nalr ompX+/ompX : : npt (Fig. 4). Microscopic observation of Y. pestis KIM6+ Nalr ompX : : npt-HEp-2 co-cultures revealed that very few bacterial cells were associated with the HEp-2 cells. In contrast, the parental and merodiploid control strains of Y. pestis, both of which express OmpX, were observed predominantly as large aggregates associated with Hep-2 cell monolayers. Bacterial cell counts from the cell-association assays were consistent with microscopic observation. Y. pestis KIM6+ Nalr ompX : : npt cell numbers associated with HEp-2 cells (representing both adherent and internalized cells) were reduced by 90 % (11-fold reduction) compared to the parental strain and merodiploid control. This led to an 98 % (65.5-fold) reduction in internalization of Y. pestis. The data indicated that internalization was more severely affected than adherence. Specifically, only 6 % of the cell-associated Y. pestis KIM6+ Nalr ompX : : npt were internalized by eukaryotic cells vs 35.75 and 32 % for Y. pestis KIM6+ Nalr and Y. pestis KIM6+ Nalr ompX+/ompX : : npt, respectively.
|
; Yang et al., 1996), standard assay protocols were used. The ompX deletion mutant was significantly more susceptible to complement-mediated killing compared to the parental and control strains (Fig. 5) grown at both 28 and 37 °C. Incubation in 50 % NHS at 37 °C was essentially 100 % lethal to Y. pestis KIM6+ Nalr ompX : : npt after 1 h. In contrast, Y. pestis KIM6+ Nalr and Y. pestis KIM6+ Nalr ompX+/ompX : : npt expressing OmpX both retained complete viability during this time period. To exclude the possibility that the autoaggregation phenotype of the parental strain may significantly distort the complement-resistance results by protecting a fraction of the bacterial population from complement by making the bacterial cells inaccessible, we observed that during longer incubation with serum, bacterial aggregates were being disrupted and after 4 h the cells were more separated (data obtained from confocal microscopy of GFP-labelled bacteria; not shown). Incubation of the bacteria with serum for 4 h and subsequent addition of fresh serum did not result in any decrease in viability, which indicates that it is OmpX, not autoaggregation, which is the major factor in serum resistance.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
In conducting this study we searched the Y. pestis KIM and CO92 databases and encountered conflicting nomenclature; the protein in the KIM6 database was designated OmpX, whereas the homologue in the CO92 strain was designated Ail. Although an Ail designation could be appropriate, because our work was conducted with Y. pestis KIM6+, we used the OmpX designation in this report to be consistent with the current database designation. Furthermore, Y. pestis has three additional ail-like genes with varying levels of predicted sequence similarity, and to designate ompX as ail would require a further qualifier as to which ail homologue was being addressed. Until a unified nomenclature is assigned, we have used ompX to limit potential confusion. However, we recommend that a standard nomenclature be assigned through future research and discussions.
Inactivation of ompX reduced epithelial cell association and internalization 90 and 98 %, respectively. At present we cannot distinguish whether the increased cell association of Y. pestis expressing OmpX is due directly to the effects of OmpX or indirectly due to OmpX-mediated autoaggregation. The residual cell association observed for the ompX deletion mutant is consistent with evidence suggesting that Y. pestis interaction with non-professional phagocytes is multifactorial. Kukleva et al. (2000) noted that a 22 kDa protein in the Y. pestis outer membrane promoted adherence to phagocytes; the protein in their study was not identified, nor were its effects on non-professional phagocytes analysed. Other proteins are involved in adhesion and invasion by Y. pestis (Cowan et al., 2000; Lahteenmaki et al., 1998, 2001a, b, 2003; Leigh et al., 2005; Liu et al., 2006). The role of the pCP1 plasmid, encoding the plasminogen activator (Pla protease), in interaction with HeLa cells has been reported (Cowan et al., 2000). Although the reduced cell association and internalization resulting from pCP1 curing was less than that caused by inactivating ompX in Y. pestis KIM6+, one cannot make direct comparisons since different cell culture systems and strains were used. Psa fimbriae are also adhesins, but have not been reported to promote internalization (Liu et al., 2006).
Y. pestis OmpX, in contrast to Y. enterocolitica Ail, mediates adherence and internalization when bacteria are grown at <30 °C. We propose two potential explanations for this difference. The first is that Y. enterocolitica Ail expression is temperature-regulated; low levels of Ail are produced at 28 °C and much higher levels are produced at 37 °C (Bliska & Falkow, 1992; Pierson & Falkow, 1993). However, Y. pestis OmpX expression is constitutive. The second is that artificial overexpression of Y. enterocolitica Ail at 28 °C does not promote internalization of Y. enterocolitica, indicating that additional factors contribute to Ail-mediated internalization (Bliska & Falkow, 1992). As shown previously, adherence conferred by Y. enterocolitica Ail is affected by the length or structure of O side-chains in lipopolysaccharide (LPS) which are temperature-regulated (Pierson, 1994). Presumably because of steric interference, only bacteria with shortened O side-chains (Y. enterocolitica grown at 37 °C) can promote interaction between Ail and the host cell (Bliska & Falkow, 1992; Pierson, 1994). In contrast to Y. enterocolitica, Y. pestis LPS, due to mutation, lacks O-antigen regardless of growth temperature (Prior et al., 2001). This lack of O-antigen interference could explain OmpX-mediated internalization at low temperature. The length of O-antigen has been previously shown to regulate functions of other proteins such as Pla and PgtE (Kukkonen et al., 2004). LPS structural diversity (Skurnik et al., 2000; Zhou et al., 2004) might also explain differences between OmpX in Y. pestis and Ail in Y. pseudotuberculosis. Despite sharing nearly 100 % sequence homology with Y. pestis OmpX, Ail fails to confer adherence of Y. pseudotuberculosis to HEp-2 cells (Yang et al., 1996). Another possible explanation of this discrepancy is the one substitution (Phe vs Val) in the predicted third (out of four) surface-exposed-loop regions of Y. pseudotuberculosis Ail. Resolution of this issue by complementing the ompX : : npt mutant with ail is a goal of future studies.
Like Y. enterocolitica Ail and S. typhimurium Rck, OmpX mediates serum resistance (Heffernan et al., 1994; Miller et al., 1989, 1990). Disruption of ompX in this study increased sensitivity of Y. pestis cultured at either 28 or 37 °C. Resistance was attributed to a direct protective effect by OmpX from complement-mediated killing, rather than shielding of the bacteria from serum components within cell aggregates. Incubating the parental strain in the presence of serum caused complete dissociation of the cell aggregates by 4 h; yet the cells remained resistant to fresh serum added to the suspension at this point (results not shown). The protection by OmpX at either 28 or 37 °C contrasts with Y. enterocolitica Ail, which confers this trait only at 37 °C (Bliska & Falkow, 1992; Pierson & Falkow, 1993; Pierson, 1994), reflecting temperature regulation of Ail expression in that organism (Bliska & Falkow, 1992). OmpX expression at both temperatures probably helps ensure Y. pestis complement resistance by organisms multiplying in humans or following growth at ambient temperatures. It is clear that OmpX plays a significant role in serum resistance. Since the strains used in this study are wild-type with respect to Pla protease, our results are consistent with a previous report which demonstrated that deletion of Pla protease, known to degrade C3, does not apparently increase sensitivity to serum (Sodeinde et al., 1992). Comparative studies of another member of the Ail family, Rck of S. typhimurium, may suggest that the mode of OmpX action is through inhibition of C9 polymerization and formation of the mature membrane attack complex (MAC) on the bacterial surface (Heffernan et al., 1992b). It has also been reported that Y. pestis binds the complement regulatory protein C4bp as observed by Ngampasutadol et al. (2005). Whether Y. pestis OmpX is directly involved in these latter two processes is currently being examined.
The autoaggregation phenotype associated with pellicle formation and flocculent growth is characteristic of certain Y. pestis strains and connected with virulence (Laird & Cavanaugh, 1980). The parental KIM6+ strain has this property, which was lost by Y. pestis KIM6+ ompX : : npt grown at either 28 or 37 °C. These phenotypes have been associated with biofilms in some bacteria such as Campylobacter jejuni (Joshua et al., 2006), Mycobacterium smegmatis (Chen et al., 2006), and Pseudomonas aeruginosa (Friedman & Kolter, 2004) and are attributed to resistance to host defence mechanisms (Schembri et al., 2003). Other Y. pestis genetic loci, including hmsHFRS (in the pgm locus) and hmsT (Hare & McDonough, 1999), have been correlated with autoaggregation. The presence of hmsHFRS and hmsT in the Y. pestis strains used in this study was confirmed by growth on Congo red agar and by PCR, respectively (data not shown), excluding the possibility that the altered autoaggregation phenotype was attributed to loss of either hmsHFRS or hmsT. We also demonstrated that Y. pestis KIM6–, lacking pgm, autoaggregated. This finding supports studies by Podladchikova & Rykova (2006) on derivatives of the Y. pestis EV76 hms– pYV strain in which an unidentified bacterial cell surface protein of 17 kDa was involved in autoaggregation.
In summary, we have demonstrated a prominent role for Y. pestis OmpX in several potential virulence processes. Of three genes (inv, yadA and ail) associated with these processes in enteropathogenic Yersinia, yadA (Rosqvist et al., 1988) and inv (Simonet et al., 1996) are inactive in Y. pestis. Experiments are ongoing to investigate other roles of OmpX in pathogenesis and its potential application as a vaccine candidate.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: P. van der Ley
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bliska, J. B. & Falkow, S. (1992). Bacterial resistance to complement killing mediated by the Ail protein of Yersinia enterocolitica. Proc Natl Acad Sci U S A 89, 3561–3565.
Bobrov, A. G., Geoffroy, V. A. & Perry, R. D. (2002). Yersiniabactin production requires the thioesterase domain of HMWP2 and YbtD, a putative phosphopantetheinylate transferase. Infect Immun 70, 4204–4214.
Bockmann, R. A. & Caflisch, A. (2005). Spontaneous formation of detergent micelles around the outer membrane protein OmpX. Biophys J 88, 3191–3204.
Caspi, R., Foerster, H., Fulcher, C. A., Hopkinson, R., Ingraham, J., Kaipa, P., Krummenacker, M., Paley, S., Pick, J. & other authors (2006). MetaCyc: a multiorganism database of metabolic pathways and enzymes. Nucleic Acids Res 34, D511–D516.
Chen, J. M., German, G. J., Alexander, D. C., Ren, H., Tan, T. & Liu, J. (2006). Roles of Lsr2 in colony morphology and biofilm formation of Mycobacterium smegmatis. J Bacteriol 188, 633–641.
Cirillo, D. M., Heffernan, E. J., Wu, L., Harwood, J., Fierer, J. & Guiney, D. G. (1996). Identification of a domain in Rck, a product of the Salmonella typhimurium virulence plasmid, required for both serum resistance and cell invasion. Infect Immun 64, 2019–2023.[Abstract]
Cowan, C., Jones, H. A., Kaya, Y. H., Perry, R. D. & Straley, S. C. (2000). Invasion of epithelial cells by Yersinia pestis: evidence for a Y. pestis-specific invasin. Infect Immun 68, 4523–4530.
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.
de Lorenzo, V., Herrero, M., Jakubzik, U. & Timmis, K. N. (1990). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172, 6568–6572.
Dupont, M., De, E., Chollet, R., Chevalier, J. & Pages, J. M. (2004). Enterobacter aerogenes OmpX, a cation-selective channel mar- and osmo-regulated. FEBS Lett 569, 27–30.[CrossRef][Medline]
Dziewanowska, K., Carson, A. R., Patti, J. M., Deobald, C. F., Bayles, K. W. & Bohach, G. A. (2000). Staphylococcal fibronectin binding protein interacts with heat shock protein 60 and integrins: role in internalization by epithelial cells. Infect Immun 68, 6321–6328.
Friedman, L. & Kolter, R. (2004). Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Mol Microbiol 51, 675–690.[CrossRef][Medline]
Gasteiger, E. H. C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D. & Bairoch, A. (2005). Protein Identification and Analysis Tools on the ExPASy Server. In The Proteomics Protocols Handbook, pp. 571–607. Edited by J. M. Walker. Totowa, NJ: Humana Press.
Gay, P., Le Coq, D., Steinmetz, M., Ferrari, E. & Hoch, J. A. (1983). Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli. J Bacteriol 153, 1424–1431.
Hare, J. M. & McDonough, K. A. (1999). High-frequency RecA-dependent and -independent mechanisms of Congo red binding mutations in Yersinia pestis. J Bacteriol 181, 4896–4904.
Heffernan, E. J., Harwood, J., Fierer, J. & Guiney, D. (1992a). The Salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail. J Bacteriol 174, 84–91.
Heffernan, E. J., Reed, S., Hackett, J., Fierer, J., Roudier, C. & Guiney, D. (1992b). Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck. J Clin Invest 90, 953–964.[Medline]
Heffernan, E. J., Wu, L., Louie, J., Okamoto, S., Fierer, J. & Guiney, D. G. (1994). Specificity of the complement resistance and cell association phenotypes encoded by the outer membrane protein genes rck from Salmonella typhimurium and ail from Yersinia enterocolitica. Infect Immun 62, 5183–5186.
Joshua, G. W., Guthrie-Irons, C., Karlyshev, A. V. & Wren, B. W. (2006). Biofilm formation in Campylobacter jejuni. Microbiology 152, 387–396.
Karp, P. D., Ouzounis, C. A., Moore-Kochlacs, C., Goldovsky, L., Kaipa, P., Ahrén, D., Tsoka, S., Darzentas, N., Kunin, V. & López-Bigas, N. (2005). Expansion of the BioCyc collection of pathway/genome databases to 160 genomes. Nucleic Acids Res 33, 6083–6089.
Katayama, H., Nagasu, T. & Oda, Y. (2001). Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 15, 1416–1421.[CrossRef][Medline]
Kukkonen, M., Suomalainen, M., Kyllonen, P., Lahteenmaki, K., Lang, H., Virkola, R., Helander, I. M., Holst, O. & Korhonen, T. K. (2004). Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica. Mol Microbiol 51, 215–225.[CrossRef][Medline]
Kukleva, L. M., Zadnova, S. P., Shcherbakov, A. A. & Protsenko, O. A. (2000 ). The role of the protein with molecular weight of 22 kD in the phagocytosis of vaccine strain of Yersinia pestis EV. Zh Mikrobiol Epidemiol Immunobiol 55–58.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[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. (2001a). The Pla surface protease/adhesin of Yersinia pestis mediates bacterial invasion into human endothelial cells. FEBS Lett 504, 69–72.[CrossRef][Medline]
Lahteenmaki, K., Kuusela, P. & Korhonen, T. K. (2001b). Bacterial plasminogen activators and receptors. FEMS Microbiol Rev 25, 531–552.[Medline]
Lahteenmaki, K., Kukkonen, M., Jaatinen, S., Suomalainen, M., Soranummi, H., Virkola, R., Lang, H. & Korhonen, T. K. (2003). Yersinia pestis Pla has multiple virulence-associated functions. Adv Exp Med Biol 529, 141–145.[Medline]
Laird, W. J. & Cavanaugh, D. C. (1980). Correlation of autoagglutination and virulence of Yersiniae. J Clin Microbiol 11, 430–432.
Lee, K., Wang, T., Paszczynski, A. J. & Daoud, S. S. (2006). Expression proteomics to p53 mutation reactivation with PRIMA-1 in breast cancer cells. Biochem Biophys Res Commun 349, 1117–1124.[CrossRef][Medline]
Leigh, S. A., Forman, S., Perry, R. D. & Straley, S. C. (2005). Unexpected results from the application of signature-tagged mutagenesis to identify Yersinia pestis genes required for adherence and invasion. Microb Pathog 38, 259–266.[CrossRef][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.
McEvedy, C. (1988). The bubonic plague. Sci Am 258, 118–123.[Medline]
Mecsas, J., Welch, R., Erickson, J. W. & Gross, C. A. (1995). Identification and characterization of an outer membrane protein, OmpX, in Escherichia coli that is homologous to a family of outer membrane proteins including Ail of Yersinia enterocolitica. J Bacteriol 177, 799–804.
Miller, V. L. & Falkow, S. (1988). Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of epithelial cells. Infect Immun 56, 1242–1248.
Miller, S. I., Kukral, A. M. & Mekalanos, J. J. (1989). A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc Natl Acad Sci U S A 86, 5054–5058.
Miller, V. L., Bliska, J. B. & Falkow, S. (1990). Nucleotide sequence of the Yersinia enterocolitica ail gene and characterization of the Ail protein product. J Bacteriol 172, 1062–1069.
Miller, V. L., Beer, K. B., Heusipp, G., Young, B. M. & Wachtel, M. R. (2001). Identification of regions of Ail required for the invasion and serum resistance phenotypes. Mol Microbiol 41, 1053–1062.[CrossRef][Medline]
Ngampasutadol, J., Ram, S., Blom, A. M., Jarva, H., Jerse, A. E., Lien, E., Goguen, J., Gulati, S. & Rice, P. A. (2005). Human C4b-binding protein selectively interacts with Neisseria gonorrhoeae and results in species-specific infection. Proc Natl Acad Sci U S A 102, 17142–17147.
Nielsen, H. & Krogh, A. (1998). Prediction of signal peptides and signal anchors by a hidden Markov model. Proc Int Conf Intell Syst Mol Biol 6, 122–130.[Medline]
Pierson, D. E. (1994). Mutations affecting lipopolysaccharide enhance Ail-mediated entry of Yersinia enterocolitica into mammalian cells. J Bacteriol 176, 4043–4051.
Pierson, D. E. & Falkow, S. (1993). The ail gene of Yersinia enterocolitica has a role in the ability of the organism to survive serum killing. Infect Immun 61, 1846–1852.
Podladchikova, O. N. & Rykova, V. A. (2006). Identification of the autoagglutination factor of Hms– cells of the plague agent. Mol Gen Mikrobiol Virusol 25–29.
Prior, J. L., Hitchen, P. G., Williamson, D. E., Reason, A. J., Morris, H. R., Dell, A., Wren, B. W. & Titball, R. W. (2001). Characterization of the lipopolysaccharide of Yersinia pestis. Microb Pathog 30, 49–57.[CrossRef][Medline]
Rebeil, R., Ernst, R. K., Jarrett, C. O., Adams, K. N., Miller, S. I. & Hinnebusch, B. J. (2006). Characterization of late acyltransferase genes of Yersinia pestis and their role in temperature-dependent lipid A variation. J Bacteriol 188, 1381–1388.
Rosqvist, R., Skurnik, M. & Wolf-Watz, H. (1988). Increased virulence of Yersinia pseudotuberculosis by two independent mutations. Nature 334, 522–524.[CrossRef][Medline]
Schembri, M. A., Kjaergaard, K. & Klemm, P. (2003). Global gene expression in Escherichia coli biofilms. Mol Microbiol 48, 253–267.[CrossRef][Medline]
Seo, K. S., Lee, S. U., Park, Y. H., Davis, W. C., Fox, L. K. & Bohach, G. A. (2007). Long-term staphylococcal enterotoxin C1 exposure induces soluble factor-mediated immunosuppression by bovine CD4+ and CD8+ T cells. Infect Immun 75, 260–269.
Shaffer, J. & Goldin, M. (1974). Clinical Diagnosis by Laboratory Methods, 15th edn. Philadelphia, PA: W.B. Saunders Company.
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. (1996). Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68, 850–858.[Medline]
Simonet, M., Riot, B., Fortineau, N. & Berche, P. (1996). Invasin production by Yersinia pestis is abolished by insertion of an IS200-like element within the inv gene. Infect Immun 64, 375–379.[Abstract]
Skurnik, M., Peippo, A. & Ervela, E. (2000). Characterization of the O-antigen gene clusters of Yersinia pseudotuberculosis and the cryptic O-antigen gene cluster of Yersinia pestis shows that the plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype O:1b. Mol Microbiol 37, 316–330.[CrossRef][Medline]
Smith, M. J. (2000). Genetic regulation of type III secretion systems in Yersinia enterocolitica, p. 148. PhD thesis, University of Idaho.
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.
Stoorvogel, J., van Bussel, M. J., Tommassen, J. & van de Klundert, J. A. (1991). Molecular characterization of an Enterobacter cloacae outer membrane protein (OmpX). J Bacteriol 173, 156–160.
Surgalla, M. J. & Beesley, E. D. (1969). Congo red-agar plating medium for detecting pigmentation in Pasteurella pestis. Appl Microbiol 18, 834–837.[Medline]
Titball, R. W. & Williamson, E. D. (2004). Yersinia pestis (plague) vaccines. Expert Opin Biol Ther 4, 965–973.[CrossRef][Medline]
Vandenbosch, J. L., Rabert, D. K. & Jones, G. W. (1987). Plasmid-associated resistance of Salmonella typhimurium to complement activated by the classical pathway. Infect Immun 55, 2645–2652.
Wren, B. W. (2003). The yersiniae – a model genus to study the rapid evolution of bacterial pathogens. Nat Rev Microbiol 1, 55–64.[CrossRef][Medline]
Yang, Y., Merriam, J. J., Mueller, J. P. & Isberg, R. R. (1996). The psa locus is responsible for thermoinducible binding of Yersinia pseudotuberculosis to cultured cells. Infect Immun 64, 2483–2489.[Abstract]
Zhou, D., Han, Y., Song, Y., Huang, P. & Yang, R. (2004). Comparative and evolutionary genomics of Yersinia pestis. Microbes Infect 6, 1226–1234.[CrossRef][Medline]
Zietz, B. P. & Dunkelberg, H. (2004). The history of the plague and the research on the causative agent Yersinia pestis. Int J Hyg Environ Health 207, 165–178.[CrossRef][Medline]
Received 31 December 2006;
revised 18 May 2007;
accepted 21 May 2007.
This article has been cited by other articles:
|
|
 |
|
  J. L. Spinner, J. A. Cundiff, and S. D. Kobayashi Yersinia pestis Type III Secretion System-Dependent Inhibition of Human Polymorphonuclear Leukocyte Function Infect. Immun., August 1, 2008; 76(8): 3754 - 3760. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Zhang, M. Skurnik, S.-S. Zhang, O. Schwartz, R. Kalyanasundaram, S. Bulgheresi, J. J. He, J. D. Klena, B. J. Hinnebusch, and T. Chen Human Dendritic Cell-Specific Intercellular Adhesion Molecule-Grabbing Nonintegrin (CD209) Is a Receptor for Yersinia pestis That Promotes Phagocytosis by Dendritic Cells Infect. Immun., May 1, 2008; 76(5): 2070 - 2079. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
|
, Y. pestis KIM6+ Nalr; 
, Y. pestis KIM6+ Nalr 
, Y. pestis KIM6+ Nalr ompX+/ompX : : npt. The number of surviving bacteria after incubation with NHS is presented as a percentage of the number of bacteria incubated in HIS (100 %). Data in parentheses represent bacterial cell numbers for each strain (x105) following incubation in HIS for 0, 0.5, and 1 h respectively. These values represent 100 % survival in HIS at each time point. Results are the mean±SEM from data derived from two assays performed on separate days (n=6). * and ** indicate statistically significant differences (P<0.002 and P<0.001, respectively) at the 0.5 and 1 h time points between the deletion mutant and either of the other two strains tested.