FEMS MicrobiologyLoiters 97 (1992) 51-56 © 1992 Federation of European MicrobiologicalSocieties 0378-11197/92/$05.00 Published by Elsevier
FEMSLE 05062
Prevalence of ompT among Escherichia coli isolates of human origin M i c h a e l D. L u n d r i g a n ~ a n d Risa M. W e b b b "Department of Microbiolol,9', and b Department of Medicine. Dicisionof h~fi'ctious Diseases. Unicersityof Mississippi Medical Center. Jackson, Mississippi. USA Received 30 June 1992 Accepted 2 July ITS)2 Key words: OmpT; Outer membrane; Plasminogen activator; Virulence factor; Escherichia coli 1. S U M M A R Y O m p T is a protease associated with the outer membrane of Escherichia coli and possesses a high degree of homology to the plasminogen activator, Pla, of Yersinia pestis. We show here that O m p T from intact cells can indeed activate plasminogen. Clinical specimens of E. coli were examined for protease activity and for the o m p T gene. Few isolates (12%) were found to be positive for O m p T activity, whereas most (77%) carried the o m p T gene and expressed the cloned protease gene. In this report we present evidence suggesting that the surface architecture of E. coli influences the activity of O m p T and that O m p T may be indicative of the pathogenic potential of the organism.
2. I N T R O D U C T I O N O m p T is the only protease clearly shown to be localized to the outer surface of Escherichia coli Correspondence to: M.D. Lundrigan, Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216. USA.
[1,2]. A number of proteins may serve as substrates for O m p T [2-7]; however, the actual physiological function of O m p T remains elusive. The gene encoding the O m p T protease has been mapped, cloned and sequenced [1,8,9]. The o m p T nucleotide sequence is similar to the plasminogen activator gene, pla, of Yersinia pestis [10]. In addition, both O m p T and Pla degrade certain outer membrane proteins [11,12]. O m p T synthesis is temperature-dependent; synthesis occurs at 37°C but not at 27°C [13]. This regulation may reflect an adaptive response necessary for the survival or pathogenicity of the microbe. Deletion of the o m p T gene has no obvious effect on the growth rate of E. coli under typical laboratory conditions [6]. The effect of different environments on an ompT-negative cell remains to be elucidated. E. coli typically inhabits the bowel. Some strains can invade epithelial cells and multiply in the underlying mucosa leading to a systemic infection, while others are commonly found in urinary tract infections. The ability to overcome physical barriers and other host defense mechanisms clearly contributes to the pathogenic potential. A role for bacterial cell surface proteases in circumventing host defense mechanisms has not
been we[[ demonstrated, although the Y. pestis plasminogen activator has been implicated as a virulence factor [12]. Sodeinde and Goguen [10] have proposed that Y. pestis requires this plasminogen to invade the bloodstream from an intradermal site of infection. In a similar fashion, the plasminogen-like activator of E. coli, OmpT, may allow invasion of a mucosal surface, resulting in bacteremia, wound infection or pyelonephritis. This report shows a correlation may exist between the presence of OmpT in E. coli and the infectivity.
3. MATERIALS AND METHODS
3.'. Bacterial strains and plasmids The laboratory strains of E. coli used in this study were RW193 (F-, azi-6 lacYl leu-6 mtl-I proC14 rpsLl09 thi-I trpE38 tsx-67 entA403) and UT4400 (RW193 AompT-fepA) [1]. The 282 clinical strains studied were isolated by the Clinical Laboratories Department of the University of Mississippi Medical Center and are designated by letters (see Table 1) indicating the source of the isolate followed by an isolate number. The characteristics of these strains relevant to this study are compiled in Table 1. These strains were mainta!ned on EMB or L-agar plates [14]. Plasmid-containing strains were grown on L-plates or broth containing ampicillin for the ompT-complementing plasmids pML19 and pML21 [9] or kanamycin at 25 p.g/ml for pML23. Plasmid pML23 was constructed by inserting a kanamycin gene cartridge as a Smal fragment from pUC4KIXX (Pharmacia LKB Biotechnology Inc.) into the Smal sites of pML19, thereby inactivating the ompT gene. 3.2. Protease assays The standard assay for OmpT activity was the cleavage of T7 RNA polymerase [2] in which cells (0.25 absorbance units) were incubated with 4 p.g intact T7 RNA polymerase (kindly provided by J.J. Dunn). After incubation of the reaction mixture for 1 h at 37°C, the cells were removed by centrifugation and the cleavage products analysed by SDS-polyacrylamide gel electrophoresis
(PAGE). The ability of OmpT to activate plasminogen was tested by the fibrin film lysis assay [15]. Overnight cultures (l ml) grown in L-broth at 37°C were harvested, washed once in borate buffer [16] and suspended in 1/10 volume of borate buffer. The washed cells were serially diluted and then spotted onto a fibrin film which had been prepared by adding 25 NIH units of bovine thrombin in 0.5 ml of borate buffer to 10 mi of 0.5% bovine fibrinogen in a Petri dish. Chioramphenicol had been added to the fibrinogen solution to prevent bacterial growth.
3.3. Outer membrane preparation Isolation of E. coli outer membrane by detergent solubilization and ultracentrifugation was according to the procedure of Filip et al. [17]. Cultures were adjusted to an A540 of 2.0 prior to membrane isolation. 3.4. Electrophoresis The Lugtenberg gel system [18] was used for electrophoresis of outer membrane proteins with 11% polyacrylamide, 0.2% sodium dodecyl sulfate (SDS), and 0.375 M Tris. HCI, pH 8.8. For electrophoresis of T7 RNA polymerase cleavage products, the polyacrylamide concentration was increased to 14%. 3.5. Genetic techniques Manipulations of plasmid DNA were performed by standard techniques [14]. Screening of E. coli strains for the presence of the ompT gene was accomplished by colony hybridization [14]. An 800-bp Hincll fragment obtained from plasmid pML21 and purified from an agarose gel was labelled with [a-32p]dCTP by 'oligolabelling' as described by Feinberg and Vogelstein [19]. This fragment contains most of the ompT coding region. Colonies were transferred to 'Colony/Plaque Screen' membranes (E.l. du Pont De Nemours & Co.) by the method of Grunstein and Hogness [20] and were hybridized at 65°C to the labelled DNA probe according to the procedures provided by the manufacturer. Washing was with 2 × SSC, 1% SDS at 65°C. After hybridization the membranes were analysed by autoradiography.
4. R E S U L T S A N D DISCUSSION
below). A derivative of RWI93, UT4400, in which the ompT gene has been deleted, failed to degrade the polymerase (lane 3). T h e effect of three clinical isolates on the cleavage on the polymerase is shown in lanes 4, 5 and 6. Isolate D U I 8 produced a cleavage pattern (lane 5) identical to RWI93, whereas the o t h e r two isolates, D U l 7 and D U I 9 (lanes 4 and 6), did not. The multitude of bands in lane 6 was due to whole cells that were carried over into the electrophoresis sample. Table l is a compilation of the results obtained by screening h u m a n isolates of E. coil for surface protease activity. Only 34 (12.1%) of the 282 h u m a n isolates were positive for O m p T protease activity. Electrophoresis of outer m e m b r a n e proteins obtained from the three clinical isolates described above (Fig. 2, lanes 5 - 1 0 ) did not reveal a thermoregulatcd polypeptide of a molecular mass close to that of O m p T (Fig. 2, lanes l, 2, and 17, 18). This was expected for isolates D U I 7 and
G r o d b e r g and D u n n [2] developed a sensitive assay for detection of the O m p T protease. This assay tests the ability of whole ceils to degrade T7 R N A polymerase and was used here to screen 282 h u m a n isolates of E. coli for the presence of O m p T proteolytic activity. Figure 1 is a typical electrophoretogram displaying the results of such assays. The T7 R N A polymerase m o n o m e r is a 100-kDa protein as shown in lane 1 of Fig. I. W h e n the polymerase was exposed to the surface of strain RW193 (lane 2), in which ompT is chromosomally encoded, specific cleavages occurred between residues 172 (lysine) and 173 (arginine) or between the two lysine residues 179 and 180, resulting in polypeptide fi'agments of about 80 kDa and 20 kDa molecular mass [2]. An additional fragment of about 15 kDa was occasionally seen and its appearance was d e p e n d e n t on the O m p T concentration in the assay (see
1
2
3
4
56
7 8
mm 66"
910111213
-,- qUD~,-,,O .
t~lm-"~
.
.
.
.
.
KD
.
IIIP
..... ~
' ~ ~
-100 -80
45" -40 35"
~
24~
.... ~
"20
18.414.3-
"15
Fig. I. Assay of OmpT protease activity on the cell surface. T7 RNA polymerase was exposed to cells and then separated as described in MATERIALSAND METHODS.Proteins were visualized with Coomassie blue. Molecular mass standards are indicated at the left and approximate molecular masses are shown at the right. Lane 1 is l ,ug of T7 RNA polymerase. Lanes: (2) RWI93: (3) UT4400; (4) DUI7; (5) DUI8; (6) DuIg; (7) DUI7/pMLIg; (8) DUI8/pMLI9: (9) DUIg/pMLI9: (10) UT4400/pMLI9; (11) UT4400/pML19 I : 10; (12) UT4400/pMLI9 I : 100; and (13) UT44()I)/pMLI9 I : 1000.
54 Table 1 Incidence of OmpT among human E. coil isolates Source :' and number h of imh,tes
Occurrence of OmpT its determined by Activity " Ilybridization d
DU (196) DS (8) C (26) A (5) B (26)
I3G
76f~,;
0r,~ Igt:~ I)"~;
I(H)C:,
8c; 5~;
IIXIr~
BC
(21)
5()C~"
73c; 73r;
" DU, urine: DS. st(~l: C. wounds; A. respiratnry: B, geMtourinary: BC. bh~d. h A total of 282 isolates were tested fi)r protease activity and for the ability to hybridize tn t.npT DNA. ¢ Protease activitywas assayed by the ability nf whole cells to cleave T7 RNA polymerase. d Radiolabelled ompT DNA was hybridized to colony blots. DUI9, which lack O m p T activity, but not for isolate DU18 that demonstrated O m p T activity. O m p T protein could not be unequivocally identified from the outer membrane protein profile of an additional 21 protease-positive and 17 protease-negative strains (data not shown). It was possible that these strains could not express O m p T to high enough levels to be detected on polyacrylamide gels. Therefore the three clinical isolates: DUI7, DUI8, and DU19 were' transformed with pML19 [9], an o m p T complementing plasmid, and outer membrane proteins examined
1 2
3
4
5
6
7
8
by PAGE. The amount of O m p T present among the pMLlg-transformed isolates was nearly the same for each one (Fig. 2, lanes l l - 1 6 ) and expression of the protease was thermorcgulated. Although there may be a decrease in o m p T expression in these isolates compared to the Kl2 strain U T 4 4 0 0 / p M L 1 9 (Fig. 2, lanes 17 and 18), there is no significant difference in the amount of O m p T protein in the outer membrane among these three clinical strains. O m p T activity from these transformants was also assayed. When carrying pMLl9, isolate DU17 produced weak protease activity (Fig. l, lane 7), i~olate D U I 9 produced moderate activity (lane 9), and isolate D U i 8 produced strong activity (lane 8) comparable to the activity of U T 4 4 0 0 / p M L 1 9 when diluted I : l0 (lane ll). Serogrouping of 50 of the isolates did not reveal a common O or K antigen among those isolates which did or did not express O m p T activity (data not shown) nor was a specific colony morphology associated with O m p T phenotype, Clearly the differences in O m p T activity were due to factors other than expression of the o m p T gene and localization of the protein to the outer membrane. Complete degradation of the T7 RNA polymerase occurred when the polymerase was exposed to cells containing high levels of O m p T (Fig. l, lane 10). When plasmid p M L I 9 transformed cells, and hence the amount of protease,
9101112131415161718
4534.7-
-Omp Z { Ompr.. ump~
-OmpA
24" Fig. 2. Outer membrane proteins of E. cnli strains. Outer membranes were prepared from cells grown at 37°C and 27°C to stationary phase in L-broth. Relevant E. coil K-12 proteins are indicated on the right. Strains examined and growth temperatures were: (1) RWI93, 37°C: (2) RW193, 27°C; (3) UT4400, 37°C; (4) UT4400, 27°C:(5) DUI7, 37°C: (6) DUI7, 27°C: (7) DU~.8, 37°C: (8) DUI8, 27°C; (9) DuIg, 37°C: (10) DuIg, 27°C; (I 1) DUI7/pMLI9, 37°C;(12) DUI7/pML19, 27°C;(13) DUIS/pMLlg, 37°C; (14) DUI8/pMLIg, 27°C;(I5) DU 19/pMLIg, 37°C; (16) DUIg/pMLIg, 27°C;(17) UT4400/pMLIg, 37°C; (18) UT4400/pMLIg, 27°C.
107
NUMBER OF CELLS 106 105
104
STRAIN RW193
UT4400
UT4400/pML19
UT4400/pML23
C8
Fig. 3. Fibrin film lysis assay for plasminogen activator activity. Washed cells were diluted and spotted onto a fibrin film prepared [~'om bovine fibrinogen and thrombin. Inoculated films were incubated at 37°C for 12 h. The number of cells contained in each 10-p.I spot is shown at the top of the figure and the strains examined are indicated at the left edge. See text.
were diluted the degradation was reduced and the specific cleavage pattern reestablished (Fig. 1, lanes 10-13). This observation suggests that this protease has two proteolytic activities; a specific e n d o p e p t i d a s e activity which proceeds very quickly and a slower non-specific peptidase activity which results in complete degradation of the protein. Lysis of fibrin films was used by Sodeinde and G o g u e n [10] to assay the plasminogen activator, Pla, of Yersinia pestis and Sabnonella typhimurium protein E, when it was present on a high copy n u m b e r plasmid. However, Soldeinde a n d G o g u e n were not able to detect plasminogen activator activity by chromosomally encoded protein E or OmpT. We show here for the first time that O m p T can dissolve a fibrin film. Figure 3 shows a fibrin film assay which demonstrates that OmpT, encoded by the plasmid pMLI9, can dissolve fibrin. The control strains UT4400 and
UT44(XI/pML23 (ompT::Kan), do not dis.~)lve the fibrin film. Additonally, UT4400 containing pUCIg, the vector from which p M L I 9 was derived, does not dissolve fibrin (data not shown). in agreement with the findings of Sodeinde and Goguen, RW193, which carries ompT on the chromosome, did not dissolve the fibrin with 107 cells per spot; thus, for fibrin lysis the protease must be present in concentrations greater than that produced by the haploid gene. In contrast to Pla, O m p T has no detectable coagulase activity even when expressed from a multicopy plasmid (data not shown). All positive and 4(I negative strains were tested for their ability to dissolve fibrin. Of these, only one O m p T ~ isolate (C8) was positive (Fig. 3). E n h a n c e d plasminogen activation by O m p T from the C8 isolate could be due to a more active enzyme conformation or the surface architecture of the cell could be such that the OmpT-active site is more accessible to the substrate, plasminogen. Because the isolates that cleaved T7 R N A polymerase did not clearly display an OmpT-like protein on electrophoretic analysis of m e m b r a n e proteins and some isolates were unable to express plasmid-encoded O m p T protease activity, we used "colony hybridization" to determine whether the strains had the ompT structural gene. O f 282 E. coil isolates tested, 217 or 77% were found to hybridize to radiolabelled ompT D N A (Table 1). Each isolate was tested for hybridization at least three times with virtually no ambiguity as to the results. The presence of the ompT gene correlated with blood culture isolates ( P = 0.0386, by Chi-square statistic), whereas the proteolytic activity was more commonly found in urine and wound isolates. Wher~ blood and respiratory isolates were considered as one group, the correlation with ompT hybridization became even more significant ( P = 0.0147). We show here that different E. coli isolates vary in their ability to express O m p T protease activity. The probable reason for this observation is that these strains differ in their surface architecture such that in the majority of strains the substrate is inaccessible to the active site. The effect of surface architecture could be: (!) to assure proper insertion and orientation of the
p r o t e a s e in the o u t e r m e m b r a n e ; (2) to provide n e e d e d interactions with o t h e r m e m b r a n e c o m p o n e n t s ; o r (3) to form a surface relatively free o f i m p e d i n g structures. A l t h o u g h not investigated h e r e , changing growth conditions (e.g. growth in s e r u m ) might alter t h e cell e n v e l o p e such that a higher p e r c e n t a g e o f strains could cleave T7 R N A polymerase o r dissolve fibrin films.
ACKNOWLEDGEMENTS W e thank J.E.L. A r c e n e a u x and S.W. C h a p m a n for their constructive c o m m e n t s c o n c e r n i n g the manuscript. In addition, we wish to t h a n k E.F. M e y d r e c h for statistical analysis o f the data. This work was s u p p o r t e d in p a r t by G r a n t 2 SO7 RR05386 A w a r d s by the Biomedical R e s e a r c h S u p p o r t G r a n t P r o g r a m , Division o f R e s e a r c h Resources, National Institutes o f Health.
REFERENCES [1] Earhart, C.F., Lundrigan, M.. Pickett, C.L. and Pierce, J.R. (1979) FEMS MicrobioL Lett. 6, 277-280. [2] Grodberg, J. and Dunn, J.J. (1988) J. Bacteriol. 170, 1245-1253. [3] Sugimura, K. and Higashi, N. (1988) J. BacterioL 170, 3650-3654.
[4] Sedgwick, B. (1989) J. Bacleriol. 17I, 2249-2251. [5] Hellebust, H., Murby, M., Abrahms~n, L., Uhl~n, M. and Enfors, S.O. (1989) Bio/Technology 7, 165-168. [6] Baneyx0 F. and Georgiou, G. (1990) J. Bacteriol. 172, 491-494. [7] Akiyama, Y. and lto, K. (1990) Biochem. Biophys. Res. Comm. 167, 711-715. [8] Rupprecht. K.R., Gordon, G., Lundrigan. M., Gayda, R.C., Markovitz, A. and Earhart, C.F. (1983)J. Bacteriol. 153, 1104-106. [9] Grodberg, J., Lundrigan, M.D., Toledo, D.L, Mangel, W.F. and Dunn, J.J. (1988) Nucleic Acids Res. 16, 1209. [10] Sodeinde, O.A. and Goguen, J.D. (1989) Infect. Immun. 57, 1517-1523. [11] Fiss, E.H., Hollifield, W.C. Jr. and Neilands, J.B. (1979) Biochem. Biophys. Res. Commun. 91, 29-34. [12] Sodeinde, O.A., Sample, A.K., Brubaker, R.R. and Goguen, J.D. (1988) Infect. Immun. 56, 2749-2752. [13] Lugtenberg, B., Peters, R., Bernheimer, H. and Berendsen, W. (1976) MoL Gem Genet. 147, 251-262. [14] Maniatis, T., Fritsch, E.F. and Sambrook, J.E. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [15] Beesley, E.D., Brubaker, R.R., Jansen, W.A. and Surgalla, M.J. (1967) J. Bacteriol. 165, 19-26. [16] Lewis, J.H. and Ferguson, J.H. (1950)J. a i m Invest. 29, 486-490. [17] Filip, C., Fletcher, G., Wulff, J.L. and Earhart, C.F. (1973) J. Bacteriol. 115. 717-722. [18] Lugtenberg, B., Meijers, J., Peters, R., Van der Hock, P. and Van Alphen, L. (1975) FEBS Left. 58, 254-258. [19] Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. [20] Grunstein. M. and Hogness, D. (1975) Proc. Natl. Aead. Sci. USA 72, 3961-3965.
FEMSLE 05062
Prevalence of ompT among Escherichia coli isolates of human origin M i c h a e l D. L u n d r i g a n ~ a n d Risa M. W e b b b "Department of Microbiolol,9', and b Department of Medicine. Dicisionof h~fi'ctious Diseases. Unicersityof Mississippi Medical Center. Jackson, Mississippi. USA Received 30 June 1992 Accepted 2 July ITS)2 Key words: OmpT; Outer membrane; Plasminogen activator; Virulence factor; Escherichia coli 1. S U M M A R Y O m p T is a protease associated with the outer membrane of Escherichia coli and possesses a high degree of homology to the plasminogen activator, Pla, of Yersinia pestis. We show here that O m p T from intact cells can indeed activate plasminogen. Clinical specimens of E. coli were examined for protease activity and for the o m p T gene. Few isolates (12%) were found to be positive for O m p T activity, whereas most (77%) carried the o m p T gene and expressed the cloned protease gene. In this report we present evidence suggesting that the surface architecture of E. coli influences the activity of O m p T and that O m p T may be indicative of the pathogenic potential of the organism.
2. I N T R O D U C T I O N O m p T is the only protease clearly shown to be localized to the outer surface of Escherichia coli Correspondence to: M.D. Lundrigan, Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216. USA.
[1,2]. A number of proteins may serve as substrates for O m p T [2-7]; however, the actual physiological function of O m p T remains elusive. The gene encoding the O m p T protease has been mapped, cloned and sequenced [1,8,9]. The o m p T nucleotide sequence is similar to the plasminogen activator gene, pla, of Yersinia pestis [10]. In addition, both O m p T and Pla degrade certain outer membrane proteins [11,12]. O m p T synthesis is temperature-dependent; synthesis occurs at 37°C but not at 27°C [13]. This regulation may reflect an adaptive response necessary for the survival or pathogenicity of the microbe. Deletion of the o m p T gene has no obvious effect on the growth rate of E. coli under typical laboratory conditions [6]. The effect of different environments on an ompT-negative cell remains to be elucidated. E. coli typically inhabits the bowel. Some strains can invade epithelial cells and multiply in the underlying mucosa leading to a systemic infection, while others are commonly found in urinary tract infections. The ability to overcome physical barriers and other host defense mechanisms clearly contributes to the pathogenic potential. A role for bacterial cell surface proteases in circumventing host defense mechanisms has not
been we[[ demonstrated, although the Y. pestis plasminogen activator has been implicated as a virulence factor [12]. Sodeinde and Goguen [10] have proposed that Y. pestis requires this plasminogen to invade the bloodstream from an intradermal site of infection. In a similar fashion, the plasminogen-like activator of E. coli, OmpT, may allow invasion of a mucosal surface, resulting in bacteremia, wound infection or pyelonephritis. This report shows a correlation may exist between the presence of OmpT in E. coli and the infectivity.
3. MATERIALS AND METHODS
3.'. Bacterial strains and plasmids The laboratory strains of E. coli used in this study were RW193 (F-, azi-6 lacYl leu-6 mtl-I proC14 rpsLl09 thi-I trpE38 tsx-67 entA403) and UT4400 (RW193 AompT-fepA) [1]. The 282 clinical strains studied were isolated by the Clinical Laboratories Department of the University of Mississippi Medical Center and are designated by letters (see Table 1) indicating the source of the isolate followed by an isolate number. The characteristics of these strains relevant to this study are compiled in Table 1. These strains were mainta!ned on EMB or L-agar plates [14]. Plasmid-containing strains were grown on L-plates or broth containing ampicillin for the ompT-complementing plasmids pML19 and pML21 [9] or kanamycin at 25 p.g/ml for pML23. Plasmid pML23 was constructed by inserting a kanamycin gene cartridge as a Smal fragment from pUC4KIXX (Pharmacia LKB Biotechnology Inc.) into the Smal sites of pML19, thereby inactivating the ompT gene. 3.2. Protease assays The standard assay for OmpT activity was the cleavage of T7 RNA polymerase [2] in which cells (0.25 absorbance units) were incubated with 4 p.g intact T7 RNA polymerase (kindly provided by J.J. Dunn). After incubation of the reaction mixture for 1 h at 37°C, the cells were removed by centrifugation and the cleavage products analysed by SDS-polyacrylamide gel electrophoresis
(PAGE). The ability of OmpT to activate plasminogen was tested by the fibrin film lysis assay [15]. Overnight cultures (l ml) grown in L-broth at 37°C were harvested, washed once in borate buffer [16] and suspended in 1/10 volume of borate buffer. The washed cells were serially diluted and then spotted onto a fibrin film which had been prepared by adding 25 NIH units of bovine thrombin in 0.5 ml of borate buffer to 10 mi of 0.5% bovine fibrinogen in a Petri dish. Chioramphenicol had been added to the fibrinogen solution to prevent bacterial growth.
3.3. Outer membrane preparation Isolation of E. coli outer membrane by detergent solubilization and ultracentrifugation was according to the procedure of Filip et al. [17]. Cultures were adjusted to an A540 of 2.0 prior to membrane isolation. 3.4. Electrophoresis The Lugtenberg gel system [18] was used for electrophoresis of outer membrane proteins with 11% polyacrylamide, 0.2% sodium dodecyl sulfate (SDS), and 0.375 M Tris. HCI, pH 8.8. For electrophoresis of T7 RNA polymerase cleavage products, the polyacrylamide concentration was increased to 14%. 3.5. Genetic techniques Manipulations of plasmid DNA were performed by standard techniques [14]. Screening of E. coli strains for the presence of the ompT gene was accomplished by colony hybridization [14]. An 800-bp Hincll fragment obtained from plasmid pML21 and purified from an agarose gel was labelled with [a-32p]dCTP by 'oligolabelling' as described by Feinberg and Vogelstein [19]. This fragment contains most of the ompT coding region. Colonies were transferred to 'Colony/Plaque Screen' membranes (E.l. du Pont De Nemours & Co.) by the method of Grunstein and Hogness [20] and were hybridized at 65°C to the labelled DNA probe according to the procedures provided by the manufacturer. Washing was with 2 × SSC, 1% SDS at 65°C. After hybridization the membranes were analysed by autoradiography.
4. R E S U L T S A N D DISCUSSION
below). A derivative of RWI93, UT4400, in which the ompT gene has been deleted, failed to degrade the polymerase (lane 3). T h e effect of three clinical isolates on the cleavage on the polymerase is shown in lanes 4, 5 and 6. Isolate D U I 8 produced a cleavage pattern (lane 5) identical to RWI93, whereas the o t h e r two isolates, D U l 7 and D U I 9 (lanes 4 and 6), did not. The multitude of bands in lane 6 was due to whole cells that were carried over into the electrophoresis sample. Table l is a compilation of the results obtained by screening h u m a n isolates of E. coil for surface protease activity. Only 34 (12.1%) of the 282 h u m a n isolates were positive for O m p T protease activity. Electrophoresis of outer m e m b r a n e proteins obtained from the three clinical isolates described above (Fig. 2, lanes 5 - 1 0 ) did not reveal a thermoregulatcd polypeptide of a molecular mass close to that of O m p T (Fig. 2, lanes l, 2, and 17, 18). This was expected for isolates D U I 7 and
G r o d b e r g and D u n n [2] developed a sensitive assay for detection of the O m p T protease. This assay tests the ability of whole ceils to degrade T7 R N A polymerase and was used here to screen 282 h u m a n isolates of E. coli for the presence of O m p T proteolytic activity. Figure 1 is a typical electrophoretogram displaying the results of such assays. The T7 R N A polymerase m o n o m e r is a 100-kDa protein as shown in lane 1 of Fig. I. W h e n the polymerase was exposed to the surface of strain RW193 (lane 2), in which ompT is chromosomally encoded, specific cleavages occurred between residues 172 (lysine) and 173 (arginine) or between the two lysine residues 179 and 180, resulting in polypeptide fi'agments of about 80 kDa and 20 kDa molecular mass [2]. An additional fragment of about 15 kDa was occasionally seen and its appearance was d e p e n d e n t on the O m p T concentration in the assay (see
1
2
3
4
56
7 8
mm 66"
910111213
-,- qUD~,-,,O .
t~lm-"~
.
.
.
.
.
KD
.
IIIP
..... ~
' ~ ~
-100 -80
45" -40 35"
~
24~
.... ~
"20
18.414.3-
"15
Fig. I. Assay of OmpT protease activity on the cell surface. T7 RNA polymerase was exposed to cells and then separated as described in MATERIALSAND METHODS.Proteins were visualized with Coomassie blue. Molecular mass standards are indicated at the left and approximate molecular masses are shown at the right. Lane 1 is l ,ug of T7 RNA polymerase. Lanes: (2) RWI93: (3) UT4400; (4) DUI7; (5) DUI8; (6) DuIg; (7) DUI7/pMLIg; (8) DUI8/pMLI9: (9) DUIg/pMLI9: (10) UT4400/pMLI9; (11) UT4400/pML19 I : 10; (12) UT4400/pMLI9 I : 100; and (13) UT44()I)/pMLI9 I : 1000.
54 Table 1 Incidence of OmpT among human E. coil isolates Source :' and number h of imh,tes
Occurrence of OmpT its determined by Activity " Ilybridization d
DU (196) DS (8) C (26) A (5) B (26)
I3G
76f~,;
0r,~ Igt:~ I)"~;
I(H)C:,
8c; 5~;
IIXIr~
BC
(21)
5()C~"
73c; 73r;
" DU, urine: DS. st(~l: C. wounds; A. respiratnry: B, geMtourinary: BC. bh~d. h A total of 282 isolates were tested fi)r protease activity and for the ability to hybridize tn t.npT DNA. ¢ Protease activitywas assayed by the ability nf whole cells to cleave T7 RNA polymerase. d Radiolabelled ompT DNA was hybridized to colony blots. DUI9, which lack O m p T activity, but not for isolate DU18 that demonstrated O m p T activity. O m p T protein could not be unequivocally identified from the outer membrane protein profile of an additional 21 protease-positive and 17 protease-negative strains (data not shown). It was possible that these strains could not express O m p T to high enough levels to be detected on polyacrylamide gels. Therefore the three clinical isolates: DUI7, DUI8, and DU19 were' transformed with pML19 [9], an o m p T complementing plasmid, and outer membrane proteins examined
1 2
3
4
5
6
7
8
by PAGE. The amount of O m p T present among the pMLlg-transformed isolates was nearly the same for each one (Fig. 2, lanes l l - 1 6 ) and expression of the protease was thermorcgulated. Although there may be a decrease in o m p T expression in these isolates compared to the Kl2 strain U T 4 4 0 0 / p M L 1 9 (Fig. 2, lanes 17 and 18), there is no significant difference in the amount of O m p T protein in the outer membrane among these three clinical strains. O m p T activity from these transformants was also assayed. When carrying pMLl9, isolate DU17 produced weak protease activity (Fig. l, lane 7), i~olate D U I 9 produced moderate activity (lane 9), and isolate D U i 8 produced strong activity (lane 8) comparable to the activity of U T 4 4 0 0 / p M L 1 9 when diluted I : l0 (lane ll). Serogrouping of 50 of the isolates did not reveal a common O or K antigen among those isolates which did or did not express O m p T activity (data not shown) nor was a specific colony morphology associated with O m p T phenotype, Clearly the differences in O m p T activity were due to factors other than expression of the o m p T gene and localization of the protein to the outer membrane. Complete degradation of the T7 RNA polymerase occurred when the polymerase was exposed to cells containing high levels of O m p T (Fig. l, lane 10). When plasmid p M L I 9 transformed cells, and hence the amount of protease,
9101112131415161718
4534.7-
-Omp Z { Ompr.. ump~
-OmpA
24" Fig. 2. Outer membrane proteins of E. cnli strains. Outer membranes were prepared from cells grown at 37°C and 27°C to stationary phase in L-broth. Relevant E. coil K-12 proteins are indicated on the right. Strains examined and growth temperatures were: (1) RWI93, 37°C: (2) RW193, 27°C; (3) UT4400, 37°C; (4) UT4400, 27°C:(5) DUI7, 37°C: (6) DUI7, 27°C: (7) DU~.8, 37°C: (8) DUI8, 27°C; (9) DuIg, 37°C: (10) DuIg, 27°C; (I 1) DUI7/pMLI9, 37°C;(12) DUI7/pML19, 27°C;(13) DUIS/pMLlg, 37°C; (14) DUI8/pMLIg, 27°C;(I5) DU 19/pMLIg, 37°C; (16) DUIg/pMLIg, 27°C;(17) UT4400/pMLIg, 37°C; (18) UT4400/pMLIg, 27°C.
107
NUMBER OF CELLS 106 105
104
STRAIN RW193
UT4400
UT4400/pML19
UT4400/pML23
C8
Fig. 3. Fibrin film lysis assay for plasminogen activator activity. Washed cells were diluted and spotted onto a fibrin film prepared [~'om bovine fibrinogen and thrombin. Inoculated films were incubated at 37°C for 12 h. The number of cells contained in each 10-p.I spot is shown at the top of the figure and the strains examined are indicated at the left edge. See text.
were diluted the degradation was reduced and the specific cleavage pattern reestablished (Fig. 1, lanes 10-13). This observation suggests that this protease has two proteolytic activities; a specific e n d o p e p t i d a s e activity which proceeds very quickly and a slower non-specific peptidase activity which results in complete degradation of the protein. Lysis of fibrin films was used by Sodeinde and G o g u e n [10] to assay the plasminogen activator, Pla, of Yersinia pestis and Sabnonella typhimurium protein E, when it was present on a high copy n u m b e r plasmid. However, Soldeinde a n d G o g u e n were not able to detect plasminogen activator activity by chromosomally encoded protein E or OmpT. We show here for the first time that O m p T can dissolve a fibrin film. Figure 3 shows a fibrin film assay which demonstrates that OmpT, encoded by the plasmid pMLI9, can dissolve fibrin. The control strains UT4400 and
UT44(XI/pML23 (ompT::Kan), do not dis.~)lve the fibrin film. Additonally, UT4400 containing pUCIg, the vector from which p M L I 9 was derived, does not dissolve fibrin (data not shown). in agreement with the findings of Sodeinde and Goguen, RW193, which carries ompT on the chromosome, did not dissolve the fibrin with 107 cells per spot; thus, for fibrin lysis the protease must be present in concentrations greater than that produced by the haploid gene. In contrast to Pla, O m p T has no detectable coagulase activity even when expressed from a multicopy plasmid (data not shown). All positive and 4(I negative strains were tested for their ability to dissolve fibrin. Of these, only one O m p T ~ isolate (C8) was positive (Fig. 3). E n h a n c e d plasminogen activation by O m p T from the C8 isolate could be due to a more active enzyme conformation or the surface architecture of the cell could be such that the OmpT-active site is more accessible to the substrate, plasminogen. Because the isolates that cleaved T7 R N A polymerase did not clearly display an OmpT-like protein on electrophoretic analysis of m e m b r a n e proteins and some isolates were unable to express plasmid-encoded O m p T protease activity, we used "colony hybridization" to determine whether the strains had the ompT structural gene. O f 282 E. coil isolates tested, 217 or 77% were found to hybridize to radiolabelled ompT D N A (Table 1). Each isolate was tested for hybridization at least three times with virtually no ambiguity as to the results. The presence of the ompT gene correlated with blood culture isolates ( P = 0.0386, by Chi-square statistic), whereas the proteolytic activity was more commonly found in urine and wound isolates. Wher~ blood and respiratory isolates were considered as one group, the correlation with ompT hybridization became even more significant ( P = 0.0147). We show here that different E. coli isolates vary in their ability to express O m p T protease activity. The probable reason for this observation is that these strains differ in their surface architecture such that in the majority of strains the substrate is inaccessible to the active site. The effect of surface architecture could be: (!) to assure proper insertion and orientation of the
p r o t e a s e in the o u t e r m e m b r a n e ; (2) to provide n e e d e d interactions with o t h e r m e m b r a n e c o m p o n e n t s ; o r (3) to form a surface relatively free o f i m p e d i n g structures. A l t h o u g h not investigated h e r e , changing growth conditions (e.g. growth in s e r u m ) might alter t h e cell e n v e l o p e such that a higher p e r c e n t a g e o f strains could cleave T7 R N A polymerase o r dissolve fibrin films.
ACKNOWLEDGEMENTS W e thank J.E.L. A r c e n e a u x and S.W. C h a p m a n for their constructive c o m m e n t s c o n c e r n i n g the manuscript. In addition, we wish to t h a n k E.F. M e y d r e c h for statistical analysis o f the data. This work was s u p p o r t e d in p a r t by G r a n t 2 SO7 RR05386 A w a r d s by the Biomedical R e s e a r c h S u p p o r t G r a n t P r o g r a m , Division o f R e s e a r c h Resources, National Institutes o f Health.
REFERENCES [1] Earhart, C.F., Lundrigan, M.. Pickett, C.L. and Pierce, J.R. (1979) FEMS MicrobioL Lett. 6, 277-280. [2] Grodberg, J. and Dunn, J.J. (1988) J. Bacteriol. 170, 1245-1253. [3] Sugimura, K. and Higashi, N. (1988) J. BacterioL 170, 3650-3654.
[4] Sedgwick, B. (1989) J. Bacleriol. 17I, 2249-2251. [5] Hellebust, H., Murby, M., Abrahms~n, L., Uhl~n, M. and Enfors, S.O. (1989) Bio/Technology 7, 165-168. [6] Baneyx0 F. and Georgiou, G. (1990) J. Bacteriol. 172, 491-494. [7] Akiyama, Y. and lto, K. (1990) Biochem. Biophys. Res. Comm. 167, 711-715. [8] Rupprecht. K.R., Gordon, G., Lundrigan. M., Gayda, R.C., Markovitz, A. and Earhart, C.F. (1983)J. Bacteriol. 153, 1104-106. [9] Grodberg, J., Lundrigan, M.D., Toledo, D.L, Mangel, W.F. and Dunn, J.J. (1988) Nucleic Acids Res. 16, 1209. [10] Sodeinde, O.A. and Goguen, J.D. (1989) Infect. Immun. 57, 1517-1523. [11] Fiss, E.H., Hollifield, W.C. Jr. and Neilands, J.B. (1979) Biochem. Biophys. Res. Commun. 91, 29-34. [12] Sodeinde, O.A., Sample, A.K., Brubaker, R.R. and Goguen, J.D. (1988) Infect. Immun. 56, 2749-2752. [13] Lugtenberg, B., Peters, R., Bernheimer, H. and Berendsen, W. (1976) MoL Gem Genet. 147, 251-262. [14] Maniatis, T., Fritsch, E.F. and Sambrook, J.E. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [15] Beesley, E.D., Brubaker, R.R., Jansen, W.A. and Surgalla, M.J. (1967) J. Bacteriol. 165, 19-26. [16] Lewis, J.H. and Ferguson, J.H. (1950)J. a i m Invest. 29, 486-490. [17] Filip, C., Fletcher, G., Wulff, J.L. and Earhart, C.F. (1973) J. Bacteriol. 115. 717-722. [18] Lugtenberg, B., Meijers, J., Peters, R., Van der Hock, P. and Van Alphen, L. (1975) FEBS Left. 58, 254-258. [19] Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. [20] Grunstein. M. and Hogness, D. (1975) Proc. Natl. Aead. Sci. USA 72, 3961-3965.
