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Journal of Bacteriology, August 1998, p. 4002-4006, Vol. 180, No. 15
Abteilung Mikrobiologie, Universität
Osnabrück, D-49069 Osnabrück,
Germany,1 and
Department of Chemical
Engineering, The University of Texas at Austin, Austin, Texas
78712-10622
Received 8 September 1997/Accepted 27 May 1998
The influence of extracytoplasmic proteases on the resistance of
Escherichia coli to the antimicrobial peptide protamine was investigated by testing strains with deletions in the protease genes
degP, ptr, and ompT. Only
In animals, bacteria are often
exposed to defensins and other cationic antimicrobial peptides
produced by the host (6, 11, 12). Groisman and coworkers
have introduced the strongly cationic antimicrobial peptide
protamine as a model compound for the action of
defensins on cells of Salmonella typhimurium
(9, 18, 19). The site of action of protamine is the
cytoplasmic membrane, where it causes membrane permeabilization,
possibly by creating a large pore in the membrane (3, 10,
20). At low protamine doses, growing cells of Escherichia
coli slowly recover from protamine treatment (20). This
effect is probably due to protamine degradation by cell proteases,
since the susceptibility of growing cells of E. coli toward
protamine increases by fivefold in a strain lacking the
extracytoplasmic proteases DegP, OmpT, and protease III
(20). Here, we examine which of the above proteases is
responsible for protamine inactivation and whether this
inactivation is due to protamine degradation. For this purpose, we
used strains possessing only a single extracytoplasmic protease
(16) and employed high-performance liquid chromatography
(HPLC) to monitor the fate of protamine in the medium of growing
E. coli cells. We show that OmpT is the protease responsible
for protamine inactivation by degradation of the toxic peptide at the
external face of the cell envelope. Our data suggest that a major
function of OmpT is to protect the cells against antimicrobial cationic
peptides from the medium.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of OmpT as the Protease That Hydrolyzes the
Antimicrobial Peptide Protamine before It Enters Growing Cells of
Escherichia coli
ABSTRACT
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ompT strains were hypersusceptible to protamine. This
effect was abolished by plasmids carrying ompT. Both at low
and at high Mg2+ concentrations,
ompT+ strains cleared protamine from the medium
within a few minutes. By contrast, at high Mg2+
concentrations, protamine remained present for at least 1 h in the
medium of an ompT strain. These data indicate that OmpT is the protease that degrades protamine and that it exerts this function at the external face of the outer membrane.
TEXT
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TABLE 1.
E. coli K-12 strains and plasmids used in
this study
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FIG. 1.
Effect of ompT and other extracytoplasmic
protease genes on protamine susceptibility of E. coli cells.
Protamine (100 mg/liter) was added at t = 0 to a
suspension of growing cells. Closed symbols, protamine-treated cells;
open symbols, control cells. (A) Circles, strain KS272; squares, strain
SF110; triangles, strain SF103; diamonds, strain KS474; inverted
triangles, strain SF115; and octagons, strain SF120; (B)
circles, strain UT5600; triangles, strain UT5600(pML19); and
squares, strain UT5600(pDS319). The last two strains grew in the
presence of 100 mg of carbenicillin and 12.5 mg of tetracycline per
liter, respectively.
The strains and plasmids used in this study are listed in Table 1. Studies of protamine susceptibility of E. coli cells growing in shaking culture flasks were carried out as described elsewhere (20). Peptide contents and protamine concentrations in the supernatant of centrifuged cells were determined by reverse-phase HPLC separation on a C18-column with an acetonitrile-H2O gradient in a 140 apparatus from Applied Biosystems (Foster City, Calif.). The N-terminal sequences of proteins or peptides were determined by microsequencing after automatic Edman degradation in an ABI-473A Instrument (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany). Protamine sulfate (from salmon milt; Calbiochem, Bad Soden, Germany) is a mixture of compounds (2). One of its major compounds, salmine AI, has the primary sequence MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR (1).
Protamine susceptibility of strains lacking extracytoplasmic proteases
was tested in growth experiments (20). Protamine was added
at a concentration of 100 mg/liter to a cell suspension with an optical
density at 578 nm (OD578) of 0.65, and the cells were grown
in a minimal mineral medium containing 20 mM K+ ions with
10 mM glucose as the carbon source. Figure 1A shows that strain SF115
lacking protease III and DegP but not OmpT was as susceptible as the
wild-type strain. These strains resumed growth at approximately 1 h after the addition of the toxic peptide. In contrast, the
ompT strains SF110 and SF120 did not recover from protamine
treatment within 6 h (Fig. 1A). Mutations in either extracytoplasmic protease gene ptr or degP in an
ompT+ background exerted no effect (strains
SF103, SF115, and KS474 [Fig. 1A]). This suggests that OmpT alone is
sufficient for protection of cells against the toxic peptide. Further
information was obtained with the nonisogenic ompT strain
UT5600 (4). As expected, this strain was hypersusceptible to
protamine (Fig. 1B, closed circles). However, two types of
ompT-containing plasmids rescued these cells. Cells
harboring plasmid pML19 (an ompT-containing derivative of
pUC19 [7, 8]) grew relatively slowly (Fig. 1B, closed
triangles). Addition of protamine to these cells hardly exerted
any effect on cell growth (Fig. 1B, triangles). The poor growth of UT5600(pML19) is due to the overexpression of OmpT from the high-copy-number pML19 plasmid. Therefore, we also tested the
effect of ompT expression from the lower-copy-number
plasmid pSD319, which is a pBR332 derivative. Strain
UT5600(pSD319) still grew more slowly than did the control strain
UT5600(pBR322) (data not shown). Moreover, addition of protamine to
cells of strain UT5600(pSD319) led to considerable cell lysis, as
indicated by the trough in the growth curve of this strain (Fig. 1B).
This effect was only partially due to the presence of tetracycline in
the growth medium of this strain and did not occur after protamine addition to cells of strain UT5600(pBR322) growing under similar conditions (data not shown). Still, the presence of ompT on
the plasmid rescued the cells of strain UT5600(pSD319), since these cells resumed growth after approximately 2 h (Fig. 1B), whereas the control culture of strain UT5600(pBR322) did not resume growth within 5 h (data not shown). Taken together, the data from Fig. 1
show that OmpT is the extracytoplasmic protease that protects E. coli cells against protamine.
More information about the fate of protamine in the cell suspension
came from HPLC analysis of the peptide contents of supernatants from
centrifuged cells. Growth medium contained several compounds, including
thiamine, with signals in the HPLC chromatogram, with a retention time
of 10 to 11 min (Fig. 2A, peak 1).
Addition of 100 mg of protamine per liter to this medium gave a major
additional peak with a retention time centered at 13 to 14 min (Fig.
2B, peak 2). The form of this peak confirms that protamine consists of
a mixture of compounds (2). Addition of protamine to a
suspension of growing cells of the ompT+ strain
KS272 led to a rapid removal of the toxic peptide from the medium (Fig.
2, traces C to E). After 75 s, the protamine peak was already much
smaller (Fig. 2C), and at 450 s, it had vanished from the medium
(see Fig. 4D). Several new products appeared in the medium (Fig. 2D and
E). Below, we show that some of them were protamine degradation
products. From these data, it is concluded that the cells removed
protamine with a half-life of 1 to 2 min from the medium. However, we
cannot differentiate whether this removal from the medium represents
protamine adsorption to the cells (10, 23), proteolysis of
the compound, or both. The ompT strain SF110 removed
protamine as rapidly from the medium as did strain KS272 (data not
shown), suggesting that adsorption to the outer membrane is important
in the process of protamine removal from the medium.
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High Mg2+ concentrations prevent adsorption of protamine to
the cell wall (10, 23). In order to obtain more information on the fate of protamine under these conditions, the experiments were
repeated at 5 mM Mg2+. At this [Mg2+],
protamine did not inhibit cell growth of the
ompT+ strain KS272 (Fig.
3A). Moreover, these cells did not lose
K+ (Fig. 3C), suggesting that under these conditions
protamine did not reach the cytoplasmic membrane. By contrast, at 5 mM
Mg2+, growth of the ompT strain SF110 slowed
down gradually and stopped completely at approximately 1 h after
protamine addition (Fig. 3B). These cells lost K+, although
much more slowly than at 0.4 mM Mg2+ (half-lives for
K+ loss, 20 min and a few minutes, respectively [Fig.
3D]), suggesting that at 5 mM Mg2+, protamine slowly
reached the cytoplasmic membrane of the ompT strain.
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Figure 4A to C shows that at 5 mM Mg2+, protamine removal by strain KS272 is almost as rapid as that in the absence of Mg2+ (half-lives of 1 to 2 min in both cases [Fig. 4A to C and Fig. 2, respectively]). At high Mg2+ concentrations, several new peptides with retention times of between 9.20 and 12.67 min were formed (e.g., Fig. 4C). These compounds remained at constant concentrations for a period of 3 h in the medium (data not shown). The peptides from peaks 3 to 7 in Fig. 4C all contained several arginines at their N terminus, suggesting that they are protamine degradation products. Peptide 6 in Fig. 4C had an N-terminal sequence, XSSRRPVRRR, indicating that it is similar to protamine 2c from rainbow trout (15). It was not yet known that salmon protamine contains this type of compound. These data suggest that at 5 mM Mg2+, protamine degradation products do not adsorb to the cell wall of the ompT+ strain.
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Protamine removal from the medium by the ompT strain
SF110 was extremely slow (half-life of approximately 30 to 60 min
[Fig. 4D to F]). Moreover, the protamine hydrolysis products observed at high Mg2+ concentrations with strain KS272 did not
appear in the medium of strain SF110 (Fig. 4D to F). We assume that
this removal of protamine from the medium represents uptake by the
cells, since its time course correlated well with inhibition of growth
and K+ loss from these cells (Fig. 3B and D, respectively).
It is not known whether under these conditions protamine is degraded at all and, if this is the case, in which cellular compartment proteolysis occurs.
Since protamine remained in the medium at high Mg2+
concentrations with the ompT strain (Fig. 4D to F), we
assume that under these conditions the major portion of protamine is
not rapidly adsorbed by the cells. The observations that under these
conditions the isogenic ompT+ strain removed
protamine rapidly from the medium and that its degradation products
appeared in the medium (Fig. 4A to C) suggest that OmpT cleaves
protamine at the external surface of the outer membrane. This idea,
i.e., that the active center of OmpT is directed outward, is supported
by observations that in intact E. coli cells OmpT
degrades T7 RNA polymerase added to the medium (8) and that
it activates plasminogen (13). However, the question
concerning the orientation of the active site of OmpT in the outer
membrane remains controversial, since other data suggest that OmpT is
active in the degradation of some periplasmic proteins that come into contact with the outer membrane (17).
OmpT is known to degrade peptides between cationic residues and between cationic residues and several apolar residues (8, 14, 17, 21, 22). Therefore, it was not unexpected that OmpT is the extracytoplasmic protease that inactivates the highly cationic peptide protamine (Fig. 1, 2, and 4). We have preliminary evidence that, in addition to protamine, OmpT detoxifies the cationic peptide melittin (data not shown). Moreover, OmpT is essential for the virulence of E. coli strains in the human urinary tract (5). We speculate that in this situation OmpT is active in the degradation of cationic peptides (defensins) excreted by epithelial cells from the urinary tract. However, at present no data that either support or contradict this hypothesis are available.
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ACKNOWLEDGMENTS |
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Eva Uhlemann and Eva Limpinsel are thanked for expert technical assistance.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB171, Teilprojekte B5 and C1) and by the Fonds der Chemischen Industrie.
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FOOTNOTES |
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* Corresponding author. Mailing address: Abteilung Mikrobiologie, Universität Osnabrück, Barbarastraße 11, D-49069 Osnabrück, Germany. Phone: 49-541-9692855. Fax: 49-541-9692870. E-mail: bakker_e{at}sfbbio1.biologie.uni-osnabrueck.de.
Present address: Institut für Molekulare Biotechnologie,
Abteilung Strukturbiologie/Kristallographie, Arbeitsgruppe
Physikalische DNA-Analytik, 07745 Jena, Germany.
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Journal of Bacteriology, August 1998, p. 4002-4006, Vol. 180, No. 15
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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