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Infection and Immunity, October 1999, p. 5538-5540, Vol. 67, No. 10
Department of Pathology and Microbiology,
School of Medical Sciences, Bristol, England1
and Biotechnology Laboratory, Vancouver, British Columbia,
Canada V6T 1Z32
Received 5 February 1999/Returned for modification 1 April
1999/Accepted 27 July 1999
Enteropathogenic Escherichia coli (EPEC) was found to
exhibit a type III secretion-dependent, contact-mediated, hemolytic activity requiring the EspA, EspB, and EspD secreted proteins. EspB and
EspD display homology to pore-forming molecules. Our data suggest a
mechanism to explain the requirement for all three Esp proteins in the
transfer of EPEC proteins, such as Tir, into target cells.
Many gram-negative pathogens encode
a type III secretion apparatus dedicated to the release of a diverse
set of virulence-associated proteins (7, 17). Some proteins
are secreted into the extracellular environment, while others are
translocated directly into target cells to subvert various host
processes (7, 17). The injection of proteins into host cells
by Shigella and Yersinia spp. has been correlated
to their in vitro abilities to lyse erythrocytes (RBC) (5,
6). Enteropathogenic Escherichia coli (EPEC)
encodes a similar type III secretion apparatus (8)
that is involved in the release of a number of EPEC-secreted proteins
(EspA, EspB, EspD, EspF, and Tir) (3, 10-12, 14, 16, 18).
EspA has been detected in bacterial-surface appendages that appear to
connect the bacteria to host cells and participate in protein delivery (15). A role for EspD, but not EspB, in the correct
formation of these EspA-associated appendages has been reported
(15), while all three Esp proteins are required for the
translocation of Tir into target cells (11). Both EspB and
Tir have been detected in the host cytoplasmic and membrane fractions
(1a, 9, 11, 12, 23, 25). Whereas the function of EspB in
host cells is unknown, Tir is modified in the host cytoplasm and
becomes inserted into the membrane, where it acts as a receptor for the bacterial outer membrane protein intimin (9, 11, 22).
Tir-intimin interaction triggers host-signalling events and leads to
the organization of host cytoskeletal proteins into characteristic
pedestal-like structures beneath the adherent bacteria (11, 13,
20). No role for EspF has been found in Tir translocation or
pedestal formation (3, 18, 24a).
To determine if the EPEC type III secretion system is associated with a
hemolytic activity, EPEC 2348/69 and its type III secretion-defective
mutants, the cfm-14 (2) and sepB
mutants (8), were analyzed for their abilities to release
hemoglobin from RBCs. EPEC strains were grown in Luria broth overnight
(without shaking), diluted into Dulbecco's modified Eagle medium
(DMEM) (phenol red minus; GIBCO-BRL), and grown at 37°C without
shaking under a 5% CO2 atmosphere to an optical density
(A600) of approximately 0.7. One-half milliliter
of culture was added to 0.5 ml of a 4% RBC-DMEM solution and
centrifuged (at 2,500 × g for 1 min) to initiate
EPEC-RBC contact. After a 30-min incubation (at 37°C in 5%
CO2), the bacterium-RBC pellet was gently resuspended to facilitate the release of hemoglobin. Cells were repelleted (at 12,000 × g for 1 min), and the supernatant was
monitored for the presence of released hemoglobin at the optical
wavelength of 543 nm. Similarly treated uninfected RBCs were used as a
spectrophotometric zero.
Figure 1A demonstrates that EPEC exhibits
a haemolytic activity, lysing approximately 15% of the RBCs. This
activity was dependent on the type III secretion apparatus, as no
significant hemoglobin release was detected with the cfm-14
or sepB mutant. Such activity was not obtained with
bacterium-free (filtered) supernatants or when EPEC organisms were
coincubated with the RBCs but separated from them by a
0.4-µm-pore-size filter (12-mm Transwell filter; Costar) (data not
shown), implying the absence of a diffusible hemolytic factor. Type III
secretion from several pathogens is activated by contact with host
cells (7). Lysis of RBCs by EPEC was found to be dependent
on de novo protein synthesis, as the addition of chloramphenicol to
EPEC prior to incubation with the RBCs prevented hemoglobin release
(data not shown). De novo protein synthesis and host cell contact are
both required for EPEC to induce host responses leading to Tir-mediated
pedestal formation (11, 21, 22), correlating hemolytic
activity with the translocation of Tir into host cells.
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Type III Secretion-Dependent Hemolytic Activity of
Enteropathogenic Escherichia coli
ABSTRACT
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FIG. 1.
EPEC-induced hemolytic activity. EPEC, various deletion
mutants, and complemented mutant strains were assayed for their
abilities to lyse sheep RBCs as described in the text. Release of
hemoglobin was assessed at an optical wavelength of 543 nm by using
RBCs incubated in the absence of bacteria as a spectrophotometeric
zero. Results from three independent experiments are shown. Error bars,
standard deviations.
To investigate the involvement of type III secreted proteins in hemolysis, EPEC mutants with single deletions in genes encoding secreted proteins were tested for their abilities to lyse RBCs. Whereas the tir and espF mutants efficiently lysed RBCs, no significant lytic activity was detected for the espA, espD, and espB mutants (Fig. 1B). The ability of the latter mutants to lyse RBCs was restored, but not to wild-type levels, by the introduction of plasmids expressing the missing gene product (Fig. 1B). The espF mutant used here was generated by J.W. and has the same phenotype as espF, described in reference 22.
These results can be rationalized in light of recent findings relating to bacterium-host cell interactions. Thus, the absence of hemolytic activity for the espA mutant may be attributable to its role in the formation of the novel appendages that link EPEC to the host cell surface and participate in protein translocation (15). Therefore, EspA may have an indirect effect on hemolytic activity by mediating the delivery of the pore-forming molecule(s) into the host membrane.
In contrast to EspA, both EspB and EspD display weak homology to the Yersinia YopB and Shigella IpaB proteins, and to each other (Fig. 2). These Shigella and Yersinia type III secreted proteins have pore-forming activity (5, 6). However, only EspB resists digestion by proteases of intact host cells (12, 24), making it the best candidate for a transmembrane pore-forming molecule that allows EPEC proteins to cross the host membrane. This would explain why disruption of espB abolishs hemolytic activity.
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EspD could potentially have an indirect, or direct, role in pore formation (hemolytic activity), as on the one hand it is required for the correct formation of the EspA-associated "delivery" appendages (15) while on the other it is found associated with the host membrane (24). EspB, EspD, YopB, and IpaB are all predicted to possess transmembrane domains (4, 19) that are located, and even align, within these homologous regions (Fig. 2). Thus, it is possible that EspB and EspD have similar functions and associate to form a pore structure in the host membrane. The presence of a putative transmembrane domain in EspA (4, 19) could enable it to participate in pore formation also. This raises the possibility that all three Esp proteins could participate, at the tips of the EspA appendages, in the formation of a functional membrane-spanning pore structure to allow the injection of EPEC proteins into the host cell.
Neither the espF nor the tir gene plays a role in hemolytic activity, as no significant reduction in RBC lysis was detected. It is already known that Tir is translocated into the host cell (1a, 9, 11), while the presence of polyproline-rich regions in EspF indicates that this protein may be targeted into host cells (3). This contrasting phenotype supports a role for EspA, EspB, and EspD in forming a structure(s) to mediate the transfer of EPEC molecules into host cells.
In conclusion, we have shown that the EPEC type III secreted proteins EspA, EspB, and EspD are essential for contact-mediated hemolytic activity that is correlated with the ability to translocate EPEC proteins, such as Tir, into host cells.
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ACKNOWLEDGMENTS |
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We thank James Kaper and Michael Donnenberg for providing EPEC UMD864 (espB) and UMD870 (espD) and the cfm-14 and sepB mutants.
This work was supported by a Medical Research Council (MRC) of Canada operating grant to B.B.F. and a Wellcome Trust Career Development Fellowship to B.K. B.B.F. is a Howard Hughes Medical Institute Institutional Scholar and an MRC Scientist.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology and Microbiology, School of Medical Sciences, University Walk, Bristol, BS8 1TD, England. Phone: (0117) 928 7530. Fax: (0117) 930 0543. E-mail: B.Kenny{at}bristol.ac.uk.
Editor: P. E. Orndorff
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