INFECTION AND IMMUNITY, Dec. 2002, p. 68466852 Vol. 70, No.

12
0019-9567/02/$04.000 DOI: 10.1128/IAI.70.12.68466852.2002
Copyright 2002, American Society for Microbiology. All Rights Reserved.

Invasion Activity of a Mycobacterium tuberculosis Peptide Presented by

the Escherichia coli AIDA Autotransporter
Nicola Casali,1 Marc Konieczny,2 M. Alexander Schmidt,2 and Lee W. Riley1*
School of Public Health, University of California at Berkeley, Berkeley, California,1 and Institut fur Infektiologie,
Zentrum fur Molekularbiologie der Entzundung, Universitatsklinikum Munster, Munster, Germany2
Received 7 November 2001/Returned for modification 7 January 2002/Accepted 8 August 2002

The mce1A gene of Mycobacterium tuberculosis was initially identified by its ability to promote uptake of
Escherichia coli into HeLa cells. It was subsequently shown that this activity was confined to a 58-amino-acid
region of the protein. A 72-amino-acid fragment (InvX) incorporating this active peptide was expressed in E.
coli as a fusion to the AIDA (adhesin involved in diffuse adherence) autotransporter translocator, and its stable
expression on the surface of the bacterium was demonstrated. Recombinant E. coli expressing InvX-AIDA
showed extensive association with HeLa cells, and InvX was shown to be sufficient for internalization. Uptake
was found to be both microtubule and microfilament dependent and required the Rho family of GTPases. Thus,
the E. coli AIDA system facilitated both the qualitative and quantitative analysis of the functional domain of
a heterologous protein.

It has been estimated that one-third of the worlds popula-
tion is infected with Mycobacterium tuberculosis, the etiologic
agent of tuberculosis, which kills approximately two million
people each year (14). In vivo M. tuberculosis is thought to
primarily invade and replicate within alveolar macrophages,
although a recent study suggests that it may persist in other
types of human lung cells (21). Additionally, it has been shown
to enter a number of different cell types in vitro (6, 37). M.
tuberculosis is known to bind many different macrophage re-
ceptors. For example, bacilli opsonized with C3b and C3bi bind
complement receptors CR1, CR3, and CR4 (35, 36) and also
bind CR3 nonopsonically via envelope polysaccharides (11). In
addition, the terminal mannosyl residues of lipoarabinoman-
nan bind mannose receptors (34) and opsonization with sur-
factant protein A enhances uptake (12). In spite of this array of
receptors, very little is known about the M. tuberculosis ligands
involved in invasion or the macrophage signaling events in-
volved in phagocytosis.
A putative M. tuberculosis virulence gene, mce1A (mycobac-
terium cell entry), was originally identified because its expres-
sion in Escherichia coli enabled this noninvasive bacterium to
enter nonphagocytic mammalian cells (1). It was subsequently
shown that latex microspheres coated with recombinant
Mce1A were also capable of entering HeLa cells (9). Compar-
ison of the ability of truncated forms of recombinant Mce1A to
induce the uptake of microspheres confined this activity to a
58-amino-acid domain located between positions 106 and 163
of the protein. Disruption of the mce1A gene homologue in
Mycobacterium bovis BCG reduced its ability to invade HeLa
cells, providing evidence that, in its native host, Mce1A plays a
role in the invasion of nonphagocytic cells (18).
Sequencing of the M. tuberculosis genome revealed that
mce1A (Rv0169) was part of an operon that encoded eight
* Corresponding author. Mailing address: University of California at
Berkeley, School of Public Health, 140 Warren Hall, Berkeley, CA
94720. Phone: (510) 642-9200. Fax: (510) 643-6350. E-mail:
lwriley@uclink4.berkeley.edu.
putative membrane-associated proteins (10, 39). The first two
genes of the operon (Rv0167-68) encode proteins with six
putative transmembrane regions and are predicted to be inte-
gral membrane proteins. The following six encoded proteins
(Rv0169-74) have putative signal sequences, indicating that
they are presented on the surface of the bacillus. Indeed,
electron microscopic analysis of immunogold-labeled M. tuber-
culosis has demonstrated the surface exposure of Mce1A,
which is consistent with a role in interaction with the host cell
(9). The mce operon is present four times in the M. tuberculosis
chromosome (10).
Autotransporters comprise a growing family of gram-nega-
tive proteins that are also referred to as type V secretion
systems (19, 30). Remarkably, in these secretion systems, all
the information required for export through the outer mem-
brane is contained within a single self-translocating polypep-
tide. The typical autotransporter consists of an amino-terminal
signal peptide, a passenger domain, a linker region, and a
core translocator at the carboxy terminus. The signal peptide
directs translocation across the inner membrane, most proba-
bly by a sec-dependent mechanism. Subsequently, the core
translocator, which consists of 14 amphipathic -strands, in-
serts into the outer membrane in a characteristic -barrel
structure. The amino-terminal passenger is then extruded
through a central pore and may undergo further proteolytic
processing at the cell surface (20, 32, 38).
Several studies have reported that plasmids expressing re-
combinant autotransporter proteins are able to present heter-
ologous passenger proteins at the bacterial cell surface. For
example, the immunoglobulin A1 protease of Neisseria gonor-
rhoeae was shown to efficiently express the cholera toxin B
subunit on the surfaces of E. coli and Salmonella enterica se-
rovar Typhimurium cells (24). The E. coli autotransporter,
known as AIDA (adhesin involved in diffuse adherence) (3, 4,
5, 27), has been used to present functional T-cell epitopes as
well as enzymatically active -lactamase on the surface of the
native host (26, 28).
This report describes the construction of a fusion protein

6846
VOL. 70, 2002 RECOMBINANT M. TUBERCULOSIS PEPTIDE MEDIATES INVASION 6847

consisting of the AIDA autotransporter translocator and a

72-amino-acid region of Mce1A, designated InvX, which con-
tains the putative active domain with short flanking regions (9).
It is shown that expression of the InvX-AIDA fusion in E. coli
results in the stable presentation of functional InvX on the
bacterial surface. The AIDA system provided a convenient way
to analyze and quantify InvX-directed uptake into HeLa cells,
demonstrating its utility in the characterization of functional
domains from heterologous proteins.

MATERIALS AND METHODS

Bacterial strains and cell lines. E. coli strain UT4400, an ompT-negative
derivative of UT2300, was the host strain used in all experiments (15). E. coli was
grown in Luria-Bertani medium supplemented with 100 g of ampicillin/ml or 50
g of kanamycin/ml to maintain plasmids. The human epithelial cell line, HeLa
(ATCC CCL-2), was maintained in Dulbeccos modified Eagle medium supple-
mented with 10 mM sodium pyruvate, 10% fetal bovine serum, and 10 g of
penicillin-streptomycin/ml.
Construction of vectors. Plasmid pMK90 is an ampicillin-resistant pBR322
derivative that expresses a recombinant AIDA protein under the control of its
own promoter (3). The AIDA coding sequence has been altered to remove the
native passenger; it consists of a 49-amino-acid signal peptide, a 78-amino-acid
linker region incorporating a multiple cloning site, and the entire 440-amino-acid
-barrel core. A 240-bp DNA fragment encoding InvX (M. tuberculosis H37Rv
genome positions 198847 to 199063) was amplified by PCR from a plasmid
containing mce1A and cloned into pMK90, generating pMK100. The correct
insert was confirmed by sequencing. The amino acid sequence of InvX is VNA
DIKATTVFGGKYVSLTTPKNPTKRRITPKDVIDVRSVTTEINTLFQTLT
SIAEKVDPVKLNLTLSAAAE. Plasmid pMS2kan expresses a kanamycin resis-
tance marker.
Invasion and attachment assays. Gentamicin protection assays were per-
formed according to the method of Elsinghorst (16). HeLa cells were seeded at
105 cells per well onto coverslips or directly into 24-well plates and cultured for
24 h until confluent. Cell culture medium was modified to contain 1% mannose
and no antibiotics. Recombinant E. coli cells were added to the monolayer at a
multiplicity of infection (MOI) of 10:1 and incubated at 37C for 3 h. To
enumerate associated (adherent and intracellular) bacteria, we washed the
monolayer three times with phosphate-buffered saline and subsequently lysed the
cells in 0.1% Triton X-100 (Bio-Rad Laboratories, Hercules, Calif.). Alterna-
tively, washing was followed by incubation with medium containing 100 g of
gentamicin (Sigma Chemical Co., St. Louis, Mo.)/ml for 1 h to kill extracellular
bacteria and permit the enumeration of intracellular bacteria. The monolayer
was again washed three times with phosphate-buffered saline and lysed. Serial
dilutions of released bacteria were plated for counting. Associated and invaded
bacteria are presented as a percentage of the inoculum; results shown are the
mean values for an experiment performed in triplicate standard deviation.
Each experiment was executed three times using independent cultures, with
similar results.
In some experiments, HeLa cells were pretreated with various concentrations
of inhibitory agents; inhibitors were kept in the medium throughout the exper-
iment. Cytochalasin D (Sigma Chemical Co.) was added at a concentration of
0.01 to 1 g/ml, and the cells were incubated at 37C for 30 min prior to infection.
Nocodazole (Sigma Chemical Co.) was used at a concentration of 0.5 to 10
g/ml, with preincubation for 1 h at 4C followed by warming to 37C for 30 min.
Clostridium difficile toxin B (Sigma Chemical Co.) was added at a concentration
of 10 ng/ml, and incubation was continued for 20 h at 37C before the addition
of bacteria. The effect of inhibitors on HeLa cell viability was assessed by using
the trypan blue exclusion assay (2), and the effect on the growth of the recom-
binant E. coli strains in supplemented Dulbeccos modified Eagle medium was
determined.
After infection, HeLa cells seeded onto coverslips were fixed in ice-cold
methanol at 4C for 10 min and stained with SureStain Wright-Giemsa (Fisher
Scientific, Pittsburgh, Pa.) at room temperature for 10 min. Coverslips were
washed three times in water and mounted onto microscope slides with CytoSeal
(Stephens Scientific, Riverdale, N.J.). Slides were viewed with a Nikon Op-
tiphot-2 microscope and photographed with a Sony DKC-5000 digital camera.
Electron microscopy. Infected cells were prepared for examination by trans-
mission electron microscopy as previously described (9). Briefly, cells were fixed
in 2% glutaraldehyde and stained with osmium tetroxide solution before dehy-
dration through graded ethanol solutions. Cells were embedded in Spurs low-
FIG. 1. Indirect immunofluorescent staining of InvX-expressing E.
coli. E. coli cells were surface labeled with a mouse polyclonal antibody
raised against Mce1A and a fluorescein isothiocyanate-conjugated an-
ti-mouse secondary antibody. Fluorescence microscopy showed that E.
coli expressing the InvX-AIDA fusion protein bound anti-Mce1A an-
tibody but not mouse preimmune serum (not shown). Neither antibody
nor preimmune serum bound to the surface of E. coli cells expressing
AIDA alone (not shown). Magnification, 1,000.
viscosity embedding medium, and ultrathin sections were stained with uranyl
acetate and lead citrate. Samples were examined with a JEOL model 100CX-II
transmission electron microscope.
Immunofluorescence microscopy. E. coli cells were fixed onto microscope
slides with 0.4% paraformaldehyde for 10 min at room temperature, and non-
specific binding was blocked by incubation in 1% (wt/vol) bovine serum albumin
for 30 min. Slides were incubated for 1 h with a 1:40 dilution of a mouse antibody
raised against Mce1A (HI5), washed, and incubated with a 1:100 dilution of
fluorescein isothiocyanate-labeled anti-mouse antibody (Sigma Chemical Co.)
for 1 h. After extensive washing, the coverslips were mounted with SlowFade
Light antifade reagent (Molecular Probes, Eugene, Oreg.). Slides were viewed
on a Nikon Eclipse TE300 inverted microscope with an epifluorescence attach-
ment, and photomicrography was performed with Nikon U-III equipment.
Statistical analyses. A two-tailed Students t test, adjusted for unequal vari-
ance, was used to compare the mean CFUs recovered after the gentamicin
protection assays.
RESULTS
InvX is presented on the surface of E. coli cells by the AIDA
autotransporter translocator. The recombinant AIDA auto-
transporter translocator was expressed from the plasmid
pMK90. A 72-amino-acid region of Mce1A (positions 106 to
177) termed InvX, which contains the 58-amino-acid putative
active domain with short flanking regions (9), was cloned into
the AIDA vector, resulting in the construct pMK100. An
ompT-negative E. coli strain, UT4400, was used to express
AIDA fusions, as OmpT is known to sometimes cleave surface-
exposed passengers (25, 38). Immunoblotting with polyclonal
antibodies raised against the core AIDA translocator or
Mce1A indicated that the expected fusion protein was ex-
pressed in E. coli UT4400(pMK100) cells (data not shown).
Surface expression of InvX was ascertained by loss of immu-
noreactivity with the anti-Mce1A antibody after trypsin diges-
tion of whole cells (data not shown) and verified by indirect
immunofluorescence of whole cells (Fig. 1).
InvX can mediate adhesion to HeLa cells and is sufficient
6848 CASALI ET AL. INFECT. IMMUN.

FIG. 2. InvX-mediated association and invasion of HeLa cells. HeLa cells were infected with E. coli harboring the InvX-expressing plasmid
pMK100 or the control plasmid pMK90 at an MOI of 10:1 for 3 h. Bars represent the mean number of associated (a) or intracellular (b) bacteria
as a percentage of the inoculum in a representative experiment performed in triplicate. Standard deviations are indicated by error bars.

for internalization. InvX-expressing E. coli cells showed a 40-
fold greater association with HeLa cells than the control strain
(P 0.008) (Fig. 2a). The recombinant E. coli strains had a
doubling time of approximately 1 h in tissue culture medium;
hence, the level of association after 3 h was calculated at
almost 400% that of the inoculum. Adherence was also as-
sessed microscopically by Giemsa staining. E. coli(pMK100)
showed extensive association with HeLa cells, forming dense
lacy networks on the monolayer, whereas the control strain
showed little adherence (Fig. 3).
We assessed the ability of InvX to mediate uptake of the
host E. coli cells by using gentamicin protection assays (16). E.
coli(pMK100) cells showed invasion levels at the 3-h time point
that were 25-fold higher than those for the control cells, with
0.8% of the inoculum protected (P 0.011) (Fig. 2b). Incu-
bation of the infected monolayer for up to 3 h in the presence
of gentamicin did not result in increased bacterial recovery,
indicating that the recombinant E. coli did not replicate inside
HeLa cells during this time period.
In order to confirm that the gentamicin-protected bacteria
truly resided intracellularly, we additionally examined infected
cells by electron microscopy (Fig. 4). After 3 h of incubation, E.
coli(pMK100) cells showed extensive adherence to the HeLa
cell surface and appeared to elicit filopodia-like membrane
protrusions. Intracellular bacteria were seen in membrane-
bound compartments, definitively demonstrating that they had
been internalized. Control E. coli(pMK90) was rarely seen
associated with HeLa cells and was never seen inside cells.
Disruption of the actin cytoskeleton inhibits invasion. To
determine whether actin microfilaments were required for
InvX-mediated invasion, HeLa cells were pretreated with cy-
tochalasin D, which specifically inhibits actin polymerization
(33). Gentamicin protection assays showed that treatment of
cells with 1 g of cytochalasin D/ml reduced the invasion of E.
coli(pMK100) to control levels (P 0.016) and that the inhi-
bition of invasion was dose dependent (Fig. 5a and b). HeLa
cell viability and bacterial viability were not affected. Bacterial
adhesion was also not affected by cytochalasin D treatment,
indicating that the role of actin microfilaments was in bacterial
uptake.
Inhibition of Rho-family GTPases prevents invasion. C. dif-
ficile toxin B inactivates Rho family GTPases by monoglucosy-
lation with a dominant negative effect (23). Toxin B treatment
resulted in the rounding up of HeLa cells but did not affect

FIG. 3. InvX-mediated attachment to HeLa cells. HeLa cells were infected with the control strain (a) or E. coli cells expressing InvX (b) at an
MOI of 10:1. After 3 h, the monolayer was washed thoroughly and stained with Giemsa to visualize bacteria associated with the HeLa cells.
Magnification, 400.
VOL. 70, 2002 RECOMBINANT M. TUBERCULOSIS PEPTIDE MEDIATES INVASION
FIG. 4. Electron micrographs of InvX-expressing E. coli invading HeLa cells. E. coli cells expressing InvX were incubated with HeLa cells for
3 h at an MOI of 10:1 before they were processed for transmission electron microscopy. The micrographs show that E. coli cells attached to the
HeLa cell surface elicit membrane protrusions (black arrows) and that intracellular bacilli are located within membrane-bound compartments
(white arrows). Scale bars are 1 m.
their viability. This pretreatment completely inhibited E. co-
li(pMK100) entry (P 0.003) (Fig. 5e) but did not affect
bacterial adhesion or viability. Thus, it appears that Rho family
GTPases are involved in the invasion mechanism of InvX-
expressing E. coli. It was not possible to determine the dose
dependency of inhibition due to the narrow concentration
range at which toxin B was active without being toxic.
Microtubule disruption inhibits invasion. Pretreatment of
HeLa cells with 10 g of nocodazole/ml, which specifically
depolymerizes microtubules, resulted in rounding of the cells
without affecting viability. Nocodazole treatment clearly inhib-
ited the uptake of E. coli(pMK100), reducing it to the level of
the control, E. coli(pMK90) (P 0.009) (Fig. 5c). It was shown
that the inhibition of invasion was dose dependent (Fig. 5d)
but that bacterial viability and association were not affected.
Hence, E. coli expressing InvX requires an intact microtubule
network for entry but not for adhesion to HeLa cells.
Invasion does not affect the uptake of bystander bacteria.
We wished to determine whether internalization of InvX-ex-
pressing E. coli resulted in the nonspecific uptake of proximal
bacilli. Thus, HeLa cells were simultaneously infected with
either E. coli(pMK100) or E. coli(pMK90) cells, which carry an
ampicillin resistance marker, and a second noninvasive recom-
binant E. coli strain expressing kanamycin resistance. Recov-
ered intracellular bacteria were plated on ampicillin and kana-
mycin to determine whether nonspecific uptake occurred. No
significant uptake of the noninvasive kanamycin-resistant
strain was observed when coinfected with either E. co-
li(pMK100) or E. coli(pMK90) (Fig. 6).
DISCUSSION
Mce1A of M. tuberculosis has previously been shown to fa-
cilitate the uptake of E. coli into HeLa cells (1). This activity
appears to reside in the amino acid region between positions
106 and 163 of Mce1A, the deletion of which rendered the
truncated protein inactive in HeLa cell invasion assays using
latex microspheres (9). In previous studies, in which E. coli was
shown to be taken up by HeLa cells (1), the mechanism by
6849
which the recombinant protein gained access to the surface of
the bacilli to induce cell uptake was not investigated. It is
possible that E. coli became coated with recombinant protein
released by low-level lysis in the growing culture, which is
consistent with the reported adhesive properties of Mce1A (9).
The role of the active domain in the cell uptake of latex
microspheres could not be readily quantified, as light micros-
copy did not distinguish internalized versus surface-associated
microspheres and electron microscopy is not a reliable quan-
titative technique. Thus, the ability of the AIDA autotrans-
porter translocator to present Mce1A-derived peptide on the
surface of E. coli cells was investigated as a system that would
enable us to more reliably characterize and quantify the inter-
action of the putative active domain with mammalian cells.
In this study, the AIDA autotransporter translocator was
shown to be capable of presenting InvX, a 72-amino-acid pep-
tide encompassing the putative active domain of Mce1A, on
the surface of E. coli cells. Surface localization was demon-
strated by the binding of InvX-specific antibodies to whole cells
by indirect immunofluorescence and confirmed by the loss of
immunoreactivity after protease treatment. E. coli cells ex-
pressing InvX-AIDA on their surface were tested for their
ability to adhere to and invade HeLa cells compared to a
control recombinant E. coli strain expressing the AIDA trans-
locator without any passenger domain. An adherence assay
revealed that, after 3 h of incubation, dense networks of InvX-
expressing bacteria were associated with the HeLa cell mono-
layer. Approximately 0.8% of the inoculum was protected from
gentamicin, implying an intracellular location, and electron
microscopic analysis of HeLa cells incubated with InvX-ex-
pressing E. coli definitively demonstrated the presence of in-
ternalized bacteria. Thus, InvX promotes the attachment of
and is sufficient for the entry of noninvasive bacteria into HeLa
cells. The ability of the AIDA autotransporter translocator to
present functional peptides establishes its potential for use in
the analysis of surface interactions between bacterial and
mammalian cells.
Invasive bacterial pathogens frequently hijack host cell cy-
6850 CASALI ET AL. INFECT. IMMUN.

FIG. 5. Effect of cytoskeletal inhibitors on InvX-mediated invasion. Gentamicin protection assays were used to determine the effects of
cytochalasin D (a and b), nocodazole (c and d), or toxin B (e) on the level of invasion of E. coli(pMK90) (filled bars) and E. coli(pMK100) (open
bars) into HeLa cells. Bars represent the mean number of internalized bacilli from a representative experiment performed in triplicate. Standard
deviations are indicated by error bars.

toskeletal components and subvert host-signaling pathways to
promote their own uptake. Numerous pathogens are able to
trigger the rearrangement of host cell actin, and a number of
bacterial virulence factors target the Rho family of small GT-
Pases, Rho, Rac, and Cdc42, which are key regulators of actin
reorganization (17). The inhibition of InvX-induced uptake in
the presence of cytochalasin D and C. difficile toxin B indicates
that internalization is actin dependent and that Rho GTPases
are involved in the signaling pathway of actin rearrangement.
Microtubules are less often exploited by invasive bacteria than
microfilaments, but there are a growing number of instances in
which invasion has been shown to be microtubule dependent
(31). Internalization of InvX-expressing E. coli was abolished
by pretreatment with nocodazole, demonstrating that an intact
microtubule network is needed for InvX-mediated invasion. It
is interesting to note that M. tuberculosis invasion of cultured
alveolar epithelial cells has also been shown to be both micro-
filament and microtubule dependent (6).
VOL. 70, 2002 RECOMBINANT M. TUBERCULOSIS PEPTIDE MEDIATES INVASION
FIG. 6. Effect of InvX-mediated invasion on bystander bacteria.
Control HeLa cells were infected with either E. coli(pMK90) (shaded
bars) or the InvX-expressing E. coli(pMK100) (white bars). In addi-
tion, HeLa cells were coinfected with the same number of noninvasive
bystander E. coli(pMS2) cells (black bars) in the presence of either E.
coli(pMK90) or E. coli(pMK100). The number of internalized bacilli
was determined in triplicate by using a gentamicin protection assay,
and the mean result was expressed as a percentage of the inoculum.
Error bars indicate standard deviations.
Strategies for the exploitation of the host cytoskeleton by
bacterial pathogens during entry can be divided into two dis-
tinct categories, termed the zipper and trigger mechanisms
(13). The zipper mechanism involves local cytoskeletal rear-
rangement and requires direct contact between bacterial li-
gands and cellular receptors, which sequentially encircle the
organism, resulting in a tight apposition between the host cell
membrane and the bacillus. The most well-studied examples of
this receptor-mediated zipper-type mechanism of entry include
the Yersinia pseudotuberculosis invasin and the Listeria mono-
cytogenes internalins InlA and InlB (7, 8, 22, 29). In contrast,
the trigger mechanism, exemplified by Salmonella and Shigella
entry, requires a large number of proteins (17). This mecha-
nism involves the induction of dramatic membrane ruffles,
which trap the bacilli in spacious vacuoles, resulting in the
passive entry of bacteria and particles in the vicinity. InvX-
induced invasion did not affect the uptake of proximal by-
stander bacteria, suggesting that this mode of entry resembles
the receptor-mediated zipper-type mechanism. The electron
micrographs showing filopodia encircling the bacilli, which are
subsequently enclosed in vacuoles with tightly fitting mem-
branes, provide additional evidence in support of this mecha-
nism. Consistent with these observations is the finding that
solubilized recombinant Mce1A is able to competitively inhibit
the uptake of Mce1A-coated microspheres (9). However, de-
spite extensive investigation, the receptor for Mce1A and InvX
remains elusive. The ability to express the active domain of
Mce1A by the AIDA system may now facilitate its identifica-
tion.
ACKNOWLEDGMENTS
We thank the Electron Microscopy Lab and the Scientific Visual-
ization Center at the University of California, Berkeley, for assistance
in preparing the images.
This work was supported by NIH grant number AI53266.
N.C. and M.K. contributed equally to this work.
6851
REFERENCES
1. Arruda, S., G. Bomfim, R. Knights, T. Huima-Byron, and L. W. Riley. 1993.
Cloning of an M. tuberculosis DNA fragment associated with entry and
survival inside cells. Science 261:14541457.
2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.
Smith, and K. Struhl. 1992. Short protocols in molecular biology. John Wiley
and Sons, New York, N.Y.
3. Benz, I., and M. A. Schmidt. 1989. Cloning and expression of an adhesin
(AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.
Infect. Immun. 57:15061511.
4. Benz, I., and M. A. Schmidt. 1992. AIDA-I, the adhesin involved in diffuse
adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is
synthesized via a precursor molecule. Mol. Microbiol. 6:15391546.
5. Benz, I., and M. A. Schmidt. 1992. Isolation and serologic characterization of
AIDA-I, the adhesin mediating the diffuse adherence phenotype of the
diarrhea-associated Escherichia coli strain 2787 (O126:H27). Infect. Immun.
60:1318.
6. Bermudez, L. E., and J. Goodman. 1996. Mycobacterium tuberculosis invades
and replicates within type II alveolar cells. Infect. Immun. 64:14001406.
7. Braun, L., B. Ghebrehiwet, and P. Cossart. 2000. gC1q-R/p32, a C1q-binding
protein, is a receptor for the InlB invasion protein of Listeria monocytogenes.
EMBO J. 19:14581466.
8. Braun, L., H. Ohayon, and P. Cossart. 1998. The InIB protein of Listeria
monocytogenes is sufficient to promote entry into mammalian cells. Mol.
Microbiol. 27:10771087.
9. Chitale, S., S. Ehrt, I. Kawamura, T. Fujimura, N. Shimono, N. Anand, S.
Lu, L. Cohen-Gould, and L. W. Riley. 2001. Recombinant Mycobacterium
tuberculosis protein associated with mammalian cell entry. Cell. Microbiol.
3:247254.
10. Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V.
Gordon, K. Eiglmeier, S. Gas, C. E. Barry, F. Tekaia, K. Badcock, D.
Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies, K. Devlin, T.
Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornby, K. Jagels, A. Krogh,
J. McLean, S. Moule, L. Murphy, K. Oliver, J. Osborne, M. A. Quail, M. A.
Rajandream, J. Rogers, S. Rutter, K. Seeger, J. Skelton, R. Squares, S.
Squares, J. E. Sulston, K. Taylor, S. Whitehead, and B. G. Barrell. 1998.
Deciphering the biology of Mycobacterium tuberculosis from the complete
genome sequence. Nature 393:537544.
11. Cywes, C., H. C. Hoppe, M. Daffe, and M. R. Ehlers. 1997. Nonopsonic
binding of Mycobacterium tuberculosis to complement receptor type 3 is
mediated by capsular polysaccharides and is strain dependent. Infect. Im-
mun. 65:42584266.
12. Downing, J. F., R. Pasula, J. R. Wright, H. L. D. Twigg, and W. J. D. Martin.
1995. Surfactant protein A promotes attachment of Mycobacterium tubercu-
losis to alveolar macrophages during infection with human immunodefi-
ciency virus. Proc. Natl. Acad. Sci. USA 92:48484852.
13. Dramsi, S., and P. Cossart. 1998. Intracellular pathogens and the actin
cytoskeleton. Annu. Rev. Cell Dev. Biol. 14:137166.
14. Dye, C., S. Scheele, P. Dolin, V. Pathania, and M. C. Raviglione. 1999.
Global burden of tuberculosis: estimated incidence, prevalence, and mortal-
ity by country. JAMA 282:677686.
15. Elish, M. E., J. R. Pierce, and C. F. Earhart. 1988. Biochemical analysis of
spontaneous fepA mutants of Escherichia coli. J. Gen. Microbiol. 134:1355
1364.
16. Elsinghorst, E. A. 1994. Measurement of invasion by gentamicin resistance.
Methods Enzymol. 236:405420.
17. Finlay, B. B., and P. Cossart. 1997. Exploitation of mammalian host cell
functions by bacterial pathogens. Science 276:718725.
18. Flesselles, B., N. N. Anand, J. Remani, S. M. Loosmore, and M. H. Klein.
1999. Disruption of the mycobacterial cell entry gene of Mycobacterium bovis
BCG results in a mutant that exhibits a reduced invasiveness for epithelial
cells. FEMS Microbiol. Lett. 177:237242.
19. Henderson, I. R., R. Cappello, and J. P. Nataro. 2000. Autotransporter
proteins, evolution and redefining protein secretion. Trends Microbiol.
8:529532.
20. Henderson, I. R., F. Navarro-Garcia, and J. P. Nataro. 1998. The great
escape: structure and function of the autotransporter proteins. Trends Mi-
crobiol. 6:370378.
21. Hernandez-Pando, R., M. Jeyanathan, G. Mengistu, D. Aguilar, H. Orozco,
M. Harboe, G. A. W. Rook, and G. Bjune. 2000. Persistence of DNA from
Mycobacterium tuberculosis in superficially normal lung tissue during latent
infection. Lancet 356:21332138.
22. Isberg, R. R., D. L. Voorhis, and S. Falkow. 1987. Identification of invasin: a
protein that allows enteric bacteria to penetrate cultured mammalian cells.
Cell 50:769778.
23. Just, I., J. Selzer, M. Wilm, C. von Eichel-Streiber, M. Mann, and K.
Aktories. 1995. Glucosylation of Rho proteins by Clostridium difficile toxin B.
Nature 375:500503.
24. Klauser, T., J. Pohlner, and T. F. Meyer. 1990. Extracellular transport of
cholera toxin B subunit using Neisseria IgA protease -domain: conforma-
tion-dependent outer membrane translocation. EMBO J. 9:19911999.
6852 CASALI ET AL.
25. Klauser, T., J. Pohlner, and T. F. Meyer. 1992. Selective extracellular release
of cholera toxin B subunit by Escherichia coli: dissection of Neisseria IgA
-mediated outer membrane transport. EMBO J. 11:23272335.
26. Konieczny, M. P., M. Suhr, A. Noll, I. B. Autenrieth, and M. A. Schmidt.
2000. Cell surface presentation of recombinant (poly-)peptides including
functional T-cell epitopes by the AIDA autotransporter system. FEMS Im-
munol. Med. Microbiol. 27:321332.
27. Konieczny, M. P. J., I. Benz, B. Hollinderbaumer, C. Beinke, M. Niederweis,
and M. A. Schmidt. 2001. Modular structure of the AIDA autotransporter
translocator: evidence for a transmembrane -sheet core stabilized by a
surface-exposed N-terminal domain. Antonie Leeuwenhoek 80:1934.
28. Lattemann, C. T., J. Maurer, E. Gerland, and T. F. Meyer. 2000. Autodis-
play: functional display of active -lactamase on the surface of Escherichia
coli by the AIDA-I autotransporter. J. Bacteriol. 182:37263733.
29. Lecuit, M., H. Ohayon, L. Braun, J. Mengaud, and P. Cossart. 1997. In-
ternalin of Listeria monocytogenes with an intact leucine-rich repeat region is
sufficient to promote internalization. Infect. Immun. 65:53095319.
30. Loveless, B. J., and M. H. Saier, Jr. 1997. A novel family of channel-forming,
autotransporting, bacterial virulence factors. Mol. Membr. Biol. 14:113123.
31. Oelschlaeger, T. A., and D. J. Kopecko. 2000. Microtubule dependent inva-
sion pathways of bacteria. Subcell. Biochem. 33:319.
32. Pohlner, J., R. Halter, K. Beyreuther, and T. F. Meyer. 1987. Gene structure
Editor: S. H. E. Kaufmann
INFECT. IMMUN.
and extracellular secretion of Neisseria gonorrhoeae IgA protease. Nature
325:458462.
33. Rosenshine, I., S. Ruschkowski, and B. B. Finlay. 1994. Inhibitors of cy-
toskeletal function and signal transduction to study bacterial invasion. Meth-
ods Enzymol. 236:467476.
34. Schlesinger, L. S. 1993. Macrophage phagocytosis of virulent but not atten-
uated strains of Mycobacterium tuberculosis is mediated by mannose recep-
tors in addition to complement receptors. J. Immunol. 150:29202930.
35. Schlesinger, L. S., C. G. Bellinger-Kawahara, N. R. Payne, and M. A. Hor-
witz. 1990. Phagocytosis of Mycobacterium tuberculosis is mediated by human
monocyte complement receptors and complement component C3. J. Immu-
nol. 144:27712780.
36. Schorey, J. S., M. C. Carroll, and E. J. Brown. 1997. A macrophage invasion
mechanism of pathogenic mycobacteria. Science 277:10911093.
37. Shepard, C. C. 1955. Phagocytosis by HeLa cells and their susceptibility to
infection by human tubercle bacilli. Proc. Soc. Exp. Biol. Med. 90:392396.
38. Suhr, M., I. Benz, and M. A. Schmidt. 1996. Processing of the AIDA-I
precursor: removal of AIDAc and evidence for the outer membrane anchor-
ing as a -barrel structure. Mol. Microbiol. 22:3142.
39. Tekaia, F., S. V. Gordon, T. Garnier, R. Brosch, B. G. Barrell, and S. T. Cole.
1999. Analysis of the proteome of Mycobacterium tuberculosis in silico. Tu-
ber. Lung Dis. 79:329342.