Escolar Documentos
Profissional Documentos
Cultura Documentos
2
3 Engineering Human Vaginal Lactobacillus for Surface Expression of Two-Domain
4 CD4
5
6 Xiaowen Liu¶1, Laurel A. Lagenaur¶1, Peter P. Lee1, 2, Qiang Xu1*
D
7
From 1Osel, Inc., 4008 Burton Drive, Santa Clara, CA 95054, 2Department of Medicine,
E
8
10
11
P T
Running Title: Surface Anchoring a Viral Receptor in Lactobacilli
E
12
C
13 * To whom correspondence should be addressed:
14
15
16
17
Osel, Inc.
19 Fax: 408-986-0019
20 E-mail: qxu@oselinc.com
1
1 Abstract
2 Women are at significant risk of heterosexually transmitted HIV infection, with the
3 mucosal epithelium of the cervix and vaginal serving as a major portal of entry. The
D
5 of lactobacilli, which may be genetically modified to serve as a more efficient protective
E
6 barrier against heterosexual transmission of HIV. We selected a vaginal strain of
P T
HIV proteins. Genomic sequencing analyses revealed that the L. jensenii 1153 encodes
several unique high-molecular-weight cell wall anchored proteins with a C-terminal cell
E
10 wall sorting LPQTG motif. In this report, we employed these proteins to express a
C
11 surface-anchored two-domain CD4 (2D CD4) in L. jensenii 1153. Our studies indicated
12 that the C-terminal cell wall sorting signal LPQTG motif alone is insufficient to drive
13
14
15
16
C
surface expression of heterologous proteins, and display of surface-anchored 2D CD4
A
required native sequences of a defined length upstream of the unique C-terminal LPQTG
cell wall sorting signal and the positively charged C-terminus in a Lactobacillus-based
expression system. The modified L. jensenii displayed 2D CD4 molecules that were
20 based surface expression system, with potential broad applicability, represents a major
21 step toward developing an inexpensive, yet durable approach to topical microbicides for
23 pathogens.
2
1 Introduction
3 worldwide is via heterosexual contact (38). Women are particularly at risk for HIV
4 infection, as the efficiency of HIV transmission from male to female is greater than vice
D
5 versa (1). There are few means by which women can actively protect themselves against
E
6 HIV infection, particularly in the absence of a protective vaccine or the inability to
7 negotiate condom use. The need to develop new methods of HIV prevention that are
P T
controlled by women is urgently recognized by health organizations worldwide.
The cervico-vaginal mucosa is the main site of HIV entry in women. While the exact
E
10 cell type and site of transmission are actively investigated (7, 17, 42, 50), it is believed
C
11 that a potential microbicide product will be most effective if it offers a broad protection
13
14
15
16
C
protective mucosa in the vagina is populated with commensal bacteria typically
A
dominated by H2O2-producing lactobacilli. The dominance of lactobacilli over
pathogenic anaerobes is positively associated with vaginal health (35). The principal
Lactobacillus species isolated from the vaginal mucosa of healthy women are L. jensenii,
17 L. crispatus, L. gasseri, and L. iners (2, 48, 51). These species are efficient colonizers of
18 the vaginal mucosa and likely exist as a natural ‘biofilm’ composed of bacteria and
20 flora has been associated with establishment of opportunistic infections in urinary tract,
21 bacterial vaginosis, and increased risk of acquiring HIV and herpes simplex virus type 2
22 in women (11, 41, 44). Thus, vaginal lactobacilli play a critical role in the maintenance
3
1 Through genetic engineering, a member of the vaginal microflora may be enhanced to
3 diseases (STD), such as HIV. Our novel approach involves genetically modifying a
4 natural human isolate of H2O2-producing lactobacilli to express the first two domains of
D
5 the high affinity HIV-binding protein, human CD4 (39). CD4, a member of the
E
6 immunoglobulin (Ig) superfamily, is the primary host receptor for HIV entry into
P T
four Ig-like domains, D1 to D4. The two N-terminal domains, 2D CD4 (K1-S183) (39),
encode and properly fold to form the gp120 binding epitope (4) and, when expressed in
E
10 the absence of the remaining domains of CD4, retain the high-affinity binding to HIV-1
C
11 gp120 (40). Given that human CD4 is an endogenous protein in the human immune
12 system, it has less potential to have immunogenic properties when expressed on the
13
14
15
16
C
mucosal surface in vivo. Importantly, glycosylation of CD4 is not required for binding to
A
gp120 (25). Soluble 2D CD4 that adopts a native disulfide-bonded conformation has
been expressed in a number of well-established systems, including L. jensenii (6, 10, 12).
17 viruses at the bacterial surface, thus impeding the access of viruses to underlying
18 epithelial cells and lymphocytes targets. These trapped viruses may be rendered unstable
20 compounds, such as lactic acid and hydrogen peroxide, secreted by the lactobacilli (21),
22 Surface expression of heterologous proteins has been achieved via the sortase-
4
1 Streptococcus gordonii, Lactobacillus paracasei, and Staphylococcus carnosus (5, 18,
2 23, 31, 43). While genetic manipulation of two human vaginal isolates of L. fermentum
3 and L. jensenii has been reported (10, 33), this is the first report on surface expression of
D
5 report, we describe a broadly applicable approach for genetic modification of lactobacilli,
E
6 enabling expression of 2D CD4 covalently linked to peptidoglycan in the cell wall of L.
7 jensenii 1153. The surface anchored 2D CD4 adopted a native conformation, recognizing
P T
two conformation dependent anti-CD4 antibodies (Leu3a and Sim.4). We demonstrated
that a native cell wall sorting signal alone was insufficient to drive surface expression of
E
10 2D CD4 and required fusion with native upstream sequences of a defined length. The
C
11 approach reported here also affords surface expression of heterologous proteins in other
12 Lactobacillus species.
A C
5
1 Materials and Methods
3 vaginal isolates of Lactobacillus, including L. jensenii 1153, L. jensenii Xna (10, 27), L.
4 gasseri 1151, and L. casei Q were routinely cultivated at 37°C and 5% CO2 in either
D
5 MRS broth or Rogosa SL broth (Difco) as described previously (10). A non-vaginal
E
6 isolate of L. paracasei 343 (from Dr. Peter H. Pouwels) was cultured similarly. Shuttle
P T
electroporated into lactobacilli (10). Transformed lactobacilli were routinely propagated
either on MRS agar plates or in liquid media containing 20 µg/ml erythromycin (10).
E
10 Identification of Protein Sequences with Cell Wall Anchor Motifs —The genome
C
11 sequence of L. jensenii 1153 was determined as described previously (27). Cell wall-
12 anchored proteins of Gram-positive bacteria share several common features that enable
13
14
15
16
C
them to be covalently anchored to the cell wall peptidoglycan (16). Among them, they
A
have a conserved C-terminal LPXTG motif, followed by a hydrophobic stretch of amino
acids and a short charged tail, which are collectively called the cell wall sorting signal
(16). Other motifs, including LPXTA in L. paracasei (19), have been identified.
17 Accordingly, a computer script was written to identify motifs similar to LPXTG and
18 LPXTA in all reading frames of the assembled contigs of the partially sequenced L.
19 jensenii 1153 genome. We used independent PCR amplification and manual sequencing
20 to verify the contigs containing putative cell wall anchor motifs. For anchor sequence
23 3’) to PCR amplify the 5712 bp coding region. The sequences were also subjected to a
6
1 BLAST search for sequence homology to cell wall-anchored proteins in Gram-positive
2 bacteria.
4 anchoring in L. jensenii, an expression cassette was constructed and subcloned into the
D
5 SacI and XbaI sites of a shuttle vector pOSEL175 (27). The expression cassette contains
E
6 four components, including a Lactobacillus-compatible P23 promoter, CbsA signal
7 sequence (10), DNA sequence encoding a heterologous protein, and cell wall anchoring
P T
domains from known or putative cell surface proteins in Gram-positive bacteria. Two–
domain CD4 (K1-S183) was synthesized and subcloned as previously described (10).
E
10 Unique restriction sites, including SacI, EcoRI, NheI, MfeI, and XbaI, were placed
C
11 between each component from 5’ to 3’ ends, respectively. Amplification of each
12 component was performed using conventional PCR with Pfu DNA polymerase.
13
14
15
upstream of the LPQTG motif in C370 sequence were amplified with flanking MfeI and
XbaI restriction sites from the genomic DNA of L. jensenii 1153. The same reverse
18 underlined) was used, in pair with the forward primers listed in Table I for each PCR
21 above to amplify the nucleotide sequences corresponding to the region containing the C-
22 terminal LPQTG domain and their upstream 200 amino acids (CWA200) in C370
23 sequence that contains the last two and a half repeats (Fig. 1A).
7
1 To drive surface expression of 2D CD4, the protein coding sequence of 2D CD4 was
3 sequence. The DNA fragments resulting from the above PCR reactions were digested
4 with both MfeI and XbaI. The resulting fragments were ligated with MfeI/XbaI double
D
5 digested pOSEL651 (10). The resulting plasmids were designated as pOSEL262 (with
E
6 no repeat), pOSEL268 (with one repeat), pOSEL278 (with two repeats), pOSEL280 (with
7 four repeats), pOSEL281 (with seven repeats) and pOSEL276 (with eight repeats).
P T
Similarly, the MfeI/XbaI digested fragment corresponding to CWA200 sequence
upstream of LPQTG motif in C370 was cloned, yielding pOSEL249 (with 2.5 repeats).
E
10 c-Myc Tagging of Putative Cell Wall Anchor Sequences of L. jensenii—
C
11 Oligonucleotide primers corresponding to c-Myc epitope (EQKLISEEDL) were designed
13
14
15
16
C
GCGCTAGCGAACAGAAACTGATCTCCGAAGAGGACCTGTTGAAGAAGGCAG
A
AAGAAGT-3’; reverse primer 5’-CCGCAATTGTTATGCTTCATCATCTTTTCT-3’),
terminal cell wall sorting signal and upstream CWA200 in C370 sequence. The
17 amplified PCR fragments were digested with both MfeI and NheI, and then subcloned
23 primers listed in Table II were used to delete the positively charged amino acids
8
1 from the C-terminus of the C370 sequence. All reverse primers contained an XbaI
2 restriction site. The amplified PCR fragments were digested with both MfeI and
3 XbaI, and then subcloned into MfeI/XbaI double-digested pOSEL249. The clones
D
5 into L. jensenii for protein analysis.
E
6 Oligonucleotide-directed Mutagenesis of LPXTG Motif of Putative Cell Wall
P T
Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Plasmid pOSEL249 (containing the
protein coding sequence of 2D CD4, and the C-terminal LPQTG domain and their
E
10 upstream CWA200 of C370 sequence) was used as a template. The mutagenic primers
C
11 were designed based on preferred codon usage in L. jensenii and the nucleotide sequences
12 corresponding to LPQTG and its flanking sequences in C370 (Table II). After the PCR
13
14
15
16
C
reaction, Dpn I enzyme was added to the amplification mixture to degrade the methylated
A
parental plasmids. Newly synthesized plasmids were introduced into chemically
competent E. coli Top10 cells (Invitrogen, Carlsbad, CA) in LB broth supplemented with
200 µg/ml erythromycin. Plasmids were subsequently isolated for DNA sequencing
17 (Biotech Core, Mountain View, CA) to identify clones with the desired mutations.
20 late log phase or close to stationary phase, containing 109 cells were centrifuged at 12,000
21 x g for 5 min. The resulting cell pellets were washed once in 20 mM HEPES, pH 7.2 and
23 The bacterial cell wall was digested in the presence of a muramidase, mutanolysin
9
1 (Sigma Chemical Co., St. Louis, MO) at a final concentration of 15 units/ml for 1 hr at
2 37°C. Afterward, the cells were centrifuged at 2,500 x g for 10 min to isolate cell wall-
3 enriched fraction from protoplast-enriched one (34). The resulting samples were heat
D
5 the cell wall- and protoplast-enriched fractions, respectively.
E
6 Western Analysis of Heterologous Protein Expression in L. jensenii—The modified
7 lactobacilli were grown at 37oC and 5% CO2 in MRS or Rogosa SL broth buffered with
P T
100 mM HEPES (pH 7.4) to late log phase or early stationary phase. Cell-free
E
10 g, 10 min) and proteins were heat denatured in the SDS-PAGE loading buffer (50 mM
C
11 Tris-HCl, pH 6.8, 10 mM DTT, 0.4% SDS, 6% sucrose, 0.01% bromophenol blue).
12 Afterward, soluble proteins were resolved and detected as describe previously (10) with
13
14
15
16
C
the rabbit polyclonal anti-CD4 antibody, T4-4 (NIH AIDS Reagents Reference Program),
A
the mouse monoclonal anti-c-Myc antibody (Invitrogen), or the rabbit polyclonal anti-
C370 antibody, OI9 (developed at AbboMax, Inc., San Jose, CA). The antigen-antibody
18 Piscataway, NJ).
23 HEPES, pH 7.4. Bacteria at early log phase were washed and suspended in phosphate
10
1 buffered saline (PBS) containing 2% fetal bovine serum (FBS). About 2 x 108 cells were
2 probed with rabbit anti-CD4 antibody T4-4 or rabbit anti-C370 anchor protein antibody
3 OI9 at 1:3000 dilutions in PBS, 2% FBS at 4ºC for 30 min. After washes in PBS
4 containing 2% FBS, the bacteria were then probed with Alexa Fluor 488 goat anti-rabbit
D
5 IgG (Molecular Probes, Eugene, OR) at 1:200 dilutions at 4ºC for 30 min. Alternatively,
E
6 the modified L. jensenii were probed with anti-CD4 monoclonal antibody (MAb), Sim.4
P T
by probing with 10 µl of neat FITC-conjugated goat anti-mouse IgG (Becton Dickinson,
San Jose, CA). The anti-CD4 MAbs, Sim.4 and Leu3a, recognize conformational
E
10 epitopes in the HIV-1 gp120-binding site (domain 1 of CD4) (39). The fluorescence of
C
11 20,000 labeled cells in triplicate samples was analyzed in a FACScan system (Becton
12 Dickinson) running with the CellQuest software. Density plot output (Side scatter or
13
14
15
16
C
forward scatter vs. fluorescence) was obtained from modified L. jensenii, with those
A
harboring plasmid pOSEL175 as background control. The shift in mean fluorescence
intensity between the plots was taken as a measure of antibody binding to bacterial
surface and calculated using FLOWJO software (Tree Star, Inc., Ashland, OR).
18 anti-CD4 MAb Leu3a directly conjugated with phycoerythrin (PE) (Pharmingen, San
19 Diego, CA) was used to label bacteria and Quantum Simply Cellular (QSC) beads (Bangs
20 Laboratories, Inc., Fishers, IN). The QSC beads contain a blank bead and 4 populations
21 of microbeads with varying capacities to bind mouse monoclonal IgG. Quantum R-PE
22 medium reference beads (Bangs Laboratories, Inc.) were also used for quantitation of the
11
1 fluorochrome (MESF) using QuickCal Sample Report software. The modified bacteria
2 were grown to OD600 of about 0.4, harvested, and washed with PBS, 1% FBS. The
3 bacteria and the QSC beads were incubated with 10 µl of anti-CD4 MAb conjugated with
4 PE for 1 hr, then washed 3 times with PBS, 1% FBS. Finally, the bacteria and both QSC
D
5 and QR-PE beads were visualized on a FACScan (Becton Dickinson).
E
6
7 harboring pOSEL175 or plasmids designed for surface expression were cultured to early
P T
log phase in Rogosa as described above. Approximately 1.4 x 108 bacteria were washed
with PBS, 1% FBS and resuspended in 50 µl of buffer. Bacteria were incubated directly
E
10 with 20 µl of Leu3a-FITC (BD Biosciences, San Jose, CA) for 20 min and washed 3
C
11 times with PBS, 1% FBS and visualized. Alternatively, bacteria were incubated with
12 rabbit anti-CD4 antibody T4-4 at 1:100 dilutions for 20 min. After 3 washes in PBS, 1%
13
14
15
16
C
FBS, the bacteria were incubated with goat anti-rabbit IgG conjugated with FITC
A
(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or anti-rabbit Rhodamine
min. After three washes, the labeled cells were visualized. In dual antibody binding
17 experiments, bacteria were first incubated with MAb Leu3A-FITC, then incubated with
18 PAb T4-4, and finally incubated with anti-rabbit Rhodamine Red X prior to visualization.
20 Microsystems, Mannheim, Germany). Standard filters were used for FITC and Texas
21 red. Rhodamine Red X was detected in the same channel as Texas red. The detector slits
22 for FITC channel were configured to collect between 500-547 nm to minimize cross talk
23 between the green and yellow channels. All compared images were detected using the
12
1 same gain. In dual antibody binding experiments, images were collected separately, and
2 then superimposed. For all negative controls, differential interference contrast (DIC)
3 images were collected simultaneously with the fluorescence images using the transmitted
4 light detector. Composed images were organized using Adobe Photoshop (Adobe
D
5 Systems, Inc., San Jose, CA).
T E
E P
C C
A
13
1 Results
3 expression of surface proteins, we sought to identify native surface anchors from vaginal
D
5 strain that is a fast grower and amendable to genetic manipulation (28). Accordingly,
E
6 genomic sequencing was conducted, with 484 contigs assembled from the sequence reads
7 that cover approximately 75% of the genome. Analyses of this partial genomic sequence
P T
database led to identification of more than 30 contigs containing putative LPQTG
sequences. A more detailed sequence homology search was performed with those in the
E
10 databases of the National Center for the Biotechnology. Six protein-coding ORFs were
C
11 selected, including a protein sequence tentatively assigned as C370 (Fig.1) that contained
12 structural features that are commonly present in known cell wall anchored proteins (30).
13
14
15
16
C
BLAST search showed that the C370 protein sequence was most similar to the EF0109
A
protein in a vancomycin-resistant Enterococcus faecalis strain (32) and shared a
moderate homology to some other cell wall associated proteins in Gram-positive bacteria
C370 contains eight nearly identical tandems (approximately 72 amino acids per repeat
17 unit), and a cell wall sorting signal (17), including a LPQTG motif preceding a
18 hydrophobic region, and a positively charged C-terminal tail (Fig. 1), which are features
19 commonly seen in cell wall anchored proteins. Indeed, the antiserum raised against
20 CWA 200 of C370 (contains 2.5 repeats, Fig. 1) readily bound to L. jensenii 1153, based
21 on flow cytometric analysis (data not shown), further suggested that the C370 protein is
14
1 Requirement of Native Sequences of a Defined Length Upstream of LPQTG Cell
2 Wall Sorting Signal for Surface display of 2D CD4—It was unknown whether the cell
3 wall sorting signal alone in C370 sequence would be sufficient to support efficient
D
5 required to maximize surface protein display. Accordingly, several constructs were
E
6 engineered, with protein coding sequence of 2D CD4 C-terminally fused to C370 cell
7 wall sorting signal sequence and the upstream sequences with various lengths.
P T
pOSEL262, 268, 278, 249, 280, 281, and 276 contain 0, 1, 2, 2.5, 4, 7, and 8 repeats of
the C370 sequence. To determine whether 2D CD4 was expressed on the bacterial
E
10 surface, flow cytometric analysis was used (Fig. 2). The bacterial cells harboring the
C
11 parental plasmid pOSEL175 had a minimal binding of anti-CD4 polyclonal antibody
12 (PAb) T4-4 (Fig. 2A). The level of anti-CD4 PAb T4-4 bound to bacterial cells
13
14
15
16
C
harboring pOSEL262 (0 repeat) was indistinguishable from those harboring the negative
A
control pOSEL175. Consistent with these observations, surface expression of 2D CD4
was not detected when cell wall sorting signal of a similar length from S. pyogenes or L.
paracasei was employed (data not shown). These data suggested that the 36-amino acid
17 cell wall sorting signal alone in C370 sequence, although native, was not sufficient to
19 intensity was detected when the number of repeats was increased to 2 copies in
22 molecules that adopt a correctly folded conformation, the transformed bacteria were
23 probed with anti-CD4 MAb Sim.4 for flow cytometric analysis (Fig. 2B). Again, the
15
1 mean fluorescence intensity in bacterial harboring pOSEL262 (0 repeat) was
D
5 upstream of the native cell wall sorting signal also enabled surface display of 2D CD4 in
E
6 L. jensenii 1153 (data not shown). Insertion of additional repeats did not yield a
P T
performed, when the modified lactobacilli harboring above-mentioned plasmids were
probed with anti-CD4 MAb Leu3a that also recognizes a conformation-dependent epitope
E
10 in CD4. These studies confirmed the above findings (data not shown). These data
C
11 suggested that sequence of a minimal defined length upstream of LPQTG is required for
13
14
15
terminus (30). These signature charged sequences serve as a critical cell surface retention
18 KKKRKDDEA1903, present in C370 sequence (Fig. 1B) serve the same function.
20 9, and pOSEL249-10 (Fig. 3). The modified L. jensenii harboring these C-terminal
21 deletion constructs were probed with anti-CD4 PAb T4-4 or MAb Sim.4 for detection by
23 bacterial cells harboring these mutant plasmids was observed relative to pOSEL249 (Fig.
16
1 3B and 3C), as a result of abolished surface display of 2D CD4. Furthermore, a 48-kDa
2 protein of 2D CD4 in fusion with CWA200 sequence was detected by Western analyses
3 using anti-CD4 PAb T4-4 in cell-free conditioned media of all of the modified L. jensenii
4 harboring C-terminal charged tail deletion constructs, suggesting the tail-deleted fusion
D
5 proteins were abundantly released instead of being anchored on cell surfaces (data not
E
6 shown).
7 Amino Acid Substitution in the LPXTG Motif and its Impact on Surface
P T
Expression of 2D CD4 in L. jensenii—It was previously reported that replacing proline
(P) with asparagine (N) in LPETG cell wall sorting motif in protein A of Staphylococcus
E
10 aureus decreased the efficiency of protein surface display (30). Conversely, replacing
C
11 threonine (T) with serine (S) has little effect (30). To determine whether the LPQTG
12 motif within C370 is indeed the critical sorting signal, the importance of P, T, and G
13
14
15
16
C
within the LPQTG sequence was investigated. Point mutations were generated by PCR
A
within the LPQTG motif in C370 sequence. The P residue was mutated to alanine (A) or
asparagine (N); the amino acid T was mutated to A, S or glycine (G); the amino acid G in
the LPQTG motif was mutated to A. Plasmids with the altered LPQTG motif were
19 determine the effect of mutagenesis of LPQTG on L. jensenii surface protein display, the
20 L. jensenii strains harboring pOSEL175 (empty vector, negative control), pOSEL651 (no
21 LPQTG motif, negative control), pOSEL249 (wildtype LPQTG motif, positive control),
22 along with the various mutants, were probed with anti-CD4 PAb T4-4 or MAb Sim.4,
17
1 analysis of antibody binding in modified bacteria detected a substantial decrease of mean
4 pOSEL249 (Fig. 4), indicating that there was a significant reduction of 2D CD4 protein
D
5 displayed on the cell surface. However, the mean fluorescence intensity in the bacterial
E
6 cells harboring pOSEL249(T→S) (with LPQSG motif), and pOSEL249(T→A) (with
7 LPQAG motif) was comparable to those harboring the wild type, demonstrating that
P T
replacing T with S or A has little effect on the efficiency of cell wall anchoring (Fig. 4).
The mean fluorescence intensity in the bacterial cells harboring pOSEL249(G→A) (with
E
10 LPQTA motif) decreased to about 40% relative to those harboring the wild type. Taken
C
11 together, modulation of surface displayed 2D CD4, as a result of either amino acid
12 substitutions in LPQTG motif or deletions in the C-terminally charged tail of the C370
13
14
15
16
C
sequence, reflected on behavior of a native surface protein in L. jensenii 1153.
A
Utility of Native Anchor Sequences of L. jensenii in Surface Display of Proteins in
of L. jensenii 1153 could function for protein surface display in other human lactobacilli
17 or L. jensenii strains, pOSEL241 was constructed as a fusion of the c-Myc epitope tag to
18 the CWA200 and the cell wall sorting signal of the C370 sequence (Figure 5A).
19 pOSEL241 or negative control plasmid pOSEL175 were introduced into L. jensenii Xna,
22 jensenii 1153 harboring pOSEL241. All of the transformed lactobacilli were digested
23 with mutanolysin, an N-acetyl muramidase that cuts the β1-4 glycosidic bond between
18
1 MurNAc-GlcNAc of the glycan strands in mature peptidoglycan. Cell wall anchored
3 treatment and SDS-PAGE separation (30). Western analyses of cell wall digests
4 followed by probing with MAb anti-c-Myc detected laddering patterns, consistent with
D
5 the pattern of anchored proteins when digested with mutanolysin (34). This pattern was
E
6 found in transformed L. jensenii Xna and L. gasseri 1151 harboring pOSEL241 and was
7 similar to those in L. jensenii 1153, and to a lesser extent in L. casei Q (Fig. 5B). We
P T
also detected an increase in fluorescence intensity in L. jensenii Xna and L. gasseri 1151
harboring pOSEL241, indicating that the c-Myc epitope was displayed on the cell surface
E
10 of these bacteria (Fig. 5C). When analyzed by Flow cytometry following
C
11 immunostaining of the bacterial cells with MAb anti-c-Myc, a very low level of
12 fluorescence was detected in all Lactobacillus species harboring pOSEL175. There was
13
14
15
16
C
approximately a 19-fold increase in fluorescence intensity of L. casei Q harboring
A
pOSEL241 relative to that of L. casei Q harboring pOSEL175 (Fig. 5B), indicating c-
Myc tagged protein was being surface-anchored, albeit at a much lower level than the
other strains. In summary, the native anchor sequence of L. jensenii 1153 clearly exhibits
17 a broad utility for displaying proteins in various Lactobacillus species of human origin,
20 order to properly engage trimeric HIV-1 gp120 in a virion, we believe that the 2D CD4
22 evenly distributed in correct conformation. Since the native anchor sequence C370
23 surface protein had never been described, and had only limited homology to other surface
19
1 proteins, we knew nothing of its surface topology. Therefore, we needed to determine
2 the pattern of distribution of 2D CD4 on the bacterial cell surface. When engineered L.
3 jensenii harboring pOSEL249 (2D CD4-CWA200- cell wall sorting signal of C370
4 sequence) were probed with anti-CD4 PAb T4-4, the fluorescence detected by flow
D
5 cytometric increased dose-dependently with the amount of available T4-4 antibody,
E
6 indicating an even distribution of the 2D CD4 protein displayed on the cell surface (Fig.
7 6A). In addition, both anti-CD4 PAb (detects both native and linear epitopes of CD4)
P T
T4-4 and MAb Leu3a (detects a native conformational epitope in gp120-binding site)
E
10 scanning microscopy. The modified L. jensenii, upon probing with antibody, were
C
11 optically sectioned with images collected every 0.2 µm through the cell wall (Z-stack).
13
14
15
16
C
protein on the cell surface (Fig. 6B). Based on the co-localization of anti-CD4 PAb T4-4
A
and MAb Leu3a in these composite images, the surface-expressed 2D CD4 molecules
18 standards with known numbers of surface molecules. In one set, the microbeads
19 contained a mixture of bead populations, which have varying capacities to bind mouse
20 MAb, and reacted with the MAb Leu3a under the same conditions as the bacterial cells.
21 The second set of microbeads was directly conjugated to phycoerythrin (PE). The results
22 showed that there were approximately 1250 correctly folded 2D CD4 molecules per
20
1 Discussion
2 The vaginal mucosal epithelium is a major natural route of HIV entry to underlying
3 target cells. Numerous avenues are currently being investigated to curtail the HIV
4 epidemic (9, 26, 27, 37, 42, 47, 49). While an effective HIV vaccine remains the most
D
5 important goal, the ability of HIV to mutate rapidly and evade the immune response has
E
6 made the development of a vaccine problematic, necessitating the pursuit of additional
P T
transmission of STD pathogens, including HIV, in women (42, 46). Here, we propose a
unique mode of impeding the transmission of HIV on vaginal and other mucosal
E
10 membranes. This approach involves the engineering of human vaginal lactobacilli to
C
11 express proteins for intercepting infectious viral particles and re-introducing the
12 lactobacilli to re-colonize the vaginal mucosa. The success of this approach largely
13
14
15
16
C
depends on whether human vaginal isolates of lactobacilli are amenable to genetic
A
manipulation for surface display of heterologous virus-binding proteins and whether the
17 positive bacteria involves unique sorting signals. One of the best-studied systems is the
18 emm6 gene of S. pyogenes that encodes the M6 structural protein (20). Since the
19 universal cell wall sorting motifs have been identified in a variety of Gram-positive
22 cell-wall sorting signals from either the PrtP protease of L. paracasei or the M6 protein
23 (emm6) of S. pyogenes, or the cell wall sorting signal from the M6 protein of S. pyogenes
21
1 plus an upstream 100-amino acid (CWA100) (14). The inefficient levels of expression
3 system employing cell wall anchor sequences native to the vaginal lactobacilli.
4 Interestingly, the LPQTG sorting motif, which accounts for only 7% of the LPXTG
D
5 motifs found in Gram-positive bacteria (30), is present in almost all putative cell wall
E
6 anchored proteins identified in L. jensenii 1153. Recently, the same LPQTG motif was
7 also identified in L. reuteri (36), L. plantarum (22), and L. gasseri (NCBI Microbial
P T
Genomes Annotation Project). We had previously attempted surface expression of
several proteins in lactobacillus using cell wall sorting signals of other Gram-positive
E
10 bacteria, but only with limited success. The presence of unique cell wall sorting signals
C
11 that do not match those in M6 of S. pyogenes and PrtP of L. paracasei (Fig. 1B) suggests
13
14
15
16
C
peptides and proteins to the cell surface of L. jensenii.
A
It was apparent from our analysis that although there is flexibility in amino acid at the
T or G position of the native LPQTG motif, there are additional sequence requirements
for efficient surface display. First, we discovered that L. jensenii requires ~120-200
17 amino acids of native cell wall anchor sequences upstream of the C-terminal cell wall
18 anchoring motif to display 2D CD4. These upstream sequences may facilitate retention
20 associated sortase. Second, we showed that the positively charged C-terminus is essential
22 were able to achieve surface display of 2D CD4 not only in L. jensenii 1153, but also in
23 L. zeae (data not shown), L. jensenii Xna, and L. gasseri 1151, which indicates a high
22
1 level of conservation in cell wall anchoring motifs and machinery among these
3 sustained when bacteria were cultured in broth at broad pH ranges or at different growth
D
5 Despite successful demonstration of surface expression of protypical 2D CD4 in this
E
6 report, we believe that the surface expression of 2D CD4 can be further optimized to
7 increase the density of 2D CD4 expression on the cell surface by employment of strong
P T
L. jensenii promoters (27), and/or to increase steric accessibility or surface extension of
cell wall anchored molecules with enhanced affinity to bind gp120. Furthermore, there
E
10 are other promising protein candidates that could more efficiently bind and neutralize
C
11 HIV within the mucosa. These include CD4-antibody fusion proteins (13), or variants of
12 CD4 capable of forming oligomers that exhibit an enhanced avidity for binding primary
13
14
15
16
C
isolates of HIV-1 (24). In a recent article by Arthos et al., a dodecameric CD4-Ig fusion
A
was constructed using a polyvalent antibody backbone to display D1 and D2 of CD4.
This multimeric form of CD4 showed potent anti-HIV activity in vitro (3). In a follow-
up paper, the dodecameric CD4-Ig, now termed D1D2-IgP was shown by cryoelectron
17 tomography to induce virion rupture of SIV upon binding to gp120 (28). Although the
18 mechanism of membrane rupture was not elucidated, Bennett et al. suggested that
19 geometric constraints of the gp120 bound to CD4 could cause destabilization of the
20 virion. Lactobacillus surface-anchored viral biding proteins may trap, immobilize, and
21 destabilize virions on the bacterial surface, thus decreasing the number of infectious viral
23 a reduction of the infectious viral load should further reduce transmission frequencies (8).
23
1 Our current proof-of-concept experiments involve the use of modified L. jensenii
2 harboring plasmids that contain antibiotic resistance markers. Since these plasmids might
4 (45), the plasmid-based experimental approach is clearly undesirable for clinical analysis.
D
5 As an important step toward development of engineered L. jensenii with desired genes
E
6 stably maintained, we identified sequences for site-specific chromosome integration by
7 homologous recombination (27) and succeeded at stably integrating the gene encoding
P T
2D CD4 into L. jensenii chromosome for surface anchoring of 2D CD4. The resulting
strain had 40-50% reduction in surface expression of 2D CD4, while the erythromycin
E
10 resistance gene was excised (data not shown).
C
11 An important element in this concept is whether surface display of heterologous
13
14
15
16
C
advantages of the engineered lactobacilli on the vaginal mucosa. While this remains to
A
be tested in vivo, evidence that modified Gram-positive bacteria or exogenously applied
lactobacilli can colonize and persist in relevant animal models already exists.
17 displaying single chain antibody or immunogen stably colonized oral or vaginal mucosa
19 The likely purpose of the native vaginal lactobacilli is to protect the reproductive tract
20 from pathogens that are introduced during intercourse. By subtle genetic modifications,
21 such as the surface expression of an anti-viral protein on the lactobacillus, the flora
22 themselves can add an additional layer of protection against HIV-1 and possibly reduce
24
1 Acknowledgements
3 directing genomic sequencing of L. jensenii 1153 and Dr. Ken Frank for the associated
4 bioinformatics support in this work, Ernie Tai for technical assistance, and Drs. Yang Liu
D
5 and Rosa Yu for critical reading of this manuscript. We also thank Dr. John Lewicki, Dr.
E
6 Jim Turpin, Dr. John Mascola and Dr. Peter Kwong for helpful discussions, and Dr.
T
7 Owen Schwartz of the Biological Image Facility, NIAID, for help with the confocal
8 microscopy.
10 ¶
E
Footnotes
C
12 Topical Microbicides grant 5U19AI060615
14
15
16
17
A
GenBankTM/EMBL data bank with accession number EU332140.
1
Abbreviations
The abbreviations used are: HIV, human immunodeficiency virus; STD, sexually
21 phosphate-buffered saline; FBS, fetal bovine serum; kb, kilobase(s); PBMC, peripheral
22 blood mononuclear cells; FITC, fluroescein isothiocyanate; CWA, cell wall associated;
25
1 Cellular; QR-PE, Quantum R-PE.
E D
P T
C E
A C
26
1 References
D
5 2. Antonio, M. A., S. E. Hawes, and S. L. Hillier. 1999. The identification
E
6 of vaginal Lactobacillus species and the demographic and microbiologic
9 3.
180:1950-1956.
P T
Arthos, J., C. Cicala, T. D. Steenbeke, T.-W. Chun, C. D. Cruz, D. B.
E
10 Hanback, P. Khazanie, D. Nam, P. Schuck, S. M. Selig, D. Van Ryk,
C
11 M. A. Chaikin, and A. S. Fauci. 2002. Biochemical and biological
13
14
15
16 A
4.
C therapeutic and vaccine strategies. J. Biol. Chem. 277:11456-11464.
22 1064.
27
1 6. Berger, E. A., T. R. Fuerst, and B. Moss. 1988. A soluble recombinant
D
5 7. Carreno, M. P., N. Chomont, M. D. Kazatchkine, T. Irinopoulou, C.
E
6 Krief, A. S. Mohamed, L. Andreoletti, M. Matta, and L. Belec. 2002.
P T
CD50) and ICAM-2 (CD102) triggers transmigration of human
E
10 epithelial cells. J Virol 76:32-40.
C
11 8. Chakraborty, H., P. K. Sen, R. W. Helms, P. L. Vernazza, S. A.
13
14
15
16 A
9.
C M. S. Cohen. 2001. Viral burden in genital secretions determines male-to-
15:621-627.
28
1 human isolate of Lactobacillus jensenii engineered to express functional
D
5 type 2 in women: effects of hormonal contraception, bacterial vaginosis,
E
6 and vaginal group B streptococcus colonization. Clinical Infectious
7 Diseases 40:1422-1428.
9
12.
P T
Deen, K. C., J. S. McDougal, R. Inacker, G. Folena-Wasserman, J.
E
10 A soluble form of CD4 (T4) protein inhibits AIDS virus infection. Nature
C
11 331:82-84.
13
14
15
16 A C
14.
Human Immunodeficiency Virus Type 1 by sCD4-17b, a single-chain
18 4166.
21 15:167-193.
29
1 16. Fischetti, V. A., V. Pancholi, and O. Schneewind. 1990. Conservation of
D
5 van Duijnhoven, J. Middel, I. L. Cornelissen, H. S. Nottet, V. N.
E
6 KewalRamani, D. R. Littman, C. G. Figdor, and Y. van Kooyk. 2000.
9 18.
trans-infection of T cells. Cell 100:587-597.
P T
Giomarelli, B., R. Provvedi, F. Meacci, T. Maggi, D. Medaglini, G.
E
10 Pozzi, T. Mori, J. B. McMahon, R. Gardella, and M. R. Boyd. 2002.
C
11 The microbicide cyanovirin-N expressed on the surface of commensal
13
14
15
16 A C
19.
20.
Holck, A., and H. Naes. 1992. Cloning, sequencing and expression of the
30
1 W. Fiers, W. Stiekema, R. M. Lankhorst, P. A. Bron, S. M. Hoffer, M.
D
5 23. Kruger, C., Y. Hu, Q. Pan, H. Marcotte, A. Hultberg, D. Delwar, P. J.
E
6 Van Dalen, P. H. Pouwels, R. J. Leer, C. G. Kelly, C. Van
9 Biotechnol 20:702-706.
P T
passive immunity by lactobacilli producing single-chain antibodies. Nat
E
10 24. Kwong, P. D., M. L. Doyle, D. J. Casper, C. Cicala, S. A. Leavitt, S.
C
11 Majeed, T. D. Steenbeke, M. Venturi, I. Chaiken, M. Fung, H.
13
14
15
16 A C
25.
Burton, E. Freire, R. Wyatt, J. Sodroski, W. A. Hendrickson, and J.
31
1 SHIV transmission in rhesus macaques through inhibition of CCR5.
2 Science 306:485-487.
D
5 P. P. Lee, and Q. Xu. 2006. Engineered vaginal Lactobacillus strain for
E
6 mucosal delivery of the human immunodeficiency virus inhibitor
9
28.
P T
McKeating, J. A., J. Bennett, S. Zolla-Pazner, M. Schutten, S.
E
10 serum-selected human immunodeficiency virus type 1 escape mutant to
C
11 neutralization by CD4 binding site monoclonal antibodies is conferred by
13
14
15
16 A C
29. Medaglini, D., G. Pozzi, T. P. King, and V. A. Fischetti. 1995. Mucosal
32
1 (lactobodies) confer protection against rotavirus-induced diarrhea. J Infect
2 Dis 194:1580-1588.
D
5 Tettelin, R. J. Dodson, L. Umayam, L. Brinkac, M. Beanan, S.
E
6 Daugherty, R. T. DeBoy, S. Durkin, J. Kolonay, R. Madupu, W.
P T
Khouri, T. Utterback, D. Radune, K. A. Ketchum, B. A. Dougherty,
E
10 vancomycin-resistant Enterococcus faecalis. Science 299:2071-2074.
C
11 33. Pavlova, S. I., A. O. Kilic, L. Topisirovic, N. Miladinov, C. Hatzos,
13
14
15
16 A C
34.
Lactobacillus fermentum KC5b and its use for constructing a stable
23 Microbiology 148:433-442.
33
1 37. Root, M. J., M. S. Kay, and P. S. Kim. 2001. Protein design of an HIV-1
3 38. Royce, R. A., A. Sena, W. Cates, Jr., and M. S. Cohen. 1997. Sexual
D
5 39. Ryu, S. E., P. D. Kwong, A. Truneh, T. G. Porter, J. Arthos, M.
E
6 Rosenberg, X. P. Dai, N. H. Xuong, R. Axel, R. W. Sweet, and et al.
9 40.
CD4. Nature 348:419-426.
P T
Salzwedel, K., E. D. Smith, B. Dey, and E. A. Berger. 2000. Sequential
E
10 CD4-coreceptor interactions in human immunodeficiency virus type 1 Env
C
11 function: soluble CD4 activates Env for coreceptor-dependent fusion and
13
14
15
16 A C
41.
on gp120. J Virol 74:326-333.
22 Microbiol 21:491-500.
34
1 44. Taha, T. E., D. R. Hoover, G. A. Dallabetta, N. I. Kumwenda, L. A.
D
5 HIV. AIDS 12:1699-1706.
E
6 45. Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic
9 46.
76:115-137.
P T
Trager, R. S. 2003. Microbicides. Raising new barriers against HIV
E
10 infection. Science 299:39.
C
11 47. Turpin, J. A. 2002. Considerations and development of topical
13
14
15
16 A C
48.
Investig Drugs 11:1077-1097.
Microbiol. 40:2746-2749.
21 438:99-102.
35
1 Veazey, D. Notermans, S. Little, S. A. Danner, D. D. Richman, D.
D
5 CD4+ T cells. Science 286:1353-1357.
E
6 51. Zhou, X., S. J. Bent, M. G. Schneider, C. C. Davis, M. R. Islam, and L.
9 Microbiology 150:2565-2573.
P T
adult healthy women using cultivation-independent methods.
E
10
C C
A
36
1 Figure legend
2
3 Fig. 1. A native protein-coding sequence containing a cell wall sorting signal
4 identified in the human vaginal isolate L. jensenii 1153. (A). Schematic structures in
5 native C370 sequence containing LPQTG motif. The boxes 1-8 represent the tandem
E D
repetitive sequences 1-8. (B). The C-terminal cell wall sorting signal in C370 and those
T
8 jensenii 1153 (this work), M6 of S. pyogenes (GenBankTM/EMBL with accession
P
9 number A26297) (20), and the protease P (PrtP) of L. paracasei (GenBankTM/EMBL
10 with accession number B44858) (19). CWA represents putative cell wall associated
11
12
C E
regions upstream of the LPQTG motif.
C
13 required for optimal surface display of 2D CD4 in modified L. jensenii 1153. Surface
14 exposed 2D CD4 molecules were either probed with anti-CD4 PAb T4-4 (A) or MAb
15
16
17
18
A
Sim.4, that recognizes a conformationally dependent epitope in CD4 (B) for flow
cytometric analysis in the bacterial cells harboring the following plasmids: 175, a
negative control; 249, two and half repeats; 262, no repeat; 268, one repeat; 278, two
repeats; 280, four repeats; 281, seven repeats; 276, eight repeats. The mean fluorescence
20 Controls consisted of unstained cells or cells probed with isotype-matched control IgG,
22 measure of antibody binding to bacterial surface was calculated using FLOWJO software.
23 Fig. 3. Deletion of the C-terminal charged tail of C370 abolished surface display of
24 2D CD4. (A). Schematic diagram of deletion constructs in the C-terminal charged tail of
37
1 C370 sequence (B and C). Bacterial cells were surface-stained by using pre-titered rabbit
2 anti-CD4 PAb T4-4 (B) or mouse MAb Sim.4 (C), followed by probing with FITC-
D
5 system. The difference between the protein displayed on the cell surface of pOSEL249
E
6 and those in bacterial cells harboring mutant constructs was expressed as mean
7 fluorescence intensity. The surface display of 2D CD4 in the bacterial cells harboring
9
pOSEL249 was arbitrarily set as 100%.
P T
Fig. 4. Amino acid substitutions in the LPQTG motif in C370 sequence affected
E
10 surface display of 2D CD4 in L. jensenii 1153. Bacterial cells were surface-stained by
C
11 using pre-titered anti-CD4 MAb Sim.4 (A) or PAb T4-4 (B), followed by probing with
13
14
15
16
C
cytometry analysis was performed in a FACScalibur system. The difference between the
A
protein displayed on the cell surface in bacterial cells harboring pOSEL249, and those
harboring mutant constructs was expressed in mean fluorescence intensity. The surface
display of 2D CD4 in the bacterial cells harboring pOSEL249 was arbitrarily set as
17 100%.
18 Fig. 5. The CWA200 plus cell wall sorting signal sequence in C370 was sufficient to
21 tagged to CWA200 and LPQTG motif of C370 sequence. (B). Western analysis of cell
23 gasseri, and L. casei. After separation in reducing SDS-PAGE, the proteins were
38
1 electroblotted to PVDF membrane for probing with mouse anti-c-Myc MAb. (C). Flow
3 reference to those harboring pOSEL175. The bacterial cells were probed with anti-c-Myc
D
5 cells or cells probed with PE-conjugated secondary antibodies.
E
6
P T
surface anchoring of 2D CD4. Approximately 4x107 bacterial cells were probed with
E
10 Controls consisted of unstained cells or cells probed with FITC-conjugated secondary
C
11 antibodies. The expression constructs contained the following elements: P23 promoter-
13
14
15
16
C
CD4-CWA200-cell wall sorting signal of C370 sequence in pOSEL249. (B).
A
Fluorescence labeled anti-CD4 antibody was used to analyze the modified L. jensenii
1153 for distribution of 2D CD4. Bacteria were immunostained with either anti-CD4
PAb T4-4 or MAb Leu3a. Then, images were collected by confocal laser scanning
17 microscopy. Representative images were presented from the modified bacteria harboring
19
20
21
39
1 Table I. Oligonuclotides used for subcloning sequences of various length upstream of
2 LPQTG cell wall sorting signal in C370 sequence for surface display of 2D CD4. Each
3 PCR reaction used the same reverse primer, in pair with a forward primer. Restriction
4 sites introduced for subcloning are underlined. Zero repeat represents the C-terminal 36-
D
5 amino acid cell wall sorting signal alone in the C370 sequence (see details in Fig. 1B).
E
Reverse Primer
5’-CCGTCTAGATTATGCTTCATCATCTTTTCT-3’
Length of Repeat Forward Primer
T
Zero repeat 5’-CGGCAATTGCCTCAAACTGGTACTGA-3’
P
One repeat 5’-CGGCAATTGGGTCAAACTACAAATAAAGAT-3’
Three repeats
Four-eight repeats
C E
5’-GCGCAATTGGGTCAAACTACAAATAAAGAT-3’
5’-CGGCAATTGGGTCAAACTACTGACAAGAGC-3’
C
Two and half repeats 5’-GCGCAATTGAAGAAGGCAGAAGAAGT-3’
40
1 Table II. Oligonuclotides used to introduce truncations in the C-terminus of C370
2 sequence and amino acid substitutions in the LPQTG motif. The C-terminal truncated
3 forms were generated by PCR using the primer pairs given in the top half of this table.
4 Restriction sites introduced for subcloning are underlined. Single amino acid exchanges
D
5 in the LPQTG motif were introduced by site-directed mutagenesis by a PCR approach
E
6 using the primers given in the bottom half of this table. Nucleotides underlined in the
7 bottom half table encode those amino acids that are substituted by site-directed
8 mutagenesis.
Truncation
249-10
P T
Reverse Primer
5’-GCGCTCTAGATTAAAAAATTCCTGCGCCTAATG-3’
249-9
249-8
C E
5’-GCGCTCTAGATTATGCAAAAATTCCTGCGCCTAATG-3’
5’-GCGCTCTAGATTACTTTGCAAAAATTCCTGCGCC-3’
A C
Mutation
249P(A)
249P(N)
Nucleotide
exchange
1868
1868
Proline
→Alaine
Proline
Primer pair
5’-CATAAGCAAACTCTATTGGCTCAAACTGGTACTGAAAC3’
5’-GTTTCAGTACCAGTTTGAGCCAATAGAGTTTGCTTATG-3’
5’-CATAAGCAAACTCTATTGAATCAAACTGGTACTGAAAC3’
→ Asparagine 5’-GTTTCAGTACCAGTTTGATTCAATAGAGTTTGCTTATG-3’
1870 5’-CAAACTCTATTGCCTCAAAGTGGTACTGAAACTAA-3’
249T(A) Threonine
→ Alanine 5’-GTTAGTTTCAGTACCAGTTTGAGGCAATAGAGTTTG-3’
1870 5’-CAAACTCTATTGCCTCAAGGTGGTACTGAAACTAAC-3’
249T(G) Threonine
→Glycine 5’-GTTAGTTTCAGTACCACCTTGAGGCAATAGAGTTTG-3’
1870 5’-CAAACTCTATTGCCTCAAAGTGGTACTGAAACT-3’
249T(S) Threonine
→Serine 5’-GTTAGTTTCAGTACCACTTTGAGGCAATAGAGTTTG-3’
1871 5’-CTCTATTGCCTCAAACTGCTACTGAAACTAACCCAC-3’
249G(A) Glycine
→Alanine 5’-GTGGGTTAGTTTCAGTAGCAGTTTGAGGCAATAGAG-3’
9
41
A.
D
C-termianl CWA 200 region
B.
T E
P
Gram-positive Cell wall sorting signal
bacteria Proteins
C E
L. jensenii 1153 C370 LPQTGTETNPLTAIGIGLMALGAGIFAKKKRKDDEA
AC
S. pyogenes M6 LPSTGETANPFFTAAALTVMATAGVAAVVKRKEEN
Fig. 1
A.
Mean Fluorescence (T4-4)
600
500
400
300
200
100
0
175 262 268 278 249 280 281 276
B.
T E
Mean Fluorescence (Sim.4)
70
P
60
50
E
40
30
C
20
10
C
0
175 262 268 278 249 280 281 276
Fig. 2
A.
Hydrophobic
domain Charged tail
2D CD4 6 7 8 LQPTG +
pOSEL249 F 1533 AKKKRKDDEA
pOSEL249-8 F 1533 AK
pOSEL249-9 F 1533 A
pOSEL248-10 F 1533
B.
2D CD4-CWA200 (%bound to T4-4)
Surface display of 2D CD4 and
120
100
E
80
60
40
20
0
P T
E
175 651 249 249-8 249-9 249-10
C
2D CD4-CWA200 (%bound to Sim.4)
C.
Surface display of 2D CD4 and
120
C
100
80
60
40
20
0
175 651 249 249-8 249-9 249-10
Fig. 3
A.
120
Surface display of CD4-CWA200
100
80
against T4-4 (%)
60
40
20
0
5 1 9 ) ) ) ) S) )
17 65 2 4 P(A P(N T(A T(G T( G(A
9 9 9 9 9
24 24 24 24 2 4 2 49
D
B.
Surface display of CD4-CWA200
120
100
T
against Sim.4 (%)
80
P
60
E
40
C
20
AC
0
5 1 9 ) ) ) ) S) )
17 65 2 4 P(A P(N T(A T(G T( G(A
9 9 9 9 9
24 24 24 24 2 4 2 49
Fig. 4
A.
Signal CWA200-cell wall sorting
Promoter sequence c-Myc signal C370
pOSEL 241 P23 CbsAss
EQKLISEEDL
B. C.
3
E D
1
na
5
5
11
11
X
Q
ii
ii
T
Mean fluorescence
i
900
n
er
se
se
i
ss
se
Bacterial strains 800
n
ga
ca
je
je
700
L.
L.
L.
L.
pOSEL construct 175 241 175 241 175 241 175 241
kDa
188
E P 600
500
400
300
200
C
98 100
0
C
62
49
1
5
s e 175
41
11 75
L . ei Q 1
75
ga i 1 -24
17
j e i i -24
4
A
-2
1
c a 1-2
er 1-1
L . s en na -
-
3-
38
L . s er na
en 53
iQ
5
L . 15
ss 15
X
X
11
i1
s
ga i i
ca
ii
28 ii
en
en
ns
s
ns
ns
n
17
je
je
ej
L.
L.
L.
L.
14
mAb against c-Myc Modified Bacteria
Fig 5.
A.
175 651 249
100
80
Count
60
40
20
0
100 101 102 100 101 102 100 101 102
Fluorescence
D
B.
Leu3a-FITC T4-4-Rhodamne Red X Dual or DIC
T E
249
E
249
P 249
C
249 249 249
Fig. 6