AEM Accepts, published online ahead of print on 6 June 2008

Appl. Environ. Microbiol. doi:10.1128/AEM.00104-08

Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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3 Engineering Human Vaginal Lactobacillus for Surface Expression of Two-Domain
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6 Xiaowen Liu¶1, Laurel A. Lagenaur¶1, Peter P. Lee1, 2, Qiang Xu1*

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From 1Osel, Inc., 4008 Burton Drive, Santa Clara, CA 95054, 2Department of Medicine,

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9 Stanford University, Stanford, CA 94305

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Running Title: Surface Anchoring a Viral Receptor in Lactobacilli

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13 * To whom correspondence should be addressed:

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18 A C Qiang Xu, PhD

Osel, Inc.

4008 Burton Drive, Santa Clara, CA 95054

Tel. 408-986-0012 ext 205

19 Fax: 408-986-0019

20 E-mail: qxu@oselinc.com

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 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

4 cervico-vaginal mucosa naturally harbors dynamic microflora composed predominantly

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5 of lactobacilli, which may be genetically modified to serve as a more efficient protective

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6 barrier against heterosexual transmission of HIV. We selected a vaginal strain of

7 Lactobacillus, L. jensenii 1153, for genetic modification to display surface-anchored anti-

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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

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10 wall sorting LPQTG motif. In this report, we employed these proteins to express a

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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

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surface expression of heterologous proteins, and display of surface-anchored 2D CD4

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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

17 uniformly distributed on the bacterial surfaces. The surface-anchored 2D CD4 was

18 recognized by a conformation dependent anti-CD4 antibody, suggesting that the

19 expressed proteins adopted a native conformation. Establishment of this Lactobacillus-

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

22 mitigation of heterosexual transmission of HIV and other mucosally transmitted viral

23 pathogens.

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 1 Introduction

2 The predominant mode of human immunodeficiency virus-1 (HIV)1 transmission

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

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5 versa (1). There are few means by which women can actively protect themselves against

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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

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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

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10 cell type and site of transmission are actively investigated (7, 17, 42, 50), it is believed

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11 that a potential microbicide product will be most effective if it offers a broad protection

12 against mucosal transmission of HIV. In healthy women of childbearing age, the

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protective mucosa in the vagina is populated with commensal bacteria typically

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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

19 extracellular matrix materials (15). Depletion or disturbance of vaginal Lactobacillus

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

23 of reproductive health in women.

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 1 Through genetic engineering, a member of the vaginal microflora may be enhanced to

2 form an efficient protective shield against the transmission of sexually transmitted

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

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5 the high affinity HIV-binding protein, human CD4 (39). CD4, a member of the

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6 immunoglobulin (Ig) superfamily, is the primary host receptor for HIV entry into

7 susceptible cells. The extracellular portion of CD4 (residues 1-371) is a concatenation of

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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

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10 the absence of the remaining domains of CD4, retain the high-affinity binding to HIV-1

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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

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mucosal surface in vivo. Importantly, glycosylation of CD4 is not required for binding to

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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).

A biologically active CD4 surface-expressed by L. jensenii could potentially trap

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

19 (28), undergo an aborted infection process and/or be inactivated locally by antiviral

20 compounds, such as lactic acid and hydrogen peroxide, secreted by the lactobacilli (21),

21 thereby significantly reduce the number of infectious viral particles.

22 Surface expression of heterologous proteins has been achieved via the sortase-

23 catalyzed cell wall anchoring mechanism in Gram-positive bacteria (30), including

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 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

4 a heterologous mammalian protein in human vaginal isolates of L. jensenii. In this

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5 report, we describe a broadly applicable approach for genetic modification of lactobacilli,

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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

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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

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10 2D CD4 and required fusion with native upstream sequences of a defined length. The

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11 approach reported here also affords surface expression of heterologous proteins in other

12 Lactobacillus species.

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 1 Materials and Methods

2 Bacterial Strains, Culture, and Transformation—Naturally occurring human

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

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5 MRS broth or Rogosa SL broth (Difco) as described previously (10). A non-vaginal

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6 isolate of L. paracasei 343 (from Dr. Peter H. Pouwels) was cultured similarly. Shuttle

7 plasmids were constructed and maintained in Escherichia coli, purified and

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electroporated into lactobacilli (10). Transformed lactobacilli were routinely propagated

either on MRS agar plates or in liquid media containing 20 µg/ml erythromycin (10).

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10 Identification of Protein Sequences with Cell Wall Anchor Motifs —The genome

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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

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them to be covalently anchored to the cell wall peptidoglycan (16). Among them, they

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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

21 C370, a forward primer (5’- ATGTTCTATCAAATTGACCCAGCTTTGG -3’) was

22 used, in pair with the reverse primer (5’- CCTGCGCCTAATGCCATCAATCCAATA -

23 3’) to PCR amplify the 5712 bp coding region. The sequences were also subjected to a

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 1 BLAST search for sequence homology to cell wall-anchored proteins in Gram-positive

2 bacteria.

3 Construction of Expression Cassettes in L. jensenii—To facilitate protein surface

4 anchoring in L. jensenii, an expression cassette was constructed and subcloned into the

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5 SacI and XbaI sites of a shuttle vector pOSEL175 (27). The expression cassette contains

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6 four components, including a Lactobacillus-compatible P23 promoter, CbsA signal

7 sequence (10), DNA sequence encoding a heterologous protein, and cell wall anchoring

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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).

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10 Unique restriction sites, including SacI, EcoRI, NheI, MfeI, and XbaI, were placed

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11 between each component from 5’ to 3’ ends, respectively. Amplification of each

12 component was performed using conventional PCR with Pfu DNA polymerase.

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16 A CSubcloning Sequences of Various Length Upstream of LPQTG Motif in C370

Sequence for Surface Display of 2D CD4—Various lengths of repetitive sequence

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

17 primer (5’-CCGTCTAGATTATGCTTCATCATCTTTTCT-3’, with restriction site

18 underlined) was used, in pair with the forward primers listed in Table I for each PCR

19 reaction. In addition, the forward primer (5’-

20 GCGCAATTGAAGAAGGCAGAAGAAGT-3’) was paired with the reverse primer used

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).

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 1 To drive surface expression of 2D CD4, the protein coding sequence of 2D CD4 was

2 C-terminally fused to sequences of a defined length upstream of LPQTG motif in C370

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

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5 digested pOSEL651 (10). The resulting plasmids were designated as pOSEL262 (with

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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).

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Similarly, the MfeI/XbaI digested fragment corresponding to CWA200 sequence

upstream of LPQTG motif in C370 was cloned, yielding pOSEL249 (with 2.5 repeats).

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10 c-Myc Tagging of Putative Cell Wall Anchor Sequences of L. jensenii—

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11 Oligonucleotide primers corresponding to c-Myc epitope (EQKLISEEDL) were designed

12 (Forward primer 5’-

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GCGCTAGCGAACAGAAACTGATCTCCGAAGAGGACCTGTTGAAGAAGGCAG

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AAGAAGT-3’; reverse primer 5’-CCGCAATTGTTATGCTTCATCATCTTTTCT-3’),

allowing fusion of c-Myc epitope to the N-terminus of sequences containing the C-

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

18 into MfeI/NheI double-digested pOSEL651, resulting in pOSEL241.

19 Deletion Analysis of Positively Charged C-terminal Sequences in C370—A

20 series of deletion mutants was generated by PCR amplification. An

21 oligonucleotide complementary to 2D CD4 sequence in pOSEL249 (5’-

22 GATCGTGCTGATTCACGTCGT-3’) was used as forward primer. Reverse

23 primers listed in Table II were used to delete the positively charged amino acids

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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

4 were verified by nucleotide sequencing, and the constructs were electroporated

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5 into L. jensenii for protein analysis.

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6 Oligonucleotide-directed Mutagenesis of LPXTG Motif of Putative Cell Wall

7 Anchor Sequences—Point mutations were generated using QuickChange XL Site-

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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

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10 upstream CWA200 of C370 sequence) was used as a template. The mutagenic primers

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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

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reaction, Dpn I enzyme was added to the amplification mixture to degrade the methylated

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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.

18 Plasmids with verified sequences were electroporated into L. jensenii 1153.

19 Enzymatic Digestion of L. jensenii Cell Wall by Muramidase—Bacterial cultures, at

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

22 suspended in 100 µL of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 25% sucrose (34).

23 The bacterial cell wall was digested in the presence of a muramidase, mutanolysin

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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

4 denatured after addition of 25 µl of 4x and 125 µl of 1x reducing SDS-PAGE buffer to

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5 the cell wall- and protoplast-enriched fractions, respectively.

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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

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100 mM HEPES (pH 7.4) to late log phase or early stationary phase. Cell-free

supernatants for evaluation of soluble proteins were collected by centrifugation (12,000 x

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10 g, 10 min) and proteins were heat denatured in the SDS-PAGE loading buffer (50 mM

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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

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the rabbit polyclonal anti-CD4 antibody, T4-4 (NIH AIDS Reagents Reference Program),

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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

reaction was then visualized by using horseradish peroxidase-conjugated secondary

17 antibody and enhanced chemiluminescence reagents (GE Healthcare Life Sciences,

18 Piscataway, NJ).

19 Analysis of Surface Expression in Modified Lactobacilli by Flow Cytometry—

20 Overnight culture of L. jensenii 1153 harboring plasmids designed for heterologous

21 protein expression or control plasmid pOSEL175 were sub-cultured at 1:50 dilutions in

22 erythromycin-containing MRS or Rogosa SL Broth that was buffered with 100 mM

23 HEPES, pH 7.4. Bacteria at early log phase were washed and suspended in phosphate

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 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

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5 IgG (Molecular Probes, Eugene, OR) at 1:200 dilutions at 4ºC for 30 min. Alternatively,

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6 the modified L. jensenii were probed with anti-CD4 monoclonal antibody (MAb), Sim.4

7 or Leu3a, at a final concentration of 6 µg/ml or anti-c-Myc MAb (Invitrogen), followed

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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

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10 epitopes in the HIV-1 gp120-binding site (domain 1 of CD4) (39). The fluorescence of

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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

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forward scatter vs. fluorescence) was obtained from modified L. jensenii, with those

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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).

17 For quantitation of CD4 molecules expressed on the modified L. jensenii surface,

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

23 fluorescence intensity of a sample in terms of number of molecules of equivalent soluble

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 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

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5 and QR-PE beads were visualized on a FACScan (Becton Dickinson).

Confocal Analysis of Surface Expressed 2D CD4—The modified L. jensenii

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7 harboring pOSEL175 or plasmids designed for surface expression were cultured to early

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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

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10 with 20 µl of Leu3a-FITC (BD Biosciences, San Jose, CA) for 20 min and washed 3

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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%

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FBS, the bacteria were incubated with goat anti-rabbit IgG conjugated with FITC

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(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or anti-rabbit Rhodamine

Red X (Jackson ImmunoResearch Laboratories) at 1:100 dilution, and incubated for 20

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.

19 Images were collected using a Leica TCS-NT/SP confocal microscope (Leica

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

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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

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 1 Results

2 Identification of Native Cell Wall Anchor Sequences—In order to optimize

3 expression of surface proteins, we sought to identify native surface anchors from vaginal

4 isolates of Lactobacillus in our collection, focusing on a H2O2-producing L. jensenii 1153

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5 strain that is a fast grower and amendable to genetic manipulation (28). Accordingly,

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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

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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

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10 databases of the National Center for the Biotechnology. Six protein-coding ORFs were

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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).

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BLAST search showed that the C370 protein sequence was most similar to the EF0109

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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

22 displayed on the cell surface.

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 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

4 surface expression of 2D CD4 in the L. jensenii or additional upstream sequence is

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5 required to maximize surface protein display. Accordingly, several constructs were

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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.

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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

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10 surface, flow cytometric analysis was used (Fig. 2). The bacterial cells harboring the

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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

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harboring pOSEL262 (0 repeat) was indistinguishable from those harboring the negative

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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

18 display 2D CD4 on cell surface. In contrast, a significant increase in fluorescence

19 intensity was detected when the number of repeats was increased to 2 copies in

20 pOSEL278. The fluorescence intensity of each construct continued to increase up to 7

21 repeats in the bacteria harboring pOSEL281. To determine the level of 2D CD4

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

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 1 mean fluorescence intensity in bacterial harboring pOSEL262 (0 repeat) was

2 indistinguishable from that in negative control pOSEL175. There was a significant

3 increase in fluorescence intensity in bacteria harboring pOSEL249 (2.5 repeats, ~200

4 amino acids in length). Employment of a similar length of non-repetitive sequences

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5 upstream of the native cell wall sorting signal also enabled surface display of 2D CD4 in

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6 L. jensenii 1153 (data not shown). Insertion of additional repeats did not yield a

7 significant increase in fluorescence intensity. Additional flow cytometric analyses were

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performed, when the modified lactobacilli harboring above-mentioned plasmids were

probed with anti-CD4 MAb Leu3a that also recognizes a conformation-dependent epitope

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10 in CD4. These studies confirmed the above findings (data not shown). These data

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11 suggested that sequence of a minimal defined length upstream of LPQTG is required for

12 surface display of 2D CD4.

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16 A CDeletion of the C-terminal Positively Charged Tail Abolished Surface Expression

of 2D CD4 in L. jensenii—The cell wall anchored proteins in Gram-positive bacteria

contain a characteristic stretch of positively charged amino acids at the extreme C-

terminus (30). These signature charged sequences serve as a critical cell surface retention

17 signal (30). It was unknown whether the positive-charged amino acids,

18 KKKRKDDEA1903, present in C370 sequence (Fig. 1B) serve the same function.

19 Therefore, a series of deletions were constructed, resulting in pOSEL249-8, pOSEL249-

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

22 flow cytometric analysis. A marked decrease of mean fluorescence intensity in the

23 bacterial cells harboring these mutant plasmids was observed relative to pOSEL249 (Fig.

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 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

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5 proteins were abundantly released instead of being anchored on cell surfaces (data not

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6 shown).

7 Amino Acid Substitution in the LPXTG Motif and its Impact on Surface

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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

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10 aureus decreased the efficiency of protein surface display (30). Conversely, replacing

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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

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within the LPQTG sequence was investigated. Point mutations were generated by PCR

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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

17 designated as pOSEL249(P→A), pOSEL249(P→N), pOSEL249(T→A),

18 pOSEL249(T→S), pOSEL249(T→G), and pOSEL249(G→A), respectively. To further

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,

23 followed by appropriate fluorochrome-conjugated secondary antibody. Flow cytometric

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 1 analysis of antibody binding in modified bacteria detected a substantial decrease of mean

2 fluorescence intensity in bacterial cells harboring pOSEL249(P→A) (with LAQTG

3 motif) and pOSEl249(P→N) (with LNQTG motif) in reference to those harboring

4 pOSEL249 (Fig. 4), indicating that there was a significant reduction of 2D CD4 protein

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5 displayed on the cell surface. However, the mean fluorescence intensity in the bacterial

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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

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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

a Variety of Lactobacillus Species—To determine if the native C370 anchor sequences

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,

20 L. gasseri 1151, and L. casei Q by electroporation. The transformed bacteria were

21 analyzed by Western and flow cytometric analyses, in reference to positive control L.

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

2 proteins typically migrate as a large spectrum of fragments, following muramidase

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,

18 though there appears to be a species preference for sorting signals.

19 Distribution and Level of Surface Displayed 2D CD4 in Modified Lactobacilli—In

20 order to properly engage trimeric HIV-1 gp120 in a virion, we believe that the 2D CD4

21 expressed on the Lactobacillus surface needed to be available in sufficient numbers, and

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)

were used to visualize the three-dimensional image reconstruction by confocal laser

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).

12 Examination of composite images revealed a very uniform distribution of the 2D CD4

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

adopt a native conformation (Fig. 6B).

In addition, we used quantitative flow cytometric analysis to determine the numbers

17 of anchored molecules on the bacterial surface in reference to two series of microbead

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

23 bacterium harboring pOSEL249 (data not shown).

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

7 innovative approaches. Topical microbicides represent a possible approach to inhibit the

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

modified bacteria can re-colonize.

Surface expression of proteins via covalent linkage with peptidoglycans in Gram-

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

20 bacterial species (30), we initially attempted a plasmid-based modular approach to anchor

21 heterologous proteins on the surface of L. jensenii by utilizing two well-characterized

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

2 using these heterologous systems necessitated the development of a surface 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

12 that regions of native sequences should be exploited to covalently anchor heterologous

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

19 or extension of substrate sequence and thus proper proteolytic cleavage by membrane-

20 associated sortase. Second, we showed that the positively charged C-terminus is essential

21 for retention of 2D CD4 on the bacterial surface. By employment of these sequences, we

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

2 lactobacilli. In our analysis, surface expression of 2D CD4 by modified L. jensenii was

3 sustained when bacteria were cultured in broth at broad pH ranges or at different growth

4 phases (data not shown).

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

22 particles. Since the cervico-vaginal transmission of HIV is already an inefficient process,

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

3 be transferred to opportunistic human pathogen such as Staphylococci and Enterococci

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

12 proteins in L. jensenii adversely affects the persistence, colonization, and competitive

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.

Recombinant strains of S. gordonii and L. zeae designed for secreting or surface-

17 displaying single chain antibody or immunogen stably colonized oral or vaginal mucosa

18 in rat or mouse models of bacterial colonization (5, 23, 29).

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

23 its ability to infect the host.

24
 1 Acknowledgements

2 We thank Dr. Jan-Fang Cheng at Lawrence Berkeley National Laboratory for

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

Both authors contributed equally to this work

P
C
11 *This work was supported in part by NIH Integrated preclinical/Clinical program for

C
12 Topical Microbicides grant 5U19AI060615

13 Nucleotide sequence(s) reported in this paper has been submitted to the

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

transmitted diseases; CbsA, S-layer protein of L. crispatus; PAGE, polyacrylamide gel

18 electrophoresis; ELISA, enzyme-linked immunosorbent assay; gp120, 120-kDa surface

19 envelope glycoprotein of HIV; HEPES, N-[2-hydroxyethyl]piperazine-N’-[2-

20 ethanesulfonic acid]; MAb, monoclonal antibody; PAb, polyclonal antibody; PBS,

21 phosphate-buffered saline; FBS, fetal bovine serum; kb, kilobase(s); PBMC, peripheral

22 blood mononuclear cells; FITC, fluroescein isothiocyanate; CWA, cell wall associated;

23 DIC, differential interference contrast; PE, phycoerythrin; QSC, Quantum Simply

25
1 Cellular; QR-PE, Quantum R-PE.

E D
P T
C E
A C

26
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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

of known cell surface proteins in Gram-positive bacteria. Sources for sequences: L.

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.

Fig. 2. Sequences of a defined length upstream of LPQTG motif in C370 are

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

19 intensity in bacterial cells harboring pOSEL175 was defined as background fluorescence.

20 Controls consisted of unstained cells or cells probed with isotype-matched control IgG,

21 and fluorochrome-conjugated secondary antibodies. The fluorescence density as a

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-

3 conjugated anti-rabbit or PE-conjugated anti-mouse antibodies. The binding of antibody

4 to cell wall-anchored proteins was analyzed by flow cytometry using a FACScalibur

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

12 PE-conjugated anti-mouse or FITC conjugated anti-rabbit antibodies. The flow

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

19 surface-anchor protein in other Lactobacillus species of human origin. (A).

20 Schematic diagram of pOSEL241, designed to analyze surface expression of c-Myc

21 tagged to CWA200 and LPQTG motif of C370 sequence. (B). Western analysis of cell

22 wall-enriched fractions following mutanolysin digestion of transformed L. jensenii, L.

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

2 cytometric analysis of human vaginal lactobacillus isolates harboring pOSEL241 in

3 reference to those harboring pOSEL175. The bacterial cells were probed with anti-c-Myc

4 MAb, and then PE-conjugated anti-mouse antibodies. Controls consisted of unstained

D
5 cells or cells probed with PE-conjugated secondary antibodies.

Fig. 6. Distribution of surface displayed 2D CD4 in modified L. jensenii 1153. (A).

E
6

7 Flow cytometric analysis of L. jensenii harboring plasmids designed for secretion or

P T
surface anchoring of 2D CD4. Approximately 4x107 bacterial cells were probed with

rabbit anti-CD4 PAb (T4-4), followed by FITC-conjugated anti-rabbit antibodies.

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-

12 CbsAss-2D CD4 (designed for protein secretion) in pOSEL651; P23 promoter-CbsAss-2D

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

18 pOSEL249 (top panel) or pOSEL175 (bottom panel).

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’

Two repeats 5’-CGCCAATTGGGTCAAACTACTGATAAGAGT-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.

1 1267 1867 1903

M 1 2 3 4 5 6 7 8 L PQTG 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

L. paracasei PrtP LPKTA ETTERPAFGFLGVIVVSLMGVLGLKRKQREE

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

A C 175 175 175

Fig. 6