Kill the corkscrew. Disable T. denticola motility at source.
A bacteriophage engineered from the LE1 spirochete prophage scaffold — with its host-targeting tail fiber
replaced to bind T. denticola MSP — and loaded with a dual CRISPR-Cas12a payload
targeting the flagellar motor genes fliA and fliG.
Designed to work synergistically with the TdVax subunit vaccine.
Conceptual · literature-supported
No known Td phage · prophage engineering required
CRISPR phage delivery proven in S. aureus + C. difficile
Core hypothesis
A chimeric phage built on the LE1 spirochete prophage scaffold, redirected via tail fiber engineering
to bind the surface-abundant MSP receptor on T. denticola, and armed with a CRISPR-Cas12a cassette
targeting the essential flagellar regulator fliA and the motor switch gene fliG,
will render periodontal T. denticola permanently immotile or non-viable,
synergising with TdVax-primed adaptive immunity to clear red-complex infection.
01 · Rationale
Why target motility — and why phage
T. denticola drills into periodontal pockets using corkscrew motility driven by periplasmic endoflagella.
Losing motility is not merely inconvenient for the bacterium — it eliminates its primary virulence mechanism.
A phage-delivered genetic kill switch targets this exactly, and creates an evolutionary trap that classical antibiotics cannot.
Motility = primary virulence
T. denticola periplasmic flagella drive corkscrew tissue invasion. Motility-null mutants
cannot penetrate the epithelial barrier or establish subgingival biofilm. Without the corkscrew,
Td is a passive bystander — its protease and LPS virulence factors cannot reach tissue.
The evolutionary trap
If Td mutates the phage receptor (MSP) to escape infection, it loses its major outer sheath protein —
becoming immunologically naked. If it accepts phage infection, the CRISPR payload cuts the motor gene.
Neither escape route restores wild-type fitness. This is a lose-lose for the pathogen by design.
CRISPR phage delivery is real
Phage-delivered CRISPR-Cas9 eliminated S. aureus in vivo (Park et al. 2017 [1]).
Phage-delivered Cas3 killed C. difficile in mouse gut colonisation (Selle et al. 2020 [2]).
The platform exists — the engineering challenge here is the T. denticola-specific delivery vehicle.
Why not antibiotics
Broad-spectrum antibiotics disrupt the oral microbiome. A phage engineered exclusively for Td
MSP receptor binding eliminates only Td. Antibiotic resistance is irrelevant — the CRISPR target
is a chromosomal essential gene with no natural mobile resistance element.
02 · The chassis
LE1 — the only characterised spirochete phage
LE1 is a 74 kb temperate prophage from Leptospira biflexa (non-pathogenic spirochete),
the only fully-sequenced spirochete phage genome in the literature (Bourhy et al. 2005 [3]).
Its injection machinery evolved specifically for spirochete outer membrane biology — the most critical
structural prerequisite for infecting T. denticola.
LE1 head + capsid
DNA packaging · from Leptospira
→
LE1 tail shaft
OM injection machinery
→
Engineered tail fiber
MSP-binding HRDR swap
→
CRISPR-Cas12a payload
fliA + fliG dual guide
LE1 genome facts
74 kb circular prophage · 79 ORFs · GC 36%
(Td = 37.8% — near-identical) · head + tail structural
protein clusters identified · replicates autonomously as
a circular replicon in L. biflexa.
The shuttle vector advantage
LE1's replication origin already powers an L. biflexa–E. coli
shuttle vector (Saint Girons et al. 2000). The phage can be
manufactured and engineered entirely in E. coli,
quality-controlled, then deployed against Td. No Td-native production system needed.
Spirochete-compatible injection
Spirochete outer membrane (OM) is biochemically unusual: sparse LPS, abundant lipoproteins,
no classical peptidoglycan layer at the OM. LE1 tail machinery evolved in this context —
unlike E. coli phages (T4, T7, λ), it penetrates the right membrane biology.
parA/parB → Borrelia homology
LE1 partitioning genes (parA/parB) are similar to Borrelia plasmid partition loci —
confirming spirochete phylogenetic compatibility across the phylum. Treponema,
Leptospira, and Borrelia share core spirochete OM architecture.
03 · The receptor
MSP — T. denticola's most abundant surface protein
The receptor chosen for the engineered tail fiber determines both host specificity and the evolutionary trap.
MSP (Major Outer Sheath Protein / MOSP) satisfies all requirements: surface-exposed, abundant,
constitutively expressed, and loss of MSP is structurally costly for the bacterium.
The MSP evolutionary trap — no viable escape path
Path ATd mutates MSP to escape phage binding
→ loses its major outer sheath protein → immune-exposed, impaired outer membrane stability,
disrupted flagellar sheath organisation → fitness collapse, killed by TdVax-primed antibodies.
VerdictTwo simultaneous independent mutations required to escape both arms. No known precedent
for this occurring in a natural periodontal biofilm setting on a clinically relevant timescale.
Evolutionary pressure always pushes toward virulence loss.
04 · Tail fiber engineering
Three routes to redirect LE1 toward MSP
The tail fiber (receptor-binding protein, RBP) tip determines host specificity. Engineering this domain
is a mature field: chimeric RBP domain swaps and HRDR mutagenesis both have peer-reviewed proof-of-concept
in live animal models. Three approaches apply here, in order of experimental maturity.
Option A · Highest experimental precedent
HRDR mutagenesis — phagebodies (Yehl et al. 2019 [4])
Identify the host-range-determining regions (HRDRs) in the LE1 tail fiber tip by AlphaFold3 structure
prediction. Mutate HRDRs via site-directed mutagenesis inspired by antibody CDR engineering.
Screen variant library against Td-MSP-coated surfaces or live Td cells. Yehl et al. demonstrated this
for T3 phage — engineered phagebodies acquired new host range and suppressed resistance development
in a murine infection model. The same workflow applies to LE1 once the tail fiber gene is identified
from the 79-ORF genome.
Option B · Most modular
Chimeric RBP domain swap (Dunne et al. 2019 [5])
Solve the LE1 RBP crystal structure (or validate an AlphaFold3 prediction by cryo-EM). Identify the
C-terminal receptor-binding carboxyl domain (analogous to Gp15 in Listeria PSA phage). Replace this
domain with an MSP-binding VHH nanobody or DARPin raised against recombinant Td MOSP-C (the
extracellular β-barrel domain, residues ~196–242). Dunne et al. demonstrated head/neck/shoulder domain
swaps between prophage-encoded RBPs produced chimeric phages with predictable, extended host ranges.
Option C · Fastest if structure is available
AI-designed de novo binding domain (RFdiffusion + ProteinMPNN)
Use RFdiffusion to generate a 40–60 aa binder domain that docks to a known extracellular surface patch
on Td MSP (AlphaFold3 model available in the Triple Vax pipeline). ProteinMPNN designs the sequence.
Graft onto the LE1 tail fiber C-terminus. No animal immunisation needed. Highest risk (computational
binders require wet-lab affinity validation) but fastest time-to-sequence. Pairs naturally with the
MSP structural data already in the redvax pipeline.
05 · CRISPR payload
The killer cassette — ranked by impact
The payload is a minimal non-replicative CRISPR cassette. Removing LE1 replication genes from the
packaged DNA makes delivery one-shot: the phage enters, cuts the chromosome, cannot self-replicate,
and cannot spread through the environment. The dual-guide strategy makes resistance essentially impossible.
Lethal · Priority 1
fliA (TDE2683) — sigma-28 transcription factor. Controls all flagellar class-3 genes.
Cannot be deleted from Td — it is an essential gene for cell survival (Kurniyati et al. 2022).
A CRISPR cut here kills the bacterium outright. The highest-value single target because lethality is
independent of motility status: even an already-immotile cell is killed.
Immotile · Priority 2
fliG (TDE—) — flagellar C-ring motor switch. Essential for flagellar rotation and assembly.
Cloned from Td ATCC 35405 via lambda library (Heinzerling et al. 1995 [6]), 73% similar to B. subtilis
FliG. A CRISPR cut here renders Td permanently immotile: motor stops, corkscrew cannot rotate, tissue
invasion halted. No lethality — but full motility loss is a severe virulence attenuation.
Backup · Priority 3
FlgM overexpression cassette — anti-sigma sequestration. Deliver a high-copy flgM
(TDE0201) expression cassette. FlgM sequesters FliA, switching off all flaA/flaB1/2/3 transcription
simultaneously. No flagellins made → filament cannot assemble → corkscrew collapses. Because FliA is
essential, saturating it with excess FlgM may push toward lethality as a side-effect.
Dual guide targeting both fliA (lethal) and fliG (immotile) simultaneously.
Resistance to one guide requires an independent resistance mutation at the other locus.
Two simultaneous CRISPR-escape mutations is effectively an insurmountable barrier in a clinical
oral biofilm context. The lytic module from LE1 is retained to lyse the cell after payload delivery,
releasing no viable phage progeny (non-replicative phagemid packaging).
06 · Build pipeline
Five experimental steps to a functional prototype
1
Annotate the LE1 tail fiber gene
Download LE1 genome (GenBank AJ564667). Identify the tail structural protein cluster
among the 79 ORFs using HHpred/Phyre2 against known phage tail fiber databases.
Express putative RBP in E. coli. Run AlphaFold3 prediction and locate the distal
receptor-binding tip (C-terminal domain — analogous to PSA Gp15 in Listeria phage).
2
Generate MSP-binding domain
Option A: Raise VHH nanobody against recombinant Td MOSP-C (extracellular β-barrel loops,
residues ~196–242) by llama immunisation + phage display selection. Validate binding affinity
by SPR or biolayer interferometry (target KD < 100 nM).
Option B: De novo design via RFdiffusion using the Td MSP AlphaFold3 structure
already in the redvax pipeline.
3
Swap HRDR → chimeric tail fiber
Replace the LE1 RBP receptor-binding tip with the MSP-binding domain via Gibson assembly.
Confirm the chimeric tail fiber still trimerises (native state for phage tail fibers — essential
for binding avidity). Validate Td-cell binding by electron microscopy or flow cytometry
with fluorescently-labelled chimeric tail fiber protein.
4
Load CRISPR payload + non-replicative packaging
Insert into the LE1 genome: P_tac → Cas12a → crRNA(fliA) + crRNA(fliG)
under a constitutive Td promoter. Remove LE1 replication genes from the packaged phagemid DNA —
non-replicative delivery (one-shot injection, no phage spread). Retain lytic module for
cell killing post-delivery. Manufacture the chimeric phage particle in E. coli
using the existing LE1 shuttle vector system.
5
Validate in Td culture → combine with TdVax
Add chimeric phage to Td ATCC 35405 liquid culture (MOI 1–100).
Readouts: (a) phase-contrast microscopy → motility score;
(b) Western blot for FlaB loss; (c) PCR for fliA/fliG genomic cut;
(d) CFU drop for lethality kinetics.
Combination experiment: TdVax-immunised serum + phage-treated Td → opsonophagocytic killing
assay against murine peritoneal macrophages.
The two modalities target orthogonal aspects of pathogenesis and operate on different timescales —
making them naturally combinatorial rather than redundant.
TdVax (adaptive immunity)
Primes B-cell and T-cell responses against surface antigens (MSP, BamA, FlgD/FlaB).
Effective after 2–4 week seroconversion lag. Requires intact antigen display — works best
when bacteria are weakened, slowed, or in transition. IgG opsonises Td for macrophage killing.
PhageVax (genetic intervention)
Acts within minutes to hours of subgingival delivery. Immobilises or kills Td before
it can penetrate tissue. Does not require prior immune priming. Works in the acute infection
window where adaptive immunity has not yet activated.
Combined delivery concept
Both modalities could be delivered subgingivally in the same irrigation gel — a standard periodontal
treatment format. Phage knocks out Td motility immediately. TdVax-primed antibodies opsonise and
clear the now-stationary, MSP-exposed bacteria. The phage also reveals more MSP epitopes on bacteria
that mutate away from MSP-targeting, paradoxically enhancing TdVax opsonisation.
Combined protection > either alone.
09 · Open questions
What we don't know yet
Can LE1 tail fiber be redirected without disrupting trimer geometry?
Tail fibers must trimerize to achieve multivalent binding avidity. C-terminal domain swaps can
disrupt trimerization if the linker geometry changes. Structural validation of the chimeric trimer
is the gating experiment.
Does LE1 inject DNA efficiently through Td OM?
Despite being a spirochete phage, LE1 evolved in Leptospira biflexa. The Td OM composition
(different lipoprotein content, different LPS organisation) may require additional baseplate engineering
for efficient DNA injection. Cross-species injection efficiency is unknown.
What are the Td-compatible promoters for Cas12a expression?
The CRISPR payload needs a promoter recognised by Td RNA polymerase. Td uses σ70 for constitutive
expression but the exact −10/−35 consensus differs from E. coli. The LE1 native promoter
sequences (expressed in L. biflexa late after infection) are the best starting candidates.
Phage stability in the oral environment
Saliva contains nucleases, proteases, pH variation (5.5–7.5), and shear forces. LE1 phage particle
stability in artificial saliva and gingival crevicular fluid needs empirical measurement before
in vivo experiments.
Off-target spirochetes in the oral microbiome
The oral cavity contains many spirochete species (Treponema vincentii, T. medium,
T. lecithinolyticum, etc.). The MSP-binding tail fiber must not cross-react with other oral
spirochetes' surface proteins. Species-specificity validation against a panel of oral spirochetes
is required before in vivo testing.
Literature
References
1Park JY et al. (2017). Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus. Sci Rep 7:44929. DOI: 10.1038/srep44929
2Selle K et al. (2020). In vivo targeting of Clostridioides difficile using phage-delivered CRISPR-Cas3 antimicrobials. mBio 11:e00019-20. DOI: 10.1128/mBio.00019-20
3Bourhy P et al. (2005). Complete nucleotide sequence of the LE1 prophage from the spirochete Leptospira biflexa and characterization of its replication and partition functions. J Bacteriol 187(12):3931–3940. DOI: 10.1128/JB.187.12.3931-3940.2005
4Yehl K et al. (2019). Engineering phage host-range and suppressing bacterial resistance through phage tail fiber mutagenesis. Cell 179(2):459–469. DOI: 10.1016/j.cell.2019.09.001
5Dunne M et al. (2019). Reprogramming bacteriophage host range through structure-guided design of chimeric receptor binding proteins. Cell Rep 29(5):1336–1350. DOI: 10.1016/j.celrep.2019.09.062
6Heinzerling HF et al. (1995). Cloning and expression of a gene, fliG, from Treponema denticola. Gene 157(1–2):213–215. DOI: 10.1016/0378-1119(95)00257-7
7Kurniyati K et al. (2022). FliA (sigma factor σ28) is essential in Treponema denticola. J Bacteriol. DOI: 10.1128/jb.00248-22
8Esteves NC et al. (2022). Flagellotropic bacteriophages: opportunities and challenges for phage therapy. Antibiotics. DOI: 10.3390/antibiotics11081019
9Mourosi JT et al. (2022). Understanding bacteriophage tail fiber interaction with host surface receptor: the key "blueprint" for reprogramming phage host range. Int J Mol Sci 23(20):12146. DOI: 10.3390/ijms232012146