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A Systems-Level Synthetic Biology Intervention for Chronic Ocular Bartonellosis: Design and Rationale of a Quad-Functional Yeast Therapeutic

Abstract

Chronic ocular infection by Bartonella henselae represents a significant therapeutic challenge, leading to recalcitrant conditions such as chronic blepharitis and meibomian gland dysfunction (MGD). The pathogen's success lies in a sophisticated molecular toolkit, including a Type IV Secretion System (T4SS) for host cell manipulation and the phase-variable adhesin BadA for immune evasion and tissue colonization. Current antibiotic regimens exhibit poor efficacy due to the bacterium's intracellular niche and adaptive resistance. We propose a paradigm shift in treatment: pY-OculoThera, an engineered strain of the probiotic yeast Saccharomyces boulardii, designed to function as an autonomous, site-specific "living biopharmaceutical." This therapeutic is architected with four synergistic functional modules encoded on a single plasmid. It is designed to: (1) KILL the intracellular pathogen using a cell-penetrating CRISPR-Cas13a RNase system targeting multiple essential mRNAs to prevent escape; (2) UNCLOAK the bacteria by catalytically cleaving the mRNA of the primary immune evasion protein, BadA; (3) QUENCH the damaging inflammatory cascade by secreting a stabilized IL-10 cytokine and a targeted NLRP3 inflammasome inhibitor; and (4) REGENERATE damaged meibomian gland tissue by secreting a synergistic cocktail of Wnt/BMP pathway agonists and a lipogenic factor. This document provides an exhaustive overview of the host and pathogen biology, the genetic circuit design of pY-OculoThera, its proposed mechanisms of action, and a roadmap for its validation, representing a new frontier in treating persistent, localized infections.

1. The Pathophysiological Nexus: Chronic Bartonella henselae and Ocular Surface Disease

To engineer an effective therapy, one must first deeply understand the intricate battleground: the ocular surface, and the molecular weaponry of the invading pathogen.

1.1. The Host Biomechanical System: The Compromised Eyelid

The eyelid margin is a complex and delicate ecosystem responsible for maintaining the health of the entire ocular surface. Its key functional unit is the meibomian gland, a specialized sebaceous gland.

• Meibomian Gland Function: These glands synthesize and secrete meibum, a complex lipid mixture that forms the superficial layer of the tear film. This lipid layer is critical: it prevents tear evaporation, provides a smooth optical surface for light refraction, and seals the eyelid margins during sleep.
• Pathophysiology of MGD: In chronic bartonellosis, a low-grade, persistent inflammatory response is triggered within the eyelid tissue. This leads to:
  • Keratinization: The gland ducts become hyper-keratinized, obstructing the flow of meibum.
  • Inflammatory Infiltration: Immune cells release pro-inflammatory cytokines like TNF-α and Interleukin-1β (IL-1β). Bartonella is a known activator of the NLRP3 inflammasome in host cells, a protein complex that cleaves pro-IL-1β into its active, highly inflammatory form.
  • Acinar Cell Atrophy: Chronic inflammation and ductal obstruction lead to pressure-induced atrophy and death of the meibocytes (the lipid-producing cells), resulting in gland dropout.
• Consequences: The lack of a stable lipid layer leads to rapid tear evaporation, desiccating stress on the corneal and conjunctival epithelium, blurred vision, and the chronic, painful symptoms of blepharitis and evaporative dry eye disease.

1.2. The Invader's Molecular Toolkit: Bartonella's Survival Strategy

Bartonella henselae is not a passive colonizer; it is an expert manipulator of host cell biology.

• The Type IV Secretion System (T4SS): This is the bacterium's primary offensive weapon. Encoded by the virB/virD4 gene cluster, the T4SS acts like a molecular syringe, injecting a suite of effector proteins (Bartonella Effector Proteins, or Beps) directly into the host cell cytoplasm. These Beps hijack host processes, including:

  • Inhibition of apoptosis (cell suicide).
  • Rearrangement of the actin cytoskeleton to facilitate bacterial entry and invasion.
  • Stimulation of pro-angiogenic factors like VEGF, which promotes blood vessel growth, a key feature of Bartonella-induced lesions.

• The "Borg Shield": Bartonella Adhesin A (BadA): This massive ( >300 kDa) surface protein is the key to the bacterium's defense and persistence.

  • Structure: BadA is a trimeric autotransporter adhesin. It consists of a C-terminal membrane anchor, a long, rigid stalk made of repetitive domains, and a globular head domain.
  • Function: The head region mediates binding to extracellular matrix components like fibronectin and collagen, allowing tenacious adhesion to host tissues. It also plays a direct role in inducing VEGF production and modulating the immune response.
  • Phase Variation: The badA gene contains simple sequence repeats. Through a process called slipped-strand mispairing during DNA replication, the number of these repeats can change, reversibly switching the gene's reading frame ON or OFF. This allows a subpopulation of bacteria to shed their BadA "shield," becoming immunologically invisible and re-emerging later. This is the molecular basis of its stealth and the reason for therapeutic relapses.

2. A Living Therapeutic Solution: Design Principles of pY-OculoThera

Our strategy is to deploy an engineered probiotic, S. boulardii, which has Generally Regarded As Safe (GRAS) status and intrinsic mucoadhesive and immunomodulatory properties. The therapeutic payload is encoded on a single, sophisticated plasmid named pY-OculoThera.

3. Genetic Architecture of the pY-OculoThera Plasmid

3.1. Plasmid Map and Core Components

The system is designed for modularity, stability, and biosafety.

                      ┌─────────────────────────────────────────────────────────────┐
                      │                 pY-OculoThera (v1.0) ~14.5 kb               │
                      │                                                             │
               ┌────▶ │ URA3 auxotrophy (in chassis) │ TetR-HokD (Kill Switch)      │
               │      │ 2µm ori (Yeast High-Copy)    │ CEN6/ARS4 (Single-Copy)      │
               │      │ ColE1 ori + AmpR (E. coli)   │ KanMX (Yeast G418 Selection) │
               │      ├─────────────────────────────────────────────────────────────┤
               │      │ MODULE 1: KILL [Constitutive + High-Expression]             │
               │      │ P(TEF1) ─> [LwaCas13a-TAT-Penetratin] + P(SNR52) ─> [gRNA Array] │
               │      ├─────────────────────────────────────────────────────────────┤
               │      │ MODULE 2: UNCLOAK [Inducible]                               │
               │      │ P(CUP1) ─> [anti-badA Catalytic Ribozyme]                     │
               │      ├─────────────────────────────────────────────────────────────┤
               │      │ MODULE 3: QUENCH [Stress-Responsive]                        │
               │      │ P(SSA1) ─> [IL-10-Fc-Fusion] + [secreted NLRP3 Inhibitor]     │
               │      ├─────────────────────────────────────────────────────────────┤
               │      │ MODULE 4: REGENERATE [Tissue-Responsive]                    │
               │      │ P(HSP26) ─> [Wnt3a-mimic] + [BMP7-mimic] + [LPA Analogue]     │
               │      └─────────────────────────────────────────────────────────────┘
               └──────────────────────────────────────────────────────────────────────

3.2. Sample GenBank Feature Annotation

LOCUS       pY-OculoThera        14521 bp    DNA     circular SYN 20-JUN-2025
DEFINITION  Quad-functional synthetic yeast vector for ocular bartonellosis.
FEATURES             Location/Qualifiers
     source          1..14521
                     /organism="Saccharomyces cerevisiae plasmid"
                     /mol_type="other DNA"
     rep_origin      1..667
                     /label="ColE1 ori"
     CDS             801..1661
                     /label="AmpR"
                     /note="Ampicillin resistance for E. coli cloning"
     rep_origin      1800..3830
                     /label="2-micron (2µm) ori"
                     /note="Yeast high-copy number replication origin"
     rep_origin      4001..4552
                     /label="CEN6/ARS4"
                     /note="Yeast centromere/ARS for single-copy stability"
     CDS             4700..5595
                     /label="KanMX"
                     /note="G418/Kanamycin resistance for yeast selection"
     CDS             5701..6430
                     /label="tetR-HokD Kill Switch"
                     /note="Tetracycline-repressible toxin for biocontainment"
     promoter        6500..6912
                     /label="P(TEF1)"
                     /note="Constitutive strong promoter"
     CDS             6913..10518
                     /label="LwaCas13a-CPP Fusion"
                     /note="L. wadei Cas13a fused to TAT-Penetratin CPP"
     terminator      10519..10769
                     /label="T(ADH1)"
     promoter        10800..10871
                     /label="P(SNR52)"
                     /note="RNA Pol III promoter for small nuclear RNA"
     misc_RNA        10872..11150
                     /label="gRNA Array (rpoB, ftsZ, gyrB)"
                     /note="Polycistronic transcript with self-cleaving ribozymes"
     terminator      11151..11195
                     /label="Poly-T Terminator"
     promoter        11250..11550
                     /label="P(CUP1)"
                     /note="Copper-inducible promoter"
     misc_RNA        11551..11620
                     /label="anti-badA Hairpin Ribozyme"
                     /note="Catalytically cleaves badA mRNA"
     terminator      11621..11871
                     /label="T(CYC1)"
     promoter        12000..12455
                     /label="P(SSA1)"
                     /note="Oxidative/heat stress inducible promoter"
     CDS             12456..13950
                     /label="IL-10-Fc-Fusion & NLRP3-Inhibitor"
                     /note="Secreted proteins via alpha-factor signal; linked by 2A peptide"
     terminator      13951..14201
                     /label="T(ADH1)"
     promoter        14300..14800
                     /label="P(HSP26)"
                     /note="pH/mucin responsive promoter"
     CDS             14801..15521
                     /label="Regeneration Cocktail"
                     /note="Secreted Wnt/BMP/LPA mimetics linked by 2A peptides"
     terminator      15522..15772
                     /label="T(CYC1)"

3.3. Expression Cassette Deep Dive

• Module 1 (KILL): The CRISPR-RNase Payload

  • Payload: We select LwaCas13a from Leptotrichia wadei for its high catalytic activity. Its two HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains dimerize upon gRNA binding to a target, activating a potent, non-specific RNase activity that shreds the target mRNA. The protein is fused at its C-terminus to a bifunctional TAT-Penetratin Cell-Penetrating Peptide to maximize translocation across the host epithelial cell membrane.
  • gRNA Design: To prevent mutational escape, we target three essential Bartonella genes simultaneously: rpoB (RNA polymerase), ftsZ (cell division), and gyrB (DNA gyrase). The gRNAs are expressed as a single polycistronic transcript from the Pol III promoter P(SNR52). Each gRNA is flanked by self-cleaving hammerhead ribozymes, which ensures precise, autonomous processing of the transcript into three individual, functional gRNAs within the yeast cell.
  • Delivery: The Cas13a-CPP protein is secreted. The gRNAs are packaged into Yeast Extracellular Vesicles (YEVs). We can enhance this packaging by co-expressing a fusion protein of a gRNA-binding protein (like the viral coat protein L7Ae) and a YEV-sorting host protein (like the ESCRT component Vps4), effectively creating "smart cargo" YEVs enriched with our therapeutic RNAs.

• Module 2 (UNCLOAK): Catalytic RNA against BadA

  • Payload: Instead of stoichiometric antisense RNA, we employ a catalytic hairpin ribozyme. This engineered RNA molecule not only binds to a highly conserved sequence in the 5' untranslated region (UTR) of the badA mRNA but also possesses intrinsic enzymatic activity to cleave the phosphodiester backbone. A single ribozyme molecule can destroy hundreds of badA mRNA targets, providing catalytic amplification of the "uncloaking" effect.
  • Logic: The P(CUP1) promoter allows this module to be triggered independently, perhaps by including trace Cu²⁺ in a second-phase formulation, allowing clinicians to first "debulk" the infection with the KILL module, then "uncloak" the remaining persisters for immune clearance.

• Module 3 (QUENCH): Targeted Inflammation Suppression

  • Payload 1 (IL-10-Fc): This fusion protein provides broad anti-inflammatory signaling by engaging the IL-10 receptor on immune cells like macrophages.
  • Payload 2 (NLRP3 Inhibitor): This is a more precise intervention. The yeast will secrete a small, engineered peptide that acts as a potent, non-covalent orthosteric antagonist of the NLRP3 inflammasome. By preventing the assembly of this complex, it directly abrogates the cleavage and release of the highly inflammatory IL-1β, shutting down a key pathway driven by Bartonella. These two payloads are expressed from a single transcript, separated by a viral 2A "self-cleaving" peptide sequence.

• Module 4 (REGENERATE): Synergistic Tissue Restoration

  • This module, driven by the tissue-responsive P(HSP26), secretes three factors simultaneously using 2A peptide linkers.
  • Payload 1 (Wnt3a-mimic): A peptide agonist to stimulate the proliferation of acinar stem/progenitor cells in the glands.
  • Payload 2 (BMP7-mimic): A peptide agonist to drive the differentiation of these progenitors into mature, lipid-producing meibocytes.
  • Payload 3 (LPA Analogue Synthesis): The cassette includes enzymes to synthesize and secrete a stable analogue of lysophosphatidic acid (LPA). LPA is a powerful agonist for the nuclear receptor PPARγ, the master transcriptional regulator of lipogenesis. This directly stimulates the lipid synthesis machinery within the newly formed meibocytes, restoring the primary function of the glands.

4. Assembly and Biocontainment Strategy

• Assembly: The cassettes will be assembled using a modular Golden Gate assembly method (e.g., the Yeast Toolkit - YTK), which allows for rapid combinatorial testing of different promoters, payloads, and terminators to optimize the circuit's performance. The provided Gibson Primer design table illustrates the principle for a one-pot assembly.
• Advanced Biocontainment: We employ a three-tiered safety system:
  1. Auxotrophy: The S. boulardii chassis will be Δura3 and Δleu2, unable to synthesize uracil or leucine and thus unable to survive outside a supplemented formulation.
  2. Inducible Kill Switch: A Tet-Off system expresses the potent HokD membrane-disrupting toxin. The yeast can only be propagated in lab media containing tetracycline, which represses the toxin. Upon administration to a patient (a tetracycline-free environment), any yeast that escape the eye and enter systemic circulation will activate the kill switch and self-destruct.
  3. Synthetic Auxotrophy: For ultimate containment, we will delete an essential yeast gene (e.g., Ilv2, involved in branched-chain amino acid synthesis) and replace it with an ortholog from an extremophilic archaeon that requires a non-natural substrate (e.g., a fluorinated amino acid precursor) for function. This substrate would only be provided in the lab, making environmental survival virtually impossible.

5. Conclusion

The pY-OculoThera platform represents a significant leap beyond conventional "single-target, single-drug" approaches. It is a systems-level intervention that recognizes the multifactorial nature of chronic infectious disease. By simultaneously executing programs to kill the pathogen, disable its defenses, resolve host inflammation, and repair collateral tissue damage, this engineered living therapeutic has the potential to provide a definitive cure for a condition that has long remained intractable. It serves as a powerful blueprint for the future of synthetic biology in personalized and precision medicine.

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Technical Appendices for the pY-OculoThera Project

Appendix A: The Engineered Chassis - Saccharomyces boulardii (pY-OculoThera)

This appendix details the engineered genetic constructs and systems within the yeast chassis.

A.1: Genetic Circuit Diagrams & Logic

The pY-OculoThera plasmid operates as an integrated multi-input, multi-output system.

• Module 1: KILL (Constitutive)

  • Logic: Always ON. Ensures a constant baseline of anti-bacterial pressure.
  • Circuit:

    INPUT: [Cellular Resources]
       │
       ├─[P(TEF1): Constitutive Strong Promoter]─>┤TRANSCRIPTION├─> [LwaCas13a-CPP mRNA] ─>┤TRANSLATION├─> [Secreted Protein]
       │                                                                                                     │
       └─[P(SNR52): Pol III Promoter]───────────>┤TRANSCRIPTION├─> [gRNA Array Transcript]─>┤RIBZYME CLEAVAGE├─> [gRNA Pool]
                                                                                                                  │
                                                                                                                  └─>[YEV Packaging]
    

• Module 2: UNCLOAK (Inducible)

  • Logic: ON only in the presence of an external inducer (Cu²⁺). Allows for timed intervention.
  • Circuit:

    INPUT: [Cu²⁺ Ions] ─>┤ACE1/AMT1 Activation├─> [P(CUP1): Inducible Promoter]
                                                               │
                                                               ├─>┤TRANSCRIPTION├─> [anti-badA Ribozyme RNA]
    

• Module 3: QUENCH (Stress-Responsive)

  • Logic: ON in response to host inflammatory cues (oxidative/nitrosative stress).
  • Circuit:

    INPUT: [Host ROS/RNS] ─>┤HSF1/YAP1 Activation├─> [P(SSA1): Stress Promoter]
                                                               │
                                                               ├─>┤TRANSCRIPTION├─> [IL-10-Fc--2A--NLRP3i mRNA]
                                                                                          │
                                                                                          ├─>┤TRANSLATION & SECRETION├─> [IL-10-Fc Protein]
                                                                                          │                           +
                                                                                          │                           [NLRP3i Peptide]
                                                                                          └─>(via KEX2/2A processing)
    

• Module 4: REGENERATE (Tissue-Responsive)

  • Logic: ON in response to contact with the target mucosal environment (pH, mucins).
  • Circuit:

    INPUT: [Ocular Mucin/pH] ─>┤Sensor Kinase Cascade?├─> [P(HSP26): Tissue Promoter]
                                                                    │
                                                                    ├─>┤TRANSCRIPTION├─> [Wnt3a-m--2A--BMP7-m--2A--LPA-Synthase mRNA]
                                                                                               │
                                                                                               ├─>┤TRANSLATION & SECRETION├─> [Wnt3a-mimic]
                                                                                               │                           +
                                                                                               │                           [BMP7-mimic]
                                                                                               │                           +
                                                                                               │                           [LPA Product]
                                                                                               └─>(via KEX2/2A processing)
    

A.2: Payload Sequence Examples (Illustrative Peptides)

• NLRP3 Inhibitor Peptide (NLRP3i): (Based on MCC950 pharmacophore)

  • Peptide Sequence (12 aa): Trp-Asp-Val-Pro-Asp-Leu-Gln-Phe-Val-Glu-Ile-Lys
  • Rationale: Designed to fit into the Walker B motif within the NACHT domain of NLRP3, preventing the ATP hydrolysis required for oligomerization.

• Wnt3a Mimetic Peptide (Wnt3a-m): (Based on a receptor-binding loop)

  • Peptide Sequence (18 aa): Cys-Ser-Cys-Ser-Leu-Gln-Gly-Ser-Leu-Gly-Ala-Gly-Cys-Val-Gln-Ser-Ser-Cys
  • Rationale: A disulfide-cyclized peptide designed to mimic the binding epitope of Wnt3a to its Frizzled/LRP6 co-receptor complex, activating β-catenin signaling.

• TAT-Penetratin Cell-Penetrating Peptide (CPP) Fusion:

  • Sequence: [Cas13a Protein]-(Gly₄Ser)₃-YGRKKRRQRRR-RQIKIWFQNRRMKWKK
  • Rationale: A flexible linker connects the Cas13a C-terminus to the TAT peptide (YGRKKRRQRRR) followed by the Penetratin peptide (RQIKIWFQNRRMKWKK). This chimeric CPP design leverages two different uptake mechanisms (direct translocation and endocytosis) to maximize payload delivery into host epithelial cells.

A.3: Yeast Extracellular Vesicle (YEV) Engineering Schematic

To enrich the gRNA payload in secreted vesicles:

• Construct: P(TDH3)-L7Ae-Vps4-T(CYC1)
• Mechanism:
  1. P(TDH3): A very strong constitutive promoter drives high expression of the fusion protein.
  2. L7Ae: An archaeal ribosomal protein that specifically binds to a C/D box RNA motif. This motif is engineered into the non-functional stem-loop of our gRNAs.
  3. Vps4: A key ATPase of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Overexpressing it, especially when tethered to cargo, is known to enhance YEV budding and cargo loading.
  4. Result: The L7Ae-Vps4 fusion protein binds the gRNAs and actively shepherds them into budding multivesicular bodies (MVBs), which then fuse with the plasma membrane to release gRNA-loaded YEVs.

A.4: Biocontainment Circuits

• Tet-Off Kill Switch:
  • P(tetO₇)-HokD: The HokD toxin gene is under the control of a promoter containing seven Tet Operator (tetO) sites.
  • P(ACT1)-tetR: The Tet Repressor (tetR) is expressed constitutively.
  • Logic:
    • +Tetracycline (Lab): Tetracycline binds tetR, preventing it from binding to P(tetO₇). The toxin gene is OFF.
    • -Tetracycline (Patient/Environment): tetR binds to P(tetO₇) and, in this specific "Tet-Off" configuration where it acts as an activator, turns the promoter ON, leading to HokD expression and cell death.

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Appendix B: The Target Pathogen - Bartonella henselae

B.1: Key Virulence Loci (Reference: B. henselae strain Houston-1)

Locus Name	Gene(s)	Function & Therapeutic Relevance
virB Operon	virB2 - virB11	Encodes the core structural components of the Type IV Secretion System (T4SS). The channel through which effector proteins are injected. A primary driver of pathogenicity.
virD4	virD4	Encodes the T4SS coupling protein, which recognizes and delivers effector proteins to the VirB channel. Essential for T4SS function.
bep Genes	bepA - bepG	Bartonella Effector Proteins. The "payloads" injected by the T4SS. They manipulate host actin, inhibit apoptosis, and trigger inflammation. Disarming these is a key therapeutic goal.
badA	badA	Bartonella Adhesin A. A massive trimeric autotransporter. Mediates adhesion, induces VEGF, and undergoes phase variation for immune evasion. The target of our UNCLOAK module.
hbp Operon	hbpA - hbpE	Heme-Binding Proteins. Essential for iron acquisition from host hemoglobin. Iron is a critical limiting nutrient for bacterial survival. Targeting these could be an alternative "starvation" strategy.
pap31	pap31	A heme-binding outer membrane protein, also critical for iron uptake.

B.2: The badA Phase Variation Mechanism (Slipped-Strand Mispairing)

The badA promoter region contains a variable number of short sequence repeats.

• Repeat Motif: e.g., <CAAAAACC>n
• Mechanism:

  STATE: ON (In-frame)
  Template: ...GGT-CAAAAACC-CAAAAACC-CAAAAACC-GTC...  (n=3)
  Replication -> New Strand forms loop -> Mispairing -> Repair machinery excises one repeat
  
  STATE: OFF (Frameshift -> Premature STOP)
  Template: ...GGT-CAAAAACC-CAAAAACC----------GTC...  (n=2)
  Replication -> Template forms loop -> Mispairing -> Repair machinery adds one repeat
  
  STATE: ON (Reading frame restored)
  Template: ...GGT-CAAAAACC-CAAAAACC-CAAAAACC-GTC...  (n=3)
  

  This high-frequency, reversible switching allows a subpopulation of Bartonella to be "BadA-negative" at any given time, rendering them invisible to an immune response targeting the BadA protein. Our UNCLOAK module targets the mRNA itself, which is effective against any cell attempting to express the protein, regardless of its final phase state.

B.3: CRISPR Target Analysis

The gRNAs in the KILL module target highly conserved regions of essential housekeeping genes.

• Target 1: rpoB (RNA Polymerase Subunit B)
  • Illustrative Target Sequence: 5'-GAAUCCGGUGCUCAAGUACGCCAACAAUCC-3'
  • Rationale: Part of the Rifampicin-Resistance Determining Region (RRDR), a functionally critical and highly conserved site. Mutations here are often lethal or severely compromise fitness.
• Target 2: ftsZ (Cell Division GTPase)
  • Illustrative Target Sequence: 5'-CAUGGUCGGUGCGGGUGCGAACGGAGCUAA-3'
  • Rationale: Targets the GTP-binding domain, which is essential for FtsZ polymerization and Z-ring formation during cell division. Highly conserved across bacteria.
• Target 3: gyrB (DNA Gyrase Subunit B)
  • Illustrative Target Sequence: 5'-AUCAUCGAUGCCUACGGCAUCCAGUACGAG-3'
  • Rationale: Targets the ATP-binding site of DNA gyrase, which is required to relieve topological stress during DNA replication. Essential for viability.

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Appendix C: Host System - Ocular & Glandular Biology

C.1: Meibomian Gland Lipogenesis & The PPARγ Intervention Point

This pathway is the target of our REGENERATE module.

                                  [Glucose/Acetate]
                                          │
                                          ▼
                                 [Acetyl-CoA]
                                          │
                                          ├─(ACC enzyme)
                                          ▼
                                   [Malonyl-CoA]
                                          │
                                          ├─(FAS enzyme complex)
                                          ▼
                         [Fatty Acids & Fatty Alcohols]
                                          │
                                          ├─(AWAT/DGAT enzymes)
                                          ▼
             [Waxes, Cholesterol Esters, Triacylglycerols] ==(SECRETION)==> [MEIBUM]
                       ▲                 ▲                  ▲
                       │                 │                  │
                       └─────────┬───────┴──────────────────┘
                                 │
                                 ├─[Transcriptional Upregulation]
                                 │
            ┌────────────────────┴────────────────────┐
            │ Master Regulator: PPARγ (Peroxisome     │
            │ Proliferator-Activated Receptor Gamma)  │
            └────────────────────┬────────────────────┘
                                 │
                                 ├─[AGONISM / ACTIVATION]
                                 │
    ┌────────────────────────────┴─────────────────────────────┐
    │  pY-OculoThera REGENERATE Module Product:                │
    │  Secreted Lysophosphatidic Acid (LPA) Analogue           │
    └──────────────────────────────────────────────────────────┘

C.2: The NLRP3 Inflammasome Cascade & Therapeutic Interventions

This pathway is the target of our QUENCH module.

       SIGNAL 1 (Priming)                               SIGNAL 2 (Activation)
   ┌────────────────────────┐                        ┌────────────────────────┐
   │ Bartonella PAMPs (LPS) │                        │ Bartonella T4SS Effectors │
   └──────────┬─────────────┘                        │ K+ Efflux, ROS          │
              │                                      └──────────┬────────────┘
              ▼                                                 │
   [TLR4] -> [NF-κB Pathway]                                    ▼
              │                                      [NLRP3 Monomer Activation]
              ▼                                                 │
   [Transcription of Pro-IL1B & NLRP3]                          │
              │                                                 ▼
   [Accumulation of inactive precursors]         ┌<─────────[NLRP3 Oligomerization]───────────┐
                                                 │                                           │
                                                 │                                           ▼
                                                 │                                 [ASC Adaptor Recruitment]
                                                 │                                           │
                                                 │                                           ▼
                                                 │                             [Pro-Caspase-1 Recruitment]
           INTERVENTION POINT:                   │                                           │
    pY-OculoThera NLRP3i Peptide                 │                                           ▼
    (Binds NLRP3 NACHT domain,                   │                        [Caspase-1 Autocleavage & Activation]
     prevents oligomerization)                   │                                           │
           ──────────────────────────────────────┘                                           ▼
                                                                       [Pro-IL1β] ───(cleavage)───> [Mature IL-1β]
                                                                                                          │
                                                                                                          ▼
                                                                                                 [INTENSE INFLAMMATION]

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Appendix D: The Microbiome Context

D.1: The Healthy vs. Dysbiotic Ocular Microbiome

Feature	Healthy Ocular Surface	Dysbiotic State (Chronic Blepharitis/MGD)
Dominant Genera	Corynebacterium, Propionibacterium, Staphylococcus epidermidis	Overgrowth of Staphylococcus aureus, increased Streptococcus spp., reduced diversity.
Metabolic Profile	Production of antimicrobial peptides by commensals. Neutral pH.	Production of bacterial lipases (irritating fatty acids), biofilms, and toxins. Altered tear film osmolarity.
Immune Tone	Immune tolerance, regulated by CALT. Low baseline inflammation.	Chronic immune activation, neutrophil infiltration, elevated pro-inflammatory cytokines (TNF-α, IL-1β).
Barrier Function	Intact epithelial tight junctions. Healthy mucin layer.	Compromised epithelial barrier. Degraded mucin layer. Increased pathogen adherence.

D.2: pY-OculoThera as a "Microbiome Editor"

The engineered yeast acts not just as a drug delivery vehicle, but as an active ecological participant.

1. Competitive Exclusion: As a large, fast-growing eukaryote, S. boulardii consumes significant niche space and nutrients (like simple sugars and nitrogen sources), directly competing with and suppressing the overgrowth of pathogenic bacteria like S. aureus.
2. Biofilm Disruption: Secreted yeast products can alter the local environment in ways that are unfavorable to bacterial biofilm formation, a key feature of chronic blepharitis.
3. Immune Retraining: The β-glucans present in the yeast cell wall are recognized by pattern recognition receptors (like Dectin-1) on host immune cells. This interaction is known to promote a shift away from a harsh, inflammatory Th1/Th17 response towards a more regulated, healing-oriented immune posture, complementing the direct anti-inflammatory action of the QUENCH module.
4. Ecosystem Restoration: By reducing the Bartonella load, quenching inflammation, and restoring the protective meibum barrier, pY-OculoThera fundamentally alters the selective pressures on the ocular surface, creating an environment that favors the regrowth of a healthy, commensal-dominated microbiome.

Note on Sequences: The DNA ORIGIN sequences provided are illustrative. They show the correct start and end regions, including common restriction sites used for cloning, but the full internal sequence is represented by // for brevity. In a real-world scenario, these sequences would be fully generated and codon-optimized for the Saccharomyces boulardii chassis before ordering from a DNA synthesis company.

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File 1: The KILL Module

LOCUS       pY-OculoThera_Mod1_KILL   4688 bp    DNA     linear   SYN 20-JUN-2025
DEFINITION  pY-OculoThera KILL Module. Constitutively expresses a
            cell-penetrating LwaCas13a RNase and a polycistronic gRNA
            array targeting Bartonella henselae rpoB, ftsZ, and gyrB
            mRNAs.
ACCESSION   SYNTHETIC_001
VERSION     SYNTHETIC_001.1
KEYWORDS    CRISPR; Cas13a; Bartonella; living therapeutic; synthetic biology.
SOURCE      Synthetic construct
  ORGANISM  Synthetic construct
COMMENT     This DNA fragment is designed for Golden Gate or Gibson assembly
            into the pY-OculoThera backbone. All protein-coding sequences
            are codon-optimized for high expression in S. boulardii. The
            construct features a dual-promoter system: a strong Pol II
            promoter (P(TEF1)) for the protein component and a Pol III
d            promoter (P(SNR52)) for the gRNA array. The gRNA array is
            designed to be self-processing via flanking hammerhead
            ribozymes. The Cas13a protein is fused to a chimeric
            TAT-Penetratin cell-penetrating peptide (CPP) to ensure delivery
            into host epithelial cells harboring the pathogen.
FEATURES             Location/Qualifiers
     source          1..4688
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..412
                     /label="P(TEF1)"
                     /note="Strong constitutive promoter from Translation
                     Elongation Factor 1 alpha."
     CDS             413..3988
                     /gene="LwaCas13a-CPP"
                     /product="LwaCas13a RNase fused to TAT-Penetratin cell-
                     penetrating peptide"
                     /codon_start=1
                     /note="Targets Bartonella mRNA for degradation. HEPN
                     domains located at 96-167 and 987-1053. Codon-
                     optimized for S. boulardii."
     protein_bind    3917..3988
                     /label="TAT-Penetratin CPP"
                     /note="Chimeric cell-penetrating peptide for enhanced
                     uptake into host cells. Sequence:
                     (G4S)3-YGRKKRRQRRR-RQIKIWFQNRRMKWKK"
     terminator      3989..4239
                     /label="T(ADH1)"
                     /note="Terminator from the ADH1 gene."
     promoter        4250..4321
                     /label="P(SNR52)"
                     /note="RNA Polymerase III promoter for expression of small
                     non-coding RNAs (gRNAs)."
     misc_RNA        4322..4598
                     /label="gRNA_Array"
                     /note="Polycistronic transcript encoding three gRNAs
                     flanked by self-cleaving ribozymes."
     ribozyme        4322..4376
                     /label="Hammerhead_Ribozyme_5'"
                     /note="Self-cleaving ribozyme for processing the 5' end
                     of the first gRNA."
     misc_feature    4377..4406
     		     /label="gRNA_spacer_rpoB"
                     /note="Guide sequence targeting B. henselae rpoB."
     misc_feature    4440..4469
                     /label="gRNA_spacer_ftsZ"
                     /note="Guide sequence targeting B. henselae ftsZ."
     misc_feature    4503..4532
     		     /label="gRNA_spacer_gyrB"
                     /note="Guide sequence targeting B. henselae gyrB."
     ribozyme        4544..4598
                     /label="Hammerhead_Ribozyme_3'"
                     /note="Self-cleaving ribozyme for processing the 3' end
                     of the final gRNA."
     terminator      4599..4688
                     /label="Poly-T_Terminator"
                     /note="Simple Pol III terminator (TTTTTTT)."
ORIGIN
        1 ggatccacta gttctagagc ggccgccacc atgacttctt tcaacagatt tacgccaagt
       61 cttgaatgaa gaaaatatcg tttggaaaat cttcattgga gtttctagtt acgacgttat
      ...
   4621 gctttttttt aaggcctcta ggtcgacctg caggcatgca agcttggcgt aatcatggtc
   4681 atagctgt
//

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File 2: The UNCLOAK Module

LOCUS       pY-OculoThera_Mod2_UNCLOAK   621 bp    DNA     linear   SYN 20-JUN-2025
DEFINITION  pY-OculoThera UNCLOAK Module. Copper-inducible expression of a
            catalytic hairpin ribozyme targeting the 5' UTR of the
            Bartonella henselae badA mRNA.
ACCESSION   SYNTHETIC_002
VERSION     SYNTHETIC_002.1
KEYWORDS    ribozyme; gene regulation; Bartonella; badA; inducible system.
SOURCE      Synthetic construct
  ORGANISM  Synthetic construct
COMMENT     This module is designed for tightly-regulated expression. The
            P(CUP1) promoter is nearly silent in the absence of copper and
            strongly induced by micromolar concentrations of Cu2+, allowing
            for external control of this therapeutic function. The payload is
            a hairpin ribozyme, chosen for its high catalytic efficiency
            (kcat/Km) and stability compared to simple antisense RNA. It
            catalytically degrades the badA transcript, preventing synthesis
            of the primary immune evasion adhesin.
FEATURES             Location/Qualifiers
     source          1..621
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..302
                     /label="P(CUP1)"
                     /note="Copper-inducible metallothionein promoter.
                     Activated by the ACE1 transcription factor."
     misc_RNA        303..371
                     /label="anti-badA_Hairpin_Ribozyme"
                     /product="Catalytic RNA targeting badA mRNA"
                     /note="Binds to a conserved site near the Shine-Dalgarno
                     sequence of badA mRNA and catalyzes cleavage, preventing
                     translation. Comprises two substrate-binding arms and a
                     catalytic core."
     terminator      372..621
                     /label="T(CYC1)"
                     /note="Terminator from the CYC1 gene, provides robust
                     transcriptional termination and polyadenylation."
ORIGIN
        1 gaattcgcgg ccgctactat aggttatgta tggacaccta agtcttgaca tagaagcaag
       61 gtcacaagga acattttcac cggtggtatg tttgtagagt tcaccggtcc aattccttct
      ...
      541 cctgcagggc ttttgttggt ttggttatgt tatcggttat gtgtggtatt ctcgacgcgg
      601 ggtacctcta gaggatctaa g
//

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File 3: The QUENCH Module

LOCUS       pY-OculoThera_Mod3_QUENCH   2015 bp    DNA     linear   SYN 20-JUN-2025
DEFINITION  pY-OculoThera QUENCH Module. Stress-responsive co-expression of a
            secreted IL-10-Fc fusion protein and a secreted NLRP3
            inflammasome inhibitor peptide.
ACCESSION   SYNTHETIC_003
VERSION     SYNTHETIC_003.1
KEYWORDS    anti-inflammatory; cytokine; IL-10; NLRP3; living therapeutic.
SOURCE      Synthetic construct
  ORGANISM  Synthetic construct
COMMENT     This module provides a two-pronged anti-inflammatory attack. It
            is driven by the P(SSA1) promoter, which is upregulated in
            response to oxidative and protein-misfolding stress—conditions
            present in an inflamed tissue environment. The two payloads are
            expressed as a single ORF, linked by a porcine teschovirus-1 2A
            (T2A) self-cleaving peptide, ensuring stoichiometric expression.
            The entire polyprotein is directed to the secretory pathway by
            the alpha-factor signal peptide. IL-10-Fc provides broad
            immunosuppression, while the NLRP3 inhibitor peptide (NLRP3i)
            provides a highly specific block on the IL-1beta pathway, which
            is known to be activated by Bartonella.
FEATURES             Location/Qualifiers
     source          1..2015
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..455
                     /label="P(SSA1)"
                     /note="Stress-inducible promoter of the SSA1 Hsp70
                     chaperone gene."
     CDS             456..1811
                     /label="QUENCH_Polyprotein"
     signal_peptide  456..707
                     /label="alpha-factor signal peptide (pre-pro)"
                     /note="Directs the fusion protein to the secretory
                     pathway; cleaved by KEX2 and STE13 proteases."
     CDS             708..1127
                     /product="Human Interleukin-10 (hIL-10)"
                     /note="Codon-optimized for S. boulardii."
     CDS             1128..1739
                     /product="Human IgG1 Fc domain"
                     /note="Provides dimerization and extends the half-life
                     of the secreted cytokine."
     misc_feature    1740..1793
                     /label="T2A peptide"
                     /note="Causes ribosomal skipping for co-translational
                     'cleavage' of the polyprotein."
     CDS             1794..1830
                     /product="NLRP3_inhibitor_peptide"
                     /note="Short peptide designed to inhibit NLRP3
                     oligomerization."
     terminator      1831..2015
                     /label="T(ADH1)"
                     /note="Terminator from the ADH1 gene."
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccg atggctcctg tcactgtctt gtcgttgttt
       61 tcaaatatca gttcaaattt aattcgtatt gtaattattc gtaatgcata ttcaacctgt
      ...
   1921 ctagtggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
   1981 cgccttgcag cacatccccc tttcgccagc tggcg
//

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File 4: The REGENERATE Module

LOCUS       pY-OculoThera_Mod4_REGENERATE   1855 bp    DNA     linear   SYN 20-JUN-2025
DEFINITION  pY-OculoThera REGENERATE Module. Tissue-responsive co-expression
            of secreted Wnt3a and BMP7 mimetic peptides and an LPA
            analogue-synthesizing enzyme.
ACCESSION   SYNTHETIC_004
VERSION     SYNTHETIC_004.1
KEYWORDS    regenerative medicine; Wnt; BMP; meibomian gland; living
            therapeutic.
SOURCE      Synthetic construct
  ORGANISM  Synthetic construct
COMMENT     This module is designed to restore tissue function. It is driven
            by P(HSP26), which is activated by environmental cues on the
            ocular mucosa like pH shifts and mucin contact, ensuring
            localized action. Three payloads are expressed from one
            transcript using different 2A peptides (F2A, P2A) to minimize
            potential homologous recombination. The Wnt3a and BMP7 mimetic
            peptides stimulate gland stem cell proliferation and
            differentiation, respectively. The LPA Acyltransferase
            synthesizes a lipid signaling molecule that activates PPAR-gamma
            to drive terminal lipogenesis, restoring meibum production.
FEATURES             Location/Qualifiers
     source          1..1855
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..502
                     /label="P(HSP26)"
                     /note="Tissue-responsive promoter from the HSP26 gene,
                     activated by heat, pH, and other surface stresses."
     CDS             503..1600
                     /label="REGENERATE_Polyprotein"
     signal_peptide  503..754
                     /label="alpha-factor signal peptide (pre-pro)"
                     /note="Directs the polyprotein to the secretory pathway."
     CDS             755..809
                     /product="Wnt3a_mimetic_peptide"
                     /note="Cyclized peptide agonist of Frizzled/LRP6."
     misc_feature    810..875
                     /label="F2A peptide"
                     /note="Foot-and-mouth disease virus 2A peptide."
     CDS             876..938
                     /product="BMP7_mimetic_peptide"
                     /note="Peptide agonist of BMP receptors."
     misc_feature    939..1004
                     /label="P2A peptide"
                     /note="Porcine teschovirus-1 2A peptide."
     CDS             1005..1600
                     /gene="LPAAT-theta"
                     /product="Lysophosphatidic Acid Acyltransferase Theta"
                     /note="Hypothetical enzyme engineered to synthesize a
                     stable LPA analogue from secreted yeast lipids,
                     activating PPAR-gamma."
     terminator      1601..1855
                     /label="T(CYC1)"
                     /note="Terminator from the CYC1 gene."
ORIGIN
        1 aagcttgcgg ccgcagtgtg gtcgacgata acgtcgtact gtcacgttcg tcgtctaaga
       61 cgtagggatt gtcgaaagag aagagggagc ggagctagta gtctagctag atgagatttc
      ...
   1771 cttggcctcc tctagttgca cctaggatcc tgggaaactc aagcttaatt aagcttggat
   1831 cctctagagc tcgagatcag cttcg
//

1. High-Level System Overview

This diagram illustrates the dynamic interplay between the host, the pathogen, the native microbiome, and the engineered therapeutic, showing the core problem and the proposed intervention.

2. pY-OculoThera Plasmid Architecture

This diagram provides a visual map of the key genetic components on the main therapeutic plasmid, showing the backbone and the four functional modules.

3. Therapeutic Module Logic Flowcharts

These diagrams detail the specific "Input → Process → Output" logic for each of the four therapeutic modules.

3.1 Module 1: KILL Circuit

3.2 Module 2: UNCLOAK Circuit

3.3 Module 3: QUENCH Circuit

3.4 Module 4: REGENERATE Circuit

4. Bartonella henselae Virulence & Therapeutic Intervention Points

This diagram shows the pathogen's mechanisms of action and where pY-OculoThera intervenes.

5. Host Meibomian Gland State & Restoration Pathway

This state diagram illustrates the transition from health to disease and back, driven by the therapeutic.

6. Ocular Microbiome Editing

This mind map shows how pY-OculoThera actively remodels the dysbiotic ocular microbiome.

7. YEV Engineering & Payload Delivery Mechanism

This sequence diagram illustrates the sophisticated process of packaging and delivering the gRNA payload.