Lineage-specific macrophage programs dictate metabolic suppression and stress responses associated with VBNC-like states in Listeria monocytogenes

This study demonstrates that the lineage-specific metabolic and immunological environments of brain-resident microglia versus infiltrating monocyte-derived macrophages dictate whether *Listeria monocytogenes* adopts a replicative cytosolic lifestyle or enters a stress-tolerant, viable but non-culturable state, thereby highlighting macrophage ontogeny as a critical determinant of infection outcome.

The Gist

The Big Picture: A Bacterial Heist with Two Different Hosts

Imagine Listeria monocytogenes (let's call it "Listeria") as a master thief trying to break into a high-security building (the brain). Usually, thieves have a standard playbook: break in, hide, and steal. But this paper reveals that Listeria is a shape-shifter. Its success depends entirely on who it breaks into.

The researchers studied Listeria inside two different types of "security guards" in the brain:

1. Microglia: The brain's native, homegrown security guards. They've lived there since birth.
2. MDMs (Monocyte-Derived Macrophages): The reinforcements called in from outside (the blood) when things get chaotic.

The study found that while both guards try to stop the thief, they do it so differently that the thief has to completely change its strategy to survive.

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Scene 1: The "Homegrown" Guard (Microglia)

The Environment: A Luxury Penthouse
When Listeria gets into the Microglia, it finds itself in a "nutrient-rich" environment. Think of this as the thief breaking into a penthouse suite with a fully stocked fridge, a gym, and a library.

• The Thief's Reaction: Listeria says, "Wow, this is easy!" It drops its disguise, eats the food, and starts multiplying rapidly. It runs around the room (the cell's cytoplasm), building a ladder of actin (a protein) to zip from cell to cell.
• The Result: The bacteria are happy, healthy, and growing fast. The Microglia are actually helping the bacteria spread by providing a safe, comfortable home.

Scene 2: The "Reinforcement" Guard (MDMs)

The Environment: A Torture Chamber
When Listeria gets into the MDMs, it finds itself in a completely different world. This is like the thief breaking into a prison cell with no food, extreme pressure, and guards constantly poking them with electric prods.

• The Thief's Reaction: Listeria realizes, "I'm going to die if I keep moving." So, it hits the pause button. It shuts down its metabolism (stops eating and growing) to save energy. It enters a "sleep mode" known as VBNC (Viable But Non-Culturable). It's alive, but it's not growing, and it's incredibly tough to kill.
• The Result: The bacteria hide in a locked room (a vacuole) inside the guard, waiting for the storm to pass. They are stressed, but they are surviving.

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The Secret Weapons: How the Bacteria Adapt

The researchers used a high-tech "listening device" (Dual RNA sequencing) to hear what the bacteria were "thinking" (gene expression) in both scenarios.

1. The Stress Toolkit (In the MDM Prison)
Inside the MDMs, Listeria turns on its "survival mode" genes.

• The SOS Signal: It activates genes like recA and rtcB. Think of these as the thief's emergency repair kit. If the prison guards damage the thief's DNA (the blueprints for making more thieves), these tools fix the damage so the thief doesn't die.
• The Sleep Mode: It turns off the "growth" genes and turns on "stress" genes. It's like a hibernating bear; it stops moving to conserve energy until the danger passes.

2. The Growth Spurt (In the Microglia Penthouse)
Inside the Microglia, Listeria turns on its "party mode" genes.

• The Feast: It activates genes for eating sugar and building DNA. It's like a construction crew working overtime to build a skyscraper.
• The Escape: It uses the host's nutrients to replicate quickly and spread.

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The "What If" Experiment: Breaking the Tools

To prove that these stress tools were important, the scientists created "broken" versions of the bacteria (mutants) that were missing specific tools like recA and rtcB.

• In the Penthouse (Microglia): When they removed the repair tools (recA or rtcB), the bacteria struggled. They couldn't escape the initial "prison cell" inside the guard to get to the "penthouse" living room. They got stuck and died.
• In the Prison (MDMs): When they removed the repair tools, the bacteria didn't die immediately, but they got stuck in a deeper sleep. They became even more dormant. This suggests that while the tools aren't strictly necessary for survival in the prison, they are crucial for escaping the prison to start growing again.

The Takeaway: The bacteria need these repair tools to survive the harsh conditions of the MDMs long enough to eventually escape and grow in the Microglia.

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The Host's Perspective: Two Different Defenses

The paper also looked at how the two types of guards reacted:

• The MDMs (Reinforcements): They are aggressive. They turn up the heat, flood the cell with inflammatory signals, and try to burn the thief out. They are like a SWAT team using tear gas and flashbangs.
• The Microglia (Homegrown): They are more subtle. They try to manage the situation, using different signaling pathways (like Type I Interferons) that actually end up helping the bacteria spread. They are like a security guard who accidentally leaves the back door open while trying to talk the intruder down.

The "Aha!" Moment

The most important discovery is this: The fate of the infection isn't just about the bacteria's strength; it's about the type of guard it meets.

• If Listeria meets a Microglia, it becomes an active, growing invader.
• If Listeria meets an MDM, it becomes a dormant, sleeping survivor.

This explains why Listeria infections in the brain are so hard to treat. The bacteria can hide in a "sleeping" state inside one type of cell, waiting for the immune system to calm down, only to wake up and multiply in another type of cell.

Summary Analogy

Imagine a spy (Listeria) infiltrating a country.

• If the spy gets caught by a local police officer (Microglia), the officer is confused and lets the spy run free to cause chaos.
• If the spy gets caught by a federal task force (MDM), the spy goes into deep cover, stops talking, stops moving, and waits for years until the task force leaves.

The paper shows us that to catch this spy, we can't just use one strategy. We have to understand that the spy changes its personality depending on who is chasing it.

Technical Summary

1. Problem Statement

Listeria monocytogenes (Lm) is a major cause of central nervous system (CNS) infections (neurolisteriosis) in humans and ruminants. While the bacterium's ability to transition between extracellular and intracellular environments is well-characterized, the molecular mechanisms governing its divergent intracellular fates within specific CNS macrophage populations remain unclear.

• The Conflict: During CNS infection, Lm encounters two distinct macrophage lineages: brain-resident microglia and infiltrating monocyte-derived macrophages (MDMs).
• The Phenomenon: Previous studies suggest Lm replicates actively in the cytosol of microglia but remains confined to phagolysosomes in MDMs, potentially entering a dormant, "Viable But Non-Culturable" (VBNC) state.
• The Gap: It is unknown how the ontogeny (lineage origin) of these macrophages shapes the bacterial transcriptome and proteome to drive these opposing outcomes, and which specific bacterial stress adaptation genes are critical for persistence in these distinct niches.

2. Methodology

The study employed an integrated multi-omics approach using bovine primary cells to model neurolisteriosis, as bovine rhombencephalitis phenocopies human CNS listeriosis.

• Cell Models: Primary bovine microglia (tissue-resident) and MDMs (differentiated from CD14+ monocytes). A human microglial cell line (HMC-3) was used for validation.
• Infection Model: Cells were infected with the wild-type Lm strain JF5203 (Lineage I) at specific time points (45 minutes and 6 hours post-infection) using a gentamicin protection assay to eliminate extracellular bacteria.
• Dual RNA-Sequencing (Dual RNA-seq): Simultaneous transcriptional profiling of both host and pathogen.
  • Time Points: 45 min (early entry) and 6 hours (established infection).
  • Controls: Mock-infected cells and extracellular Lm grown in FBS.
  • Analysis: Differential Gene Expression (DGE) analysis using DESeq2, KEGG pathway enrichment, and Gene Set Enrichment Analysis (GSEA).
• Proteomics: Quantitative untargeted LC-MS/MS performed on infected cells at 6 hours to correlate protein abundance with transcriptomic data.
• Functional Validation:
  • Generation of deletion mutants for key stress-response genes (recA, rtcB, mecA, vanZ, mdrT, yneA) using homologous recombination.
  • Phenotypic Assays: Colony Forming Unit (CFU) counts, LIVE/DEAD BacLight viability staining (SYTO9/PI), and immunofluorescence (IF) microscopy to assess actin polymerization (cytosolic escape) and LAMP1 association (vacuolar confinement).

3. Key Contributions

1. First Integrated Multi-Omics View: Provides the first genome-wide snapshot of host-pathogen interactions in CNS macrophages, combining dual RNA-seq with proteomics to reveal lineage-specific infection dynamics.
2. Lineage as a Determinant: Demonstrates that macrophage ontogeny (microglia vs. MDM) is a primary driver of infection outcome, overriding canonical virulence gene expression differences.
3. Metabolic Rewiring: Maps the specific metabolic shifts Lm undergoes: active replication in microglia vs. metabolic shutdown in MDMs.
4. Identification of Persistence Factors: Identifies RecA (DNA recombinase) and RtcB (RNA ligase) as critical mediators of intracellular persistence and the transition to VBNC states.

4. Key Results

A. Divergent Bacterial Transcriptional Programs

• In Microglia (Permissive Niche):
  • Phenotype: Lm escapes the vacuole, polymerizes actin, and replicates in the cytosol.
  • Transcriptome: Upregulation of metabolic pathways supporting growth, including nucleotide salvage (deoC), oxidative phosphorylation (OXPHOS), fatty acid biosynthesis, and glycolysis.
  • Stress Response: Minimal stress response; canonical virulence genes (prfA, hly, actA) are expressed but not differentially regulated compared to MDMs.
• In MDMs (Restrictive Niche):
  • Phenotype: Lm remains confined to the phagolysosome, failing to escape into the cytosol.
  • Transcriptome: Global metabolic suppression. Downregulation of OXPHOS, PTS (carbohydrate uptake), fatty acid biosynthesis, and pyrimidine de novo synthesis.
  • Stress Response: Robust upregulation of SOS response (recA, lexA, yneA, dinB), heat shock proteins (clpB, groL), and non-coding RNAs (T-box riboswitches, rli53).
  • VBNC State: The transcriptional profile in MDMs correlates with a shift toward a dormant, stress-tolerant state.

B. Host Response Divergence

• MDMs: Adopt a pro-inflammatory M1-like phenotype. They upregulate glycolysis, apoptosis, mitophagy, and inflammatory signaling (NF-κB, IL-17, TNF). They express high levels of lysozyme and antimicrobial effectors, creating a hostile vacuolar environment.
• Microglia: Maintain a homeostatic/reparative phenotype. While they induce Type I Interferon (IFN-I) signaling (due to cytosolic detection of Lm), they upregulate lipid metabolism and phagolysosomal processing markers (e.g., RAB33B, CTSK) rather than broad inflammatory effectors. They provide a nutrient-rich cytosol conducive to bacterial growth.

C. Functional Role of Stress Genes (recA and rtcB)

• In MDMs: Deletion of recA significantly reduced intracellular CFUs and increased the fraction of VBNC bacteria. Deletion of rtcB had a less pronounced effect, suggesting functional redundancy in the highly stressed MDM environment.
• In Microglia: Deletion of either recA or rtcB caused a drastic reduction in recoverable CFUs (up to 2-log reduction) and impaired vacuolar escape.
  • Mechanism: Mutants in microglia showed high SYTO9+ (viable) counts but low CFUs, indicating they entered a VBNC-like state prematurely. They were trapped in LAMP1+ vacuoles, unable to escape to the cytosol.
• Conclusion: recA and rtcB are essential for bacterial fitness during the critical transition from the phagosome to the cytosol and for maintaining persistence under stress.

D. Proteomic Correlation

• Proteomic data confirmed the transcriptomic findings: MDMs showed enrichment in glycolytic enzymes and inflammatory markers, while microglia showed enrichment in interferon-stimulated genes and lipid metabolism proteins.
• Lysozyme levels were significantly higher in MDMs, but in vitro lysozyme treatment alone was insufficient to induce the VBNC state, suggesting the VBNC phenotype is driven by the complex intracellular stress environment rather than a single factor.

5. Significance

• Paradigm Shift: The study challenges the view that bacterial virulence is solely determined by pathogen-encoded factors. Instead, it highlights that macrophage ontogeny dictates the intracellular niche, which in turn forces the pathogen to adopt specific survival strategies (replication vs. dormancy).
• VBNC Mechanism: It elucidates the molecular basis of the VBNC state in a CNS context, identifying recA and rtcB as key players in stress tolerance and persistence.
• Therapeutic Implications:
  • The persistence of Lm in MDMs via a VBNC state may explain the difficulty in eradicating CNS listeriosis with antibiotics (which often target replicating bacteria).
  • Targeting stress adaptation pathways (e.g., SOS response, RNA repair) or modulating macrophage lineage polarization could offer new therapeutic avenues to prevent bacterial persistence in the CNS.
• One Health Relevance: By using bovine models that closely mimic human disease, the findings bridge veterinary and human medicine, offering insights applicable to foodborne pathogens affecting both populations.