Bacillus velezensis-derived muropeptide promotes growth of zebrafish via NOD2-mediated induction of IGF1 signaling

This study demonstrates that the gut bacterium *Bacillus velezensis* T23 promotes zebrafish growth by releasing muropeptides that activate the NOD2 receptor, thereby triggering the IGF1-AKT/mTOR signaling pathway and enhancing intestinal cell renewal to drive systemic growth.

The Gist

The Big Picture: A Tiny Bacteria with a Big Job

Imagine your body is a bustling city. Inside this city, there is a massive construction crew working 24/7 to build and repair buildings (your muscles and organs). Usually, we think this crew only works when you eat a big meal. But this study discovered a secret "foreman" living in your gut that can tell the construction crew to work harder, even if you're just eating a normal diet.

That foreman is a tiny bacterium called Bacillus velezensis.

The Story of the Zebrafish Experiment

Scientists used zebrafish as their test subjects because they are like "miniature humans" in a fish tank. They are easy to watch grow, and their genes are very similar to ours.

1. The Growth Spurt
The researchers fed some zebrafish a diet containing this special Bacillus bacteria. The result? The fish grew bigger, heavier, and had more muscle than the fish that didn't get the bacteria. It was like giving the fish a secret growth hormone boost without changing their food.

2. The "Construction Signal" (IGF1)
How did the fish grow? The bacteria triggered a signal in the fish's body called IGF1.

• The Analogy: Think of IGF1 as a megaphone carried by the fish's liver. When the megaphone is loud, it tells the muscles, "Hey! Start building more protein! Get bigger!"
• The study found that the Bacillus bacteria turned up the volume on this megaphone, causing the fish to pack on muscle mass.

The Detective Work: How Did the Bacteria Do It?

The scientists wanted to know exactly how the bacteria sent this signal. They played a game of "elimination" to find the culprit.

• Clue #1: It wasn't the "Secret Weapons."
  Many bacteria shoot out chemical weapons (like lipopeptides) to fight off enemies. The scientists removed these weapons from the bacteria. Result: The fish still grew. So, the weapons weren't the key.

• Clue #2: It was the "Armor."
  Bacteria have a hard outer shell called a cell wall, made of a material called peptidoglycan. When the scientists took just this shell (and even smaller pieces of it called MDP) and fed it to the fish, the fish still grew!

  • The Analogy: It's like the bacteria didn't need to send a letter; they just needed to leave a piece of their armor behind. The fish's body recognized this armor piece and started the growth process.

The Receiver: The "NOD2" Antenna

The fish needed a way to "hear" this armor piece. The scientists found a specific receptor in the fish's gut called NOD2.

• The Analogy: Think of NOD2 as a security camera or an antenna on the fish's intestinal wall.
• When the armor piece (MDP) bumped into the antenna (NOD2), the antenna sent a signal down the line to the liver.
• The Proof: The scientists created fish that were missing this antenna (NOD2 knockouts). When they fed these "deaf" fish the bacteria, nothing happened. The fish didn't grow. This proved that the antenna is absolutely necessary for the growth signal to work.

The Gut-Liver Connection: A Relay Race

Here is the most fascinating part: The bacteria live in the gut, but the growth signal happens in the liver and muscles. How do they talk?

1. The Gut gets excited: When the antenna (NOD2) in the gut detects the bacteria's armor, it doesn't just shout "Grow!" immediately. Instead, it tells the gut cells to renew and repair themselves.
   • Analogy: The gut is like a highway. The signal tells the road crew to fix potholes and widen the lanes.
2. Better Absorption: With a healthier, wider highway, the fish absorbs nutrients much better.
3. The Liver Responds: Because the gut is working so efficiently, the liver gets the message: "We have plenty of fuel! Time to build!" The liver then turns on the IGF1 megaphone, which tells the muscles to grow.

Why Does This Matter?

This study is a big deal for a few reasons:

• Probiotics: It explains why certain probiotics (good bacteria) help animals and humans grow and stay healthy. It's not magic; it's a specific chemical handshake between bacteria and our immune system.
• No Antibiotics Needed: It suggests we might be able to help livestock (like chickens or pigs) grow bigger and healthier just by feeding them the right bacteria, without using antibiotics or hormones.
• Human Health: Since humans have the same "antenna" (NOD2) and the same "megaphone" (IGF1), this discovery could help us understand how to treat growth problems or malnutrition in children.

The Summary in One Sentence

A tiny bacterium called Bacillus velezensis wears a special "armor" that bumps into a sensor in our gut, which tells our body to build better roads for nutrients, leading to a signal that makes our muscles grow stronger and bigger.

Technical Summary

1. Problem Statement

While the role of the gut microbiome in regulating vertebrate metabolism and immune function is well-established, the specific mechanisms by which gut bacteria directly influence host growth remain less understood. Previous studies have shown that certain probiotics (e.g., Lactobacillus plantarum) can promote growth in malnourished models via the Insulin-like Growth Factor 1 (IGF1) pathway, mediated by bacterial cell wall components interacting with the host receptor NOD2. However, it is unclear if this mechanism applies to normally nourished animals and whether it extends to other prevalent probiotic genera, specifically Bacillus. Bacillus velezensis is a widely used probiotic in aquaculture and agriculture known for its growth-promoting effects, but the molecular mechanism driving these effects has not been elucidated.

2. Methodology

The study utilized zebrafish (Danio rerio) as a vertebrate model due to their genetic homology with mammals, rapid development, and amenability to gene editing. The experimental design included:

• Bacterial Strains: Wild-type B. velezensis T23, a mutant strain lacking secondary metabolite production (B. velezensis T23-Δsfp), and other commensal strains (Plesiomonas shigelloides, Aeromonas veronii) for comparison.
• Animal Models:
  • Standard zebrafish fed diets supplemented with live bacteria, cell walls, or purified components.
  • Germ-free (GF) zebrafish: Used to test direct bacterial-host interactions via mono-colonization and microbiota transplantation.
  • nod2 knockout (nod2-/-) zebrafish: Generated via CRISPR/Cas9 to verify the necessity of the NOD2 receptor.
  • Morpholino knockdown: Used to transiently suppress NOD2 expression in larvae.
• Interventions:
  • Feeding live bacteria, whole cell walls (WCW), purified peptidoglycan (PGN), and synthetic Muramyl Dipeptide (MDP).
  • Inhibition of secondary metabolites (lipopeptides/polyketides) using the sfp mutant.
• Analytical Techniques:
  • Growth Metrics: Weight gain, feed conversion ratio (FCR), muscle protein/moisture content, and histology (H&E staining).
  • Molecular Biology: qRT-PCR for gene expression (IGF1, AKT, mTOR), Western Blotting for protein phosphorylation (p-AKT, p-mTOR), and ELISA for IGF1 quantification.
  • Omics: 16S rRNA sequencing for microbiota composition and RNA-seq (Transcriptomics) of intestinal tissue for pathway analysis (KEGG, GSEA, WGCNA).
  • In Vitro: Zebrafish liver cell (ZFL) lines treated with MDP to test direct hepatic effects.

3. Key Contributions

• Mechanistic Elucidation: Identified that the growth-promoting effect of B. velezensis is mediated specifically by its cell wall peptidoglycan, and more precisely, the Muramyl Dipeptide (MDP) fragment.
• Receptor Specificity: Demonstrated that this effect is strictly dependent on the host NOD2 receptor, establishing a MDP-NOD2-IGF1 signaling axis.
• Independence from Secondary Metabolites: Proved that the growth effect is not driven by the well-known lipopeptides or polyketides produced by Bacillus, shifting the focus to structural cell wall components.
• Gut-Liver Axis: Revealed that the mechanism operates via a gut-liver axis where intestinal NOD2 stimulation triggers systemic IGF1 production, rather than direct action on hepatocytes.
• Applicability to Well-Nourished Hosts: Extended the known NOD2-IGF1 growth mechanism from malnourished models to normally fed vertebrates.

4. Key Results

• Growth Promotion: Supplementation with B. velezensis T23 significantly increased body weight, weight gain rate, and muscle protein content in zebrafish, while reducing feed conversion ratio (FCR).
• Activation of IGF1/AKT/mTOR Pathway: T23 treatment increased IGF1 levels in the liver, serum, and muscle. It upregulated igf1 and receptor genes (igf1ra/b) and enhanced the phosphorylation of AKT and mTOR, key regulators of protein synthesis and cell growth.
• Role of Cell Wall Components:
  • The growth-promoting effect persisted in the sfp mutant (lacking lipopeptides/polyketides), ruling out secondary metabolites as the primary driver.
  • Feeding whole cell walls or purified peptidoglycan recapitulated the growth and IGF1 activation effects of live bacteria.
  • MDP supplementation alone was sufficient to induce growth and IGF1 signaling.
• NOD2 Dependency:
  • In nod2-/- zebrafish and morpholino-knockdown larvae, T23 and MDP failed to increase IGF1 levels, activate AKT/mTOR, or promote growth.
  • Colonization levels of T23 and MDP content in the gut were unchanged in nod2-/- fish, confirming the defect was in signal transduction, not bacterial persistence.
• Site of Action (Gut-Liver Axis):
  • MDP did not directly stimulate IGF1 production in zebrafish liver cells (ZFL) in vitro, indicating the liver is not the primary site of NOD2 sensing.
  • Transcriptomic analysis of the intestine revealed that T23/MDP upregulated genes related to the cell cycle, DNA replication, and HIF1α signaling, while downregulating differentiation inhibitors (e.g., notch3).
  • Histology confirmed increased villus height and goblet cell numbers, suggesting enhanced intestinal renewal and nutrient absorption capacity.
• Microbiota Modulation: While T23 increased the relative abundance of Firmicutes and decreased Proteobacteria, the growth effect was observed even in mono-colonized germ-free fish, indicating the effect is direct and not dependent on complex microbiota restructuring.

5. Significance

• Probiotic Mechanism: This study provides a definitive molecular mechanism for how Bacillus species function as probiotics, moving beyond general "gut health" claims to a specific MDP-NOD2-IGF1 signaling pathway.
• Therapeutic Potential: The findings suggest that peptidoglycan-derived muropeptides (or NOD2 agonists) could be developed as feed additives or therapeutics to enhance growth in aquaculture and potentially treat growth disorders in humans and livestock, independent of nutritional status.
• Gut-Liver Communication: It reinforces the concept of the gut-liver axis, showing that intestinal immune sensing (via NOD2) can directly modulate systemic endocrine growth pathways (IGF1).
• Evolutionary Conservation: The study supports the hypothesis that the NOD2-mediated growth mechanism is a conserved evolutionary strategy across vertebrates, applicable to both malnourished and well-nourished states.