Do ‘single-cell’ Mtb preps change macrophage results?
Gentle sonication made Mtb hyperinflammatory (TLR2-high IL-1β/TNF) yet still replicative in macrophages, while filtering blunted inflammation and attenuated intracellular survival — so prep method can rewrite host–pathogen readouts.
Source
Single cell preparations of <i>Mycobacterium tuberculosis</i> damage the mycobacterial envelope and disrupt macrophage interactions
Study at a glance
- Design
- Animal / in-vitro — In-vitro Mtb single-cell prep (sonication vs 5 μm filter vs spin) infecting mouse BMDMs; RNA-seq, cytokines, CFU, TEM; literature survey of 119 papers
- N
- N=5 · RNA-seq n=5 BMDM infections per condition (so/sp vs 5μmF, MOI 5, 72 h); plus TEM/CFU and 119-paper methods survey
- Population
- Mycobacterium tuberculosis (H37Rv and others) and mouse bone marrow-derived macrophages, including Tlr2−/−
- Outcome
- Envelope integrity, macrophage inflammatory gene expression/cytokines, and intracellular Mtb survival
Structured fields used in claim comparison tables when every cited study has a complete layer.
Inspect source passages
Do ‘single-cell’ Mtb preps change macrophage results?
These passages occur verbatim in the stored paper text. They support the points listed below; they do not verify every statement in the explanation.
RNA-seq used n=5 per condition at MOI 5, 72 hpi.
BMDMs were uninfected or infected with Mtb prepared by sonication and spin (so/sp) or filtration (5μmF) at an MOI of 5 and analyzed 72 hpi by RNA-seq (n=5 per condition).
Only 39.5% of 119 papers reported single-cell methods.
Of the 119 papers, only 39.5% reported how they generated single cell suspensions.
What they did
Compared routine disaggregation (sonication, 5 μm filtering, spinning) for envelope damage (TEM), infected mouse BMDMs (RNA-seq n=5, cytokines, CFU), tested Tlr2−/− BMDMs, and surveyed how 119 papers reported single-cell methods.
What they found
Both sonication and filtering damaged the envelope. Sonicated bacilli drove higher TLR2-dependent transcription and IL-1β/TNF-α but replicated normally; filtered Mtb were hypo-inflammatory and attenuated. PDIM’s apparent anti-inflammatory effect depended on prep method. Only 39.5% of 119 papers reported how they made single-cell suspensions.
The limits
What it doesn't show
This is mouse BMDM work in vitro, not a clinical TB trial; other labs’ strains, detergents, or human macrophages may differ, and no single ‘correct’ prep is proven for all questions.
Key terms
- Single-cell preparation
- Disaggregating clumpy Mtb (sonication, filtering, syringing, spins) before macrophage infection.
- PDIM
- Phthiocerol dimycocerosate, a cell-envelope lipid thought to dampen macrophage inflammation — here prep-dependent.
- TLR2
- Host receptor driving much of the hyperinflammatory transcriptional response to sonicated Mtb.
- BMDM
- Mouse bone marrow-derived macrophages used as the infection model.
- MOI
- Multiplicity of infection; RNA-seq used MOI 5.
Flashcards
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Quiz yourself
Sonicated Mtb were:
Common questions
What did sonication do?
Damaged the envelope and made bacilli hyperinflammatory (TLR2, IL-1β, TNF-α) without blocking replication.
What did filtering do?
Little inflammatory response and attenuated intracellular survival.
How often do papers report the method?
Only 39.5% of 119 surveyed papers.
Does PDIM always dampen inflammation?
Its impact depended on how Mtb was prepared.
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