Microfluidics to study Phenotypic

Heterogeneity
in Mycobacteria

Giulia Manina
Chargé de Recherche – Junior Group Leader
Microbial Individuality and Infec7on
Ins1tut Pasteur, Paris
 Microbial populations are highly heterogeneous
STRES
S
Cell Number

Coefficient of Variation = SD / Mean

Cell parameter

Sources of phenotypic heterogeneity / varia;on:

SLOW ☞ Gene;c
FAST ☞ Non-gene;c
➪ Age and history ➪ Unequal par@@on
➪ Growth rate ➪ Gene expression
➪ Cell cycle ➪ Molecular noise
➪ Asymmetry ➪ Cell-cell interac@on
2 MANINA, Giulia - Microfluidics to study Phenotypic Heterogeneity in Mycobacteria
 How and why study phenotypic varia3on?
! (nm)

Fluorescent proteins
300 400 500 600 700

1 cm

Fluorescent reporters — Real-3me microscopy — Microfluidics

Advantages:
✓ Life-compatible
✓ Single-cell events (1-103)
✓ Spatiotemporal resolution
✓ Tight environmental control
✓ Varied technologies
Challenges:
✗Growth rate, snap-division, clumps
✗Costs and ‘clean room’ facilities
✗Automated image analysis
✗Integration and multiplexing
✗Downstream assays

3 MANINA, Giulia - Microfluidics to study Phenotypic Heterogeneity in Mycobacteria

Microfluidics to trap, grow and track bacteria in 2D

Fluids at micro and macro

scale behave differently Laminar
1 cm

Polydimethylsiloxane (PDMS)
Turbulent
✪ Biocompatible
✪ Transparent IN OUT

✪ Gas permeable Air

✪ Elastic
1 cm SU8 PDMS
IN OUT

Design Mask Wafer Mold Chip Assembly

4 MANINA, Giulia - Microfluidics to study Phenotypic Heterogeneity in Mycobacteria
Asymmetric division and partitioning
contribute to mycobacterial heterogeneity
Bipolar ? 0 ≥1 B [PMID : 24036848;
?
25691587; 28517109;
Old New Old 0 ≥1 24998739; 25620549]
0 ≥1
0 ≥1 C
Nucleoid
DnaN-replisome 0 ≥1
ParA-walker 0 ≥1
ParB-segrosome 78%
FtsZ-septum 0 ≥1 D
Wag31-division
0 x ≥1
Oxidized proteins
C
1 0 ≥2 B

1 0 ≥2 C
5 MANINA, Giulia - Microfluidics to study Phenotypic Heterogeneity in Mycobacteria
 Mycobacterial Isoniazid (INH) persistence
depends on pulsing expression of KatG
KatG Mycolic acids - InhA [PMID: 1501713; 8284673]

7H9 medium INH 7H9 medium INH

[Wakamotoet al, Science 2013 -PMID: 23288538]

!H
H
Resisters ▶ INH-persisters display
normal growth rate.
CFU/mL

!H
H
Persisters more likely to die.

Sensitive
Time of INH Exposure
6 MANINA, Giulia - Microfluidics to study Phenotypic Heterogeneity in Mycobacteria
 Stress enhances phenotypic heterogeneity
and the formation of NGMA subsets
CV 0.27 0.58 0.55 0.53 0.88
(a.u.) (a.u.)

****
[ PMID: 25543231;27837741]
1000
Fluorescence Fluor

100
rRNA::GFP

FRAP

10

1
Optimal Aged Drug Mφ Mouse Dead

5 µm
NGMA

7 MANINA, Giulia - Microfluidics to study Phenotypic Heterogeneity in Mycobacteria

 More µ-Fluidics for Tuberculosis
« Targeted cell separation »

Dielectrophoresis-based isola?on Tandem affinity microfluidic chip

of INH-treated M. smegma)s to assess CD4+/CD8+ T cell ratio

[Elitaset al, Lab Chip 2014 -PMID: 24756475] [Wenjieet al, Bio Microdev2015 -PMID: 26559198]

8 MANINA, Giulia - Microfluidics to study Phenotypic Heterogeneity in Mycobacteria

 More µ-Fluidics for Tuberculosis
« Automated molecular detection »

µ-Fluidic electrochemical biosensor GeneXpert: Rapid detecBon of

for M. tuberculosis gDNA detecBon TB and rifampin resistance

[Zribiet al, Biomicrofluidics2016 -PMID: 26865908] [Boehmeet al, Lancet 2010 -PMID: 20825313]

9 MANINA, Giulia - Microfluidics to study Phenotypic Heterogeneity in Mycobacteria

 Conclusions & Take-home

Time-lapse Microfluidic Microscopy for Tuberculosis

! Robust live long-term imaging

✔ Dynamic tracking of single-cell lineages and behaviors
✔ Spa;otemporal resolu;on unreachable in bulk assays
✔ Study rare events under fluctua;ng condi;ons
✔ Phenotypic heterogeneity promotes popula;on fitness
✔ Target subsets instead of average for enhanced killing

10 MANINA, Giulia - Microfluidics to study Phenotypic Heterogeneity in Mycobacteria

11 MANINA, Giulia - Microfluidics to study Phenotypic Heterogeneity in Mycobacteria