ZFIN Image: Saelens et al., 2022, Fig. 5

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Figure Caption

Fig. 5

Reintroduction of ancestral EsxM into modern Mtb reprograms macrophage motility through cytoskeletal alterations

(A) RAW264.7 cell migration during infection with Mtb strains mc²6020 (Mtb₆₀₂₀ msp12:cerulean) or mc²6020 constitutively expressing esxM (Mtb₆₀₂₀ hsp60p:esxM/msp12:cerulean) from an episomal plasmid across transwell membranes in the presence and absence of chemokine. Mean ± SD shown. One-way ANOVA with Bonferroni’s correction.

(B) Quantitation of the presence of filopodia on BLaER1 cells infected with Mtb₆₀₂₀ msp12:cerulean or Mtb₆₀₂₀ hsp60p:esxM/msp12:cerulean. Two-tailed paired Student’s t test, mean ± SD shown.

(C) Measured circularity of BLaER1 cells infected with Mtb₆₀₂₀ msp12:cerulean or Mtb₆₀₂₀ hsp60p:esxM/msp12:cerulean.

(D) BLaER1 cells infected with Mtb₆₀₂₀ hsp60p∷esxM/msp12:cerulean demonstrate increased filopodia and reduced circularity compared to BLaER1 cells infected with Mtb₆₀₂₀ msp12∷cerulean. Bacteria in cyan and actin in red. Scale bar, 8 μm.

(E) Electron micrographs of BLaER1 cells infected with Mtb₆₀₂₀ hsp60p∷esxM/msp12∷cerulean demonstrate increased filopodia compared to BLaER1 cells infected with Mtb₆₀₂₀ msp12∷cerulean. Scale bar, 2 μm.

(F) BLaER1 cells infected with Mtb₆₀₂₀ msp12∷cerulean demonstrate peripheral localization of Arp2, compared to interior localization of Arp2 in cells infected with Mtb₆₀₂₀ hsp60p∷esxM/msp12∷cerulean. Insets display fluorescence intensity of Arp2 and actin along the green line, with interior localization in Mtb₆₀₂₀ hsp60p∷esxM/msp12∷cerulean infections.

(G) Quantification of Arp2 localization from (F). Plot of the percentage of the area under the curve (AUC) for fluorescence in the Arp2 channel for the interval 0% to 25% of the normalized distance across the longest cell axis normalized to AUC from the middle 50% (25%–75%) of the total cell length. Each data point represents a single cell. Statistics from Student’s t test. Scale bar, 8 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

All images are representative of at least two biologically independent experiments.

See also Figure S4.

Acknowledgments

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Reprinted from Cell, 185(24), Saelens, J.W., Sweeney, M.I., Viswanathan, G., Xet-Mull, A.M., Jurcic Smith, K.L., Sisk, D.M., Hu, D.D., Cronin, R.M., Hughes, E.J., Brewer, W.J., Coers, J., Champion, M.M., Champion, P.A., Lowe, C.B., Smith, C.M., Lee, S., Stout, J.E., Tobin, D.M., An ancestral mycobacterial effector promotes dissemination of infection, 4507-4525.e18, Copyright (2022) with permission from Elsevier. Full text @ Cell