Figure 3. Attenuation of ESX-1-deficient strain is surfactant independent.

Snapshots from live-cell imaging in deficient surfactant (DS) conditions at 4 days post-infection for (A) wild-type Mtb, (B) an attenuated ESX-1-deficient strain (esx-1), and (C) at 6 days post-infection for the avirulent Δicl1Δicl2 strain infected at a higher inoculum. The calibration bar (inset in A-C) indicates the corresponding absolute intensities in the Mtb channel for a direct comparison across strains. Growth of the ESX-1-deficient strain is attenuated relative to wild-type Mtb (~10-fold decrease in intensity) and the Δicl1Δicl2 strain is unable to grow. Scatter plots indicate Mtb growth rate in individual ATs (D, H) and macrophages (F, J) and bar graphs indicate the non-growing fraction (NGF) of bacteria for infected ATs (E, I) and macrophages (G, K) in normal surfactant (NS) and DS conditions for wild-type and ESX-1-deficient strains. ‘nd’, not detected. Each dataset was fitted with a non-parametric kernel density estimation characterized by the mean (black) and median (red) values and whiskers represent the 1-99 percentile interval. For wild-type Mtb, DS conditions significantly increase growth rates and lower the NGF in both ATs (D, E) (n=122 for NS, n=219 for DS, p=3.1E-39) and macrophages (F, G) (n=185 for NS, n=122 for DS, p=1.0E-40). For the ESX-1-deficient strain, differences between NS and DS conditions are not significant in ATs (E) (n=61 for NS, n=25 for DS, p=0.10); for macrophage infection, differences are statistically significant (J) (n=93 for NS, n=55 for DS, p=5.1E-3), but a significant number of bacteria remain non-growing in DS conditions (K).

Figure 3—figure supplement 1. Additional characterization of a DS LoC infected with the Δicl1Δicl2 Mtb strain at 6 dpi.

(A, B, C) Snapshots from three representative fields of view taken with a widefield microscope at 6 days post-infection from a DS LoC infected with a high dose of the Δicl1Δicl2 strain of Mtb. An overwhelming majority of macrophages (false-colored magenta) remain infected with multiple single Δicl1Δicl2 Mtb bacteria (false-colored green, indicated with yellow arrows) despite the absence of net bacterial growth over time. Confocal microscopy provides higher resolution images. (D) A 3D projected view from the top and side (E) of a field of view on the LoC confirms the presence of single Δicl1Δicl2 Mtb bacteria in multiple macrophages. (F) A zoomed-in 3D projection view from the top and side (G) of a macrophage infected by multiple (left) and a single (right) Δicl1Δicl2 bacteria. The underlying AT layer is not shown.

Figure 3—figure supplement 2. Additional characterization of the Mtb growth rates in ATs vs. macrophages under DS and NS conditions.

Scatter plots of growth rates of intracellular bacterial microcolonies and bar charts for the corresponding non-growing fraction (NGF) from Figure 3 for infected ATs and macrophages under normal surfactant (NS) conditions (A, C) and deficient surfactant (DS) conditions (B, D) for wild-type and ESX-1-deficient strains of Mtb, respectively. Under NS conditions, ATs are a more permissive niche than macrophages for both strains (A, C). Under DS conditions, macrophages are equally permissive as ATs for Mtb growth for wild-type Mtb (B) but not for ESX-1-deficient Mtb (D). The number of samples for each strain and condition is given in Table 1. p-Values were calculated using the Kruskal Wallis one-way ANOVA test.