Uptake of bacteria is independent of one another.

<p>(<b>A</b>) Flow cytometry measurements of bacterial uptake at various MOI. HeLa cells were labeled with β₁-integrin antibody for 60 minutes, washed, co-incubated with GFP-expressing <i>E</i>. <i>coli</i> grown in 0.1% arabinose to induce high-level of invasin expression from the <i>pBACr-AraInv</i> plasmid, along with fluorescent-conjugated secondary antibody (Alexa₆₄₇) for 90 minutes prior to washing. Scatter plots show the relationship between uptake (Log GFP) and β₁-integrins (Log Alexa₆₄₇) in individual cells. Each uptake distribution for MOI>0 is bimodal with infected cells (GFP+ mode) coexisting with those devoid of bacteria (GFP- mode). The two modes are designated by a Gaussian mixture model (see (B)), each mode parameterized by a mean and a standard deviation. Colored and black points indicate GFP+ and GFP- subpopulations, respectively. (<b>B</b>) Processing of data for MOI = 50 in (A). The expectation-maximization (EM) algorithm was used to probabilistically assign cells into either GFP- sub-population (black) or GFP+ sub-population (red). Histograms summarize data from each respective channel; Principle components represented by ellipse and major axis superimposed on scatter plot (circle). The half-length of the major axis (i.e. from the center to a vertex) represents twice the standard deviation in that direction. (<b>C</b>) Correlation between uptake and host receptor levels. Using flow cytometry data in (A), each line approximates the dependence of the bacterial uptake in the GFP+ subpopulation on the host receptor concentration for the corresponding MOI. The length of each line is 4 times the standard deviation along the major axis. Mean indicated by circle. Inset shows linear fit on means. (<b>D</b>) Uptake probability. Using flow cytometry data in (A), GFP values were scaled by their respective MOI and then processed as in (C). These curves approximate the dependence of the uptake probability per bacterium on the host receptor concentrations. (<b>E</b>) Simulations showing the dependence of uptake on the host receptor number per cell at bacterial MOI of 250 for two values of the invasin dissociation constant (K_D = k_rB1/k_fB1). (<b>F</b>) Experimental modulation of invasin expression. HeLa cells were incubated with <i>E</i>. <i>coli</i> (GFP/Invasin) grown in the indicated concentrations of arabinose to vary invasin expression, which leads to enhanced binding of bacteria to the host receptors. Shown are the major axes and mean of GFP+ subpopulations scaled by their respective MOI.