The PittII Msf-KO strain has decreased survival in macrophages.

<p>Polymyxin B protection assays showing the number of viable bacteria recovered from THP-1 macrophage monolayers 2, 24, 48 and 72 hours after inoculation. Each result represents the mean of 3 wells in 3 biological replicate experiments. <b>(A)</b> Bacterial uptake and survival of: PittII (clinical OM isolate), PittII Msf-KO, the single <i>msfA1</i> gene inserted into the Msf-KO at the <i>ompP1</i> locus: PittII Msf-COMP, and a PittII OMPP1-KO control. Dotted line indicates the limit of detection for the Msf-KO strain. * p<0.05 by one-way weighted ANOVA for independent samples and p<0.05 by Tukey HSD post-hoc test for Msf-KO compared to WT, Msf-COMP and OMPP1-KO. <b>(B)</b> Differentiated THP-1 macrophage monolayers were infected with PittII expressing GFP. At each time-point the cells were washed and fixed. Samples were stained using rabbit anti-NTHi and Alexa Fluor secondary antibodies (red). Red staining indicates extracellular bacteria that are dead. Yellow/orange indicate extracellular bacteria that are viable (expressing GFP). Intracellular bacteria appear green since macrophages were not permeabilized. After 24 hours differential staining of extracellular and intracellular bacteria shows that the majority of the bacteria are inside the macrophages. <b>(C)</b> Bacterial uptake and survival of: NTHi strain 86-028NP (clinical OM isolate) and 86–028 Msf-KO. Dotted line indicates the limit of detection for the Msf-KO strain. * p<0.05 by two-tailed <i>t</i>-test for two independent means. <b>(D)</b> Bacterial uptake and survival of: Rd KW20 (non-encapsulated variant of a type D strain that lacks any Msf gene) and Rd Msf-INS (A mutant with the PittII <i>msfA1</i> gene inserted at the <i>ompP1</i> locus). Dotted line indicates the limit of detection for the Rd KW20 strain. * p<0.05 by two-tailed <i>t</i>-test for two independent means.