08 5.35×10-5 4.77×10-3 Glycerol metabolism Genes of unknown function Gene Log 2 fold p -value FDR Comment HI0997 1.34 8.95×10-4 5.51×10-2 Hypothetical protein HI1427 1.31 4.17×10-7 5.72×10-5 Transmembrane protein Genes down-regulated at pH 8.0 compared to 6.8 Gene Log 2 fold p -value FDR Comment HI1349 -1.23 5.14×10-6 5.10×10-4 Ferritin ahpD -1.72 1.24×10-7 2.01×10-5

Stress response Conclusions H. influenzae can adapt to the physical and chemical properties that BTK inhibitor mouse exist in different anatomical niches (such as the nasopharynx, lung, blood and the middle ear mucosa). Various strains of this pathogen adapt to these niches differently, such growing rapidly and planktonically or alternatively by forming a biofilm. The different niches are known

to vary in a range of properties, the pH being one of these that subtly but significantly shifts from about neutral in the blood to pH 8.0 in the middle ear [31, 32]. The pH does not remain constant within a niche and even in the blood there can various reasons for the pH to shift. While blood pH is tightly regulated at around pH 7.4, there are other parts of the body encountered by H. influenzae as a result of systemic infection ARRY-438162 chemical structure starting in the blood that can include conditions that do reach pH 8.0. A capsular isolate taken from the blood would therefore need to be able to exist in the pH range of 6.8-8.0 but in this lifestyle it is rarely associated with a biofilm. A NTHi isolate from the middle ear (R3264) would predominantly encounter pH 8.0 and its processes of colonization would occur at this pH (although once again the pH is thought not to be constant Cediranib (AZD2171) in this niche,

but varying within a range of 7.0-9.0). In this niche as part of its colonization, the bacterial cell would form a biofilm. Indeed some studies have shown that biofilm is induced in the middle ear as a very likely consequence of the increased pH (this was presented as a function of the induction of type IV pili but does not exclude other pathways not examined in this study) [33]. The type IV pili genes are more likely to be highly regulated in the biofilm cells themselves and not the planktonic cells we analysed. Not all H. influenzae MEK inhibitor cancer isolates respond to the changes in physical and chemical properties between the niches that H. influenzae can occupy with the same capacity or in the same manner. We show that H. influenzae isolates respond differently to the subtle and yet physiologically relevant changes in pH from 6.8 to 8.0. These changes are slight in regards to the observed growth rates but the changes are underpinned by lifestyle changes, such as modes of growth or biofilm formation. A capsular isolate (Eagan), continues to grow, with variation from pH 6.8 to 8.0 and does not form a biofilm while a NTHi isolate known to colonize the middle ear, does form a biofilm at pH 8.0.