Abstract
Bacteria mostly live as multicellular communities, although they are unicellular organisms, yet the mechanisms that tie individual bacteria together are often poorly understood. The adhesin involved in diffuse adherence (AIDA-I) is an adhesin of diarrheagenic Escherichia coli strains. AIDA-I also mediates bacterial auto-aggregation and biofilm formation and thus could be important for the organization of communities of pathogens. Using purified protein and whole bacteria, we provide direct evidence that AIDA-I promotes auto-aggregation by interacting with itself. Using various biophysical and biochemical techniques, we observed a conformational change in the protein during AIDA-AIDA interactions, strengthening the notion that this is a highly specific interaction. The self-association of AIDA-I is of high affinity but can be modulated by sodium chloride. We observe that a bile salt, sodium deoxycholate, also prevents AIDA-I oligomerization and bacterial auto-aggregation. Thus, we propose that AIDA-I, and most likely other similar autotransporters such as antigen 43 (Ag43) and TibA, organize bacterial communities of pathogens through a self-recognition mechanism that is sensitive to the environment. This could permit bacteria to switch between multicellular and unicellular lifestyles to complete their infection.
Highlights
Istic of many multicellular behaviors such as swarming [8]
The extracellular domain of AIDA-I is mainly composed of imperfect repeats of a 19-amino acid sequence that is characteristic of self-associating autotransporters (SAAT) [6]
Unexpectedly, we demonstrated that AIDA-AIDA interactions and bacterial auto-aggregation can be modulated through induction of reversible conformational change in AIDA-I
Summary
Bacterial Strains and Growth Conditions—The E. coli K-12 strain C600 (F- thr-1 leuB6 thi-1 lacY1 supE44 rfbD1 fhuA21) obtained from New England Biolabs was used in this study and does not contain the locus coding for AIDA-I. To study the effect of salts, the protein samples was diluted in TBS with different concentrations of NaCl or sodium deoxycholate prior to measurement. 1 mM Tris-HCl, pH 8, 150 mM NaCl. The samples were diluted These aggregates settle at the bottom of culture tubes, which in 2ϫ SDS-PAGE loading buffer containing -mercaptoetha- can be followed with a spectrophotometer by measuring nol and denatured by heating at 100 °C for 10 min. We observed that auto-aggregation is totally inhibited by the addition of a lysate or a membrane extract of bacteria expressing AIDA-I but, by contrast, the addition of an identical amount of cell lysate or a membrane extract of bacteria harboring an empty vector had no significant effect (Fig. 2A) This experiment suggested that AIDA-I does not mediate autoaggregation by interacting with another cell surface molecule
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