Dokument

Summary:
Bacteria are commonly found in their natural environments not as single cells, but as part of communities called biofilms. For biofilm formation, bacteria typically have to attach to a surface. This attachment is also important for the establishment of infections. In order to interact with the surfaces, bacteria have a series of appendages usually known as adhesins. Biofilm formation, as well as attachment, have been extensively studied. However, the role of each adhesins in attachment to different surfaces and the signaling involved in surface sensing is not fully understood. This work aims to further understand the roles of motility and the main adhesins present in E. coli in attachment to both hydrophobic and hydrophilic surfaces under static conditions and under flow. Furthermore, the influence of chemotaxis and the second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) in the bacterial initial attachment was also studied. In this work, motility mediated by the flagella was shown to be essential for cells to reach the surface and non-motile bacteria showed an impaired surface attachment and colonization. However, though flagella have been reported to play a role on attachment beyond motility, their relevance in attachment was strongly dependent on the experimental setup and phase of attachment. Thus, they were shown to be important as secondary adhesins for attachment to mannosylated surfaces and for attachment under flow. However, flagella did not improve the attachment to abiotic surfaces and, under certain conditions, the presence of a non-rotating flagella did impair attachment instead of favoring it. Motility in E. coli is controlled by the chemotaxis machinery and the second messenger c-di-GMP. The role of chemotaxis was studied by using chemotaxis deficient strains and chemoattractant stimulation and was shown to improve attachment by reducing the tumbling and increasing smooth swimming near the surface. In the case of c-di-GMP, mutants of diguanylate cyclases and phosphodiesterases that are responsible for the c-di-GMP pool in the cell, as well as strains lacking the flagellar brake YcgR, were used. Though c-di-GMP is normally considered a biofilm promoting signal, the results presented in this work show that lower levels of this second messenger improve bacterial surface attachment and colonization by increasing bacterial swimming speed. Furthermore, type 1 fimbriae were found to mediate the increased attachment of these fast swimming bacteria. Both the effect of chemotaxis and c-di-GMP were observed on abiotic and mannosylated, biomimetic surfaces. In the last part of these work, type 1 fimbriae were also observed to be crucial for long term surface attachment and growth on the surface. The need for fimbriae at later time points of attachment is abolished in the absence of protein synthesis and cell division. Though differences in flagella expression were hypothesized to be responsible for these fimbrial requirement, flagella were shown to not be able to compensate for the lack of fimbriae. However, fimbriae were not essential for growth on the surface in the absence of motility. Non-motile flagellated cells were shown to be capable of attachment and growth at the surface. Although whole proteome analysis revealed an increased in fimbrial proteins upon attachment of motile cells, indicating a possible downstream effect of flagella surface sensing, it failed to explain the difference between attachment phenotype of wild-type and motility deficient but flagellated cells. Therefore, possible explanations of how lack of motility could bypass the need for fimbriae in long term attachment were proposed.

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