The role of the galU gene of uropathogenic Escherichia coli in modulating macrophage TNF-α response

Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTI). These bacteria are equipped with an arsenal of virulence factors, such as siderophores and adhesins enabling UPECs to sufficiently colonize the urinary tract of humans and animals. Such virulence factors manipulate and impair the recognition of UPECs by the host's innate immune system. Among those, factors like the TIR domain containing proteins in E. coli (TcpC) have been described to interfere with the Toll-like receptor 4 signaling cascade. Nevertheless, some UPECs such as strain UTI89 lack TcpC, but also manipulate the innate immune response. By a random mutant-library approach we identified the galU gene of strain UTI89 to be responsible for a reduced immune response of macrophages. Consequently, we created a site directed knockout mutant of the galU gene in strain UTI89. This mutant caused a significantly increased cytokine response when co-incubated with J774A.1 macrophages. This phenotype could be recomplemented in trans by insertion of a galU-expressing plasmid. No differences in the viability of macrophages co-incubated with either the wild-type (WT) or the ΔgalU mutant strain could be observed. Nor could any growth impairment be detected in the ΔgalU mutant compared to WT strain. Hence, the increased cytokine response was not due to differences in the bacterial cytotoxicity or bacterial counts in the assay. Our results also demonstrated a reduction of intracellular counts of UTI89ΔgalU in the infection model. We were able to show a loss of the O-polysaccharide side chain of the ΔgalU mutant LPS. A comparable LPS structure could be generated by the deletion of the waaL gene in the UTI89. This also caused an impaired immune modulation. In contrast, purified LPS was not sufficient to impair cytokine release of macrophages. Moreover, no differences could be detected by applying bacteria inactivated with heat or formalin treatment. From this, we assume that the aberration of the LPS structure caused by the knockout of the galU gene is an important but not the exclusive cause for the loss of UPEC's immune modulating properties.