Biochemical and Biophysical Research Communications
Roles of CytR, an anti-activator of cyclic-AMP receptor protein (CRP) on flagellar expression and virulence in uropathogenic Escherichia coli
Introduction
Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). When UPEC enters a urinary tract, the bacteria adhere to and invade the host bladder epithelial cells, where they aggregate and form biofilm-like microbial colonies that protect the UPEC from antimicrobial agents and the host immune system [1]. These processes allow UPEC to not only establish infection but also ensure that the infection becomes refractory to treatment.
Fimbriae and flagella are major proteins contributing to the virulence of UPEC. Fimbriae are required to adhere to and internalize into bladder epithelial cells, whereas flagellum-mediated motility contributes to bacterial fitness and migration to infection sites as the bacteria colonize the bladder [[2], [3], [4]].
We have been conducting studies on the control mechanisms involved in the expression of proteins responsible for UPEC virulence, including fimbriae and flagella. Previously, we conducted a genetic screening using a transposon library to identify genes that contribute to bacterial adhesion and aggregation in a 96-well polystyrene plate. We found a strain that had a transposon insert in the cytR gene. This strain exhibited a higher level of adhesion and aggregation on a 96-well plate than the wild-type parent.
The cytR gene product was originally characterized as a repressor protein. It represses the expression of subsets of genes encoding proteins involved in the uptake and catabolism of ribonucleosides and deoxyribonucleosides such as deoCABD, nupG, nupC, cytX, tsx and udp, whose expression is activated by CRP [5]. The CytR protein binds adjacent DNA site to CRP binding site on the upstream region of target genes, subsequently attenuating the action of CRP by repositioning CRP binding site on the target DNA. Therefore, CytR is regarded as an anti-activator of CRP [6].
In this study, to characterize the role of CytR on the regulation of genes associated with UPEC virulence, we constructed an in-frame cytR gene deletion mutant, and found that the mutant, which had higher motility and flagellar expression, also exhibited higher levels of internalization and aggregation within bladder epithelial cells compared with the parental strain in the presence of crp. The purified CytR, together with CRP bound to upstream region of flhD which encodes the master regulator for flagellar expression. CytR functions as an anti-activator of CRP for UPEC virulence-associated flagellar expression.
Section snippets
Bacterial strains, host cells and culture conditions
The bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were grown in Luria-Bertani (LB) or RPMI1640 (Sigma-Aldrich, St. Louis, MO, United States). Optical density at 600 nm (OD600) was measured as an indicator of cell growth. Antibiotics were added to the growth media for marker selection and maintaining plasmids, concentrations of ampicillin (150 μg/mL), chloramphenicol (45 μg/mL) and kanamycin (50 μg/mL). HTB-9 bladder epithelial cells were cultured, as
Deletion of the cytR gene promotes bacterial aggregation on polystyrene and glass surfaces and within the bladder epithelial cells
The strain with a transposon insert in its cytR gene exhibited a higher degree of attachment to the 96-well polystyrene plates than the wild-type parent. To confirm this effect, we constructed an in-frame deletion mutant of the cytR gene. Similarly, we found that this mutant exhibited a stronger adhesion compared with its parent strain (Fig. 1A). The cell attachment strength of the mutant reduced to that of the parent level when pTrc99AcytR, a heterologous cytR expression plasmid, was
Discussion
CytR was originally characterized as a transcriptional repressor of genes which encode proteins involved in the uptake and catabolism of nucleosides [18]. In some of these genes, repression by CytR occurs only in the presence of CRP, which also activates the transcription of those genes. Therefore, CytR acts as an anti-activator of CRP [6]. In this study, we showed that the regulation of the flhD gene occurs likewise. Nucleosides are used for nucleotide syntheses and as carbon and nitrogen
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