Roles of CytR, an anti-activator of cyclic-AMP receptor protein (CRP) on flagellar expression and virulence in uropathogenic Escherichia coli

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Highlights

  • Deletion of cytR promotes bacterial internalization and aggregation.

  • The cytR mutant exhibited an elevated flagellar expression and motility.

  • The CytR bound to upstream region of flhD with the CRP activator protein.

  • CytR is an anti-activator of CRP for UPEC virulence and motility.

Abstract

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infection (UTI), a common bacterial infectious disease. This bacterium invades the urinary tract cells, where it aggregates, and subsequently forms multicellular colonies termed intracellular bacterial communities (IBCs). The motility of the bacteria plays a key role in the mechanism of virulence in the host bladder. Here, we show that CytR is a modulator of bacterial internalization and aggregation within the bladder epithelial cells sustained by CRP in UPEC. Mutational analyses and gel-shift assays indicated that CytR represses the expression of flhD, thereby encoding a master regulator for flagellar expression that is responsible for bacterial motility when CRP is present, whereas CRP is an activator of flhD expression. Thus, elevated flagellar expression was involved in promoted virulence in the cytR mutant. These combined observations suggest another regulatory layer of flagellar expression and the role of CytR in UPEC virulence.

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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