Prokaryotes have elaborate mechanisms to deal with the regulation of the gene expression process by the action of the transcription factors (Pabo and Sauer, 1992, Browning and Busby, 2004, Flores-Bautista et al., 2020). Transcription factors (TFs) are the regulatory proteins that bind to a specific DNA sequence (operator region) and function to activate or, at times, inhibit gene transcription according to the need of the cell (Gralla, 1996, Barnard et al., 2004). Catabolite repressor activator (Cra) is a member of the LacI family, known to positively regulate the genes encoding gluconeogenic enzymes and negatively regulate genes encoding glycolytic enzymes and the energy-coupling proteins of the bacterial phosphotransferase system (Njoroge et al., 2013, Shimada et al., 2011). In addition, it is also a global regulator of the genes for carbon metabolism (Ishihama, 2010, Saier and Ramseier, 1996, Ramseier et al., 1993) and virulence in Escherichia coli; Salmonella typhi, and Shigella flexneri (Njoroge et al., 2013, Yoon et al., 2009, Gore, 2010, Allen et al., 2000, Gore and Payne, 2010).

The human pathogen Enterohemorrhagic Escherichia coli (EHEC) employs Cra transcriptional factor (initially named as fruR) for the maintenance of bacterial homeostasis and to regulate virulence gene expression (Njoroge et al., 2013, Mellies et al., 2007, Njoroge et al., 2012, Carlson-Banning and Sperandio, 2016). One crucial characteristic of EHEC virulence is its ability to form attaching and effacing (AE) lesions on epithelial cells. Most of the genes necessary for AE lesion formation are located within a pathogenicity island (PI) known as the locus of enterocyte effacement (LEE) (McDaniel et al., 1995). The first gene of the LEE operon is a transcription factor named LEE regulator (LER), which enhances the expression of other genes of the same operon. To regulate the expression of the LEE operon, the Cra transcription factor activates the expression of the LER gene under gluconeogenic conditions, which in turn enhances the expression of the LEE operon. The LEE operon genes encode the structural components of a type three secretion system (T3SS), as well as some of its effectors (McDaniel et al., 1995, Jarvis et al., 1995). Through T3SS, bacteria transfer the virulence factors in the host cytoplasm that affect the changes in the host cells, allowing the invading pathogen to colonize, multiply, and in some cases, chronically persist in the host (Jarvis et al., 1995). Previously, a study has shown that the knockout mutants of the Cra transcription factor were unable to cause infection (Njoroge et al., 2013).

The TFs involved in the regulation of virulence gene expression are considered promising targets for the development of antivirulence drugs (Hung et al., 2005, Rasko et al., 2008, Lee et al., 2019). The potential advantage of drugs inhibiting TFs over antibiotics is that antivirulence drugs do not impose a high selective pressure on a bacterial population to emerge as drug-resistant and repress the dissemination of antibiotic resistance and virulence genes (Ogawara, 2021). In this regard, many studies have aimed to identify small molecule inhibitors against TFs (Hung et al., 2005, Lee et al., 2019, Kim et al., 2009, Garrity-Ryan et al., 2010, Greenberg et al., 2018, Catlin et al., 2020), however, the Cra transcription factor has not been targeted to identify antivirulence molecules.

The structural information of Cra has been elucidated earlier (Neetu et al., 2021, Chavarría et al., 2011). The structural architecture of Cra is comprised of the DNA binding domain and the effector molecule binding domain. The fructose-1-phosphate (F1P) is the reported effector molecule of Cra (Chavarría et al., 2011, Chavarría et al., 2014). The F1P molecule binds to the effector binding domain of Cra and consequently induces conformational changes, which makes Cra incompetent to bind with the DNA (Chavarría et al., 2011, Chavarría et al., 2014). Despite these structural information, antivirulence molecules against the Cra transcription factor have not been identified.

In this study, we have determined the crystal structure of Cra in complex with HEPES molecule which paved the way for screening potential inhibitors against EcCra. A potential inhibitor, sulisobenzone was identified and its binding to EcCra was observed with EMSA, in vitro transcription assay, and ITC experiments. Finally, a crystal structure of EcCra-sulisobenzone complex was determined that revealed a mechanism for high-affinity binding to the EcCra.