Locus of Adhesion and Autoaggregation (LAA) pathogenicity island genes hes and sisA are involved in virulence and biofilm formation in LEE-negative Shiga toxin-producing Escherichia coli (STEC)

: Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen capable of causing severe diseases such as hemolytic uremic syndrome (HUS), particularly in children. While the locus of enterocyte effacement (LEE) has long been associated with STEC virulence, an increasing number of LEE-negative strains are being linked to human infections. Some strains harbor the locus of adhesion and autoaggregation (LAA), a pathogenicity island comprising genes implicated in adhesion and biofilm formation. In this study, we investigated the role of two LAA-associated genes, hes and sisA, in the virulence of the LEE-negative STEC strain UC4224 isolated from raw milk cheese. Using lambda Red recombineering, we generated mutant strains with deletions in stx1, stx2, and/or the hes-sisA region. Galleria mellonella larvae were employed as an in vivo infection model to evaluate pathogenicity. Mutants were also assessed for their ability to produce biofilm and adhere to epithelial cell lines. Our results showed that deletion of hes-sisA significantly decreased larval mortality, biofilm formation, and adhesion to Caco-2 intestinal cells. These findings suggest that the hes and sisA genes may have a role in STEC colonization and persistence, representing potential determinants of human infection. This work highlights the importance of LAA-associated hes and sisA in the pathogenicity of LEE-negative STEC strains and suggests that alternative virulence factors beyond Shiga toxins are critical to disease progression. Further studies are warranted to explore the functional roles of these and other genes within the LAA region across different STEC backgrounds and to improve their contribution to human infection.IMPORTANCEShiga toxin-producing Escherichia coli (STEC) is the third most common cause of gastrointestinal illness in Europe. Cattle act as asymptomatic reservoirs, transmitting infection to humans mainly through contaminated raw milk and meat. Several outbreaks linked to raw milk products have been reported in recent years, prompting EU Rapid Alert System notifications, including in 2024. These infections represent a major public health and economic concern. In 2019, the European Food Safety Authority recommended enhanced surveillance and a broader assessment of STEC pathogenicity, emphasizing virulence factors beyond Shiga toxins and intimin. This study investigates a specific genomic region within a pathogenicity island to elucidate its role in virulence and biofilm formation. The findings highlight additional molecular markers that may contribute to STEC pathogenic potential. Incorporating these markers into molecular screening and surveillance can improve the identification of high-risk STEC clones, supporting more accurate risk assessment and targeted public health interventions.