<i>Cronobacter sakazakii</i> lysozyme inhibitor LprI mediated by HmsP and c-di-GMP is essential for biofilm formation and virulence

<i>Cronobacter sakazakii</i> poses a significant threat, particularly to neonates and infants. Despite its strong pathogenicity, understanding of <i>C. sakazakii</i> biofilms and their role in infections remains limited. This study investigates the roles of HmsP and c-di-GMP in biofilm formation and identifies key genetic and proteomic elements involved. Gene knockout experiments reveal that HmsP and c-di-GMP are linked to biofilm formation in <i>C. sakazakii</i>. Comparative proteomic profiling identifies the lysozyme inhibitor protein LprI, which is downregulated in <i>hmsP</i> knockouts and upregulated in c-di-GMP knockouts, as a potential biofilm formation factor. Further investigation of the <i>lprI</i> knockout strain shows significantly reduced biofilm formation and decreased virulence in a rat infection model. Additionally, LprI is demonstrated to bind extracellular DNA, suggesting a role in anchoring <i>C. sakazakii</i> within the biofilm matrix. These findings enhance our understanding of the molecular mechanisms underlying biofilm formation and virulence in <i>C. sakazakii</i>, offering potential targets for therapeutic intervention and food production settings.IMPORTANCE<i>Cronobacter sakazakii</i> is a bacterium that poses a severe threat to neonates and infants. This research elucidates the role of the lysozyme inhibitor LprI, modulated by HmsP and c-di-GMP, and uncovers a key factor in biofilm formation and virulence. The findings offer crucial insights into the molecular interactions that enable <i>C. sakazakii</i> to form resilient biofilms and persist in hostile environments, such as those found in food production facilities. These insights not only enhance our understanding of <i>C. sakazakii</i> pathogenesis but also identify potential targets for novel therapeutic interventions to prevent or mitigate infections. This work is particularly relevant to public health and the food industry, where controlling <i>C. sakazakii</i> contamination in powdered infant formula is vital for safeguarding vulnerable populations.