Genome-wide high-throughput transposon mutagenesis unveils key factors for acidic pH adaptation of Corynebacterium diphtheriae

<i>Corynebacterium diphtheriae,</i> a notable pathogen responsible for the life-threatening disease diphtheria, encounters harsh intracellular environments within the host, particularly within macrophages where acidic conditions prevail. To elucidate the genetic and molecular mechanisms underlying its acid stress response, we employed a Transposon Directed Insertion-site Sequencing approach. This comprehensive study identified crucial genes and pathways facilitating <i>C. diphtheriae</i>'s survival at low pH. In subsequent experiments, the Ktr potassium transport system was identified as a putative key factor for maintaining pH homeostasis and growth under acidic stress. A <i>ktrBA</i> deletion strain exhibited significantly reduced growth at pH 5, which could be restored by <i>ktrBA</i> expression in <i>trans</i>. The deletion strain showed unchanged uptake and survival in macrophages compared to the wild-type, indicating that the Ktr system is not crucial for the survival of <i>C. diphtheriae</i> in phagocytes. These findings advance our understanding of <i>C. diphtheriae</i>'s pathophysiology, further delineating the intricate survival strategies of <i>C. diphtheriae</i> in hostile environments.