The lipoprotein NlpD in <i>Cronobacter sakazakii</i> responds to acid stress and regulates macrophage resistance and virulence by maintaining membrane integrity

<i>Cronobacter sakazakii</i>, an emerging opportunistic pathogen, is implicated in severe foodborne outbreak infections in premature and full-term infants. Generally, acid tolerance is vital for the pathogenesis of foodborne pathogens; however, its role in <i>C. sakazakii</i> virulence remains largely unknown. To screen out acid-tolerance determinants from transposon mutants, anovel counterselection method using gentamicin and acid was developed. Using the counterselection method and growth assay, we screened several acid-sensitive mutants and found that <i>nlpD</i> encodes an acid-resistance factor in <i>C. sakazakii</i>. Compared to the wild-type strain, the <i>nlpD</i> mutant exhibited attenuated virulence in a rat model. Using macrophage THP-1 cells and a pH probe, we verified that <i>nlpD</i> enables bacteria to resist macrophages by resisting acidification. Finally, we confirmed that <i>nlpD</i> maintains <i>C. sakazakii</i> membrane integrity in acid using propidium iodide permeabilization assays via flow cytometry. Our results confirm that <i>nlpD</i> is a novel virulence factor that permits <i>C. sakazakii</i> to survive under acid stress conditions. Considering that NlpD is a conserved lipoprotein located in the bacterial outer membrane, NlpD could be used as a target for drug development.