The lipoprotein NlpD in Cronobacter sakazakii responds to acid stress and regulates macrophage resistance and virulence by maintaining membrane integrity: Running Title: Identification and characterization of a novel factor involved in acid tolerance and virulence in Cronobacter sakazakii

Cronobacter sakazakii, 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 C. sakazakii 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 nlpD encodes an acid-resistance factor in C. sakazakii. Compared to the wild-type strain, the nlpD mutant exhibited attenuated virulence in a rat model. Using macrophage THP-1 cells and a pH probe, we verified that nlpD enables bacteria to resist macrophages by resisting acidification. Finally, we confirmed that nlpD maintains C. sakazakii membrane integrity in acid using propidium iodide permeabilization assays via flow cytometry. Our results confirm that nlpD is a novel virulence factor that permits C. sakazakii 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.