Flavin affinity for the reductase HpaC differentially sensitizes Neisseria gonorrhoeae during Type IV pilus-dependent killing

The Neisseria gonorrhoeae Type IV pilus is a dynamic fiber involved in host cell attachment, DNA transformation, twitching motility, and evading the innate immune system. We previously reported that pilus expression affects iron homeostasis and sensitivity to killing by oxidative (iron-dependent antibiotic streptonigrin and hydrogen peroxide and non-oxidative (antimicrobial peptide LL-37) agents. Here, we use in vitro evolution to identify genes involved in N. gonorrhoeae susceptibility to streptonigrin. We identified a mutation in the NGO0059 locus that encodes HpaC that results in a glycine to cysteine change in position 93. Although HpaC homologs are known as part of a two-component FAD-dependent monooxygenase system consisting of an hpaC reductase and an hpaB monooxygenase, Neisseria lack the monooxygenase. While HpaC increases streptonigrin sensitivity, HpaC also promotes hydrogen peroxide and LL-37 resistance. We tested whether the HpaC effect in streptonigrin, hydrogen peroxide and LL-37 sensitivity involved the Type IV pilus. We determined that HpaC affects streptonigrin independently of the pilus while hydrogen peroxide- and LL-37-mediated killing involves both HpaC and the pilus. We demonstrate that the Gly93Cys change conferred enhanced affinity for FAD and resulted in a loss-of-function phenotype in streptonigrin susceptibility. These data suggest that HpaC's role in FAD oxidation and reduction impacts pilus-dependent and -independent resistance against neutrophil-mediated killing.