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

## TL;DR

This study explores how a bacterial protein called HpaC helps Neisseria gonorrhoeae resist immune defenses, both with and without the Type IV pilus.

## Contribution

The study identifies a mutation in HpaC that enhances flavin affinity and reveals its dual role in pilus-dependent and -independent resistance mechanisms.

## Key findings

- A Gly93Cys mutation in HpaC increases FAD affinity and reduces streptonigrin sensitivity.
- HpaC contributes to resistance against hydrogen peroxide and LL-37 via the Type IV pilus.
- HpaC's role in FAD oxidation and reduction impacts resistance to neutrophil-mediated killing.

## Abstract

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.

Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease, gonorrhea. Neutrophils are first host immune responders that activate multiple potent antimicrobial defences including reactive oxygen species and antimicrobial peptides. Yet, N. gonorrhoeae is highly resistant to these host protection mechanisms. The Type IV pilus is known to promote resistance to these antibacterial agents; however, how the pilus does this was not known. We show that an FAD reductase HpaC has pilus-dependent and pilus-independent functions that contribute to this bacterium’s resistance through its interaction with flavin.