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Flavin affinity for the reductase HpaC differentially sensitizes Neisseria gonorrhoeae during Type IV pilus-dependent
Linda I Hu1, Egon A Ozer2, H S Seifert1
1Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.
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.
Insights
Neisseria gonorrhoeae HpaC protein impacts bacterial resistance. A mutation enhances FAD binding, affecting susceptibility to killing by streptonigrin, hydrogen peroxide, and LL-37, with pilus involvement varying by agent.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Neisseria gonorrhoeae utilizes Type IV pili for various functions, including immune evasion.
- Pilus expression influences bacterial iron homeostasis and susceptibility to oxidative and non-oxidative killing agents.
- Previous work linked pilus expression to altered sensitivity to streptonigrin, hydrogen peroxide, and LL-37.
Purpose of the Study:
- To identify genes influencing N. gonorrhoeae susceptibility to the antibiotic streptonigrin using in vitro evolution.
- To characterize the function of the HpaC protein and its role in bacterial resistance mechanisms.
- To elucidate the interplay between HpaC, Type IV pili, and resistance to different antimicrobial agents.
Main Methods:
- In vitro evolution of N. gonorrhoeae to identify resistance determinants.
- Genetic analysis of mutations in the NGO0059 locus encoding HpaC.
- Biochemical assays to assess FAD binding affinity of HpaC variants.
- Phenotypic characterization of bacterial susceptibility to streptonigrin, hydrogen peroxide, and LL-37, with and without Type IV pilus involvement.
Main Results:
- A mutation in the NGO0059 locus (HpaC) conferring increased streptonigrin sensitivity was identified.
- The identified HpaC Gly93Cys mutation enhanced FAD binding and resulted in a loss-of-function for streptonigrin susceptibility.
- HpaC independently affects streptonigrin sensitivity, but is involved with Type IV pili in resistance to hydrogen peroxide and LL-37.
- HpaC's FAD oxidation-reduction activity impacts both pilus-dependent and -independent resistance.
Conclusions:
- HpaC plays a dual role in N. gonorrhoeae resistance, modulating susceptibility to distinct antimicrobial agents.
- The HpaC Gly93Cys mutation alters FAD binding, impacting bacterial defense mechanisms.
- Interactions between HpaC and Type IV pili are crucial for resistance against certain host immune components, like neutrophil-mediated killing.
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