The Neisseria gonorrhoeae type IV pilus promotes resistance to hydrogen peroxide- and LL-37-mediated killing by modulating the availability of intracellular, labile iron

Abstract

The Neisseria gonorrhoeae Type IV pilus is a multifunctional, dynamic fiber involved in host cell attachment, DNA transformation, and twitching motility. We previously reported that the N. gonorrhoeae pilus is also required for resistance against hydrogen peroxide-, antimicrobial peptide LL-37-, and non-oxidative, neutrophil-mediated killing. We tested whether the hydrogen peroxide, LL-37, and neutrophil hypersensitivity phenotypes in non-piliated N. gonorrhoeae could be due to elevated iron levels. Iron chelation in the growth medium rescued a nonpiliated pilE mutant from both hydrogen peroxide- and antimicrobial peptide LL-37-mediated killing, suggesting these phenotypes are related to iron availability. We used the antibiotic streptonigrin, which depends on free cytoplasmic iron and oxidation to kill bacteria, to determine whether piliation affected intracellular iron levels. Several non-piliated, loss-of-function mutants were more sensitive to streptonigrin killing than the piliated parental strain. Consistent with the idea that higher available iron levels in the under- and non-piliated strains were responsible for the higher streptonigrin sensitivity, iron limitation by desferal chelation restored resistance to streptonigrin in these strains and the addition of iron restored the sensitivity to streptonigrin killing. The antioxidants tiron and dimethylthiourea rescued the pilE mutant from streptonigrin-mediated killing, suggesting that the elevated labile iron pool in non-piliated bacteria leads to streptonigrin-dependent reactive oxygen species production. These antioxidants did not affect LL-37-mediated killing. We confirmed that the pilE mutant is not more sensitive to other antibiotics showing that the streptonigrin phenotypes are not due to general bacterial envelope disruption. The total iron content of the cell was unaltered by piliation when measured using ICP-MS suggesting that only the labile iron pool is affected by piliation. These results support the hypothesis that piliation state affects N. gonorrhoeae iron homeostasis and influences sensitivity to various host-derived antimicrobial agents.

Development and implementation of a Type I-C CRISPR-based programmable repression system for Neisseria gonorrhoeae

“…We targeted the pilE gene, which is not essential in vitro and encodes the major subunit of the type four pilus (Tfp). Expression of the Tfp is necessary for transformation competence, so we expected CRISPRi-mediated pilE repression to diminish DNA transformation ( 50 – 53 ). To control for Tfp antigenic variation, we performed these experiments in N-1-60, an FA1090 strain derivative defective for pilE antigenic variation and pilC phase variation ( 53 ).…”

“…Expression of the Tfp is necessary for transformation competence, so we expected CRISPRi-mediated pilE repression to diminish DNA transformation ( 50 – 53 ). To control for Tfp antigenic variation, we performed these experiments in N-1-60, an FA1090 strain derivative defective for pilE antigenic variation and pilC phase variation ( 53 ). We used pilus-dependent colony morphology ( 54 ) to determine whether the CRISPRi- pilE strain yields a nonpiliated colony morphology when induced with IPTG.…”

“…Strains and their details are listed in Table S1. All strains are FA1090 derivatives, either in the OpaD nv or N-1-60 backgrounds ( 40 , 53 ). The OpaD nv is a phase-locked OpaD constitutively expressing strain in an opaA-K deleted (Opaless) background ( 40 ).…”

“…The OpaD nv is a phase-locked OpaD constitutively expressing strain in an opaA-K deleted (Opaless) background ( 40 ). The N-1-60 strain contains multiple point mutations in the pilE G4 and is pilC2 phase-locked “on” with the 1-81-S2 pilin variant ( 53 ). Strains were grown on Gonococcal (Gc) Medium Base (BD Difco) (36.25 g/L), agar (1.25 g/L), Kellogg Supplement I (22.2 mM glucose, 0.68 mM glutamine, 0.45 mM cocarboxylase), and Kellogg Supplement II [1.23 µM Fe(NO 3 ) 3 ] ( 54 ).…”

Type IV pilus retraction is required for Neisseria musculi colonization and persistence in a natural mouse model of infection

“…Tfp contributes to the virulence of human pathogens Neisseria meningitidis (Nme) and Neisseria gonorrhoeae (Ngo) ( 2 – 7 ). The pilus fiber, consisting of the major subunit pilin (PilE) and minor pilins ( 8 – 10 ), promotes initial contact of Ngo and Nme with human epithelial cells ( 11 – 14 ).…”