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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.

SUBMITTER: Hu LI

PROVIDER: S-EPMC9246397 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Retraction of the type IV pilus (Tfp) mediates DNA uptake, motility, and social and infection behavior in a wide variety of prokaryotes. To date, investigations into Tfp retraction-dependent activities have used a mutant deleted of PilT, the ATPase motor protein that causes the pilus fiber to retract. ΔpilT cells are nontransformable, nonmotile, and cannot aggregate into microcolonies. We tested the hypothesis that these retraction-dependent activities are sensitive to the strength of PilT enzymatic activity by using the pathogen Neisseria gonorrhoeae as a model. We constructed an N. gonorrhoeae mutant with an amino acid substitution in the PilT Walker B box (a substitution of cysteine for leucine at position 201, encoded by pilTL201C). Purified PilTL201C forms a native hexamer, but mutant hexamers hydrolyze ATP at half the maximal rate. N. gonorrhoeae pilTL201C cells produce Tfp fibers, crawl at the same speed as the wild-type (wt) parent, and are equally transformable. However, the social behavior of pilTL201C cells is intermediate between the behaviors of wt and ΔpilT cells. The infection behavior of pilTL201C is also defective, due to its failure to activate the epidermal growth factor receptor (EGFR)-heparin-binding EGF-like growth factor (HB-EGF) pathway. Our study indicates that pilus retraction, per se, is not sufficient for N. gonorrhoeae microcolony formation or infectivity; rather, these activities are sensitive to the strength of PilT enzymatic activity. We discuss the implications of these findings for Neisseria pathogenesis in the context of mechanobiology. Type IV pili are fibers expressed on the surface of many bacteria. Neisseria gonorrhoeae cells crawl, take up DNA, and communicate with each other and with human cells by retracting these fibers. Here, we show that an N. gonorrhoeae mutant expressing an enzymatically weakened type IV pilus retraction motor still crawls and takes up DNA normally. However, mutant cells exhibit abnormal social behavior, and they are less infective because they fail to activate the epidermal growth factor receptor. Our study shows that N. gonorrhoeae social and infection behaviors are sensitive to the strength of the retraction motor enzyme.

Project description:Study questionDo the truncated LL-37 peptides, GI-20 and GF-17, have spermicidal activity and microbicidal effects on the sexually transmitted infection (STI) pathogen Neisseria gonorrhoeae with equivalent potency to LL-37?Summary answerGI-20 and GF-17 exhibited spermicidal effects on both mouse and human sperm as well as microbicidal action on N. gonorrhoeae with the same efficacy as LL-37.What is known alreadyThe antimicrobial peptide LL-37 exerts microbicidal activity against various STI pathogens as well as spermicidal effects on both mouse and human sperm.Study design, size, durationSpermicidal activities of GI-20 and GF-17 were evaluated in vitro in mouse and human sperm and in vivo in mice. Finally, in vitro antimicrobial effects of LL-37, GI-20 and GF-17 on an STI pathogen, N. gonorrhoeae were determined. All experiments were repeated three times or more. In particular, sperm samples from different males were used on each experimental day.Participants/materials, setting, methodsThe plasma membrane integrity of peptide-treated sperm was assessed by cellular exclusion of Sytox Green, a membrane impermeable fluorescent DNA dye. Successful mouse in vitro fertilization was revealed by the presence of two pronuclei in oocytes following co-incubation with capacitated untreated/peptide-pretreated sperm. Sperm plus each peptide were transcervically injected into female mice and the success of in vivo fertilization was scored by the formation of 2-4 cell embryos 42 h afterward. Reproductive tract tissues of peptide pre-exposed females were then assessed histologically for any damage. Minimal inhibitory/bactericidal concentrations of LL-37, GI-20 and GF-17 on N. gonorrhoeae were determined by a standard method.Main results and the role of chanceLike LL-37, treatment of sperm with GI-20 and GF-17 resulted in dose-dependent increases in sperm plasma membrane permeabilization, reaching the maximum at 18 and 3.6 ?M for human and mouse sperm, respectively (P < 0.0001, as compared with untreated sperm). Mouse sperm treated with 3.6 ?M GI-20 or GF-17 did not fertilize oocytes either in vitro or in vivo. Moreover, reproductive tract tissues of female mice pre-exposed to 3.6 ?M GI-20 or GF-17 remained intact with no lesions, erosions or ulcerations. At 1.8-7.2 ?M, LL-37, GI-20 and GF-17 exerted bactericidal effects on N. gonorrhoeae.Large scale dataN/A.Limitations, reasons for cautionDirect demonstration of the inhibitory effects of GI-20 and GF-17 on human in vitro and in vivo fertilization cannot be performed due to ethical issues.Wider implications of the findingsLike LL-37, GI-20 and GF-17 acted as spermicides and microbicides against N. gonorrhoeae, without adverse effects on female reproductive tissues. With lower synthesis costs, GI-20 and GF-17 are attractive peptides for further development into vaginal spermicides/microbicides.Study funding/competing interest(s)This work was supported by Canadian Institutes of Health Research (MOP119438 and CCI82413 to N.T.) and NIH (R01 AI105147 to G.W.). There are no competing interests to declare.

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

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