Generation of Metal-Depleted Conditions for In Vitro Growth of Neisseria gonorrhoeae

Cornelissen, Cynthia Nau

doi:10.1007/978-1-4939-9496-0_14

Cited by 2 publications

(2 citation statements)

References 23 publications

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“…CP-dependent growth of N. gonorrhoeae. Cells were grown to the log phase in Zn-restricted CDM as described above being diluted to an OD 600 of ϳ0.02 in a microtiter plate containing appropriately diluted mixtures (premixes) of supplements and/or metals (23,52). The positive-control premix had 5 M ZnSO 4 as the sole Zn source and also lacked TPEN.…”

Section: Methodsmentioning

confidence: 99%

Molecular Insight into TdfH-Mediated Zinc Piracy from Human Calprotectin by Neisseria gonorrhoeae

Kammerman

Bera

et al. 2020

mBio

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Neisseria gonorrhoeae, responsible for the sexually transmitted infection gonorrhea, is an obligate human pathogen exquisitely adapted for survival on mucosal surfaces of humans. This host-pathogen relationship has resulted in evolution by N. gonorrhoeae of pathways that enable the use of host metalloproteins as required nutrients through the deployment of outer membrane-bound TonB-dependent transporters (TdTs). Recently, a TdT called TdfH was implicated in binding to calprotectin (CP) and in removal of the bound zinc (Zn), enabling gonococcal growth. TdfH is highly conserved among the pathogenic Neisseria species, making it a potentially promising candidate for inclusion into a gonococcal vaccine. Currently, the nature and specificity of the TdfH-CP interaction have not been determined. In this study, we found that TdfH specifically interacted with human calprotectin (hCP) and that growth of the gonococcus was supported in a TdfH-dependent manner only when hCP was available as a sole zinc source and not when mouse CP was provided. The binding interactions between TdfH and hCP were assessed using isothermal titration calorimetry where we observed a multistate model having both high-affinity and low-affinity sites of interaction. hCP has two Zn binding sites, and gonococcal growth assays using hCP mutants deficient in one or both of the Zn binding sites revealed that TdfH exhibited a site preference during Zn piracy and utilization. This report provides the first insights into the molecular mechanism of Zn piracy by neisserial TdfH and further highlights the obligate human nature of N. gonorrhoeae and the high-affinity interactions occurring between TdTs and their human ligands during pathogenesis. IMPORTANCE The dramatic rise in antimicrobial resistance among Neisseria gonorrhoeae isolates over the last few decades, paired with dwindling treatment options and the lack of a protective vaccine, has prompted increased interest in identifying new bacterial targets for the treatment and, ideally, prevention of gonococcal disease. TonB-dependent transporters are a conserved set of proteins that serve crucial functions for bacterial survival within the host. In this study, binding between the gonococcal transporter, TdfH, and calprotectin was determined to be of high affinity and host restricted. The current study identified a preferential TdfH interaction at the calprotectin dimer interface. An antigonococcal therapeutic could potentially block this site on calprotectin, interrupting Zn uptake by N. gonorrhoeae and thereby prohibiting continued bacterial growth. We describe protein-protein interactions between TdfH and calprotectin, and our findings provide the building blocks for future therapeutic or prophylactic targets.

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Section: Methodsmentioning

confidence: 99%

Molecular Insight into TdfH-Mediated Zinc Piracy from Human Calprotectin by Neisseria gonorrhoeae

Kammerman

Bera

et al. 2020

mBio

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“…Briefly, an overnight culture of bacteria was used to inoculate 10 mL of supplemented GC broth without ferric nitrate; the bacterial mixture was adjusted to an optical density (OD) of 0.1 at l595 nm and incubated at 37°C with shaking (200 rpm) for 2 h or until OD had doubled. Iron stress was initiated by adding Desferal to a final concentration of 50 µM followed by incubation for a further 4 h (Cornelissen, 2019). Bacteria were also grown in normal supplemented GC broth (with ferric nitrate) and without Desferal.…”

Section: Bacteria and Growth Conditionsmentioning

confidence: 99%

An immunoproteomics study of antisera from patients with gonorrhea identifies novel Neisseria gonorrhoeae proteins

Dijokaite-Guraliuc,

Humbert,

Skipp

et al. 2023

Front. Bacteriol.

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BackgroundNeisseria gonorrhoeae (gonococcus) is the causative agent of the sexually transmitted disease gonorrhea, for which no vaccines exist. Efforts are being made to identify potential vaccine protein antigens, and in this study, an immunoproteomics approach was used to identify protein signatures in gonococci that were recognized by sera from patients with gonorrhea.MethodsSera from patients with uncomplicated gonorrhea and from controls were reacted on Western blot with gonococcal whole-cell lysate separated by 2D electrophoresis. Reactive bands were excised and digested, and peptides were analyzed by mass spectrometry to identify protein hits. Proteins were analyzed with in-silico bioinformatics tools (PSORTb v3.0, CELLO, SOSUI-GramN, LipoP 1.0, SignalP 5.0, TMHMM 2.0, eggNOG-mapper 5.0) to select for surface-exposed/outer membrane proteins (OMPs) and exclude cytoplasmic proteins and most periplasmic proteins. Sera were tested for bactericidal activity against homologous and heterologous gonococcal strains.ResultsPatient sera reacted with 180 proteome bands, and 18 of these bands showed ≥2-fold increased reactivity compared with sera from individuals (n = 5) with no history of gonococcal infection. Mass spectrometry produced peptide signatures for 1,107 proteins, and after bioinformatics analyses, a final collection of 33 proteins was produced that contained 24 OMPs/extracellular proteins never previously studied to our knowledge, 6 proteins with homologs in Neisseria meningitidis that can generate functional immune responses, and 3 unknown proteins. The sera showed little or no significant bactericidal activity, which may be related to the immunoproteomic identification of contraindicated proteins Rmp and H.8 that can generate blocking antibodies.ConclusionStudies on the vaccine potential of these newly identified proteins deserve consideration.

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Generation of Metal-Depleted Conditions for In Vitro Growth of Neisseria gonorrhoeae

Cited by 2 publications

References 23 publications

Molecular Insight into TdfH-Mediated Zinc Piracy from Human Calprotectin by Neisseria gonorrhoeae

Molecular Insight into TdfH-Mediated Zinc Piracy from Human Calprotectin by Neisseria gonorrhoeae

An immunoproteomics study of antisera from patients with gonorrhea identifies novel Neisseria gonorrhoeae proteins

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