Lightning-Link® Fluorescein and Lightning-Link® PE/Cy5 in a Study of Virulence Factors
November 13th, 2018
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Neisseria meningitidis is a commensal bacterium that is usually a harmless resident of the human nasopharynx. However, it has the capability to cause devastating bacterial meningitis and septicemia. Neisseria meningitidis presents on many cell surfaces where it secretes multiple virulence factors that permit host colonization and infection.
Two of eight autotransporter proteins identified in meningococcal bacteria are adhesion and penetration protein (App) and meningococcal serine protease A (MspA). Studies have suggested that both have a role as in vivo virulence factors that mediate adherence of meningococci to human cells thus facilitating colonization and infection of the human host. However, their exact contribution to the pathogenesis of meningococcal infection has not been fully elucidated.
In this study the authors aimed to further characterize and confirm roles for App and MspA in the pathogenesis of in vivo meningococcal infection using a human CD46 transgenic mouse model (hCD46e). They used the Lightning-Link® Fluorescein and Lightning-Link® PE/Cy5 conjugation kits to label recombinant App (rpdApp) and MspA (rpdMspA) from both dendritic cells (DCs) and human brain microvascular endothelial cells (HBMECs), with fluorescein isothiocyanate (FITC) and cyanine-5 (Cy5) respectively, prior to analysis with confocal microscopy.
Outcomes of confocal microscopy imaging studies confirmed that both App and MspA share several commonalities that include mediation of bacteria–host cell adhesion, internalization and translocation to the host cell nucleus.
In initial studies, the authors confirmed roles for App and MspA as in vivo meningococcal virulence factors. Using the serogroup B meningococcal strain MC58, and a series of mutations whereby either one or both genes encoding App and MspA were deleted (MC58Δapp, MC58ΔmspA and MC58ΔappΔmspA), they infected groups of ten hCD46e transgenic mice. A further group of mice received resuspension fluid alone, i.e. without MC58, and were used as the negative control group. Survival rates for infected transgenic mice showed that higher survival rates were obtained in mice with either one or both deletions, compared with 0% survival in mice infected with wild type MC58 and 100% survival in the negative control group, at 72 hours following infection.
Cell internalization studies were also conducted with recombinant proteins, rpdApp and rpdMspA. These were constructed from the passenger domain for the extracellular forms of App and MspA respectively, with each protein fused to the chaperone trigger factor (TF) for Escherichia coli. (E. coli). Recombinant TF was used as the control for these studies. FITC labeling of rpdApp and rpdMspA, was performed using Lightning-Link® Fluorescein conjugation kit, prior to coincubation with human DCs and analysis by confocal microscopy. These studies confirmed internalization of App and MspA within human cells and their translocation to the nucleus. Cy5-labeling of rpdApp and rpdMspA was performed using using Lightning-Link® PE/Cy5 conjugation kit, prior to coincubation with HMBECs and analysis by confocal microscopy. Similar results were obtained for HBMECs as those obtained for DCs.
Other Experiments Within This Study
Crosslinking and enzyme linked immunoassay (ELISA) studies also confirmed that the mannose receptor (MR), transferrin receptor 1 (TfR1) and histones interact with App and MspA. Dendritic cell (DC) uptake could be blocked using mannan and transferrin, the specific physiological ligands for MR and TfR1, whereas in vitro clipping assays confirmed the ability of both App and MspA to proteolytically cleave the core histone H3. In apoptosis assays analysed by flow cytometry, App and MspA were also shown to induce DC cell death in a dose-dependent manner via a caspase dependent pathway.
Summary and Conclusion
In this study, the authors were able to elucidate several roles and characteristics for both App and MspA, two of eight autotransporter proteins identified in meningococcal bacteria, in the pathogenesis of meningococcal infection.
App and MspA were confirmed as in vivo virulence factors since transgenic mice expressing human CD46 that were infected with meningococcal mutants lacking either App, MspA or both, achieved improved survival rates compared with mice infected with wild type MC58.
Cell internalization studies were conducted with recombinant App and MspA (rpdApp and rpdMspA). Their strategy utilized Expedeon’s Lightning-Link® Fluorescein and Lightning-Link® PE/Cy5 conjugation kits to label DCs with FITC and HBMECs with Cy5, respectively. Once labeled, each cell line was used for internalization studies, analysed by confocal microscopy. The outcomes confirmed that App and MspA were internalized within both DC and HBMEC cell lines and translocated to the nucleus.
Lightning-Link® Fluorescein and Lightning-Link® PE/Cy5 Conjugation Kits to Visualize Internalization and Translocation of App and MspA.
In this study the authors relied on the Lightning-Link® Fluorescein and Lightning-Link® PE/Cy5 conjugation kits to form stable conjugates with recombinant proteins, rpdApp and rdpMspA. The Lightning-Link® Fluorescein and PE/Cy5 conjugation kits both offer several advantages:
- Direct conjugation of antibodies, proteins and peptides, or any other biomolecule with an available amine group
- Quick and easy to use – Save time (30 seconds hands-on time), no special knowledge required
- No separation steps – 100% recovery – No antibody / protein loss
- Can be used in a wide range of applications – Flexible
- Freeze dried – Ships at ambient temperature, long shelf life
- Fully scalable (10ug to 1g or more) – Easy transfer from R&D to manufacturing
- Stringently QC tested – Consistent high quality, excellent batch to batch reproducibility.
All the above are key for developing sensitive and specific antibodies for use in applications such as confocal microscopy and flow cytometry studies.
Khairalla, AS., Omer, SA., Mahdavi, J., Aslam, A., Dufailu, OA., Self, T., Jonsson, AB., Geörg, M., Sjölinder, H., Royer, PJ., Martinez-Pomares, L., Ghaemmaghami, AM., Wooldridge, KG., Oldfield, NJ., Ala’Aldeen, DA. Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters. Cell Microbiol. 2015 Jul;17(7):1008–20. doi: 10.1111/cmi.12417. Epub 2015 Feb 8. Available at: https://www.ncbi.nlm.nih.gov/pubmed/?term=Nuclear+trafficking%2C+histone+cleavage+and+induction+of+apoptosis+by+the+meningococcal+App+and+MspA+autotransporters Accessed November 2018.