Binding of Borrelia Peptidoglycan to PGLYRP1 and CD55

Low passage B. burgdorferi were cultured to a density of ~10⁶−10⁷ cells/mL, washed two times with PBS and incubated with either recombinant human PGLYRP1 (with 8X-His tag), recombinant mouse PGLYRP1 (with 6X-His tag) (Sino Biological, #50115-M08H), recombinant human CD55 (with 8X-His tag), or murine CD55 (with 8X-His tag) at room temperature for 1 hour. Varying concentrations of purified B. burgdorferi PG [11 (link)] were used for this assay. PG preparation had been sonicated prior to use and consisted of fragmented sacculi. Borrelia PG was pre-incubated with either murine or human PGLYRP1 and was subsequently added to Borrelia to test for binding. After a co-incubation period of 1 hour, spirochetes were fixed in 4% PFA, washed three times with PBS and blocked in 1% BSA overnight at 4°C. The spirochetes were probed anti 6X-His monoclonal antibody- conjugated to Alexa Fluor 488 (ThermoFisher, #MA1-21315-488) and run through SA3800 Spectral Analyzer (Sony Biotechnology). The data was analyzed by FlowJo.

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Gupta A., Arora G., Rosen C.E., Kloos Z., Cao Y., Cerny J., Sajid A., Hoornstra D., Golovchenko M., Rudenko N., Munderloh U., Hovius J.W., Booth C.J., Jacobs-Wagner C., Palm N.W., Ring A.M, & Fikrig E. (2020). A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis. PLoS Pathogens, 16(11), e1009030.

Publication 2020

Alexa Fluor 488 SA3800 Spectral Analyzer

Alexa fluor 488 Assay Biological Borrelia Cultured cells Human Monoclonal antibody Murine Sacculi Sino Spirochetes

Corresponding Organization : Howard Hughes Medical Institute

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Variable analysis

independent variables

Recombinant human PGLYRP1 (with 8X-His tag)
Recombinant mouse PGLYRP1 (with 6X-His tag)
Recombinant human CD55 (with 8X-His tag)
Murine CD55 (with 8X-His tag)
Varying concentrations of purified B. burgdorferi PG

dependent variables

Binding of B. burgdorferi PG to B. burgdorferi cells

control variables

Low passage B. burgdorferi were cultured to a density of ~10^6−10^7 cells/mL
B. burgdorferi cells were washed two times with PBS
Incubation time of 1 hour at room temperature
PG preparation was sonicated prior to use and consisted of fragmented sacculi
B. burgdorferi cells were fixed in 4% PFA, washed three times with PBS and blocked in 1% BSA overnight at 4°C

controls

Positive control: B. burgdorferi PG was pre-incubated with either murine or human PGLYRP1 and subsequently added to B. burgdorferi to test for binding
No negative control was explicitly mentioned

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