Human neuroglial cells internalize Borrelia burgdorferi by coiling phagocytosis mediated by Daam1

Abstract

Borrelia burgdorferi, the agent of Lyme borreliosis, can elude hosts’ innate and adaptive immunity as part of the course of infection. The ability of B. burgdorferi to invade or be internalized by host cells in vitro has been proposed as a mechanism for the pathogen to evade immune responses or antimicrobials. We have previously shown that B. burgdorferi can be internalized by human neuroglial cells. In this study we demonstrate that these cells take up B. burgdorferi via coiling phagocytosis mediated by the formin, Daam1, a process similarly described for human macrophages. Following coincubation with glial cells, B. burgdorferi was enwrapped by Daam1-enriched coiling pseudopods. Coiling of B. burgdorferi was significantly reduced when neuroglial cells were pretreated with anti-Daam1 antibody indicating the requirement for Daam1 for borrelial phagocytosis. Confocal microscopy showed Daam1 colocalizing to the B. burgdorferi surface suggesting interaction with borrelial membrane protein(s). Using the yeast 2-hybrid system for identifying protein-protein binding, we found that the B. burgdorferi surface lipoprotein, BBA66, bound the FH2 subunit domain of Daam1. Recombinant proteins were used to validate binding by ELISA, pull-down, and co-immunoprecipitation. Evidence for native Daam1 and BBA66 interaction was suggested by colocalization of the proteins in the course of borrelial capture by the Daam1-enriched pseudopodia. Additionally, we found a striking reduction in coiling for a BBA66-deficient mutant strain compared to BBA66-expressing strains. These results show that coiling phagocytosis is a mechanism for borrelial internalization by neuroglial cells mediated by Daam1.

Lyme Disease Frontiers: Reconciling Borrelia Biology and Clinical Conundrums

“…In vitro co-culture studies providing evidence of adhesion and internalization across a range of host materials and cell types are summarized in Table 3. Several phagocytic cell types have been co-incubated with Borrelia leading to bacterial detection and spirochete internalization via a coiling phagocytic mechanism [213,220]. Monocytes and dendritic cells degraded the internalized spirochetes as expected [209,218], whereas in macrophages, there were occasional live Borrelia observed within the cell, which were able to be re-cultured [217].…”

“…After co-incubation with lymphocytes, Borrelia was internalized and observed to be motile within vacuoles one to two hours later, and some killing of lymphocytes was observed after one day [180]. Borrelia has also been observed to be viable after 20 h co-incubation with neuronal and glial cells following antibiotic challenge [219,220]. When cultured alongside synovial cells, intact spirochetes have been observed at 7 weeks of co-culture and 63 days post-antibiotic challenge [223,224].…”

The Intergenic Small Non-Coding RNA ittA is Required for Optimal Infectivity and Tissue Tropism in Borrelia burgdorferi

“…A more recent report showed that BBA66 mediated the attachment of B. burgdorferi to these cells via the Daam1 protein [82]. Interestingly, the bba66 mutant exhibited reduced levels of internalized B. burgdorferi while borrelial cells that produced greater amounts of BBA66 were phagocytosed more efficiently [82]. In the RNA-seq data shown (Fig.…”

“…Mutants in bba66 are acquired by larvae and persist through molting, but were significantly impaired in their ability to infect mice when introduced by tick bite compared to that of mice fed upon by ticks seeded with wild type B. burgdorferi , suggesting a role for BBA66 in transmission [66]. Recently, an additional function of BBA66 was found, whereby BBA66 binds to the neuroglial and macrophage protein Daam1 [82]. Daam1 is in the formin family of proteins involved in regulating cytoskeletal reorganization in mammalian cells [83].…”

“…A prior study showed that Daam1 co-localized to pseudopods on macrophages that processed B. burgdorferi by coiling phagocytosis [84]. A more recent report showed that BBA66 mediated the attachment of B. burgdorferi to these cells via the Daam1 protein [82]. Interestingly, the bba66 mutant exhibited reduced levels of internalized B. burgdorferi while borrelial cells that produced greater amounts of BBA66 were phagocytosed more efficiently [82].…”