Complement-mediated serum sensitivity among spirochetes that cause Lyme disease

Abstract

Borrelia burgdorferi-related isolates were tested for their sensitivity to normal human serum (NHS) and their ability to activate complement. By dark-field microscopy, electron microscopy, and subsurface plating, it was shown that exposure of a Borrelia garinii isolate to 10% or more NHS resulted in immobilization, blebbing, and killing of the spirochetes. These effects were mediated by complement, since they were not seen after heat treatment of NHS, in the presence of EDTA, or in an agammaglobulinemic serum. All seven B. garinii type 5 or 6 and all four VS116/M19 strains were serum sensitive, whereas all eight Borrelia afzelii, five of eight B. garinii type 4, and three of seven B. burgdorferi sensu stricto isolates were serum resistant. The other isolates were partially serum sensitive. Four serum-sensitive B. garinii isolates had been isolated from human cerebrospinal fluid. Most likely, activation of both the alternative pathway and the classical pathway of complement was involved, since bactericidal activity was diminished in properdin-deficient sera as well as in a C1q-depleted serum and in a C4-deficient serum. Bactericidal activity could be restored when a serum lacking C1q or C4 was mixed with a properdin-deficient serum. Isolates with various genetic backgrounds were equally able to activate C3 as measured by enzyme-linked immunosorbent assay. In the presence of Mg-EGTA, C3 was activated by all isolates after exposure to >10% NHS. This study shows that B. burgdorferi-related spirochetes can be either serum sensitive or serum resistant in vitro and that this characteristic is associated with their genetic background. Lyme borreliosis (LB) is a multisystem disorder caused by the tick-borne spirochetes Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. Erythema migrans (EM), a skin lesion surrounding the site of the initial tick bite, is often the presenting symptom of LB (54). Subsequently, spirochetes can disseminate to other skin sites, resulting in multiple EM lesions and finally acrodermatitis chronica atrophicans (ACA), or to various organs, such as the heart, the joints, the eyes, or the central nervous system (54). Dissemination of Borrelia from the skin to distant sites is generally considered to occur hematogenously. After needle or tick-borne inoculation of mice with B. burgdorferi, all inoculated mice had positive blood cultures up to 6 weeks postinoculation (p.i.), while after 1 year only 1 of 10 mice was positive (6). Spirochetes have been cultured in low frequency from blood from rabbits 2 weeks p.i. (26) and from rhesus monkeys 1 to 6 weeks p.i. (41), although in another study blood cultures from such animals were negative (37). Culture of spirochetes from human blood has been successful in a few cases (7, 34, 58) but is usually negative (35). These findings suggest that spirochetes are present in the circulation in the early stage of infection but that they disappear in later stages of the disease. Whether the humoral immune system plays a role in the prevention o...

Blood treatment of Lyme borreliae demonstrates the mechanism of CspZ ‐mediated complement evasion to promote systemic infection in vertebrate hosts

“…We found that blood treatment is essential to demonstrate the CspZ-mediated FH-binding activity to confer spirochete survival in sera (Figure 4). It is noteworthy that we count immotile cells as the indicator of cell death by dark-field microscopy, a method that has been commonly used to identify killed cells (Alitalo et al, 2001;Alitalo et al, 2005;Brooks et al, 2005;Kraiczy, Hunfeld, Peters, et al, 2000;McDowell et al, 2011;van Dam et al, 1997). Furthermore, the lack of motility is the most apparent sign of complement-mediated bacterial killing, which allows determination of cell viability microscopically without the need for the extended duration of time to cultivate spirochetes.…”

“…Approximately 5 × 10 6 cells of mid-log B. burgdorferi were cultivated in BSK-II complete medium with 5% human blood with the buffy coat removed. This concentration of human blood does not reduce the growth and motility of serum-sensitive strains (Breitner-Ruddock, Wurzner, Schulze, & Brade, 1997;Hart, Nguyen, et al, 2018;Kenedy & Akins, 2011;van Dam et al, 1997). The human blood was supplemented with a cocktail of antibiotics (final concentration: 50 μg ml −1 of rifampicin, 20 μg ml −1 of phosphomycin, and 2.5 μg ml −1 of amphotericin) to prevent potential bacterial and fungal contamination.…”

Sensitivity ofBorreliagenospecies to serum complement from different animals and human: a host–pathogen relationship

“…Considering the close relation of dog and cat with human, it is important to know whether co-feeding can take place in these carnivores. In human, studies are available [17,26] and B. afzelii, and its influence on the pathogenic and clinical manifestations. Our study is an extension of previous reports by assessing the complement sensitivities of more genospecies.…”

“…To inhibit the classical pathway of complement activation, some aliquots from each animal species were incubated with 10 mM EGTA, 4 mM MgCl 2 in veronal buffer saline (VBS) at 37°C for 30 min. Some serum aliquots from each animal species were incubated with 10 mM EDTA in VBS at 37°C for 30 min to inactivate both pathways (classical and alternative) [17].…”

“…Complement activation was measured according to van Dam et al, [17]. In brief, Borrelia strains were grown in BSK-II medium at 33°C for one week.…”