The oxidizing agent, paraquat, is more toxic to Wolbachia than to mosquito host cells

Abstract

Cultured cells provide an important in vitro system for examining metabolic interactions between the intracellular bacterium, Wolbachia pipientis, and its insect hosts. To test whether Wolbachia-associated changes in antioxidant activities could provide a tool to select for infected cells, we tested the effects of paraquat (PQ) on Aedes albopictus mosquito cells. Like mammalian cells, mosquito cells tolerate PQ over a wide range of concentrations, and for considerable lengths of time, depending on cell density at the time of treatment. When mosquito cells were plated at low density and allowed to grow in the presence of PQ, we measured an LC50 of approximately 1–2 µM. Unexpectedly, cells persistently infected with Wolbachia strain wStr, from the planthopper Laodelphax striatellus, grew to higher densities in the presence of 1.5 µM PQ than in its absence. This effect of PQ was similar to the improved growth of host cells that occurs in the presence of antibiotics that suppress the Wolbachia infection. A more detailed examination of growth and metabolic sensitivity indicated that wStr is about 10-fold more sensitive to PQ than the mosquito host cells. Microscopic examination confirmed that Wolbachia levels were reduced in PQ-treated cells, and DNA estimates based on the polymerase chain reaction (PCR) indicated that Wolbachia abundance decreased by approximately 100-fold over a 10-d period. Although Wolbachia genomes encode superoxide dismutase, inspection of annotated genomes indicates that they lack several genes encoding products that ameliorate oxidative damage, including catalase, which converts the PQ byproduct, hydrogen peroxide, to molecular oxygen and water. We suggest that loss of multiple genes that participate in repair of oxidative damage accounts for increased sensitivity of Wolbachia to PQ, relative to its host cells.

Effects of mimosine on Wolbachia in mosquito cells: cell cycle suppression reduces bacterial abundance

“…To compensate for higher cell densities attained with antibiotic suppression of Wolbachia (Fallon et al . 2013a, b), cell counts were normalized by defining values for mimosine-free controls as 100% for each population. Visual inspection by microscopy indicated that the abundance of fluorescently stained Wolbachia appeared unaffected by mimosine (not shown), and metabolic activity assessed by MTT values of both control and mimosine-treated C/ w Str1 cells reached maximal levels at about 5 d after plating (Fig.…”

Flow cytometric evaluation of the intracellular bacterium, Wolbachia pipientis, in mosquito cells

“…In previous studies, we used Coulter cell counts, MTT assays and PCR reactions to demonstrate a reduction in Wolbachia levels in host cells exposed to the oxidizing agent, paraquat (Fallon et al, 2013b), and the riboflavin transport inhibitor, lumiflavin (Fallon et al, 2014). As a final check on the reliability of FC date, these experiments were repeated by quantifying WP and CP events.…”

“…In insect tissues, Wolbachia are pleomorphic in size and shape (Hertig, 1936) and cannot be physically separated from mitochondria (Sun et al, 2001), which have a DNA genome ~75-fold smaller than that of Wolbachia . Because Wolbachia are no longer recoverable by centrifugation after treatment with 2X PI-MM, presumably due to detergent-associated damage to the bacterial cell wall, we validated the presence of Wolbachia in the WP by showing that material from filtered cell culture supernatant recovered by centrifugation is associated with a cell-free WP with 99% of events in the lower left quartile of the SSlog/FL3 histogram (Fig.…”

“…To suppress Wolbachia growth, tetracycline and rifampicin were added to the culture medium at final concentrations of 5 μg/ml, and 0.4 μg/ml, respectively. Paraquat (Fallon et al, 2013b) and lumiflavin (Fallon et al, 2014) were used as described previously.…”

“…To facilitate evaluation of conditions that may favor or suppress Wolbachia growth in these C/wStr1 cells (Fallon et al 2013b, 2014), we developed a flow cytometric (FC) protocol for simultaneous evaluation of host cell number and Wolbachia abundance in persistently-infected host cells.…”