Molecular Biology Methods

25 fractions with and without FCS were incubated at 26 °C for 12 days. A negative control containing Wolbachia incubated in cell culture medium was added. The initial Wolbachia concentration was between 2 and 4 x 10³ 16S rRNA gene copies/µl and the final concentration of insect cells in the membrane fraction was equivalent to 0.95 x 10⁶ insect cells/ml as calculated from the amount of cells counted prior to lysis. Growth was monitored on days 3, 6, 9 and 12 by qPCR.

2.2.1.4 Infection of C6/36 insect cells with Wolbachia

The ability of Wolbachia incubated in cell-free culture to infect new C6/36 insect cells was determined by infection of uninfected C6/36 insect cells. A cell-free Wolbachia culture with a concentration of 10³ 16S rRNA gene copies/µl supplemented with the membrane fraction obtained from cell lysate of uninfected insect cells was prepared as described in sections 2.2.1.2 and 2.2.1.3. Wolbachia were incubated at 26 °C for 12 days. On day 9 uninfected insect cells were seeded in a 24-well plate with a concentration of 10⁵ insect cells/well. Infection was performed in triplicate. On day 12 medium was removed from uninfected insect cells and 750 µl from the insect cell-free Wolbachia culture was added. As a negative control Wolbachia culture was heated at 95 °C for 10 min, cooled down and subsequently applied to uninfected insect cells. The uninfected insect cells covered with cell-free Wolbachia culture were centrifuged at 2,000 g for 1 h at 15 °C and subsequently incubated at 26 °C over night. On the next day, cells were harvested and transferred into a 6-well plate containing 1.5 ml cell culture medium supplemented with 10 % FCS and incubated at 26 °C. After 6 days the cells were harvested in 2 ml freshly added cell culture medium, transferred to culture slides (300 µl suspension/well) and incubated at 26 °C for 1 day. To verify the success of Wolbachia-infection the infected insect cells grown on culture slides were examined by immunofluorescence microscopy (section 2.2.4.1).

2. Materials and Methods

26

2.2.2.2 RNA extraction

Wolbachia of filarial nematodes cannot be successfully maintained in cell culture, for this reason total RNA was extracted from wAlb B infected and uninfected A.

albopictus C6/36 cell lines. Insect cells were grown in plug-sealed 75 cm² culture flasks to

~90 % confluence. RNA isolation followed a standard Trizol reagent protocol. For this, cells were harvested at 2,500 g for 8 min at 4 °C and pellets were suspended in 1 ml Trizol reagent and mixed vigorously with 0.1 ml 1-Bromo-3-chloro-2-propanol for 15 seconds. The mixture was incubated for 10 min at room temperature and centrifuged afterwards for 15 min at 12,000 g at 4 °C. The upper aqueous phase containing the nucleic acids was retained and the nucleic acids were precipitated by the addition of 0.5 ml isopropanol and by incubation at -20 °C for 10 min. After a centrifugation step at 12,000 g for 15 min at 4 °C the supernatant was discarded and the pellet was washed in 1 ml 75 % ethanol. The ethanol was removed by centrifugation at 7,500 g for 4 min at 4 °C and the pellets were dried at 37 °C and subsequently suspended in nuclease free water.

To remove contaminating DNA the extracted RNA was treated with the DNA-free Kit according to the manufacturer’s protocol. The yield of total RNA was measured using a Nanovue and aliquots were stored at -80 °C.

2.2.2.3 Two-step reverse transcriptase PCR

The transcription of specific genes was analyzed by a two-step reverse transcriptase PCR (RT-PCR). Reverse transcription was performed using the Omniscript Reverse Transcriptase Kit according to the manufacturer’s protocol. 75 ng/µl RNA was reverse transcribed to cDNA using 10 µM random decamer primers. 200 – 300 bp fragments of genes involved in peptidoglycan metabolism were amplified with gene specific primers (Table 2.1) using cDNA as template and genomic DNA as a control. PCR was carried out in 20 µl reactions using 0.5 U Hot Star Taq polymerase, 3 mM MgCl2, 200 µM dNTPs, 0.5 µM primer, 1 x PCR buffer and 2 µl template. The PCR conditions included a heat activation step for 15 min at 95 °C following 35 cycles of 30 sec at 94 °C, 30 sec at a primer dependent temperature (Table 2.1), 1 min at 72 °C and a final extension step for 10 min at 72 °C. PCR products were analyzed on 2 % agarose gels, cloned into a TOPO vector using TOPO^® TA cloning kit according to the manufracturer´s protocol and sequenced at Seqlab sequencing laboratories (Goettingen, Germany) using a T7 promotor primer. The

27 analysis of the genes murA - murF, rodA, metC and glyA was performed as part of a bachelor thesis supervised during this PhD project (Jülicher 2012).

2.2.2.4 Quantitative real-time PCR

Wolbachia cell numbers were calculated by quantification of 16S rRNA gene copies using qPCR. Performance of qPCR was supported by technical assistance. A qPCR reaction contained 1 x HotStar Taq polymerase buffer, 3 mM MgCl2, 200 µM dNTPs, 0.2 µl SYBR Green (1:1000 dilution in DMSO), 0.5 µM 16S rRNA gene specific forward and reverse primer, respectively, and 0.5 U HotStar Taq polymerase. 2 µl DNA extracted from 200 µl Wolbachia suspension was used as template. qPCR conditions included a heat activation step at 95 °C for 15 min followed by 45 cycles of 95 °C for 10 sec, 55 °C for 15 sec and 72 °C for 20 sec. Melt curve analysis showed a specific peak for all positive samples.

Data were analyzed using Rotorgene 6000 software version 1.7.

2.2.2.5 BrdU cell proliferation assay

Besides qPCR, replication of insect cell-free Wolbachia was detected using the BrdU cell proliferation assay kit. In this assay, the thymidine analog bromodeoxyuridine (BrdU) is used, which is incorporated into newly synthesized DNA during replication.

Subsequently, incorporation is detected by an ELISA based method. For detection of Wolbachia replication BrdU was supplemented to cell-free Wolbachia culture to a final dilution of 1:10,000. A negative control of cell-free Wolbachia culture without BrdU was included in the experiment. Reactions of 0.2 to 2 ml culture were incubated in 24-well plates or 25 cm² for 12 days and triplicate samples were harvested every 3 days by centrifugation at 18,400 g for 5 min at 4 °C. Pelleted Wolbachia were incubated in 200 µl fixative/denaturing solution for 30 min at room temperature to fix the cells and to denature DNA necessary for proper antibody binding. Subsequent to centrifugation at 18,400 g for 5 min at room temperature, the supernatant was discarded and the pellet was incubated with 40 µl a BrdU monoclonal antibody diluted 1:100 in antibody dilution buffer for 1 h at room temperature. Unbound antibody was removed by 3 washing steps in 300 µl wash buffer accompanied by repeated centrifugations at 18,400 g for 5 min. In the next step 40 µl of peroxidase-conjugated secondary antibody was added in a 1:1000 dilution and incubated for 30 min at room temperature. After 3 washing steps with wash

2. Materials and Methods

28 buffer and 1 washing step with demineralised water accompanied by centrifugation at 18,400 g for 5 min, 100 µl substrate solution containing the peroxidase substrate tetra-methylbenzidine was added and incubated for 15 min in the dark. The reaction was stopped by the addition of 100 µl stop solution. Samples were transferred to a 96-well plate and absorbance was detected immediately in an ELISA plate reader at 450 nm wavelength.