2.1 Identifying Carriers Able to Deliver Antisense Molecules to B. saltans and Its Intracellular Symbiont

Initially, we determined the best delivery protocol to introduce antisense molecules into B. saltans and Cbv cells. We treated B. saltans cells with fluorophore-conjugated synthetic PNAs, as these molecules have been most thoroughly tested in intracellular bacteria. For intracellular delivery, we used two different protocols: incubation of B. saltans cells for 24 h and electroporation of B. saltans with 50 µM PNA molecule PNA00218_TMR (Table 1).

We imaged B. saltans cells after 24 h to ascertain localization of the fluorescent antisense molecule within hosts and symbionts (Figure 1A). In our experiment, incubation resulted in much higher fluorescence intensity within the B. saltans cells compared to electroporation (Figures 1A,B and S1). This impact might be related to the fact that incubated cells are in contact with antisense molecules for 24 h, while the electroporation protocol allows for just a very brief contact with antisense molecules (during electroporation).

Next, we determined whether PNA00218_TMR can enter bacterial cells within the B. saltans cytoplasm. To this end, we quantified the fluorescence intensity within the intracellular bacteria of B. saltans and compared it to the fluorescence intensity of the cell cytoplasm (excluding bacteria and nucleus). Intracellular bacteria had slightly higher average fluorescence intensity (19,383 AU vs. 17,192 AU) than the surrounding cell cytoplasm, meaning that antisense molecules can enter Cbv cells but do not disproportionately accumulate within the symbiont.

In the process of optimizing the delivery, we also assessed other antisense molecules chemistries, including PNAs attached to peptide and lipid carriers, 2'ome RNAs, and phosphothiorate oligos (Table 1). However, they all consistently yielded weaker intracellular fluorescence by both incubation and electroporation (Figure S2) in B. saltans cells. Incubation of B. saltans with PNAs became our protocol of choice for subsequent B. saltans and Cbv targeting experiments.

2.2 Antisense Inhibition of Host and Symbiont Genes

To achieve antisense inhibition of B. saltans and intracellular symbiont, we designed antisense oligonucleotides spanning the start codon and ribosome binding site of three host and two symbiont-encoded genes (Good 2002). The choice was dictated by the availability of specific antibodies for the host, while for the symbiont, we targeted proteins with readily predictable function. In B. saltans, our targets were PFR2C and PFR3 (paraflagellar rod protein isoforms putatively involved in B. saltans feeding) (Gomaa et al. 2022) and tubulin. For Cbv, we used an antitoxin from one of the putative toxin–antitoxin operons (CPBP_00218 gene) and a putative cell division protein ftsZ. Bacterial antisense targeting is best achieved with 10-nucleotide-long antisense molecules (Good 2002). For B. saltans, genome complexity precludes the design of such short yet specific antisense molecules. Thus, we focused on 14 and 20 nucleotide antisense molecules. For all experiments, we have also used scrambled nontargeting oligos as a control.

First, we treated B. saltans cells with antisense PNAs for 24 and 48 h designed to silence PFR2C and PFR3 (together for Western blot, as our antibody detects both protein isoforms, and separately for qPCR as we can detect each mRNA/cDNA separately) and tubulin (Figure 2). We did not find evidence of antisense inhibition either by Western blot or by qPCR against targeted transcripts. Although protein samples were quantified and normalized for input, we observed stochasticity in PFR and tubulin protein levels. Therefore, we have additionally confirmed that our Western blots were quantitative (Figure S3) and that the differences in protein levels visible in Figure 2A reflect B. saltans biology.

Due to the lack of specific antisense effect on B. saltans cells and the stochastic B. saltans protein abundance (especially in loading controls in the above experiments [Figure 2]), we checked whether the cell numbers of B. saltans were altered upon antisense PNA treatment (Figure 3). To our surprise, B. saltans proliferated massively in culture upon antisense PNA addition. Although the strength of the effect depended on the oligonucleotide length and sequence, it was universal for the B. saltans and Cbv-targeting and nontargeting PNAs.

4.1 B. saltans Culture

B. saltans was cultured in a cerophyl-based medium enriched with 3.5 mM sodium phosphate dibasic (Na₂HPO₄) (Gomaa et al. 2022). Cultures were incubated at 22°C in T25 tissue culture flasks containing 20 mL of media bacterized with K. pneumoniae subsp. pneumoniae (ATCC 700831). 3–4-day-old cultures were used for experiments.

4.2 Antisense Molecules

The intended mode of action of our antisense constructs was physical protection of the targeted mRNA from being subjected to posttranscriptional processing and/or translation of proteins. PNAs are synthetic DNA mimics in which the deoxyribose phosphate backbone is replaced by a pseudo-peptide polymer (N-(2-aminoethyl)glycine units) to which the unmodified nucleobases are linked (Nielsen et al. 1991). PNAs hybridize with complementary DNA or RNA with high affinity and specificity (Hyrup and Nielsen 1996). Although the most recent application of PNA is as hybridization probes in fluorescent in situ hybridization (FISH) experiments, their best-described use is antisense inhibition of target genes (Good and Nielsen 1998; Pellestor and Paulasova 2004; Koppelhus and Nielsen 2003). PNAs can interfere with transcription and translation of genes by tightly binding to DNA or mRNA and are thought to act as blocking agents. In vitro studies have demonstrated PNA binding to complementary DNA, effectively inhibiting transcriptional elongation and preventing the binding of transcription factors (Boffa et al. 1996; Nielsen et al. 1994). For antisense design, we followed a previously described strategy (Dryselius et al. 2003), with short oligomers against bacterial transcripts and longer ones for eukaryotic gene blocking (Good and Nielsen 1998). For phosphorothioate antisenses targeting B. saltans genes, we followed the design of antisense oligos for the related trypanosomatid T. cruzi (Málaga and Yoshida 2001). We used phosphorothioate antisenses and 2'-O-Methyl (2'OMe) RNA-DNA design with modified nucleotides at the extremities and inside the sequence to protect the construct from both exo- and endonucleases (Lohman 2024). Antisense PNAs were synthesized by Panagene, South Korea. Other oligonucleotides were synthesized by IDT. All antisense oligonucleotides were ordered with HPLC purification and > 90% purity and subjected to the manufacturer's quality checks.

4.3 Incubation of B. saltans Cells With Antisense Molecules

B. saltans cultures were filtered through 100 and 8 µm filters. Cells were harvested by centrifugation at 1200 × g for 12 min at 19°C, washed with 10–15 mL sterile filtered (SF) 1× PBS, and cells re-suspended in 5 mL SF 1× PBS. Single treatment cell aliquots containing 5 × 10⁵ cells were centrifuged again, and PBS was replaced with SF cerophyll medium. Antisense molecules were added in a final concentration of 50 µM. After 24 h and 48 h, B. saltans cells were harvested for Western blot and qPCR. The detailed protocol can be found here: dx.doi.org/10.17504/protocols.io.6qpvr8pqblmk/v1.

4.4 Electroporation of Fluorescent Antisense Molecules Into B. saltans and Live Imaging

We electroporated antisense molecules into B. saltans cells according to the published protocol (https://www.protocols.io/view/electroporation-of-fluorescent-antisense-molecules-e6nvwk8e2vmk/v1). To prepare B. saltans cells for electroporation, the culture was first filtered through 100 µm and 8 µm filters. The cells were then harvested by centrifugation at 1200 × g for 12 min at 19°C. Following this, the cells were washed with 10 mL PBS and centrifuged again under the same conditions. The cells were resuspended in 5 mL PBS, counted using a hemacytometer, and the volume containing 5 × 10⁵ cells was taken as recommended for the Neon transfection kit (Thermo Fisher Scientific) with a 10 µL tip. The cells were centrifuged once more at 1200 × g for 12 min at 19°C. After removing the PBS, the cells were resuspended in electroporation buffer. An antisense molecule was then added to the B. saltans cells at a final concentration of 50 µM and mixed by pipetting. Finally, the mixture was aspirated into a Neon pipette and electroporated with a single pulse at 1800 V with a 10 ms pulse width.

The antisense molecule concentration was chosen based on our preliminary experiments showing that the concentration used in the literature (20 µM) was not effective in our system (Málaga and Yoshida 2001; Kurupati et al. 2007). Given our experimental design, this was also the highest achievable antisense molecule concentration, in the range of concentrations used for other experiments involving trypanosomatids (Hashimoto et al. 2014; Málaga and Yoshida 2001) and 10× to 100× more than in previous experiments showing antisense inhibition in bacteria (Good and Nielsen 1998).

4.5 Imaging

To image B. saltans cells, we mixed, incubated, or electroporated cells with 1% low melting temperature agarose (Thermo Fisher Scientific) in a 1:1 ratio and let it set for a few seconds in a well of a 96-well plate. We stained DNA with Hoechst 33342 (Thermo Fisher Scientific) diluted 1:2000 in PBS by adding the solution to the agarose-embedded B. saltans and incubating it for 10 min at room temperature (RT). We washed the agarose with PBS (2 × 5 min) at RT. We removed the agarose from the well with clean forceps, placed it on a microscope slide, added a drop of mounting medium (Vectashield, Vector Laboratories), and flattened the agarose using the coverslip. We proceeded with confocal imaging at the Centre for Cell Imaging using an LSM 880 Laser Scanning Confocal (Zeiss) equipped with a 63× (1.4 NA) objective, and parameters listed in Table S1. Because the bright-field images were used to localize the B. saltans and for general reference only, the acquisition and display parameters were adjusted individually for each image. Images were analyzed using custom Fiji (Schindelin et al. 2012) Script provided by Marie Held at the Centre for Cell Imaging, University of Liverpool. The protocol for analysis can be found here: dx.doi.org/10.17504/protocols.io.6qpvr8pqblmk/v1, and the script for automation of analysis can be found here: https://github.com/Marien-kaefer/General_Fiji_macros/tree/main/BodoSaltans.

4.6 Western Blot

We performed Western blots according to the published protocol (https://www.protocols.io/view/protein-extraction-quantification-and-western-blot-5qpvobqodl4o/v2). Cells were harvested as above, and protein was extracted according to Newton et al. (2015). Subsequently, protein was quantified with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. All concentrations were normalized to those of the most diluted samples. Samples were reduced, separated on polyacrylamide gels (Bolt Bis-Tris Plus, Thermo Fisher Scientific), and then transferred to PVDF membranes using a mini blot module (Thermo Fisher Scientific). The membranes were blocked for 30 min at RT in 5% skimmed milk diluted in wash buffer (PBS with 0.1% Tween 20). Subsequently, the membranes were incubated overnight at 4°C with primary antibodies (Thermo Fisher Scientific, diluted 1:100 in 5% milk in wash buffer, Table 2). After incubation, the membranes were washed three times for 10 min each with wash buffer. They were then incubated for 1 h at RT with horseradish peroxidase (HRP)-labeled secondary antibodies (diluted 1:5000 in 5% milk in wash buffer). The membranes were washed again three times for 10 min each with wash buffer. Finally, the signal was developed using an HRP substrate (e.g., Immobilon western kit, Merck) and scanned for chemiluminescent signal using an imaging system (e.g., ImageQuant LAS4000 or Bio-Rad Chemidoc). The exposure time was chosen for each membrane separately to the highest value possible while avoiding saturated pixels, and results were saved as 16-bit images.

4.7 RNA Extraction, cDNA Synthesis, and qPCR

B. saltans cells were homogenized in Trizol Reagent (Invitrogen) and RNA was extracted using a Direct-zol RNA MiniPrep kit (Zymo Research), including a DNAse digestion step according to the manufacturer's instructions. RNA was eluted in 25 µL DNase/RNase-Free Water (Zymo Research), and the concentration was determined using a NanoDrop Spectrophotometer. cDNA was prepared from 1 µg of total RNA using Random Primers and M-MLV Reverse Transcriptase (both Promega). Primers were allowed to bind to template RNA for 5 min at 70°C, followed by 25°C for 10 min before M-MLV was added and incubated at 37°C for 60 min and then 80°C for 10 min.

Real-time qPCRs were carried out in a LightCycler 480 Instrument II (Roche) with the Power SYBR Green PCR Master Mix (ThermoFisher Scientific). Each reaction contained 6 µL of SYBR Green PCR Master Mix, 0.5 µL of each primer solution at 3.6 µM, and 5 µL of diluted DNA. Each plate contained three technical replicates of every sample for each set of primers. Primers used are listed in Table 3. Relative expression levels were calculated by the Pfaffl method (Pfaffl 2001), with PFR2C relative to tubulin and tubulin relative to actin.