Strains and growth conditions. Organisms used in this study are listed in Supplementary Table 1. Bacterial strains were routinely grown in lysogeny broth (LB) and on LB agar plates. V. cholerae mutants were constructed by splicing by overlap extension PCR18 and natural transformation19. Complementation was done using the expression vector pBAD24. Bacteria carrying the expression vector were grown in LB broth at 37ºC containing ampicillin 100 µg ml− 1 and, 0.2% arabinose for gene expression.
T. pyriformis was routinely passaged in 10 ml growth medium containing peptone-yeast-glucose (PYG) (20 g l− 1 proteose peptone, 1 g l− 1 yeast extract) supplemented with 0.1 × M9 minimal medium (6 g l− 1 NaH2PO4, 3 g l− 1 K2PO4, 0.5 g l− 1 NaCl, 1 g l− 1 NH4Cl) and 0.1 M sterile-filtered glucose in 25 cm2 tissue culture flasks with ventilated caps (Sarstedt Inc., Nümbrecht, Germany) and incubated statically at room temperature (RT).
Prior to experiments, 250 µl of T. pyriformis were passaged in 10 ml of ASW medium (5.6 g l− 1 NaCl, 0.470 g l− 1 Na2SO4, 0.026 g l− 1 NaHCO3, 0.08 g l− 1 KCl, 0.013 g l− 1 KBr, 0.600 g l− 1 MgCl2.6H2O, 0.130 g l− 1 CaCl2.2H2O, 0.002 g l− 1 SrCl2.6H2O and 0.002 g l− 1 H3BO3) supplemented with 1% heat- killed Pseudomonas aeruginosa PAO1 (HKB) in a 25 cm2 tissue culture flask, and further incubated at RT statically for 24 h before enumeration and use. This process is necessary to remove the nutrient media and to acclimatise the ciliate to phagotrophic feeding.
To prepare heat-killed bacteria (HKB), P. aeruginosa was grown overnight in LB at 37°C with shaking at 200 rpm and adjusted to (OD600 = 1.0; 109 cells ml− 1) in ASW. The tubes were then transferred to a water bath at 65°C for 2 h, and then tested for viability by plating on LB agar plates at 37°C for 2 d. HKB stocks were stored at -20°C.
Co-incubation of V. cholerae and T. pyriformis. V. cholerae A1552 was co-incubated with T. pyriformis in ASW. Briefly, T. pyriformis were enumerated by microscopy and adjusted to 103 cells ml− 1 and added to co-cultures of V. cholerae A1552 adjusted to 108 cells ml− 1 in ASW using a spectrophotometer (OD600 nm). Co-incubation was performed for 5 h at RT, 60 rpm. After incubation, TritonX-100 was added at a final concentration of 1% to release intracellular and EFVs-encased bacteria. Cells were centrifuged for 5 min (3220 × g) at 4°C, and the cell pellet was resuspended in a freshly made 4% ice-cold paraformaldehyde solution. Fixed cells were incubated overnight at 4°C.
Cell permeabilization. Fixed cells were centrifuged (5500 × g) for 10 min. All centrifugation steps were performed at 5500 × g, 4°C, 10 min. After centrifugation, cells were washed with 1 ml of 0.1 M Tris-HCl pH7 (Sigma) and centrifuged again. Cells were then resuspended in 250 µl of cold-PBS (pH 7.4, Sigma) supplemented with RNAase inhibitors (Enzymatics RI and Superase-IN) as indicated by the MicroSPLiT protocol8. Following resuspension, 250 µl of 100% cold ethanol was added and incubated for 1 min at RT. Cells were again centrifuged and resuspended in a solution containing 0.04% Tween20 and 50 mM EDTA. The suspension was incubated for 3 min on ice. Following incubation, 750 µl of cold PBS supplemented with RNAase inhibitors was added. After centrifugation, cells were resuspended in 100 µl of a lysozyme buffer (0.1 M Tris pH7, 50 mM EDTA, 1 mg ml− 1 lysozyme) and incubated at RT for 15 min. Permeabilization was assessed by mixing 50 µl of permeabilized cells with Ovalbumin Alexa Fluor 488 conjugate (Thermofisher), following manufacturer’s recommendations.
In situ cDNA barcoding and NGS library preparation. In situ polyA tailing, reverse transcription, ligation barcoding and library preparation were performed following the steps published in the MicroSPLiT protocol8.
Data processing. Single-cell gene expression matrices were obtained by mapping the cDNA reads (Read 1) to the reference genome of V. cholerae O1 El Tor strain N16961 using STAR aligner (v2.7.10b) with addition of several STARsolo parameters (--genomeDir, --readFilesIn, --soloType, --soloCBposition, --soloUMIposition, --soloCBwhitelist, --soloCBmatchWLtype, --soloUMIdedup, --soloFeatures, --soloMultiMappers)20. The resulting matrices were analysed using standard workflow of the R package Seurat (v4.3.0) with default parameters except otherwise indicated21. The data were log-transformed using the ′NormalizeData(normalization.method = ‘LogNormalize’, scale.factor = 10000)′ function. Five hundred most variable genes were selected using ′FindVariableFeatures(selection.method = ‘vst’, nfeatures = 500)′ and z-scored using ′ScaleData′. Linear dimensional reduction was performed using principal component analysis (PCA). Louvain clustering algorithm was used to generate clusters using the function ′FindNeighbors(dims = 1:10)′ and ′ FindClusters(resolution = 0.5)′. Uniform manifold approximation and projection (UMAP) non-linear dimensional reduction techniques was used to visualize and explore the datasets using ′RunUMAP(dims = 1:10)′ function. Differential markers in each cluster was identified using the ′FindAllMarkers(logfc.threshold = 0.2)′ function. The average expression of top ten differentially expressed markers in each cluster were used to generate a heatmap using the ′DoHeatMap′ function. FeaturePlot function was used to visualize specific marker expression in the clustered population.
Killing of T. pyriformis. To assess the number of dead T. pyriformis, co-incubations were performed as described before. Briefly, V. cholerae and T. pyriformis were co-incubated in ASW for 5 h at RT. After approximately 5 h, dead cells accumulated at the bottom of the well and were counted using an inverted epifluorescence microscope (Nikon Eclipse Ti inverted microscope).
Survival of V. cholerae in EFVs. To produce EFVs, V. cholerae strains, ΔlacZ (WT) and mutant, were co-incubated with T. pyriformis independently in ASW. Briefly, T. pyriformis were enumerated by microscopy and adjusted to 103 cells ml− 1 and added to co-cultures of V. cholerae adjusted to 108 cells ml− 1 in ASW using a spectrophotometer (OD600 nm). After overnight incubation at RT, samples were analyzed using an inverted epifluorescence microscope (Nikon Eclipse Ti inverted microscope) to detect the presence of EFVs in the supernatant. To purify V. cholerae-EFVs, supernatants were filtered (by gravity) several times through 8 µm filters (Millipore) and the filters containing EFVs suspended in 1 ml ASW. The EFVs were incubated for 1 h with gentamicin 300 µg ml− 1 at RT to kill any remaining extracellular bacteria. After gentamicin treatment, V. cholerae-EFVs pellets were collected by centrifugation (3220 × g for 20 min), washed three times in ASW and suspended in 1 ml of ASW. Finally, Triton X-100 was added to a final concentration of 1% to lyse the EFVs and release bacterial cells. The lysed EFV aliquots (WT and mutant) were mixed 1:1 and plated on LB supplemented with X-Gal (60 µg ml− 1) to differentiate between the V. cholerae ΔlacZ (white colonies) and the mutant strains (blue colonies).
Data analysis. Statistical analysis was performed using GraphPad Prism version 8.4.3 for Windows, GraphPad Software, La Jolla California USA, (www.graphpad.com). Data that did not follow Gaussian distribution was determined by analysing the frequency distribution through the Saphiro-Wilk normality test. Two-tailed student’s t-tests were used to compare means between experimental samples and controls using Welch’s t test correction. For experiments including multiple samples, one-way ANOVA was used for the analysis and using the Kruskal-Wallis test to compare the medians of non-normally distributed data, and Tukey’s test to compare the average of normally distributed data.