Toxoplasma gondii infection of fibroblasts causes the production of exosome-like vesicles containing a unique array of mRNA and miRNA transcripts compared to serum starvation

Figures & data

Fig. 1 Electron microscopy of vesicle preparations. SS and Toxo vesicle-enriched samples were negative stained and revealed extracellular vesicles of approximately 80–90 nm, the correct dimensions for exosomes.

 mRNA expression in ELVs comparing vesicular mRNA at 24- and 72-hours versus whole cell lysate expression.

	ELVs fold-expression Toxo/SS		Whole cell fold-expression Toxo/SS
mRNA	72 hours	24 hours	24 hours	Identified function
Metallothionein 2A (MT2A)	130.5	47.5	2.4	Frequently observed in invasive human breast cancer (20).
Ferritin, heavy polypeptide 1 (FTH1)	87.0	38.1	−1.2	Iron binding (21).
Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1)	67.6	18.5	−1.2	Multiple functions: role in apoptosis inhibition and linked to autism (22, 23).
Protein transport protein Sec61 subunit gamma (Sec61G)	53.2	21.2	1.1	A subunit of the protein translocation channel at the endoplasmic reticulum (24).
Rab13 (RAS member)	88.6	28.5	1.3	Multiple functions: neuronal regeneration and actin cytoskeletal rearrangements (25, 26).
SET nuclear oncogene (SET)	34.7	9.0	−1.1	Proto-oncogene (27, 28).
Zinc finger protein 253 ( ZNF253)	40.3	29.8	−2.3	Unknown: Assumed DNA binding
RNA, U1 small nuclear 1	33.1	−2.7	1.1	Multiple functionality
Thymosin beta 4, X-linked (TMSB4X)	24.7	4.4	−1.4	Multiple functions: actin sequestration and neuroprotection following traumatic brain injury protection (29–31).
H2A histone family, member Z (H2AFZ)	26.3	11.0	1.4	Histone protein subunit
LLP homolog (LLPH)	5.1	17.8	1.3	Long-term synaptic facilitation (Aplysia) (32, 33).

Values are reported as fold-expression Toxo/SS.

Fig. 2 To confirm the microarray data PCR analysis was performed for highly represented genes in Toxo compared to SS ELVs. cDNA from 2 experimental replicates of SS and Toxo ELVs RNA preparations was used to confirm the presence of mRNA transcript for Metallothionein 2A (MTH2A), protein transport protein Sec61 subunit gamma (Sec61G), SET nuclear oncogene (SET), thymosin beta 4, X-linked (TMSB4X), and Rab 13. (Please see Supplementary File for primer sets). The ethidium bromide–treated PCR products were run on a 2% agarose gel and visualized with UV light. Bands of the expected base-pair size are present for these representative mRNA in the Toxoplasma-infected ELVs and are absent from the ELVs of the SS cells except for MTH2A, which shows limited expression in one of the SS samples.

Fig. 3 PCR analysis of Rab13 mRNA in Toxo compared to SS ELVs confirmed the presence of full-length transcript in ELVs from TOXO and not in SS ELVs. PCR was performed on cDNA made from SS and Toxo ELVs RNA preparations with primers that were close to full length mRNA and still maintain specificity giving an expected product size of 976 bp. Red arrows indicate location of the 100, 500 and 1,000 bp markers. In lanes 2 and 3 a band of the expected 976 bp size for full length Rab13 can clearly be seen in the ELVs from TOXO and not SS. Also present in lanes 3 and 4, as a positive control, is the 200 bp Rab13 PCR product that was used to confirm the micro array RNA data.

Table II. miRNA expression in ELVs at 24- and 72-hours versus whole cell lysate expression.

	ELVs fold-expression Toxo/SS		Whole cell fold-expression Toxo/SS
miRNA	72 hours	24 hours	24 hours	Identified function
miR-23b	4.6	10.8	−1.1	Cytoskeletal rearrangements and IL-17 regulation (41, 42).
miR-92a	7.5	8.7	1.5	Regulator of apoptosis. Associated with many cancers (43–47).
miR-595	1.3	7.3	−2.2	Upregulated in quiescence and mandibular prognathism (48, 49).
miR-125b	6.5	7.0	−1.4	Regulates cell survival. Associated with many cancers (38, 39) (40, 50) .
miR-199a-3p	3.3	7.0	−1.3	Cytoskeletal remodelling, motility and metastasis (42).
miR-125a-5p	2.3	6.5	−1.4	Activates apoptosis via p53 and other oncogenes (ERBB2) (51, 52).
miR-503	1.3	5.8	−1.2	Inhibits tumour angiogenesis and growth (53).
miR-320d	4.5	5.7	−1.3	Inhibits HSV induced Kaposi sarcoma Herpes Virus (HHV-8) mediated proliferation by inhibiting Replication and Transcription Activator (RTA) (54).
miR-1183	1.9	5.6	1.1	unknown
miR-99a-star	3.2	5.4	1.5	Inhibits cell proliferation (55, 56).

Values are reported as fold-expression Toxo/SS.

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