Genomics

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Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics


ABSTRACT: Recent studies revealed that several vibrio species have evolved the capacity to survive inside host cells. However, it is still often ignored if intracellular stages are required for pathogenicity. Virulence of Vibrio tasmaniensis LGP32, a strain pathogenic for Crassostrea gigas oysters, depends on entry into hemocytes, the oyster immune cells. To identify the mechanisms involved in LGP32 resistance to the hemocyte killing machinery and phagocytosis-dependent cytotoxicity, we used a global comparative RNAseq approach early after vibrio entry into hemocytes (1h after phagocytosis). Transcripts of intracellular LGP32 were obtained from hemocyte monolayers and their relative level of expression was compared to that of transcripts of extracellular LGP32 obtained from vibrios kept in SSW alone. LGP32 was grown at 20°C in Zobell medium for 10h and washed twice in SSW by centrifugations (10min, 1000g, 20°C). Bacteria were then600 of 0.5 (1.10^9 cfu/mL equivalent). Hemolymph was collected from the adductor muscle of oysters using a 2 mL syringe with a 23-G needle. Freshly collected hemolymph was dispensed in a 6-well plate to obtain monolayers of 6 × 106 hemocytes per well. One hour after plating, vibrios were added at a multiplicity of infection of 100:1, and plates were centrifuged for 5min at 400g for binding synchronization. After 1h of co-incubation, wells were washed extensively three times with SSW to remove extracellular bacteria and 500 µl Trizol reagent (Invitrogen) was added to every well for total RNA extraction. The efficiency of vibrios internalization in hemocytes was verified by microscopy showing that about 40% of hemocytes internalized about 50 to 70 bacteria, with rare bacteria remaining extracellular. As a control, washed bacteria were incubated in triplicates in SSW for 1h and resuspended in Trizol after centrifugation. RNA from three independent experiments of LGP32 phagocytosis by hemocytes were extracted using the Trizol reagent protocol (Life Technologies). RNA concentration was measured using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific Inc.) and RNA quality was monitored by agarose gel electrophoresis and a 2100 Bioanalyzer (Agilent Technologies Inc.). The bioanalyzer analysis showed that bacterial RNAs represented approximately 10% of the total RNA in the intrahemocyte samples. Accordingly, control samples were prepared by mixing total RNA extracted from LGP32 incubated in SSW with C. gigas hemocyte RNA with a ratio of 1:9. For each sample, 7 µg of starting material was treated with DNaseI (4 U) (Ambion’s DNA-free™) following the manufacturer instructions. Samples were then enriched in bacterial RNA using the MICROBEnrich™ Kit (Ambion) and bacterial rRNA were then removed by the MICROBExpress™Kit (Ambion) following the manufacturer instructions. Because the MICROBEnrich™ Kit is based on oligonucleotides that are designed to capture rRNAs from mammal species, it is not fully efficient to remove invertebrate rRNAs. Accordingly, we performed a further depletion step using a 5’-phosphate-dependent exonuclease (Terminator, Epicentre), that degrades processed transcripts, following instructions from the manufacturer. For cDNA sequencing, for each sample, a directional cDNA library was constructed and sequenced on an illumina Hiseq 1000, in paired-end reads of 2x100bp. 3 samples were multiplexed per lane giving ~ 60 x 10^6 reads per sample. Out of 50-60 M reads obtained from the RNA-seq, 1.2 M reads from the SSW samples and 2.3 M reads from the intrahemocyte samples were successfully mapped onto the genome of LGP32.

ORGANISM(S): Vibrio tasmaniensis

PROVIDER: GSE73260 | GEO | 2015/12/01

SECONDARY ACCESSION(S): PRJNA296479

REPOSITORIES: GEO

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