Comparative Transcriptomics of the pVA1 Plasmid in Vibrio parahaemolyticus, the Causative Agent of Acute Hepatopancreatic Necrosis Disease in Shrimp, Across Different Nutrient Conditions

Vibrio parahaemolyticus strain M0904 harbors the pVA1 plasmid, which encodes virulence factors, including the Bacillus thuringiensis Cry (δ-endotoxin)-like binary toxins PirA and PirB, responsible for acute hepatopancreatic necrosis disease (AHPND) in shrimp. This study evaluated genome-wide pVA1 expression across three culture media differing in nutrient availability (Tryptic Soy Broth [TSB], M9 minimal medium [MM9], and seawater + glucose) using RNA-seq. Cultures were harvested at different times and growth phases (TSB at 4 h, exponential; MM9 and seawater at 6 h, early plateau), so the medium is partly confounded with growth state. pVA1 genes were highly expressed (1403.93 TPM, the arithmetic mean of the three per-medium means) with no significant plasmid-level difference between media in any analysis. The toxin–antitoxin genes (hokC/pndA and hok/gef) were the most highly expressed, while pirA and pirB were highly expressed relative to housekeeping genes (4.7- to 161.0-fold and 9.11- to 124.59-fold higher, respectively; 34.3- and 38.0-fold in TSB without the outlier library TSBr4). A difference in pirAB between media (lower in seawater than in TSB, adjusted p < 0.01) was detected only when TSBr4, a genome-wide outlier library, was retained; it was absent without that library (adjusted p ≥ 0.12), and no difference was detected between MM9 and seawater, which were harvested at the same time. Among ten secretion-related loci identified in the genome, a chromosome II Type I Secretion System locus (T1SSa(i): mean 69–158 TPM across media) and the chromosome I Type II Secretion System core were both expressed in all three media. T1SSa(i) is proposed as a candidate PirAB export route because PirA and PirB lack a cleavable Sec/Tat signal peptide, which makes the also-constitutive T2SS an unlikely route; the flagellar type III export apparatus meets the same criteria and cannot be excluded by transcriptomic data alone. Both are hypotheses that require genetic and proteomic confirmation.