Environmental conditions steer phenotypic switching in acute hepatopancreatic necrosis disease‐causing Vibrio parahaemolyticus, affecting PirA^VP/PirB^VP toxins production

Abstract

Bacteria in nature are widely exposed to differential fluid shears which are often a trigger for phenotypic switches. The latter mediates transcriptional and translation remodelling of cellular metabolism impacting among others virulence, antimicrobial resistance and stress resistance. In this study, we evaluated the role of fluid shear on phenotypic switch in an acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus M0904 strain under both in vitro and in vivo conditions. The results showed that V. parahaemolyticus M0904 grown at lower shaking speed (110 rpm constant agitation, M0904/110), causing low fluid shear, develop cellular aggregates or floccules. These cells increased levan production (as verified by concanavalin binding) and developed differentially stained colonies on Congo red agar plates and resistance to antibiotics. In addition, the phenotypic switch causes a major shift in the protein secretome. At 120 rpm (M0904/120), PirA VP /PirB VP toxins are mainly produced, while at 110 rpm PirA VP / PirB VP toxins production is stopped and an alkaline phosphatase (ALP) PhoX becomes the dominant protein in the protein secretome. These observations are matched with a very strong reduction in virulence of M0904/110 towards two crustacean larvae, namely, Artemia and Macrobrachium. Taken together, our study provides substantial evidence for the existence of two phenotypic forms in AHPND V. parahaemolyticus strain displaying differential phenotypes. Moreover, as aerators and pumping devices are frequently used in shrimp aquaculture facilities, they can inflict fluid shear to the standing microbial agents. Hence, our study could provide a basis to understand the behaviour of AHPND-causing V. parahaemolyticus in aquaculture settings and open the possibility to monitor and control AHPND by steering phenotypes.

Biofloc-Based Enhanced Survival of Litopenaeus vannamei Upon AHPND-Causing Vibrio parahaemolyticus Challenge Is Partially Mediated by Reduced Expression of Its Virulence Genes

“…parahaemolyticus) at 12 and 24 h post treatment. This observation suggests that the biofloc environment indeed facilitates phenotypic switching in V. parahaemolyticus leading to a decreased transcription of virulence related genes and increased expression of phenotype switching marker gene, AlkPhoX gene (Kumar et al, 2020). To study further the transcriptional modifications occurring in biofloc system, the in vivo temporal expression of flagellarelated motility genes, VP AHPND plasmid related virulent genes and phenotype switching marker gene of V. parahaemolyticus AHPND strain from control shrimp (SW group) in clear seawater (SW to SW; +V.…”

“…The expressions of 3 flagella-related motility genes, including regulators, structural, and chemotaxis genes, 2 VP AHPND plasmid related virulent genes including toxin and coding genes of virulent plasmid and AlkPhoX gene, marker gene that indicates the presence of non-virulent AHPND V. parahaemolyticus phenotype, were measured by RT-qPCR with pair of specific primers using StepOnePlus Real-time PCR systems (Applied Biosystems; Supplementary Table S1; Han et al, 2015;Yang and Defoirdt, 2015;Kumar et al, 2020). The Ct values from the two reference genes rpoA (RNA polymerase A submit) and toxR mRNA, used as the internal control, were subjected to geomean and expression of the genes was calculated relative to the rpoA and toxR mRNA levels.…”

“…Based on observations on the one hand of V. parahaemolyticus phenotype switching in altered environmental conditions (Kumar et al, 2020) and results of Hostins et al (2019) on the other hand on protective potential of biofloc system against AHPND-causing V. parahaemolyticus, it is hypothesized that a biofloc environment is involved in setting the phenotypic status of AHPND-causing V. parahaemolyticus under both in vitro and in vivo conditions, altering its virulence. This hypothesis was verified using a biofloc-based L. vannamei and V. parahaemolyticus model.…”

Effects of salinity and shaking condition on the growth and virulence of Vibrio parahaemolyticus

“…So far, while hemolysins such as thermostable direct hemolysin (tdh) and TDHrelated hemolysin (trh) are usually characterized as important virulence factors of V. parahaemolyticus in human enteritis, plasmidencoded binary toxins PirA vp /PirB vp are the noted ones for AHPND (Letchumanan et al, 2014). Besides, it is well noted that host and environmental factors have strong impact on the expression of bacterial virulence factors (Do et al, 2019;Figueroa-Angulo et al, 2012;Kumar et al, 2019). In case of V. parahaemolyticus, a marine and estuarine bacterium, it is no doubt that its growth as well as virulence is greatly influenced by dissolve oxygen and salinity.…”

“…Data indicated that most of the strains can grow in the range of 1-9% NaCl but have optimal salinity of 2-3% (Liu et al, 2016;Whitaker et al, 2010;Nishina et al, 2004). Dissolved oxygen or its associate, shaking condition has not been as well studied as salinity but recent data indicated that virulence of V. parahaemolyticus cultured at 120 rpm was significantly higher than at 110 rpm towards Artemia and Macrobrachium larvae (Kumar et al, 2019).…”