Structural Insights into the Cytotoxic Mechanism of Vibrio parahaemolyticus PirAvp and PirBvp Toxins.
ABSTRACT: In aquaculture, shrimp farming is a popular field. The benefits of shrimp farming include a relatively short grow-out time, high sale price, and good cost recovery. However, outbreaks of serious diseases inflict serious losses, and acute hepatopancreatic necrosis disease (AHPND) is an emerging challenge to this industry. In South American white shrimp (Penaeus vannamei) and grass shrimp (Penaeus monodon), this disease has a 70-100% mortality. The pathogenic agent of AHPND is a specific strain of Vibrio parahaemolyticus which contains PirAvp and PirBvp toxins encoded in the pVA1 plasmid. PirAvp and PirBvp have been shown to cause the typical histological symptoms of AHPND in infected shrimps, and in this review, we will focus on our structural understanding of these toxins. By analyzing their structures, a possible cytotoxic mechanism, as well as strategies for anti-AHPND drug design, is proposed.
Project description:Acute hepatopancreatic necrosis disease (AHPND) is a newly emergent penaeid shrimp disease which can cause 70-100% mortality in Penaeus vannamei and Penaeus monodon, and has resulted in enormous economic losses since its appearance. AHPND is caused by the specific strains of Vibrio parahaemolyticus that harbor the pVA1 plasmid and express PirAvp and PirBvp toxins. These two toxins have been reported to form a binary complex. When both are present, they lead to the death of shrimp epithelial cells in the hepatopancreas and cause the typical histological symptoms of AHPND. However, the binding mode of PirAvp and PirBvp has not yet been determined. Here, we used isothermal titration calorimetry (ITC) to measure the binding affinity of PirAvp and PirBvp. Since the dissociation constant (Kd = 7.33 ± 1.20 ?M) was considered too low to form a sufficiently stable complex for X-ray crystallographic analysis, we used alternative methods to investigate PirAvp-PirBvp interaction, first by using gel filtration to evaluate the molecular weight of the PirAvp/PirBvp complex, and then by using cross-linking and hydrogen-deuterium exchange (HDX) mass spectrometry to further understand the interaction interface between PirAvp and PirBvp. Based on these results, we propose a heterotetrameric interaction model of this binary toxin complex. This model provides insight of how conformational changes might activate the PirBvp N-terminal pore-forming domain and should be helpful for devising effective anti-AHPND strategies in the future.
Project description:Acute hepatopancreatic necrosis disease (AHPND) is a lethal shrimp disease. The pathogenic agent of this disease is a special Vibrio parahaemolyticus strain that contains a pVA1 plasmid. The protein products of two toxin genes in pVA1, pirAvp and pirBvp, targeted the shrimp's hepatopancreatic cells and were identified as the major virulence factors. However, in addition to pirAvp and pirBvp, pVA1 also contains about ~90 other open-reading frames (ORFs), which may encode functional proteins. NCBI BLASTp annotations of the functional roles of 40 pVA1 genes reveal transposases, conjugation factors, and antirestriction proteins that are involved in horizontal gene transfer, plasmid transmission, and maintenance, as well as components of type II and III secretion systems that may facilitate the toxic effects of pVA1-containing Vibrio spp. There is also evidence of a post-segregational killing (PSK) system that would ensure that only pVA1 plasmid-containing bacteria could survive after segregation. Here, in this review, we assess the functional importance of these pVA1 genes and consider those which might be worthy of further study.
Project description:Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of Vibrio parahaemolyticus that have acquired a "selfish plasmid" encoding the deadly binary toxins PirAvp/PirBvp To better understand the repertoire of virulence factors in AHPND-causing V. parahaemolyticus, we conducted a comparative analysis using the genome sequences of the clinical strain RIMD2210633 and of environmental non-AHPND and toxic AHPND isolates of V. parahaemolyticus Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1), which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer to AHPND-causing V. parahaemolyticus a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we found highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control.IMPORTANCE Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Vibrio parahaemolyticus Although the plasmid-encoded binary toxins PirAvp/PirBvp have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in V. parahaemolyticus and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND, and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to what was seen with the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections.
Project description:We report the first draft genome sequence of an acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus strain isolated from a Penaeus vannamei sample from the Philippines. The strain carries the genes encoding the Pir-like toxin pair PirAvp and PirBvp.
Project description:Acute hepatopancreatic necrosis disease (AHPND) has caused sharp declines in aquaculture industries of whiteleg shrimp Penaeus vannamei in Asia and the Americas since 2010. Vibrio parahaemolyticus, V. campbellii, V. owensii, and V. punensis have been proved to cause AHPND. However, the mechanisms underlying the burgeoning number of Vibrio species that cause AHPND is not known. All of AHPND-causing Vibrio bacteria (V AHPND) harbor a highly homologous plasmid (designated as pVA1-type) carrying pirAB vp toxin genes. In this study, we demonstrate conclusively that the pVA1-type plasmid can be transferred from V AHPND to non-pathogenic bacteria. We constructed a pVPGX1-Cm r plasmid (a pVA1-type plasmid) by adding a chloramphenicol resistance gene as a marker in a donor AHPND-causing V. parahaemolyticus 20130629002S01 (Vp2S01). Horizontal transfer of this plasmid was successfully performed from the AHPND-Vp2S01 to a non-pathogenic strain of V. campbellii at the transfer efficiency of 2.6×10-8 transconjugant/recipient, and DNase I treatment did not eliminate the transfer. The recipient V. campbellii acquired the pVA1-type plasmid and was shown to produce pirAB vp RNA and proteins. Challenge studies using the transconjugant caused 100% mortality in exposed groups of P. vannamei. The challenged shrimp, infected with the transconjugant bacteria, showed typical gross signs and histological lesions of AHPND. These results demonstrated the conjugative transfer of an AHPND pVA1-type plasmid. It provides timely information for explaining the increased species of AHPND-causing Vibrio bacteria and will be useful in the development of management strategies leading to the prevention and control of AHPND.
Project description:Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirA Vp and PirB Vp These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirA Vp but carried pirB Vp Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB Vp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirA Vp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirA Vp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirA Vp gene sequence and in the downstream pirB Vp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirAB Vp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no Pir Vp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions.IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor the pVA1 plasmid encoding toxins PirA Vp and PirB Vp The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ?50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VPAHPND Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VPAHPND Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.
Project description:Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA(vp) and PirB(vp)) proteins and found that the overall structural topology of PirA(vp)/PirB(vp) is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB(vp) heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB(vp) may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.
Project description:The acute hepatopancreatic necrosis disease (AHPND) of Penaeus vannamei shrimp is caused by Vibrio parahaemolyticus carrying toxin genes, pirA and pirB We report the complete genome sequence of the novel V. parahaemolyticus strain R14, which did not display AHPND symptoms in P. vannamei despite containing the binary toxin genes.
Project description:Vibrio parahaemolyticus is an important foodborne pathogen and has recently gained particular notoriety because it causes acute hepatopancreatic necrosis disease (AHPND) in shrimp, which has caused significant economic loss in the shrimp industry. Here, we report a whole-genome analysis of 233 V. parahaemolyticus strains isolated from humans, diseased shrimp, and environmental samples collected between 2008 and 2017, providing unprecedented insight into the historical spread of AHPND. The results show that V. parahaemolyticus is genetically diverse and can be divided into 84 sequence types (STs). However, genomic analysis of three STs of V. parahaemolyticus identified seven transmission routes in Asia since 1996, which promoted the transfer of an AHPND-associated plasmid. Notably, the insertion sequence (ISVal1) from the plasmid subsequently mediated the genetic exchange among V. parahaemolyticus STs and resulted in the deletion of an 11-kb region regulating cell mobility and the production of capsular polysaccharides. Phenotype assays confirmed that this deletion enhanced biofilm formation, providing a novel mechanism for environmental adaptation. We conclude that the transmission mode of AHPND consists of two steps, the transmission of V. parahaemolyticus and the subsequent horizontal transfer of the AHPND-associated plasmid. This plasmid allows ISVal1 to mediate genetic exchange and improve pathogen fitness in shrimp ponds. Current shrimp farming practices promoted such genetic exchanges, which highlighted a risk of the emergence of new virulent populations, with potentially devastating consequences for both aquaculture and human health. This study addressed the basic questions regarding the transmission mechanism of AHPND and provided novel insights into shrimp and human disease management.IMPORTANCE Global outbreaks of shrimp acute hepatopancreatic necrosis disease (AHPND) caused by V. parahaemolyticus represent an urgent issue for the shrimp industry. This study revealed that the transmission mode of AHPND consists of two steps, the transregional dissemination of V. parahaemolyticus and the horizontal transfer of an AHPND-associated plasmid. Surprisingly, the introduction of the AHPND-associated plasmid also offers a novel mechanism of genetic exchange mediated by insertion sequences, and it improved the fitness of V. parahaemolyticus in a harsh environment. The results presented herein suggest that current shrimp farming practices promote genetic mixture between endemic and oceanic V. parahaemolyticus populations, which introduced the plasmid and accelerated bacterial adaptation by the acquisition of ecologically important functions. This entails a risk of the emergence of new virulent populations both for shrimp and humans. This study improves our understanding of the global dissemination of the AHPND-associated plasmid and highlights the urgent need to improve biosecurity for shrimp farming.
Project description:Acute hepatopancreatic necrosis disease (AHPND) is a severe shrimp disease originally shown to be caused by virulent strains of Vibrio parahaemolyticus (VPAHPND). Rare cases of AHPND caused by Vibrio species other than V. parahaemolyticus were reported. We compared an AHPND-causing V. campbellii (VCAHPND) and a VPAHPND isolate from the same AHPND-affected pond. Both strains are positive for the virulence genes pirABvp . Immersion challenge test with Litopenaeus vannamei indicated the two strains possessed similar pathogenicity. Complete genome comparison showed that the pirABvp -bearing plasmids in the two strains were highly homologous, and they both shared high homologies with plasmid pVA1, the reported pirABvp -bearing plasmid. Conjugation and DNA-uptake genes were found on the pVA1-type plasmids and the host chromosomes, respectively, which may facilitate the dissemination of pirABvp . Novel variations likely driven by ISVal1 in the genetic contexts of the pirABvp genes were found in the two strains. Moreover, the VCAHPND isolate additionally contains multiple antibiotic resistance genes, which may bring difficulties to control its future outbreak. The dissemination of the pirABvp in non-parahaemolyticus Vibrio also rises the concern of missing detection in industrial settings since the isolation method currently used mainly targeting V. parahaemolyticus. This study provides timely information for better understanding of the causes of AHPND and molecular epidemiology of pirABvp and also appeals for precautions to encounter the dissemination of the hazardous genes.