PirAB from Vibrio parahaemolyticus exhibits a binary toxin ac-tion with bactericidal activity and cytotoxicity in Penaeus vannamei

: PirAB is a binary protein complex secreted by specific strains of Vibrio parahaemolyticus ( Vp ) that harbor the pVA1 virulence plasmid and express PirA Vp and PirB Vp toxins. PirAB Vp causes acute hepatopancreatic necrosis disease (AHPND), a newly emergent penaeid shrimp disease that can cause 70–100% mortality and has resulted in great economic losses since its appearance. The cytotoxic effect of PirAB Vp on the epithelial cell of the shrimp hepatopancreas has been extensively reported. Our studies found that the PirB Vp subunit has lectin activity and recognizes mucin-like O glycosidic structures in the shrimp hepatopancreas. The PirA Vp subunit may have a stabilization function of the binary complex. However, we also found that Vp AHPND changes the water microbiota community structure and causes a significant reduction in several bacteria, especially Neptuniibacter spp. We propose that the PirAB vp toxin could exhibit a dual role: damage the shrimp hepatopancreas and kill surrounding bacteria. bacterial cell and how it influences the pathogenesis of AHPND. According to our studies, we suggest that Vp AHPND strains could use the T6SS1 as a selective advantage during


Introduction
In 2013, Vibrio parahaemolyticus (Vp) strains were first reported as the causal agent of the emerging shrimp disease, acute hepatopancreatic necrosis disease (AHPND) [1], a highly infectious, OIE-listed, enteric disease, threatening farmed shrimp [2]. This lethal disease of marine shrimp emerged in China in 2009, originally known as early mortality syndrome (EMS). It has been a devastating disease and has caused great economic losses in the shrimp industry of Asian countries ( [3][4][5], Mexico ( [6], South America [7], and the USA [8]. China, Thailand, and Mexico reported losses of $11, $8, and nearly $1 billion US dollars, respectively, due to AHPND between 2009 and 2016 [9]. Virulence of Vp strains (Vp AHPND) is due to a conjugative plasmid of approximately 70 kbp (pVA1) that expresses a binary PirAB toxin, homologous to the Pir toxin secreted by Photorhabdus spp., which is responsible for the characteristic lesions in the shrimp hepatopancreas ( [1,10,11]. Later, other AHPND-causing Vibrio spp. were identified from affected shrimp, including Vibrio harveyi, V. owensii, V. campbellii, and V. punensis ( [4,[12][13][14][15][16][17]. AHPND can be caused by strains of several Vibrio spp. because the toxin genes pirAB Vp reside in a conjugative plasmid, allowing for horizontal transfer between bacterial species [18][19][20]. AHPND is a disease caused by toxigenic bacteria that produce the PirAB toxin, however, as the disease progresses, there is a secondary bacterial colonization of the damaged hepatopancreas. The disease represents a special threat to the penaeid shrimp culture due to its diverse etiology, complexity, rapid pathogenesis, and the widespread nature of this disease. In addition, to date, mechanisms of the AHPND toxigenesis are far to be understood.
In this review, we present an overview of AHPND, including the disease-associated signs and the progression of histopathology lesions, the degree of virulence of Vp strains, and the current knowledge of virulent pVA1 plasmid, as well as the status of the changes in the bacterial community structure caused by Vp AHPND and possible factors that could induce or inhibit toxin production. Finally, we present new research on putative membrane receptors and potential inhibitors of the PirAB V p toxin.

Acute hepatopancreatic necrosis disease in penaeid shrimp
To date, AHPND continues to be the bacterial disease of greatest economic importance that affects the tiger shrimp (Penaeus monodon) and the Pacific white shrimp (Penaeus vannamei) [21]. Although the crustacean decapod´s hepatopancreas (Hp) has been the target organ of AHPND, the Australian red claw crayfish (Cherax quadricarinatus) is not susceptible to Vp AHPND in cohabitation bioassays [22]. The authors hypothesize that there are differences in the putative receptor binding sites between penaeid shrimp and crayfish, such as the PirAB binary toxin could not bind to the hepatopancreatic epithelium of the crayfish's receptor(s), which does not cause intoxication.
In the early life stages, shrimp are more susceptible to AHPND strains intoxication with a threshold infective density of > 10 4 CFU mL -1 [11], and there is an increased mortality rate of P. vannamei inoculated with Vp AHPND at higher salinity levels. AHPND is characterized by severe dysfunction of the shrimp Hp accompanied by clinical signs and particular histopathological changes in the acute disease stage [23]. The shrimp affected with AHPND exhibits expanded chromatophores, lethargy, anorexia, empty digestive tract, and pale to white hepatopancreas. However, these clinical signs are also commonly observed in other bacterial diseases, such as necrotizing hepatopancreatitis (NHP-B) and septic hepatopancreatic necrosis (SHPN) [24]. Hence, confirmatory diagnosis of AHPND in shrimp should include, in addition to clinical signs, i.e., the histopathological lesions observed in the acute stage of the disease, molecular detection of pirA Vp and pirB Vp genes coupled with bioassays. Diagnosis of AHPND through only pirA Vp and pirB Vp genes detection may be inadequate due to instability of these genes, as observed in strains isolated from different geographical regions [25].

Degree of virulence
The term virulence is used to determine the relative ability of a microorganism to cause disease in a susceptible host, better known as degree of pathogenicity [26]. This ability allows evaluating the virulence quantitatively; to achieve this, some mechanisms of pathogenicity are evaluated like evasion of host defense mechanisms, antibiotic resistance, severity degree of lesions, percentage of induced death, invasiveness, and toxigenic capacity. Bacterial strains also possess different degrees of virulence [27,28], which may come from phenotypic or genotypic variations. For example, the genome of a virulent Listeria monocytogenes strain contains a large number of anti-sense RNA, responsible for its virulence, contrary to the non-pathogenic L. monocytogenes strains [29].
The expression of pirA Vp and pirB Vp genes can influence the degree of virulence [5] found low pirA gene expression (< 0.4 of relative expression) from Vp AHPND strains with moderate virulence, whereas finding high gene expression (2.1 of relative expression) from less virulent strains. As pirA Vp and pirB Vp genes are located in the same operon in the virulent plasmid (pVA1) [30], theoretically, both genes must be expressed constitutively, so that strains should have a similar virulence. However, it is evident that other factors are also involved. Our studies show that the copy number of pVA1 is related to the bacterial density [31]. The PirA and PirB proteins (or the PirAB Vp complex) can be differentially secreted by the bacterial cells affecting their virulence for shrimp [3,5,32] or be affected by post-translational modifications. Through western blot was observed a wider band of PirA and PirB proteins from more virulent strains [33]. However, to date, the role of pVA1 is not clear nor that of the secreted toxins in the virulence degree of bacterial AHPND strains.
Lastly, the Vp AHPND strains have different lifestyles during experimental infections that could influence pathogenesis; whereas the more virulent Vp M0904 adhered preferably to the bottom surface of the experimental units, the less virulent Vp M0607 strain adhered to the bottom and remained suspended in the water column [34]. In Mexico, during the shrimp mortality events associated with Vp AHPND, several strains were isolated with different degrees of virulence in terms of time of death [11]. In experimental infections, using similar laboratory conditions, i.e., immersion assays with P. vannamei challenged at bacterial density of 10 6 CFU mL -1 in natural or synthetic seawater (between 8 and 35 g L -1 ), shrimp reached 100% mortality starting at 17 h post-inoculation (p.i.) to 72 h p.i. or shrimp did not reach this mortality along the whole experiment (Table 1). Our observations with Mexican strains indicate that the shrimp size in relevant during experimental infections, small shrimp are more susceptible to AHPND than larger ones, possibly the PirAB toxin is dose-dependent. As Table 1 shows, some studies did not do histopathological analysis of infected shrimp to confirm the disease development and, in certain cases, histological observations do not report AHPND or do not correspond to the acute stage of AHPND lesions. The next section will describe the progression of the histopathological lesions during AHPND.

Histopathology of AHPND
Since the first reports [41], the pathological manifestations of AHPND have been described by histopathological examinations [1,3,6,8,11,15,17,23]. Although there are currently several methods for AHPND detection (clinical signs, histopathology and molecular techniques), the histopathological analysis remains the primary method to confirm positive cases [2] and to evaluate the cytotoxic effects of the PirAB toxin [32]. AHPND causes clinical signs like lethargy, erratic swimming, empty gut, discoloration, and atrophy of Hp, and progressive tissue changes that include massive sloughing of epithelial cells of hepatopancreatic tubules [1].
Histologically, three stages (initial, acute, and terminal) have been commonly reported as part of the pathogenic course of AHPND ( Figure 1) [1,11,23]. However, because of the shrimp's capacity to tolerate the effect of PirAB toxin, a remission stage of the disease has been observed in surviving shrimp, where the E (embryonic) cells are bio indicators for this stage [34]. In the initial stage of AHPND, there is a decrease in vacuoles of the R and B cells and elongation of the epithelial cells, which signal the cell sloughing ( Figure  1a, b) in absence of the pathogenic bacteria [11]. In the acute stage, a massive sloughing of epithelial cells (R, F, and B) occurs, which accumulate in the lumen of the affected tubules (Figure 1c, d). This is the most remarkable histopathological aspect used for the clinical diagnosis of AHPND; furthermore, the mitotic activity in the E cells is absent [1,11,41].
In the terminal stage, the hepatopancreatic tubules' epithelium is entirely necrotic, dead cells are in different degrees of lysis within the lumen, and there is a proliferation of bacteria associated with the necrotic material, indicating a secondary infection (Figure 1e, f) [1,41]. The inflammatory response increases over time causing melanization, hemocytic nodules and hemocytic capsules around the affected tubules, which delimit disease progression, but spread the secondary infection. Recently, we found that surviving shrimp after 3 d post-infection (p.i.) with Vp AHPND are able to develop larger melanized necrotic lesions similarly to septic hepatopancreatic necrosis (SHPN) [34,42]. Thus, surviving shrimp can decrease the cytotoxic effect caused by the PirAB Vp toxin and enter into a remission stage of the disease. This stage is characterized by the reactivation of the mitotic activity in E cells, declination of the clinical signs associated with AHPND, and a decrease of mortality [34]. In addition, the necrotic lesions associated with the terminal stage of AHPND quickly decrease over p.i. time (Figure 2a). The histopathological evidence suggests that the development of AHPND lesions, under experimental conditions, can follow three main routes from the terminal stage to disease recovery [34]. Surviving shrimp after 5 d p.i. show different histopathological conditions ( [34,42], which include recovered shrimp, displaying normal hepatopancreas without evidence of lesions ( Figure   The development of lesions in shrimp affected with AHPND is associated with the bacterial density, degree of strain virulence [3,11,23,35], PirA and PirB toxins concentration [32], and infection time [34]. We observed a delay in the time of the acute stage lesions of AHPND in P. vannamei infected with strains Vp at 10 5 CFU mL −1 , which was dependent on the degree of virulence [34]. With the most virulent strain, the acute stage occurred in the first 4 h p.i., whereas with the less virulent strain it was 8 h later. The terminal stage was present at 24 h p.i. for both strains, but it remained until 48 h p.i. only for the most virulent strain. Shrimp displayed a marked immune response such as hemocytic infiltration, hemocytic nodules, and melanization to delimit the bacterial proliferation and damage caused by PirAB toxins. We suggest that development and persistence of the terminal stage depend on the damage degree, genetic line, and immunologic status of shrimp. Previously was reported that a minimum concentration of 10 µg g -1 of PirA and PirB toxin is necessary to induce the typical lesions of AHPND [32]; below 5 µg g -1 toxins do not cause the disease, but that concentration can induce collapse (atrophy) of the tubular hepatopancreatic epithelium [32]. The atrophied epithelium has also been observed in shrimp under experimental infections at Vp AHPND doses lower than the infective threshold (< 10 4 CFU mL -1 ) [11], with strains of low virulence [3,11,36,40], and in surviving shrimp [34]. An explanation for the atrophied epithelium of surviving shrimp could be that, under experimental infection, after the acute stage of AHPND, there is a decrease in the PirAB production/secretion, which reduces the lesions caused by the disease and favors the shrimp's survival. Likewise, we suggest that the atrophied epithelium might be the combined effect of low concentration and continuous exposure to the PirAB toxin. Studies should be carried out to quantify and to know the dynamics of toxin production of Vp AHPND during an in vivo experiment.

Virulence plasmid pVa1
AHPND is mainly caused by Vp that harbors a plasmid of ~70 kbp pVA1 containing the pirAVp and pirBVp genes, which encode a delta-endotoxin responsible for the typical lesions in shrimp Hp [1,10). The genomes of pVA1-harboring Vp revealed a large pan genome, high genetic diversity grouped into three main clades, and specific structural differences, in addition to the instability of the pirAB Vp region of the pVA1 plasmid [25]. The structural differences found in pVA1 are likely due to horizontal propagation of the plasmid to other Vibrio species [19], such as V. harveyi [4,18], V. campbellii and V. owensii [18], and V. punensis [17. The above processes might cause the appearance of new pathogenic AHPND strains and a major threat for the shrimp industry. Likewise, this variability in the structural elements could eventually influence their ability to adapt to niches, growth behavior, and virulence/pathogenesis.
Recently we observed different growth of two strains of Vp AHPND, with distinct genetic elements and virulence during an experimental infection. The most virulent strain was adhered to the bottom surface and the moderate virulent strain was present on the bottom and suspended in the column of seawater [34]. Large variability of the plasmid copy number per bacterial cell of Vp AHPND strains was registered, from 7 to 121 copies, analyzed by qPCR [30,43], and, although, it has been reported that virulence is not dependent on the copy number of pirA Vp /pirB Vp genes [33], to date, no clear evidence of their role in AHPND exists. A study using a shotgun metagenomics approach to the bottom seawater during an experimental infection with juvenile shrimp (P. vannamei) inoculated with two Vp AHPND strains registered more than one copy of the pVA1 per bacterial cell (from 1.9 to 13.5 copies per bacterial cell) throughout the experimental infection time [31]. In this study, we found that the copy number of the virulent plasmid is not dependent on the degree of virulence of the Vp AHPND strain, but dependent on the bacterial density ( Figure 3). There is a lack of studies about the variability of the plasmid copy number per bacterial cell in relation with the degree of virulence and bacterial density, and how it influences the pathogenesis of AHPND.

Changes in the microbiota of seawater
To date, there are few works about microbial communities present in the seawater of cultured shrimp ( [44,45]. Most studies have focused on bacterial community structures of the intestinal microbiota ( [46][47][48] or effects of environmental factors on the microbial community in shrimp farms [49]. Proteobacteria have dominated the penaeid shrimp gut microbiota, and the microbiome is involved in the regulation of shrimp health and disease [50]. Most studies of P. vannamei affected with AHPND are focused on the bacterial community characterization of the Hp, stomach, intestine or sediment, using 16S rRNA amplicons [51][52][53]. Toxins affect the microbial communities of their host [54], but there are few studies about the effects on the surrounding microbiota of the seawater by the presence of diseased aquatic organisms [55]. The type III secretion system (T3SS) has been suggested as the mechanism for which the PirAB Vp toxin is secreted [32]. However, the type VI secretion system (T6SS) is involved in the virulence of human pathogenic Vp strains through the secretion of effector proteins, which are toxic to surrounding bacteria [56]). The T6SS1 of a Vp AHPND isolate was functional during the challenge of P. vannamei [57].
Recently, our research team studied the changes in the water microbiome of juvenile P. vannamei inoculated with a moderate virulent Vp strain (M0607) and a highly virulent Vp strain (M0904), using the shotgun metagenomics sequencing approach [31]. We found dominance of the Proteobacteria phylum in the water according to the bacterial community associated with AHPND [58]. At the family level, Rhodobacteraceae was the most predominant taxon that has already been detected in both healthy and diseased shrimp microbiota and in the culture water [45,51,58]. Oceanospirillaceae appear related to environmental conditions [45] and Vibrionaceae are significant during AHPND infection ( [52,58].
A dominance of the Neptuniibacter complex shows high genetic variation in the initial community structure [31]. Neptuniibacter spp. are common in seawater and associated with farmed organisms [59]. A significant marked reduction was observed in the reads assigned to Neptuniibacter spp. after the inoculation of both strains, particularly with Vp M0904 from 4 h p.i. on, with a gradual increase at 48 h of Pseudoalteromonas stutzeri, Halomonas sp., and Marinobacter adhaerens.
The depletion pattern in the Neptuniibacter complex suggests that these species could be highly affected by bacterial toxins secreted from both Vp strains [60], specially the PirB Vp subunit due to its lectin-like activity [61]. The reduction in abundance of the Neptuniibacter complex suggests that bacterial competition could be mediated by the T6SS, which regulates bacterial interactions [60,62]. Some Vp AHPND strains contain active T3SS1, T6SS1, and T6SS2 [63]. We found an enrichment of the functions associated with these systems related to inoculation with Vp strains [31], and these functions are closely associated with bacterial pathogenesis [64]. The T6SS represents a complex secretion machinery and contributes to competitive survival or pathogenesis in many Gram-negative bacteria [57]. Three effector proteins of T6SS were detected only in inoculated treatments, mainly in the M0904 strain: a) the cytotoxin Hcp; b) the temperature-dependent protein that activates the T6SS according to certain environmental conditions [60]; and c) the antitoxin serine/threonine protein kinase [31], which is a type of immunity protein that protects the bacterial community against self-intoxication of effector proteins from T6SS [65]. The T6SS1 system is active under specific conditions of temperature (30 °C) and salinity (3% NaCl), which were maintained during experimental infections [60], so it could be functional in both Vp AHPND strains. This antibacterial system, found in 12 strains of Vp AHPND, mediates interspecies and intraspecies competition, promoting shrimp infection [63]. It is strongly suggested that both Vp AHPND strains could use the T6SS1 as a selective advantage during shrimp intoxication by killing surrounding bacteria.

Quorum sensing
Quorum sensing (QS) is a cell-to-cell signaling mechanism in response to an increase of bacterial cell-population [66]. Bacterial QS produce, release, and recognize molecular autoinducers (AI) that bind to surface bacterial receptors, triggering signal transduction cascades that alter the expression of genes related with survival and infection factors, like sporulation, luminescence, biofilm formation, and virulence, among others [67]. The QS mechanism is widely distributed in Vibrionaceae members, being the acyl-homoserine lactones (AHLs) one of the common AIs. For example, AHLs are implicated in the signaling mechanisms that activate the production of luciferase in V. fischeri [66]. V. harveyi produces and responds to other three AIs, a) HAI-1, [N-(3-hydroxy butyryl)-homoserine lactone], an intra-species AI; b) CAI-1, [(Z)-3-aminoundec-2-en-4one] that is restricted to the Vibrio genera; and c) the inter-spices AI-2 ((2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran-borate). These three AIs act in parallel to regulate over 600 target genes through complex signaling cascades ( [66,68]. The Vibrionaceae's capacity to "sensing self" and "sensing others" allows for both competition and cooperation in complex microbial communities [69]. Virulence gene expression regulated by QS has been studied extensively in V. harveyi and may serve as a basis for the understanding of QS mechanisms in Vp since this pathogen contains the central conserved components of the QS pathway known in V. harveyi [70]. For example, a LuxT homolog of V. harveyi, the SwrT, activates genes that encode for translocation across surfaces, swarming, and is lateral-flagellar-driven in Vp [71,72]. V. harveyi and, presumably, Vp produce three types of AIs called auto inducer 2 (AI-2), harveyi auto inducer 1 (HAI1), and cholerae auto inducer 1 (CAI1), which are recognized by the surface membrane receptors LuxP/LuxQ, LuxN, and CqsS, respectively [73]. In a preliminary study, [74] showed that the production of PirAB Vp binary toxin is regulated by the AI-2 QS process. They tested the effect of a cell-free supernatant from V. harveyi containing AI-2 (CFS-VH) on an AHPND-causing Vp strain. The AI-2-containing supernatant accelerated the production time and yield of both PirA Vp and PirB Vp toxins, whereas the application of the furanone [(5Z)-4-bromo5-(bromomethylene)-2(5H)-furanone] AI-2 antagonist delayed the AHPND toxin production or secretion. This study opens new perspectives on the knowledge of QS mechanisms in Vp and on possible treatments for the management strategies to control AHPND infection in shrimp culture. Interestingly, AI-2 is synthetized by numerous bacterial species and can facilitate inter-species cell-cell signaling [75], resulting in changes of Vp behavior in complex microbial communities.

Environmental factors
Bacterial adaptation and survival depend on their capacity to properly respond to changes in their internal and external environments. Vibrio spp. survival in marine environments follows responses to carbon and energy sources, dissolved oxygen, water pH, salinity, temperature, and starvation [76]. Environmental stress can increase horizontal gene transfer mechanisms in AHPND-causing Vp strains, promoting their growth [77,78] and, therefore, increasing the risk of AHPND outbreaks and disease dispersion in tropical zones. Recently was investigated the effect of temperature shifts on the pirA Vp and pirB Vp genes expression of AHPND-Vp AAHMRU04 strain isolated from white shrimp exhibiting clinical signs of AHPND [79]. Tested bacteria were grown at 30 °C for 24 h and subsequently exposed to a set of different temperature trials for 4 days. pirA Vp and pirB Vp genes were induced when the temperature shifted from high (26-32°C) to lower (22-28°C) temperatures [79]. Changes in temperature due to global warming are a growing concern for aquaculture due to the risk of the increase of Vp-caused diseases.
The relation between salinity and AHPND in P. vannamei was studied by [80]. Pathogen-free shrimp cultures (5, 10, 15, and 20 g L -1 of NaCl) were challenged with a Vp AHPND broth. Vp AHPND caused infection in shrimps at all salinity treatments as confirmed by histological damage and the presence of pirAB Vp toxin genes by PCR analysis. However, cumulative mortality was different, showing higher survival in shrimp maintained at lower salinity. Since Vp reproduces more efficiently in higher salinity environments, the production of a greater amount of PirAB Vp toxin is probable, which resulted in a higher cumulative mortality in P. vannamei maintained at these conditions. A different behavior was observed when challenging P. vannamei, growing at different salinity conditions, with Vp AHPND-E9 [81]. In this study, mortality was higher at lower saline concentration, finding a positive correlation with the expression of pirA Vp gene. Although more experiments are needed to determine the influence of salinity on the expression of pirAB Vp , these experiments corroborate that the toxin can be expressed at different salinities [82] and that the management of saline concentration in shrimp culture can be an important factor to control Vp infectivity.
Another environmental factor studied in relation to the production of the PirAB vp binary toxin is related to fluid shear and hydrodynamic forces to which Vp could be subjected either by natural influences or by the use of aquaculture equipment to enhance shrimp productivity, such as blowers or aerators [83]. The effect of shaking conditions on the AHPND-causing Vp M0904 was studied [84]. At 110 rpm constant agitation, bacteria develop cellular aggregates together with the levan (branched polymeric fructans)-containing biofilm formation and acquisition of tolerance against antimicrobial agents (kanamycin, ampicillin, rifampicin, and tetracycline), possibly due to high biofilm production. In addition, a significant decrease was observed not only in the PirA Vp /PirB Vp toxins production but also in the virulence of Vp M0904 to Artemia and Macrobrachium larvae. An increase in shaking speed to 120 rpm produced an increase in the PirA Vp /PirB Vp toxins production, in the virulence of Vp M0904 to Artemia and Macrobrachium larvae, and in the expression of polar flagellin (flaA), polar flagellin-specific chaperone (fliS), and chemotaxis protein (CheR). This type of study provides valuable information for understanding the behavior of Vp AHPND in aquaculture environments [84].

Biofilm formation
Formation of bacterial biofilms represents one of the most important survival mechanisms, attachment strategies, and host colonization of bacteria [85].This phenomenon is influenced by abiotic and biotic factors regulated by QS [86]. ToxR is an important virulence regulator implicated in the synthesis of Vp biofilm formation and also controls the expression of virulence factors found in human pathogenic Vp, including thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), type III secretion system T3SS [87,88]. Expression of these factors is regulated by QS through the production and response to AI-2 ( [86,89,90]. Under these conditions, biofilm and toxin production appear to be simultaneous activities.
There is a lack of information about the relationship between biofilm formation and the production of PirAB vp binary toxin in Vp AHPND. The only study found is that of [84] who observed an inverse relationship between the production of biofilms and that of the PirAB Vp toxin. This behavior refers to the formation of abiotic films in response to fluid shear and hydrodynamic forces. However, the regulation, growth kinetics, and characteristic of Vp AHPND biofilms in the host and their relation with PirAB vp toxin production are yet to be characterized.

Biological activities of the PirA Vp and PirB Vp subunits
Bacterial protein toxins, like PirAB Vp , are molecular self-governing virulence factors that target specific host cells, triggering different damaging processes involved in the disease of the infected organism. Binding of bacterial toxins to the plasma cell surface receptors is an essential first step for shrimp intoxication. Knowing the structure of these receptors can contribute to a better understanding of the pathogen's infection mechanisms and to prevent host disease by blocking the toxin-receptor interaction using a mimetic antagonist [91].
It is known that PirA Vp and PirB Vp form a heterodimeric complex that binds to receptors located on the cells of the shrimp Hp [92,93]. However, the precise nature of the toxin receptors is still not known. The first crystal structures for Vp PirA Vp /PirB Vp showed a similar structural topology to Bacillus thuringiensis (Bt) Cry insecticidal toxin [10]. This evidence suggests that PirA Vp /PirB Vp is a binary pore-forming toxin and that PirA Vp /PirB Vp possesses similar structural mechanisms of receptor binding to those of the Bt Cry insecticidal toxin. Structural alignment of both toxins indicates that the PirA Vp subunit is similar to the lectin-like recognition domain III of Bt toxin whereas PirB Vp would correspond to pore forming I and II domains ( [10,92,94,95]. In this context, the initial interaction of the PirA Vp /PirB Vp toxin would be through a lectin-carbohydrate recognition between Pi-rA Vp and the glycans exposed on the surface of the plasma membrane of Hp cells [92]. Structural features and molecular docking of the PirA Vp subunit shows a potential sugarbinding cavity for glycans containing the GalNac molecule whereas the PirB Vp subunit structure contains a C-terminal receptor domain similarily to the Cry domain II for protein-protein ligand interactions and an N-terminal consistent with other membrane poreforming toxins, including Cry I domain [10,95]. However, experiments conducted with recombinant proteins, rPirA Vp and rPirB Vp , showed that only the PirAB Vp complex and the rPirB Vp displayed a Mg 2+ or Ca 2+ independent hemagglutinating activity (HA) toward rat red cells, whereas rPirA Vp was not able to agglutinate erythrocyte from several animal species [61].
In a first attempt to determine the putative PirB Vp lectin´s sugar specificity, subsequent competition experiments were conducted using a wide battery of mono-and disaccharides and glycoproteins. GalNH2 and GlcNH2 monosaccharides were better sugar inhibitors for rPirB Vp than the rest of mono and disaccharides tested. Among glycoconjugates, the fetuin glycoprotein showed the strongest rPirB Vp HA inhibition capacity, whereas egg white chicken ovalbumin and heparin showed a relative inhibitory potency. With this in mind, the PirB Vp subunit binds to a glycoconjugate glycan moiety containing amino sugars [61]. Further experiments conducted by the same group showed the existence of different glycan receptors for PirB Vp , at least a mucin-like receptor located at the surface membrane of the cell hepatopancreas and an internal hexosaminidase glycoprotein receptor, possibly involved in toxin-causing cell damage to shrimp tissues [96]. Betahexosaminidase (β-N-acetyl hexosaminidase) is a ubiquitous lysosomal enzyme with multiple roles in protein glycosylation and synthesis and metabolism of glycoconjugates [97]. This glycoprotein plays an important role in arthropod molting and chitin degradation, and also in the defense system of P. vannamei against parasites ( [98,99]. Extracellular betahexosaminidases secreted by eukaryotes occur as dimers and possess N-glycosidicallylinked glycans with oligomannosidic and complex-type glycan structures [100,101]. The possibility that PirB Vp could recognize N-linked oligosaccharides expressed by endosomal or secreted beta-hexosaminidase, allowing increased pathogenesis of Vp in crustaceans, cannot be excluded.
Previous data suggest a putative lectin-like PirB Vp subunit activity [61,96] that contrasts with the function of domains I and II proposed for the Cry toxin and also for the proposed function of PirA Vp subunit because with the experiments carried out to date, it was not possible to verify that this subunit has the ability to recognize carbohydrates. In light of this new evidence, the PirA Vp subunit could be playing an initial stabilizing role allowing PirB Vp to bind with higher affinity to the glycan receptors located at the surface of hepatopancreas cells.

Expression of mucin-like O-glycosidic structures in shrimp
O-glycans are critical for the development and function of multicellular organisms. Mucin-type glycans are widely found on the cell surface and secreted glycoconjugates of invertebrates [102]. These O-glycans may serve as receptor-binding sites for a variety of pathogenic bacteria and their toxins [103]. A small unit of P. vannamei hemocyanin had Oglycans closely associated with its agglutination activity toward Vibrio fluvialis, V. alginolyticus, and V. parahaemolyticus ( [104,105).
A mucin-like peritrophin-like gene from fleshy shrimp (Fenneropenaeus chinensis) could bind Gram-negative bacteria [106], while another mucin-like peritrophin-like gene from the shrimp Exopalaemon carinicauda is involved in the white spot syndrome virus infection [107]. In addition, a mucin-like peritrophin is implicated in the V. harveyi infection in the black tiger shrimp P. monodon ( [108]. Abiotic characteristics, such as the decrease of temperature and type of diet, increase the expression of several mucin-like proteins in P. vannamei [109][110][111]. These modifications could be related in the pathologic development of Vibrio infection, increasing their binding targets on the shrimp digestive system. Searching for possible receptors for the lectin-like PirB Vp [96] found some correspondence with a mucin-like protein expressed in the shrimp Hp of P. vannamei. These studies are the beginning of a better understanding of the infection mechanisms of Vp in shrimp.

Receptor on shrimp hemocytes
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 14 January 2022 doi:10.20944/preprints202201.0200.v1 The PirAB Vp toxin is known to mainly target the epithelial cells of the shrimp hepatopancreatic tubules. In addition, [112] found that the PirAB Vp toxin binds to epithelial cells of the digestive tract and produces similar lesions in the midgut and hindgut regions in germ-free brine shrimp, Artemia. Moreover, the dysregulation of apoptosis-related genes in Vp AHPND-challenged P. vannamei hemocytes suggests that Vp AHPND induces apoptosis in hemocytes [113]). For the Bt Cry toxin, apoptosis is induced by a series of processes that start with the interaction between the Cry1A toxin and the carbohydrate moiety (surface receptor binding) of an N aminopeptidase (APN) [114]. In the transcriptome of Vp AHPND-challenged P. vannamei was identified an aminopeptidases N1 (LvAPN1) gene [115]. DNA sequence analysis of the LvAPN1 gene showed a putative C-terminal transmembrane domain and various putative N-and O-glycosylation sites. The expression of LvAPN1 increases in hemocytes, after challenging P. vannamei with either Vp AHPND or the partially purified Vp AHPND toxins. Silencing of LvAPN1 significantly reduced LvAPN1 transcription levels in the stomach, hepatopancreas, and hemocytes, and increased the survival of adult P. vannamei that were challenged with the partially purified Vp AHPND toxins. These observations suggest the putative role of LvAPN1 as a PirAB Vp toxin receptor located in the surface of hemocytes [115]. Other putative carbohydrate receptors for the PirAB Vp toxin could be located in the surface of P. vannamei hemocytes as these cells express a plethora of glycoconjugates. Using commercial lectins with different carbohydrate specificity was proved the presence of carbohydrate moieties containing mainly N-acetyl-glucosamine (GlcNAc) and N-acetylneuraminic acid (sialic acid) [116]. In other study, these carbohydrates were recognized by the rPirB Vp subunit [61].

Search of potential inhibitors of the PirAB Vp toxin.
Understanding the structural biology of PirAB Vp becomes essential in finding or developing antiadhesive agents or receptor analogs that could prevent adhesion and subsequent cell entry of the toxin and inhibit its activity. In particular, it is important to decipher the role and structure features of complex carbohydrates that serve as toxin receptors. According to research by our group, the PirB subunit presents lectin activity and its adhesion can be inhibited in the presence of fucosylated glycans and by those that contain N-acetyl glucosamine [61,96].
In addition to glycans, the study of peptides that can interact with PirAB Vp also acquires importance. Computational tools like molecular docking can play an important role in the search of antiadhesive peptides or in the design of antiadhesive peptides analogs through construction of precise structural models of peptide-toxin complexes and calculation of binding free energies [117,118]. An interesting aspect is the search for bifunctional peptides that can be used to improve shrimp growth performance and, at the same time, protect them from the PirAB Vp toxin. An example are the oilseed peptides that contribute to improved shrimp health and growth performance when used as shrimp feed ingredients [119]. In-silico studies reveal six dual-target peptides from different oilseed proteins capable of intercepting the PirA Vp /PirB Vp complex formation. Such peptides (1139-2977 Da in mass and 10-28 residues in length), are possible candidates for the future development of peptide-based anti-AHPND agents [118].

Concluding Remarks and Future Perspectives
To date, the mechanism(s) of the PirAB Vp that cause AHPND in penaeid shrimp is far to be known. However, although, we currently know more about the pVA1 virulent plasmid, there is a lack of studies about the variability of the plasmid copy number per bacterial cell and how it influences the pathogenesis of AHPND. According to our studies, we suggest that Vp AHPND strains could use the T6SS1 as a selective advantage during shrimp intoxication by killing surrounding bacteria. Future studies on the type and activity of effector proteins of the T6SS in Vp AHPND during an infection process can help to design strategies to control AHPND-causing strains. Although studies have reported that environment factors, like salinity, can affect the production of toxins and shrimp survival, more experiments are needed to determine the influence of salinity on the expression of pirAB Vp genes and the pathogenesis of AHPND. Furthermore, there is evidence that the PirA Vp subunit could be playing an initial stabilizing role allowing PirB Vp to bind with higher affinity to different glycan receptors, at least a mucin-like receptor, located at the surface membrane of the cell hepatopancreas, and an internal hexosaminidase glycoprotein receptor that could be involved in the PirAB Vp toxin. These studies are the beginning of a better understanding of the infection mechanisms of Vp in shrimp.
The structural biology of PirAB Vp becomes essential to find or develop antiadhesive agents or receptor analogs that could prevent adhesion and subsequent cell entry of the toxin. It is important to elucidate the role and structure features of complex carbohydrates that serve as toxin receptors. The PirB Vp subunit presents lectin activity and, therefore, its adhesion can be inhibited by glycans. In this sense, research with putative glycomimetic antagonists, like fucoidan, will provide new directions for the future development of PirAB Vp inhibitors.
Data Availability Statement: The study did not report any data.