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. 2014 Mar 11;9(3):e91794.
doi: 10.1371/journal.pone.0091794. eCollection 2014.

A galectin from the kuruma shrimp (Marsupenaeus japonicus) functions as an opsonin and promotes bacterial clearance from hemolymph

Xiu-Zhen Shi  1 , Lei Wang  1 , Sen Xu  1 , Xiao-Wen Zhang  1 , Xiao-Fan Zhao  1 , Gerardo Raul Vasta  2 , Jin-Xing Wang  1
Affiliations

A galectin from the kuruma shrimp (Marsupenaeus japonicus) functions as an opsonin and promotes bacterial clearance from hemolymph

Xiu-Zhen Shi et al. PLoS One. .

Abstract

Galectins are a lectin family characterized by a conserved sequence motif in the carbohydrate recognition domain, which preferential binds to galactosyl moieties. However, few studies about the biological roles of galectins in invertebrates have been reported except for the galectin (CvGal1) from the eastern oyster Crassostrea virginica. Furthermore, galectins have been described in only a few crustacean species, and no functional studies have been reported so far. In this study, we identified and functionally characterized a galectin from the kuruma shrimp Marsupenaeus japonicus, which we designated MjGal. Upon Vibrio anguillarum challenge, expression of MjGal was up-regulated mostly in hemocytes and hepatopancreas, and the protein bound to both Gram-positive and Gram-negative bacteria through the recognition of lipoteichoic acid (LTA) or lipopolysaccharide (LPS), respectively. By also binding to the shrimp hemocyte surface, MjGal functions as an opsonin for microbial pathogens, promoting their phagocytosis. Further, as shown by RNA interference, MjGal participates in clearance of bacteria from circulation, and thereby contributes to the shrimp's immune defense against infectious challenge. Elucidation of functional and mechanistic aspects of shrimp immunity will enable the development of novel strategies for intervention in infectious diseases currently affecting the shrimp farming industry worldwide.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phylogenetic analysis of MjGal.
The tree was constructed with the MEGA 5.05 software. One thousand bootstraps were used to check the repeatability of the results. The galectin sequences of selected invertebrate and vertebrate species were obtained from NCBI GenBank. MjGal is labeled with a solid triangle.
Figure 2
Figure 2. Temporal and spatial expression of MjGal.
Total RNAs from different tissues of unchallenged and V. anguillarum or PBS challenged shrimp were reverse transcribed to cDNAs that served as templates for PCR amplification. (A) Expression of MjGal in different tissues of naïve animals as assessed by RT-PCR. The time-course expression of MjGal was upon V. anguillarum challenge was analyzed by qRT-PCR in hemocytes (B) and hepatopancreas (C), with β-actin serving as the reference gene. The histograms show the statistical analysis of the quantitative real-time PCR results. The asterisks indicate significant differences (**P<0.01, ***P<0.001) between the V. anguillarum-challenged and PBS-injected group. Error bars represent ± SD of three independent PCR amplifications and quantifications.
Figure 3
Figure 3. Expression of recombinant MjGal (rMjGal), rMjGalΔ102–106 and their bacteria binding assay by Western blot.
(A) Lane 1, purified rMjGal; lane 2, purified rMjGalΔ102–106; lane 3, purified Trx-His which was used as the control;lane 4, protein markers. (B) Schematic diagram of the five deleted residues (Cys102-Cys106) in mutant MjGalΔ102–106. (C) Purified rMjGal (500 µg/ml) was pre-incubated with either lactose (200 mM), glucose (200 mM), or TBS alone for 1 h, and suspensions of four Gram-positive (G+) bacteria (S. aureus, B. subtilis, B. megaterium and B. thuringiensis) and four Gram-negative (G−) bacteria (V. anguillarum, K. pneumoniae, E. coli and P. aeruginosa) were added and incubated at room temperature for 1 h. The bacteria were centrifuged, washed four times with TBS, and treated with 7% SDS for 1 min. The eluate was collected by centrifugation, subjected to SDS-PAGE, and the eluted rMjGal detected by Western blot using anti-His antibody. (D) The purified rMjGal was pre-incubated with excess LPS or LTA before the bacteria were added, and the binding activity for bacteria was tested by western blot. (E) Purified rMjGalΔ102–106 or Trx-His were also used as controls for the bacteria binding activity.
Figure 4
Figure 4. rMjGal strongly bound to lipopolysaccharide (LPS), lipoteichoic acid (LTA).
rMjGalΔ102–106 weakly bound to LPS, LTA. Three polysaccharides LPS, LTA and peptidoglycan (PGN) were used in this binding assay. Microtiter plates were coated with 2 µg of LPS (A) or LTA (B), and incubated with increasing concentrations of rMjGal or rMjGalΔ102–106 (0, 1, 2, 5, 10, 20 µg/ml). Binding of rMjGal or rMjGalΔ102–106 was detected using anti- Histidine antibody. Data shown are the mean±SEM derived from three repeats. No binding to PGN could be detected at any of the rMjGal concentrations tested (results not shown).
Figure 5
Figure 5. The activity of rMjGal in the clearance of V. anguillarum.
Bacteria (2×107 cells) pre-incubated with rMjGal (15 µg) were injected into the shrimp. Hemolymph was collected, diluted 1∶1000, and plated on 2216E agar plates. Bacterial colonies were counted after 37°C cultivation for 12 h. Results represent as the number of bacteria from three independent repeats and expressed as mean values ±S.D. Asterisks indicate significant differences (*P<0.05, **P<0.01, ***P<0.001).
Figure 6
Figure 6. In vitro binding of rMjGal to shrimp hemocytes.
(A) Shrimp hemocytes (2×104 cells/ml) were added to the slide glass pre-coated with rMjGal, rMjGal with lactose, Trx-His, or Trx-His with lactose. One hour later, the slide was washed three times with PBS and observed under the microscope. The number of hemocytes counted at 400× magnification under microscope. Figures showed the hemocytes which were adhered to the slides after the washing. Assays were performed in triplicate with three different hemocytes suspensions from each shrimp. Bar = 100 µm. (B) Statistical analysis of the data from (A) using GraghPad software. Significant differences were analyzed by one way ANOVA by a Trukey’s multiple comparison test and indicated by asterisks (**P<0.01, ***P<0.001). The bar was 100 µm.
Figure 7
Figure 7. In vivo binding of rMjGal to shrimp hemocytes.
rMjGal, Trx-His, or PBS were injected into shrimp and the hemocytes were collected and analyzed by immunohistochemistry. The left panel indicated the binding signal detected by the anti-Histidine antibody for the recombinant protein (Green), the middle panel showed the hemocyte nucleus location (Blue), and the right panel showed the merge of previous two panels. Bar = 20 µm.
Figure 8
Figure 8. MjGal promoted bacterial clearance from hemolymph.
Bacteria phagocytosis assay: fluorescently labeled V. anguillarum (5×107 cells) were coated with either Trx-His (A, the control) or rMjGal (B) and injected into shrimp. The hemocytes were collected after 30 min and placed onto the glass slides. Subsequently, trypan blue solution (2 mg/ml PBS) (Amresco) was added to quench the fluorescence of non-phagocytosed bacteria. The phagocytosis by hemocytes was observed at 400× magnification, and phagocytosed bacteria counted at 200×magnification under the fluorescence microscope. (A) Hemocytes from shrimp injected with V. anguillarum coated with Trx-His. (B) Hemocytes from shrimp injected with V. anguillarum coated with rMjGal. (C) The phagocytic percentages of two groups (*P<0.05). Bar = 50 µm. (D) RNA interference assay: shrimp were injected with dsRNAs twice to silence MjGal expression, and or with GFP dsRNAs as control. Vibrio anguillarum (2×107 cells) were injected into shrimp after the second injection. Hemolymph was collected from the two groups at 5, 15, 60 min post-infection, and serially diluted with PBS. Fifty µl of each sample was plated onto 2216E agar plates and incubated at 37°C overnight. RNA was extracted from hemocytes and gills of two groups, and tested by RT-PCR to confirm silencing of MjGal expression. (E) The bacterial counts in hemolymph samples collected at 5, 15 and 60 min from shrimp from the dsMjGal and dsGFP groups.
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References

    1. Loker ES, Adema CM, Zhang SM, Kepler TB (2004) Invertebrate immune systems–not homogeneous, not simple, not well understood. Immunol Rev 198: 10–24. - PMC - PubMed
    1. Janeway CA Jr, Medzhitov R (2002) Innate immune recognition. Annu Rev Immunol 20: 197–216. - PubMed
    1. Lemaitre B, Hoffmann J (2007) The host defense of Drosophila melanogaster. Annu Rev Immunol 25: 697–743. - PubMed
    1. Robinson MJ, Sancho D, Slack EC, LeibundGut-Landmann S, Reis e Sousa C (2006) Myeloid C-type lectins in innate immunity. Nat Immunol 7: 1258–1265. - PubMed
    1. Vasta GR, Quesenberry M, Ahmed H, O’Leary N (1999) C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway. Dev Comp Immunol 23: 401–420. - PubMed

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