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The anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) has previously been shown to have very active in vitro antimicrobial activity against a broad range of Gram-positive and Gram-negative bacteria, certain fungi and viruses, including known pathogens of P. monodon shrimp. With respect to the strong bactericidal effect on Gram-negative and Gram-positive bacteria, the ALFPm3 binds to their principal cell wall components, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), with a high affinity. The aim of this study was, therefore, to reveal the effects of treating ALFPm3 on membrane of Vibrio harveyi, a P. monodon pathogenic Gram-negative bacterium. The recombinant (r)ALFPm3 protein was found to localize on the V. harveyi cells in vivo, followed by inducing membrane permeabilization and leakage of cytoplasmic components. Moreover, the effect of rALFPm3 treatment on the bacterial cell morphology was confirmed by scanning and transmission electron microscopy. Membrane disruption and damage, bleb and pore formation, and the leakage of cytoplasmic contents were all clearly observed. Taken together, these results suggested that ALFPm3 effectively kills bacteria through bacterial membrane permeabilization., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The protein expression profiles of the lymphoid organ, taken from mock and systemic Vibrio harveyi-infected Penaeus monodon at 6 and 48 h post infection, were revealed. The considerable changes in the expression level of several proteins were observed between the mock and V. harveyi-infected shrimps. From 30 analyzed protein spots with 27 differentially expressed, 21 were known proteins with the most common of these being cytoskeleton proteins (33%) which were all down-regulated upon systemic bacterial infection. Other six proteins including four proteins that are involved in the shrimp immunity (alpha-2-macroglobulin, transglutaminase, heat shock protein 1 and hemocyanin subunit Y), and two proteins that are involved in metabolism (triosephosphate isomerase) and cell signaling (14-3-3 like protein), displayed significantly decreased expression levels. There was, however, an increase in the expression level of the ATP synthase beta subunit, a protein involved in energy balance. Transcription levels of ATP synthase beta subunit and 14-3-3 like protein were up- and down-regulated, respectively, in accord with the observed protein expression levels, but the alpha-2-macroglobulin transcript levels were significantly increased in contrast to the decreased protein expression levels. Interestingly, partial gene silencing of ATP synthase beta subunit revealed a high cumulative mortality of the knockdown shrimps (73.3%) and a dramatic reduction of the total hemocyte numbers in the survival shrimps. These altered proteins are likely to play essential roles in shrimp defense against the pathogenic bacterium V. harveyi.

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides previously identified in various crustaceans. Out of five isoforms identified in Penaeus monodon, ALFPm3 is the best characterized, exhibits antibacterial and antifungal activities and can protect the shrimp from viral infections. Herein, the most recent identified ALFPm, called ALFPm6, is characterized for its potential role in the shrimp's immunity. RNA interference-mediated gene silencing was used to study the function of ALFPm6 in comparison to ALFPm3. Knockdown of ALFPm3 gene led to rapid death with a cumulative shrimp mortality of 86% within 7 days, accompanied by a 12- and 50-fold higher bacterial count after 2 days in the haemolymph and hepatopancreas, respectively, compared to the control shrimp injected with GFP dsRNA. In contrast, gene silencing of ALFPm6 alone had no effect on the shrimp mortality, but led to a significant increase in the cumulative mortality and a faster mortality rate following Vibrio harveyi and white spot syndrome virus (WSSV) infections, respectively. These results support the roles of ALFPm6 and ALFPm3 in the protection of shrimp against microbial infections., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis.

Differential Display PCR technique (DD-PCR) was used for the analysis of altered gene expression in hemocytes of Vibrio harveyi-infected Penaeus monodon. Forty-four combinations of arbitrary and oligo(dT) primers were used to screen for differentially expressed genes. A total of 79 differentially expressed bands could be identified from 33 primer combinations. These included 48 bands (61%) whose expression level increased and 31 bands (39%) decreased after V. harveyi challenge. Subsequently, forty-eight differential display fragments were successfully reamplified and cloned. A total of 267 clones were randomly selected and sequenced. The sequence analysis showed that 85 (31%) out of 267 clones were matched with sequences in the GenBank database which represented 24 different genes with known functions. Among the known genes, glucose transporter 1, interferon-related developmental regulator 1, lysozyme, profilin, SERPINB3, were selected for further confirmation of their differentially expression patterns by real-time PCR. The results showed increasing in expression level of the selected genes in shrimp hemocytes after microbial challenge suggesting the involvement of such genes in bacterial response in shrimp. The anti-lipopolysaccharide factor type 3 (ALFPm3) gene, previously reported in P. monodon (Supungul et al., 2002) was found among the up-regulated genes but diversity due to amino acid changes was observed. Increase in ALFPm3 transcripts upon V. harveyi injection is in accordance with that found in the previous study.