Functional Divergence in Shrimp Anti-Lipopolysaccharide Factors (ALFs): From Recognition of Cell Wall Components to Antimicrobial Activity

Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded β-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a heterologous expression system. Group D ALFs were found to have impaired LPS-binding activities and only limited antimicrobial activity compared to Group B ALFs. Interestingly, all four ALF groups were shown to be simultaneously expressed in an individual shrimp and to follow different patterns of gene expression in response to a microbial infection. Group B was by far the more expressed of the ALF genes. From our results, nucleotide sequence variations in shrimp ALFs result in functional divergence, with significant differences in LPS-binding and antimicrobial activities. To our knowledge, this is the first functional characterization of the sequence diversity found in the ALF family.     

日本沼虾两种抗脂多糖因子的特性研究

为了研究抗脂多糖因子ALFs在日本沼虾先天性免疫中的功能作用, 研究从日本沼虾中克隆了2种抗脂多糖因子MnALF1、MnALF2。MnALF1 cDNA 全长1008 bp, 编码121个氨基酸; MnALF2 cDNA 全长836 bp, 编码124个氨基酸。这2种氨基酸均包含有一个信号肽序列和一个LPS结合位点, 并且在结合位点的两端(N-端和C-端)都有2个保守的半胱氨酸残基。这2种MnALFs与之前发现的甲壳动物的ALFs是非常相似的。qRT-PCR结果显示MnALFs在所有被检测的组织中均有表达。其中MnALF1主要在心脏和小肠内表达, 而MnALF2则主要在血细胞和肝胰脏中表达。在用嗜水气单胞菌刺激之后发现2种MnALFs在心脏、小肠、血细胞、肝胰脏中都呈现出明显的时间依赖表达模式(MnALF1在刺激之后呈现出先减少后增加的趋势, 之后分别在不同组织的不同时间点达到最大值; 然而, 对于MnALF2, 在心脏和小肠中先减少后增加, 在血细胞和肝胰脏中呈现出先增加后减少, 最后都在24h达到最大值)。结果提示这2种MnALF具有不同的组织特异性, 并且在细菌侵染的免疫防御中起着重要的保护作用。     

Prawn community structure in the subtropical Mfolozi–Msunduzi estuarine system,KwaZulu-Natal,South Africa

The life cycles of many marine and freshwater prawn species are closely associated with estuarine habitat. Here, the prawn community of the Mfolozi–Msunduzi estuarine system is described and the system's potential as an alternative nursery for prawns during prolonged closure of the adjacent St Lucia estuarine system is examined. Sampling was conducted at five localities in summer and winter in 2007–2012. The Mfolozi–Msunduzi Estuary is a river-dominated system with mouth condition, sediment grain size and turbidity being the most important factors affecting the structure of its prawn community. Twelve prawn species were recorded, including five marine penaeid and five freshwater palaemonid species. The prawn community was numerically dominated by freshwater Macrobrachium equidens and two penaeids, Fenneropenaeus indicus and Metapenaeus monoceros. Significant differences were recorded between summer and winter samples, with freshwater species dominating the catch in summer, while penaeid species were dominant in winter. The marked seasonal change in the prawn community was related to strong river flows during summer creating low-salinity conditions, unsuitable for penaeid postlarval development, throughout most of the system. Compared to St Lucia, the Mfolozi–Msunduzi Estuary can be regarded as a poor alternative nursery area for penaeids during the summer peak postlarval recruitment period.     

SpALF4: A newly identified anti-lipopolysaccharide factor from the mud crab Scylla paramamosain with broad spectrum antimicrobial activity

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides with binding and neutralizing activities to lipopolysaccharide (LPS) in crustaceans. This study identified and characterized a novel ALF homolog (SpALF4) from the mud crab Scylla paramamosain. The complete cDNA of SpALF4 had 756 bp with a 381 bp open reading frame encoding a protein with 126 aa. The deduced protein contained a signal peptide and a LPS-binding domain. SpALF4 shared the highest identity with PtALF5 at amino acid level but exhibited low similarity with most of other crustacean ALFs. Furthermore, different from the previously identified three SpALF homologs and most of other ALFs, SpALF4 had a low isoelectric point (pI) for the mature peptide and the LPS-binding domain with the values of 6.93 and 6.74, respectively. These results indicate that SpALF4 may be a unique ALF homolog with special biological function in the mud crab. Similar to the spatial structure of ALFPm3, SpALF4 contains three α-helices packed against a four-strand β-sheet, and an amphipathic loop formed by a disulphide bond between two conserved cysteine residues in LPS-binding domain. SpALF4, mainly distributed in hemocytes, could be upregulated by Vibrio harveyi, Staphylococcus aureus, or white spot syndrome virus. Recombinant SpALF4 could inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio anguillarum, Vibrio alginolyticus, Aeromonas hydrophila, Pseudomonas putida), Gram-positive bacteria (S. aureus and Bacillus megaterium), and a fungus Candida albicans to varying degrees. Further study showed that it could also bind to all the aforementioned microorganisms except S. aureus. These results demonstrate that SpALF4 is a unique ALF homolog with potent antimicrobial activity against bacteria and fungi. This characteristic suggests SpALF4 plays an essential function in immune defense against pathogen invasion in mud crab.     

Molecular cloning of crustins from the hemocytes of Brazilian penaeid shrimps

Crustins are antimicrobial peptides initially identified in the hemocytes of the crab Carcinus maenas (11.5-kDa peptide or carcinin) and recently also recognized in penaeid shrimps and other crustacean species. The aim of this study was to identify sequences encoding for crustins from the hemocytes of four Brazilian penaeid species: Farfantepenaeus paulensis, Farfantepenaeus subtilis, Farfantepenaeus brasiliensis and Litopenaeus schmitti. Using primers based on consensus nucleotide alignment of crustins from different crustaceans, cDNA sequences coding for crustins in all indigenous penaeid species were amplified. The obtained four crustin sequences encoded for peptides containing a hydrophobic N-terminal region rich in glycine repeats and a C-terminal part with 12 cysteine residues and a conserved whey acidic protein domain. All obtained crustin sequences showed high amino acidic similarity among each other and with crustins from litopenaeid shrimps (76-98%). This is the first report of crustins in native Brazilian penaeid shrimps.     

Molecular cloning and characterization of cortical rod protein in the giant freshwater prawn Macrobrachium rosenbergii, a species not forming cortical rod structures in the oocytes

The formation of cortical rod structures is a characteristic of fully mature oocytes in penaeid prawns, but such structures are absent from oocytes of giant freshwater prawn Macrobrachium rosenbergii. In the present study, we first demonstrated the presence of a 30-kDa protein, which was immunologically related to kuruma prawn cortical rod protein (CRP), in the ovary of giant freshwater prawn, and subsequently purified this protein. Furthermore, a cDNA encoding the CRP-like protein was isolated. Based on the high homology (98%) in the amino acid sequence with kuruma prawn CRP, the 30-kDa protein has been identified as a CRP homologue of giant freshwater prawn, designated mrCRP. The RT-PCR analysis revealed that mrCRP mRNA was present in the ovary from a prawn with a gonadosomatic index (GSI) of 0.2. Western blot analysis revealed the presence of a CRP-immunoreactive band of 30kDa in the ovary with GSI of 1.6. By immunocytochemistry, CRP-immunopositive signals were detected in the ovary with GSI of 0.9, that had started to accumulate considerable amounts of vitellins and lipids in the peripheral cytoplasm. With progress of vitellogenesis, mrCRP was apparently accumulated in the mature oocytes, although it was not detectable, presumably because a relatively small amount of mrCRP was masked with large amounts of vitellin and lipids. In giant freshwater prawn without forming cortical rod structures, our findings indicate that the oocytes produce mrCRP, a homologue of CRP found in penaeid prawns.     

Molecular and Functional Characterization of an Anti-lipopolysaccharide Factor Mm-ALF from Speckled Shrimp Metapenaeus monoceros

Probiotics and Antimicrobial Proteins - Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides of approximately 100 amino acid residues with a broad spectrum of antimicrobial activity....     

Anti-lipopolysaccharide Factor from Crucifix Crab Charybdis feriatus,Cf-ALF2: Molecular Cloning and Functional Characterization of the Recombinant Peptide

Antilipopolysaccharide factors (ALFs) are important effectors of innate immunity in crustaceans with broad spectrum antimicrobial activity. Present study deals with the molecular and functional characterization of a 98-amino acid ALF isoform from, crucifix crab, Charybdis feriatus termed as Cf-ALF2. The ALF isoform Cf-ALF2 exhibits characteristic features of an AMP including a cationic net charge of + 9 and a total hydrophobic ratio of 34%. Recombinant peptide rCf-ALF2 showed remarkable antimicrobial activity against Gram-negative and Gram-positive bacteria especially against Staphylococcus aureus (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 5 µM) and Escherichia coli (MIC 10 µM and MBC 20 µM). Using scanning electron microscopy, bacterial membrane blebbing, disruption, and cell content leakage were observed in peptide treated E. coli. The recombinant peptide was found to be non-hemolytic and non-cytotoxic in NCI-H460 cell line at the highest tested concentration (20 µM). Thus, this study identified a novel isoform of ALF from C. feriatus and revealed the potent antimicrobial property of the recombinant peptide Cf-ALF2 and the future prospects of using the peptide for therapeutic applications in the future.

     

Isolation and characterization of two pigment-dispersing hormones from the whiteleg shrimp, Litopenaeus vannamei

In crustaceans, the pigment-dispersing hormone (PDH) is released from the X-organ/sinus gland complex located in the eyestalks, and controls pigment dispersion in the chromatophores. Knowledge concerning the structure and activity of PDH in penaeid shrimps is remains limited, since natural PDH has been purified from only the Kuruma prawn, Marsupenaeus japonicus. In this study, two PDHs (Liv-PDH-A and -B) were purified from the sinus gland extracts of another penaeid species, the whiteleg shrimp, Litopenaeus vannamei, by two steps of reversed-phase HPLC, and their amino acid sequences were determined. They both consist of 18 amino acid residues, with a free N-terminus and an amidated C-terminus, the sequences of Liv-PDH-A and -B being NSELINSLLGIPKVMNDAamide and NSELINSLLGLPKVMNDAamide, respectively. These sequences are identical to those of mature PDHs deduced from cDNAs encoding L. vannamei PDH precursors cloned previously by other workers. Liv-PDH-A and -B showed significant pigment-dispersing activity in melanophores by in vivo bioassay.     

Isolation and characterization of microsatellite loci in the Malaysian giant freshwater prawn, Macrobrachium rosenbergii

Eight single locus microsatellite markers were developed to characterize the Malaysian giant freshwater prawn, Macrobrachium rosenbergii. These microsatellites were isolated from an enriched genomic library contained by using a 5'-anchored polymerase chain reaction technique. Primers were designed to flank the repeat sequences and subsequently used to characterize 30 unrelated individuals of the giant freshwater prawn. The polymerase chain reaction amplification products of these eight microsatellite loci were polymorphic with the number of alleles ranging from two to 10 alleles per locus while the levels of heterozygosity ranged from 0.6333 to 0.8667.     

Detection and genetic profiling of infectious hypodermal and haematopoietic necrosis virus (IHHNV) infections in wild berried freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> collected for hatchery production

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) has been detected widely in penaeid culture facilities in Asia and the Americas. IHHNV infection on sub-adult and postlarvae of the giant freshwater prawn, Macrobrachium rosenbergii which had caused up to 80% mortalities was first reported in Southeast Taiwan in 2006. In Malaysia, although, there has been no report on IHHNV infections in M. rosenbergii, preliminary work suggests that there is an urgent need to setup a screening protocol for IHHNV for both wild and cultured populations. In this study, polymerase chain reaction based screening was carried out on 30 randomly sampled berried wild M. rosenbergii before and after spawning. All samples did not showed any sign of IHHNV infection. However, the results showed that 20% of the samples were IHHNV positive. Sequence analysis of the amplified band using NCBI-BLAST showed that the putative IHHNV sequence had 98% nucleotide sequence (388 bp) identity with the IHHNV isolate AC-05-005 non-structural protein 1 gene and seven other IHHNV strains in the data bank further affirming the suggestion on the presence of IHHNV in wild freshwater prawn populations in Malaysia.