Identification and functional characterization of a superoxide dismutase (CuZnSOD) from Pinctada fucata martensii.

Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS)，avoid damage from O2 to the body, and maintain O2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.

Clinical Characteristics of Patients with Micrococcus luteus Bloodstream Infection in a Chinese Tertiary-Care Hospital.

Few pieces of research have focused on Micrococcus luteus bloodstream infection (BSI) because of its low incidence; hence data is needed to illustrate this uncommon infection. This study aimed to explore the clinical characteristics of patients with M. luteus BSI. From January 2010 to December 2019, inpatients that met the criteria for M. luteus BSI were included in this study. Data was collected by reviewing electronic records. Ninety-seven patients were enrolled in this study. Sixty-three percent of the patients have a higher neutrophil percentage (NEUT%). The average blood C-reactive protein (CRP) concentration was 5.5 ± 6.4 mg/dl. 48.5% of the patients had malignancy, and 40.2% underwent invasive surgeries. Linezolid was found to have the largest average diameter of the inhibition zone (36 mm), while erythromycin was found to have the smallest average zone diameter (15 mm). However, some M. luteus strains had a potentially broad antimicrobial resistance spectrum. Cephalosporins (59.2%) and quinolones (21.4%) were the most commonly used antibiotics for empirical therapies. In conclusion, M. luteus BSI mainly happens in immunocompromised patients or those with former invasive surgeries or indwelling catheters. M. luteus strains are less responsive to erythromycin. Cephalosporins and quinolones are effective empirical antibiotics for M. luteus BSI; however, vancomycin and teicoplanin should be considered for potentially broadly drug-resistant M. luteus strains.

lncRNA-CR46018 positively regulates the Drosophila Toll immune response by interacting with Dif/Dorsal.

The Toll signaling pathway is highly conserved from insects to mammals. Drosophila is a model species that is commonly used to study innate immunity. Although many studies have assessed protein-coding genes that regulate the Toll pathway, it is unclear whether long noncoding RNAs (lncRNAs) play regulatory roles in the Toll pathway. Here, we evaluated the expression of the lncRNA CR46018 in Drosophila. Our results showed that this lncRNA was significantly overexpressed after infection of Drosophila with Micrococcus luteus. A CR46018-overexpressing Drosophila strain was then constructed; we expected that CR46018 overexpression would enhance the expression of various antimicrobial peptides downstream of the Toll pathway, regardless of infection with M. luteus. RNA-seq analysis of CR46018-overexpressing Drosophila after infection with M. luteus showed that upregulated genes were mainly enriched in Toll and Imd signaling pathways. Moreover, bioinformatics predictions and RNA-immunoprecipitation experiments showed that CR46018 interacted with the transcription factors Dif and Dorsal to enhance the Toll pathway. During gram-positive bacterial infection, flies overexpressing CR46018 showed favorable survival compared with flies in the control group. Overall, our current work not only reveals a new immune regulatory factor, lncRNA-CR46018, and explores its potential regulatory model, but also provides a new perspective for the effect of immune disorders on the survival of Drosophila melanogaster.

A fibrinogen-related protein mediates the recognition of various bacteria and haemocyte phagocytosis in oyster Crassostrea gigas.

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like (FBG) domains, which play important roles as pattern recognition receptors (PRRs) in the innate immune responses. In the present study, a fibrinogen-like protein was identified from the oyster Crassostrea gigas (defined as CgFREP1). The open reading frame of CgFREP1 was of 966 bp that encoded a predicted polypeptide of 321 amino acids comprising a signal peptide and a fibrinogen-like domain. The mRNA expression of CgFREP1 was detected in all the examined tissues. The recombinant CgFREP1 (rCgFREP1) displayed binding activities to lipopolysaccharide (LPS), mannose (MAN), as well as Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram-negative bacteria (Vibrio splendidus and Escherichia coli). The rCgFREP1 displayed the agglutinating activity towards M. luteus, V. splendidus and E. coli in the presence of Ca2+. rCgFREP1 was able to enhance the phagocytic activity of haemocytes towards V. splendidus, and exhibited binding activity to the CUB domain of CgMASPL-1. These results suggest that CgFREP1 not only serves as a PRR to recognize and agglutinate different bacteria but also mediates the haemocytes phagocytosis towards V. splendidus.

Scavenger receptor B1 mediates phagocytosis and the antimicrobial peptide pathway in the endoparasitic wasp Micropilits mediator.

Scavenger receptors (SRs) are a family of pattern recognition receptors (PRRs) in the immune system. They are required for phagocytosis and act as co-receptors of Toll-like receptors to regulate immune signaling pathways in the fight against pathogens. Little is known about the function of SRs in insects. Here, we reported on a member of the SR family from the parasitic wasp Micropilits mediator (designated MmSR-B1) that is responsive to bacterial infection. The recombinant extracellular CD36 domain of MmSR-B1 produced in Escherichia coli cells is capable of binding to peptidoglycans and bacterial cells, causing agglutination of bacteria. Furthermore, we demonstrated that double-stranded RNA-mediated knockdown of MmSR-B1 impedes hemocyte phagocytosis and downregulates the expression of antimicrobial peptide (AMP) genes defensins and hymenoptaecins. Knockdown of MmSR-B1 led to increased death of the wasps when challenged by bacteria. Our study suggests that MmSR-B1 mediates phagocytosis and the production of AMPs in M. mediator wasps.

A Pattern Recognition Receptor C-type Lectin-S6 (CTL-S6) is Involved in the Immune Response in the Silkworm (Lepidoptera: Bombycidae).

Insect innate immunity is initiated by the special recognition and binding of the foreign pathogens, which is accomplished by the pattern recognition receptors (PRRs). As an important type of PRRs, C-type lectins (CTLs) play various roles in insect innate immunity, including pathogen recognition, stimulation of prophenoloxidase, regulation of cellular immunity and so on. In this study, we have cloned the full-length cDNA of a CTL gene named CTL-S6 from the silkworm, Bombyx mori. The open reading frame (ORF) of B. mori CTL-S6 encodes 378 amino acids, which contain a secretion signal peptide. The mRNA of CTL-S6 exhibited the highest transcriptional level in the midgut. Its transcriptional level increased dramatically in fat body and hemocytes upon Escherichia coli or Micrococcus luteus challenge. Purified recombinant CTL-S6 could bind to bacterial cell wall components, including peptidoglycan (PGN, from Bacillus subtilis) and lipopolysaccharide (LPS, from E. coli 0111:B4), and recombinant CTL-S6 was involved in the encapsulation and melanization of hemocytes. Furthermore, the addition of recombinant CTL-S6 to the hemolymph of silkworm resulted in a significant increase in phenoloxidase activity. Overall, our results indicated that B. mori CTL-S6 may serve as a PRR for the recognition of foreign pathogens, prophenoloxidase pathway stimulation and involvement in the innate immunity.

Lysozyme c-1 gene is overexpressed in Anopheles albimanus pericardial cells after an immune challenge.

Different evidences suggest that pericardial cells play an important role during the immune response against pathogens that invade the mosquito hemocoel. Previously, we identified two lysozyme genes in Anopheles albimanus heart transcriptome. The present study showed that one of these genes (IDVB: AALB004517) has high percentage of identity to mosquito lysozyme genes related to immunity, suggesting its possible participation during the mosquito immune response. This An. albimanus gen, constitutively expressed lysozyme c-1 mRNA (albLys c-1) in mosquito heart; however, it was overexpressed in bacteria-injected mosquitoes. In heart extract samples, we identified a protein of approximately 14 kDa (likely lysozyme c-1), which lysed M. luteus. In addition, mRNA-FISH assay in heart samples, showed specific fluorescent hybridization signal in pericardial cells from M. luteus-injected mosquitos. We conclude that for the first time an inducible immune factor (lysozyme c-1) is identified in Anopheles albimanus mosquito pericardial cells, which could be a key component in the response against pathogens that interact with the mosquito heart.

A fas apoptotic inhibitory molecule from Ruditapes philippinarum: Investigation on molecular characterization and functional analysis.

In the present study, a fas apoptotic inhibitory molecule (FAIM) was identified from Ruditapes philippinarum (designated as RpFAIM). Multiple alignments and phylogenetic analysis strongly suggested that RpFAIM was a new member of the FAIMs family. The RpFAIM transcripts were constitutively expressed in a wide range of tissues, and dominantly expressed in hemocytes. After V. anguillarum or M. luteus challenge, the expression level of RpFAIM transcripts was significantly induced and reached the maximum level at 6 h and 24 h, respectively. Knockdown of RpFAIM down-regulated the transcript levels of NF-κB signaling genes (e.g. RpIKK, RpIκB, RpNF-κB). The results were roughly similar to those under bacterial stimulation. Moreover, RpFAIM primarily localized in the cell cytoplasm, and its over-expression inhibited the apoptosis of HeLa cells. These results revealed that RpFAIM perhaps regulated the NF-κB signaling pathways positively, which provided a better understanding of RpFAIM in innate immunity.

Submicrometer-Sized Roughness Suppresses Bacteria Adhesion.

Biofilm formation is most commonly combatted with antibiotics or biocides. However, proven toxicity and increasing resistance of bacteria increase the need for alternative strategies to prevent adhesion of bacteria to surfaces. Chemical modification of the surfaces by tethering of functional polymer brushes or films provides a route toward antifouling coatings. Furthermore, nanorough or superhydrophobic surfaces can delay biofilm formation. Here we show that submicrometer-sized roughness can outweigh surface chemistry by testing the adhesion of E. coli to surfaces of different topography and wettability over long exposure times (>7 days). Gram-negative and positive bacterial strains are tested for comparison. We show that an irregular three-dimensional layer of silicone nanofilaments suppresses bacterial adhesion, both in the presence and absence of an air cushion. We hypothesize that a 3D topography can delay biofilm formation (i) if bacteria do not fit into the pores of the coating or (ii) if bending of the bacteria is required to adhere. Thus, such a 3D topography offers an underestimated possibility to design antibacterial surfaces that do not require biocides or antibiotics.

Identification, characterization, and expression analysis of clip-domain serine protease genes in the silkworm, Bombyx mori.

Clip-domain serine proteases (CLIPs), characterized by regulatory module clip domains, constitute an important serine protease family identified in insects and other arthropods. They participate in host immune response and embryonic development in a cascade-activated manner. Here, we present a genome-wide identification and expression analysis of CLIP genes in the silkworm, Bombyx mori. A total of 26 CLIP genes were identified in the silkworm genome. Bioinformatics analysis indicated that these CLIPs clustered into four subfamilies (CLIPA-D), and exhibit a close evolutionary relationship with CLIPs of Manduca sexta. Tissue expression profiling revealed that silkworm CLIP genes are mainly expressed in the integument, head, fat body, and hemocytes. Temporal expression profiles showed that 15 CLIP genes were predominantly expressed during the fifth-instar larval stage, early and later period of the pupal stage, and adult stage, whereas 10 CLIP genes were mainly expressed in the wandering stage and middle to later period of the pupal stage in the integument. Pathogens and 20-hydroxyecdysone (20E) induction analysis indicated that 14 CLIP genes were positively regulated by 20E, 9 were negatively regulated by 20E but positively regulated by pathogens, and 5 were positively regulated by both factors in the integument. Together, these results suggested that silkworm CLIP genes may play multiple functions in integument development, including melanization of new cuticle, molting and immune defense. Our data provide a comprehensive understanding of CLIP genes in the silkworm integument and lays a foundation for further functional studies of CLIP genes in the silkworm.

A Universal Stress Protein That Controls Bacterial Stress Survival in Micrococcus luteus.

Bacteria have remarkable mechanisms to survive severe external stresses, and one of the most enigmatic is the nonreplicative persistent (NRP) state. Practically, NRP bacteria are difficult to treat, and so inhibiting the proteins underlying this survival state may render such bacteria more susceptible to external stresses, including antibiotics. Unfortunately, we know little about the proteins and mechanisms conferring survival through the NRP state. Here, we report that a universal stress protein (Usp) is a primary regulator of bacterial survival through the NRP state in Micrococcus luteus NCTC 2665, a biosafety level 1 (BSL1) mycobacterial relative. Usps are widely conserved, and bacteria, including Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, have multiple paralogs with overlapping functions that have obscured their functional roles. A kanamycin resistance cassette inserted into the M. luteus universal stress protein A 616 gene (ΔuspA616::kanM. luteus) ablates the UspA616 protein and drastically impairs M. luteus survival under even short-term starvation (survival, 83% wild type versus 32% ΔuspA616::kanM. luteus) and hypoxia (survival, 96% wild type versus 48% ΔuspA616::kanM. luteus). We observed no detrimental UspA616 knockout phenotype in logarithmic growth. Proteomics demonstrated statistically significant log-phase upregulation of glyoxylate pathway enzymes isocitrate lyase and malate synthase in ΔuspA616::kanM. luteus We note that these enzymes and the M. tuberculosis UspA616 homolog (Rv2623) are important in M. tuberculosis virulence and chronic infection, suggesting that Usps are important stress proteins across diverse bacterial species. We propose that UspA616 is a metabolic switch that controls survival by regulating the glyoxylate shunt.IMPORTANCE Bacteria tolerate severe external stresses, including antibiotics, through a nonreplicative persistent (NRP) survival state, yet the proteins regulating this survival state are largely unknown. We show a specific universal stress protein (UspA616) controls the NRP state in Micrococcus luteus Usps are widely conserved across bacteria, but their biological function(s) has remained elusive. UspA616 inactivation renders M. luteus susceptible to stress: bacteria die instead of adapting through the NRP state. UspA616 regulates malate synthase and isocitrate lyase, glyoxylate pathway enzymes important for chronic Mycobacterium tuberculosis infection. These data show that UspA616 regulates NRP stress survival in M. luteus and suggest a function for homologous proteins in other bacteria. Importantly, inhibitors of UspA616 and homologs may render NRP bacteria more susceptible to stresses, including current antibiotics.

Endophytic Bacteria Improve Root Traits, Biomass and Yield of Helianthus tuberosus L. under Normal and Deficit Water Conditions.

Drought is more concerned to be a huge problem for agriculture as it affects plant growth and yield. Endophytic bacteria act as plant growth promoting bacteria that have roles for improving plant growth under stress conditions. The properties of four strains of endophytic bacteria were determined under water deficit medium with 20% polyethylene glycol. Bacillus aquimaris strain 3.13 showed high 1-aminocyclopropane-1-carboxylate (ACC) deaminase production; Micrococcus luteus strain 4.43 produced indole acetic acid (IAA). Exopolysaccharide production was high in Bacillus methylotrophicus strain 5.18 while Bacillus sp. strain 5.2 did not show major properties for drought response. Inoculation of endophytic bacteria into plants, strain 3.13 and 4.43 increased height, shoot and root weight, root length, root diameter, root volume, root area and root surface of Jerusalem artichoke grown under water limitation, clearly shown in water supply at 1/3 of available water. These increases were caused by bacteria ACC deaminase and IAA production; moreover, strain 4.43 boosted leaf area and chlorophyll levels, leading to increased photosynthesis under drought at 60 days of planting. The harvest index was high in the treatment with strain 4.43 and 3.13 under 1/3 of available water, promoting tuber numbers and tuber weight. Inulin content was unchanged in the control between well-watered and drought conditions. In comparison, inulin levels were higher in the endophytic bacteria treatment under both conditions, although yields dipped under drought. Thus, the endophytic bacteria promoted in plant growth and yield under drought; they had outstanding function in the enhancement of inulin content under wellwatered condition.

Transcriptome analysis of the molecular mechanism underlying immunity- and reproduction trade-off in Locusta migratoria infected by Micrococcus luteus.

Immune response and reproductive success are two vital energy-consuming processes in living organisms. However, it is still unclear which process is prioritized when both are required. Therefore, the present study was designed to examine this question arising for one of the world's most destructive agricultural pests, the migratory locust, Locusta migratoria. Transcripts from the ovaries and fat bodies of newly emerged locusts were analyzed, using RNA-seq based transcriptome and qualitative real-time PCR, at 4 h and 6 d after being infected with the gram-positive bacteria Micrococcus luteus. Changes in the main biological pathways involved in reproduction and immunization were analyzed using bioinformatics. After 4 h of infection, 348 and 133 transcripts were up- and down-regulated, respectively, whereas 5699 and 44 transcripts were up- and down-regulated, respectively, at 6 d after infection. Moreover, KEGG analysis indicated that vital pathways related with immunity and reproduction, such as Insulin resistance, FoxO signaling, Lysosome, mTOR signaling, and Toll-like receptor signaling pathways were up-regulated. Among the differentially expressed genes, 22 and 17 were related to immunity and reproduction, respectively. The expression levels of PPO1 and antimicrobial peptide defensin 3 were increased (log2FC = 5.93 and 6.75, respectively), whereas those of VgA and VgB were reduced (log2FC = -17.82 and -18.13, respectively). These results indicated that locust allocate energy and resources to maintain their own survival by increasing immune response when dealing with both immune and reproductive processes. The present study provides the first report of expression levels for genes related with reproduction and immunity in locusts, thereby providing a reference for future studies, as well as theoretical guidance for investigations of locust control.

Gene identification and antimicrobial activity analysis of a novel lysozyme from razor clam Sinonovacula constricta.

Lysozymes are important immune effectors present in phylogenetically diverse organisms. They play vital roles in bacterial elimination during early immune responses. In the present study, a second invertebrate-type (i-type) lysozyme gene from razor clam Sinonovacula constricta (denoted as ScLYZ-2) was cloned by RACE and nested PCR methods. The full-length cDNA sequences of ScLYZ-2 were 1558 bp, including a 5' untranslated region (UTR) of 375 bp, an open reading frame of 426 bp, and a 3'-UTR of 757 bp with polyadenylation signal sequence (AATAAA) located upstream of the poly(A) tail. SMART analysis showed that ScLYZ-2 contains a signal peptide in the first 16 amino acid (AA) sequences and a destabilase domain located from 24 to 134 AA sequences. The deduced AA sequences of ScLYZ-2 were highly similar (42%-58%) to other known lysozyme genes of bivalve species. Multiple alignments of AA sequences showed that ScLYZ-2 possesses the classical i-type lysozyme family signature of two motifs ["MDVGSLSCGP(Y/F)QIK" and "CL(E/L/R/H)C(I/M)C"] and two catalytic residues (Glu35 and Asp46). Moreover, phylogenetic analysis showed that ScLYZ-2 is a new member of the i-type lysozyme family. In healthy razor clams, ScLYZ-2 was highly expressed in the hepatopancreas, followed by the gills, water pipes, and abdominal foot. Lysozyme activity and ScLYZ-2 expression levels were significantly upregulated in the hepatopancreas and gills after being infected with V. splendidus, V. harveyi, V. parahaemolyticus and S. aureus and M. luteus. Moreover, the recombinant ScLYZ-2 had strong antimicrobial activities against V. splendidus, V. harveyi, and V. parahaemolyticus. Furthermore, the minimal inhibitory concentration of the recombinant ScLYZ-2 against V. parahaemolyticus was 7.2 µmol/mL. Taken together, our results show that ScLYZ-2 plays an important role in the immune defense of razor clam by eliminating pathogenic microorganisms.

Extracellular DNA Provides Structural Integrity to a Micrococcus luteus Biofilm.

Force spectroscopy was used to show that extracellular DNA (eDNA) has a pre-eminent structural role in a biofilm. The adhesive behavior of extracellular polymeric substances to poly(ethylene terephthalate), a model hydrophobic surface, was measured in response to their degradation by hydrolytic enzymes known for their biofilm dispersion potential: DNaseI, protease, cellulase, and mannanase. Only treatment with DNaseI significantly decreased the adhesive force of the model bacterium Micrococcus luteus with the surface, and furthermore this treatment almost completely eliminated any components of the biofilm maintaining the adhesion, establishing a key structural role for eDNA.

Characterization and functional analysis of a novel mannose-binding lectin from the swimming crab Portunus trituberculatus.

Mannose-binding lectin (MBL) is a pattern recognition receptor (PRR) that plays an important role in the innate immune response. In this study, a novel mannose-binding lectin was cloned from the swimmimg crab Portunus trituberculatus (designated as PtMBL). The complete cDNA of PtMBL gene was 1208 bp in length with an open reading frame (ORF) of 732 bp that encoded 244 amino acid proteins. PtMBL shared lower amino acid similarity with other MBLs, yet it contained the conserved carbohydrate-recognition domain (CRD) with QPD motif and was clearly member of the collectin family. PtMBL transcripts were mainly detected in eyestalk and gill with sexually dimorphic expression. The temporal expression of PtMBL in hemocytes showed different activation times after challenged with Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtMBL protein revealed antimicrobial activity against the tested Gram-negative and Gram-positive bacteria. It could also bind and agglutinate (Ca2+-dependent) both bacteria and yeast. Furthermore, the agglutinating activity could be inhibited by both d-galactose and d-mannose, suggesting the broader pathogen-associated molecular patterns (PAMPs) recognition spectrum of PtMBL. These results together indicate that PtMBL could serve as not only a PRR in immune recognition but also a potential antibacterial protein in the innate immune response of crab.

A color-based competition assay for studying bacterial stress responses in Micrococcus luteus.

Competition assays measure differences between populations of bacteria after stress adaptation, populations of different bacteria and mutations in antibiotic resistance genes. We have developed a competition-based assay to evaluate if genes upregulated under starvation are important for bacterial survival. Stress responses are critical for survival in non-pathogenic and pathogenic bacteria alike including Mycobacterium tuberculosis, Enterococcus fecaelis, Escherichia coli and Staphylococcus aureus. Unfortunately, most stress-survival proteins are poorly understood because suitable model bacteria and techniques are limited. To address this problem, we have engineered Micrococcus luteus NCTC 2665 (M. luteus) for competition assays by inactivating the sarcinaxanthin biosynthesis gene crtE (ΔcrtE), changing M. luteus colonies from yellow to white. This change allows easy identification in mixed cultures. The crtE knockout is relatively neutral for growth in complex and minimal acetate media and shows a measured fitness of one in competition with yellow wild-type bacteria. The ΔcrtE M. luteus competition assay identified a competition defect in a M. luteus strain when a specific universal stress protein was inactivated, suggesting a negative survival phenotype for this protein. We anticipate this competition assay can identify defects in other gene knockouts and mutational studies in M. luteus and will enhance our understanding of bacterial survival mechanisms.

A QM protein from Bombyx mori negatively regulates prophenoloxidase activation and melanization by interacting with Jun protein.

The QM gene that encodes for the ribosomal protein L10 was firstly identified from human tumour cells as a tumour suppressor. In this study, a QM gene was identified in silkworm Bombyx mori (BmQM) and its immunomodulatory function was explored. BmQM messenger RNA (mRNA) and protein were highly expressed in the silk gland and fat body, and expressed in all stages of silkworm growth. After challenged with four different microorganisms, the expression levels of BmQM mRNA in fat body or haemocytes were significantly upregulated compared with the control. After knock-down of BmQM gene, the expressions of some immune genes (PGRPS6, Gloverin0, Lysozyme and Moricin) were affected, and the transcripts of prophenoloxidase1 and prophenoloxidase2 have different degrees of change. The phenoloxidase activity was significantly reduced when the purified recombinant BmQM protein was injected. Recombinant BmQM protein inhibited systemic melanization and suppressed prophenoloxidase activation stimulated by Micrococcus luteus, but it did not affect phenoloxidase activity. Far-western blotting assays showed that the BmQM protein interacted with silkworm BmJun protein, which negatively regulates AP-1 expression. Our results indicated that BmQM protein could affect some immune gene expression and negatively regulate the prophenoloxidase-activating system, and it may play an important role in regulation of the innate immunity in insects.

Identification and functional characterization of a c-type lysozyme from Fenneropenaeus penicillatus.

Lysozyme is an important defense molecule of the innate immune system and possess high antimicrobial activities. In this study, a full-length c-type lysozyme cDNA (Fplysc) was cloned and characterized from Fenneropenaeus penicillatus. The cDNA contains an open reading frame of 477 bp encoding 158 amino acids, with 53-94% identity with those of other crustaceans. The recombinant Fplysc had antibacterial activities against Gram-positive bacteria (Streptococcus agalactiae and Micrococcus luteus) and Gram-negative bacteria (Vibrio alginolyticus and Escherichia coli), and showed antiviral activity against WSSV and IHHNV. The qRT-PCR analysis showed that Fplysc expression levels were most abundant in hemocytes and less in eyestalk. The expression levels of Fplysc were significantly upregulated in gill, intestine and hemocytes when challenged with WSSV and V. alginolyticus. Fplysc-silencling suppressed Fplysc expression in cephalothoraxes and increased mortality caused by WSSV and V. alginolyticus, and exogenous rFplysc led to a significant decrease of shrimp mortality by injecting rFplysc into Fplysc silenced shrimp, suggesting Fplysc is the important molecule in shrimp antimicrobial and antiviral response. In conclusion, the results provide some insights into the function of Fplysc in shrimp against bacterial and viral infection.

An MBT domain containing anti-lipopolysaccharide factor (PtALF8) from Portunus trituberculatus is involved in immune response to bacterial challenge.

Anti-lipopolysaccharide factors are effective antimicrobial peptides that can bind and neutralize lipopolysaccharide (LPS). In the present study, a new sequence encoding for ALF (designated as PtALF8) was cloned by suppression subtractive hybridization method using ovary of swimming crab Portunus trituberculatus as material. The full-length cDNA of PtALF8 consisted of 531 bp with an ORF of 348 bp encoding a peptide of 115 amino acids containing a putative signal peptide of 19 amino acids. The mature PtALF8 had a predicted molecular weight (MW) of 11.28 kDa and theoretical isoelectricpoint (pI) of 5.11. The PtALF8 contains an MBT domain which was not found in the other 7 isoforms of ALF reported in P. trituberculatus. Unlike most ALFs expressed in hemocytes, PtALF8 transcript was predominantly detected in hepatopancreas. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF8 transcript in hemocytes reached the highest level at 3 h, then decreased to the lowest level at 24 h, and started to increase at 48 h. The recombinant protein showed antimicrobial and bactericidal activity against several bacteria, such as Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Gram-negative bacteria, V. alginolyticus, indicated that the PtALF8 isoform might play protective function against invading bacteria in P. trituberculatus.