AMP Genes Research Articles

Comprehensive genomic analysis of Bacillus subtilis and Bacillus paralicheniformis associated with the pearl millet panicle reveals their antimicrobial potential against important plant pathogens

BackgroundPlant microbiome confers versatile functional roles to enhance survival fitness as well as productivity. In the present study two pearl millet panicle microbiome member species Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 found to have beneficial traits including plant growth promotion and broad-spectrum antifungal activity towards taxonomically diverse plant pathogens. Understanding the genomes will assist in devising a bioformulation for crop protection while exploiting their beneficial functional roles.ResultsTwo potential firmicute species were isolated from pearl millet panicles. Morphological, biochemical, and molecular characterization revealed their identities as Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36. The seed priming assays revealed the ability of both species to enhance plant growth promotion and seedling vigour index. Invitro assays with PBs 12 and PBl 36 showed the antibiosis effect against taxonomically diverse plant pathogens (Magnaporthe grisea; Sclerotium rolfsii; Fusarium solani; Alternaria alternata; Ganoderma sp.) of crops and multipurpose tree species. The whole genome sequence analysis was performed to unveil the genetic potential of these bacteria for plant protection. The complete genomes of PBs 12 and PBl 36 consist of a single circular chromosome with a size of 4.02 and 4.33 Mb and 4,171 and 4,606 genes, with a G + C content of 43.68 and 45.83%, respectively. Comparative Average Nucleotide Identity (ANI) analysis revealed a close similarity of PBs 12 and PBl 36 with other beneficial strains of B. subtilis and B. paralicheniformis and found distant from B. altitudinis, B. amyloliquefaciens, and B. thuringiensis. Functional annotation revealed a majority of pathway classes of PBs 12 (30) and PBl 36 (29) involved in the biosynthesis of secondary metabolites, polyketides, and non-ribosomal peptides, followed by xenobiotic biodegradation and metabolism (21). Furthermore, 14 genomic regions of PBs 12 and 15 of PBl 36 associated with the synthesis of RiPP (Ribosomally synthesized and post-translationally modified peptides), terpenes, cyclic dipeptides (CDPs), type III polyketide synthases (T3PKSs), sactipeptides, lanthipeptides, siderophores, NRPS (Non-Ribosomal Peptide Synthetase), NRP-metallophone, etc. It was discovered that these areas contain between 25,458 and 33,000 secondary metabolite-coding MiBiG clusters which code for a wide range of products, such as antibiotics. The PCR-based screening for the presence of antimicrobial peptide (cyclic lipopeptide) genes in PBs 12 and 36 confirmed their broad-spectrum antifungal potential with the presence of spoVG, bacA, and srfAA AMP genes, which encode antimicrobial compounds such as subtilin, bacylisin, and surfactin.ConclusionThe combined in vitro studies and genome analysis highlighted the antifungal potential of pearl millet panicle-associated Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl36. The genetic ability to synthesize several antimicrobial compounds indicated the industrial value of PBs 12 and PBl 36, which shed light on further studies to establish their action as a biostimulant for crop protection.

BmPxt1 mediated immune response by regulating PGE2 in silkworm, Bombyx mori

Prostaglandins (PGs) mediates the immune response of insects to multiple stimuli. Mammalian cyclooxygenase (COXs) is a key enzyme in the synthesis of PGs, and peroxinectin (Pxt) may have similar functions in some sequenced insect genomes. As a representative of Lepidoptera, the silkworm also contains PGs, but its synthetic pathway is not clear. We cloned a full-length cDNA encoding a Pxt, designated as BmPxt1, from silkworm. Sequence alignment analysis showed that the protein encoded by BmPxt1 has a conserved domain similar to Pxts, and its catalytic site is shared with the Pxt of Manduca sexta, which also produces PGs. The expression of BmPxt1 gene was the highest in the hemocytes and was induced by Nuclear Polyhedrosis Virus (NPV) challenge in the detected tissues. Moreover, we found that dsPxt1 treatment deficiency down-regulated BmPxt1 transcript levels and efficiently inhibiting hemocyte-spreading and nodule formation in silkworm. Hemocyte-spreading, nodule formation, phenoloxidase (PO) and AMP genes (attacin, defencin and moricin) were also inhibited by aspirin, a COX inhibitor. Treatment by PGE2 but not arachidonic acid (AA) rescued the immunosuppression; PGs concentrations was also inhibited by aspirin. PGE2, but not AA, treatment rescued the PGs concentrations. The COX inhibitor, aspirin, impaired the innate immune response including nodulation, encapsulation, and melanization in silkworm, while PGE2, but not arachidonic acid (AA), partially reversed these effects of aspirin. Recombinant BmsPxt1 significantly induced PO activation in larvae hemolymph, PGs concentrations and encapsulation of agarose beads. Injection of recombinant BmsPxt1 into larvae resulted in increased transcript levels of AMP genes. Our results confirmed that BmPxt1 was involved in the synthesis of PGs in the innate immune response of silkworm larvae, and provided new information for the role of BmsPxt1 secreted by silkworm in activating PO and antimicrobial peptides.

Genome-Wide Identification of Trachinotus ovatus Antimicrobial Peptides and Their Immune Response against Two Pathogen Challenges.

Golden pompano, Trachinotus ovatus, as a highly nutritious commercially valuable marine fish, has become one of the preferred species for many fish farmers due to its rapid growth, wide adaptability, and ease of feeding and management. However, with the expansion of aquaculture scale, bacterial and parasitic diseases have also become major threats to the golden pompano industry. This study, based on comparative genomics, shows the possibility of preferential evolution of freshwater fish over marine fish by analyzing the phylogenetic relationships and divergence times of 14 marine fish and freshwater fish. Furthermore, we identified antimicrobial peptide genes from 14 species at the genomic level and found that the number of putative antimicrobial peptides may be related to species evolution. Subsequently, we classified the 341 identified AMPs from golden pompano into 38 categories based on the classification provided by the APD3. Among them, TCP represented the highest proportion, accounting for 23.2% of the total, followed by scolopendin, lectin, chemokine, BPTI, and histone-derived peptides. At the same time, the distribution of AMPs in chromosomes varied with type, and covariance analysis showed the frequency of its repeat events. Enrichment analysis and PPI indicated that AMP was mainly concentrated in pathways associated with disease immunity. In addition, our transcriptomic data measured the expression of putative AMPs of golden pompano in 12 normal tissues, as well as in the liver, spleen, and kidney infected with Streptococcus agalactiae and skin infected with Cryptocaryon irritans. As the infection with S. agalactiae and C. irritans progressed, we observed tissue specificity in the number and types of responsive AMPs. Positive selection of AMP genes may participate in the immune response through the MAPK signaling pathway. The genome-wide identification of antimicrobial peptides in the golden pompano provided a complete database of potential AMPs that can contribute to further understanding the immune mechanisms in pathogens. AMPs were expected to replace traditional antibiotics and be developed into targeted drugs against specific bacterial and parasitic pathogens for more precise and effective treatment to improve aquaculture production.

Analysis of Osmotic Tolerance, Physiological Characteristics, and Gene Expression of Salmonella enterica subsp. enterica Serotype Derby.

Salmonella is an important foodborne pathogen, and recent epidemiological studies have shown high infection rates of Salmonella enterica subsp. enterica serotype Derby (S.Derby) in poultry in western China and other regions. S.Derby presents increasing concerns with the development of resistance to hypertonic environments; however, there are few reports investigating the mechanism of resistance. Therefore, in this study, we examined hypertonic adaptation in S.Derby at the physiological and molecular levels. The K-B paper method, wiping glass bead method, crystal violet staining, and RT-PCR combined with comparative genomics analysis were employed to characterize virulence, drug resistance, biofilm formation, and changes in gene expression of genes related to hypertonic adaptation in S.Derby. Hypertonic-adapted S.Derby exhibited resistance to OXA, AMP, PEN, and CEP antibiotics, and biofilm-forming ability was 1.25 times that of nonadapted S.Derby. RT-PCR results showed that compared with nonadapted S.Derby, the expression of virulence-related genes in hypertonic-adapted S.Derby increased by 2-3 times, that of biofilm-related genes increased by 2-4 times, and that of OXA, AMP, PEN, and CEP-related drug resistance genes was relatively high. Four hypertonic tolerance-related genes (otsA, proV, proW, omsV) were preliminarily identified in S.Derby. The expression of proW was always relatively high in hypertonic-adapted S.Derby, the expression of otsA gradually became higher than that of proW with increasing time of osmotic stress, and the expression of proV and omsV was only high in non-hypertonic-adapted S.Derby.

Effectiveness of dietary heat-killed Bacillus subtilis harboring plasmid containing 60 copies of CpG-ODN 1668 against Vibrio harveyi in Penaeus vannamei.

The cost of the purification process hinders the extensive use of cytosine phosphate guanosine-oligodeoxynucleotides (CpG-ODNs) for shrimp culture. Therefore, this study used a shuttle vector plasmid to carry 60 copies of CpG-ODN 1668 (pAD43-25_60CpG), which can replicate in Escherichia coli and Bacillus subtilis strain RIK1285. The first experiment used a reverse gavage procedure to deliver a substance (PBS [CK], pAD43-25 [P0], and pAD43-25_60CpG [P60], respectively) directly into the anterior midgut of Penaeus vannamei and transcriptome sequence analysis with a reference genome was performed to examine the expression of well-known immune-related genes. The results showed that the expression levels of immune-related genes in P60 group were significantly increased, particularly those associated with AMPs. In addition, using RT‒qPCR, the expression levels of AMP genes (LvALF, LvPEN-2, and LvPEN-3) in the P60 group may vary depending on the tissue and time point. The second experiment used dietary supplementation with three kinds of heat-killed B. subtilis (HKBS, HKBS-P0, and HKBS-P60) in 28 days of feeding experiments. The results showed that dietary supplementation with HKBS-P60 did not significantly improve shrimp growth performance and survival. However, on days 14 and 28 of the feeding regimens, alkaline phosphatase (AKP) and acid phosphatase (ACP) activity were considerably higher than in other treatments. In addition, following infection with Vibrio harveyi, AKP and ACP activity in the HKBS-P60 group was significantly higher than in other treatments, particularly at the early stage of bacterial infection. Moreover, HKBS-P60 was found to be better protected against V. harveyi infection with lower cumulative mortality (60%) compared to HKBS (90%) and HKBS-P0 (100%) at 7 days after infection. Overall, these findings confirmed that P60 could increase immunological responses in the shrimp midgut, and HKBS-P60 could be used as an effective tool to enhance the immune response and disease resistance in shrimp.

Identification and in silico structural and functional analysis of a histone H2A-derived antimicrobial peptide from teleost fishes

BackgroundAntimicrobial peptides (AMPs) are small peptides that form the first line of defence in all living organisms. In addition to conventional AMPs, there exist molecules that are often not regarded as part of the innate defences, but possess antimicrobial activity. Histone H2A-derived peptides are a group of unconventional AMPs that represent very ancient defence factors, re-used in new ways during evolution. The present study attempts to compare histone H2A-derived AMPs from four commercially important teleost fishes inhabiting various habitats, viz. Asian sea bass (Lates calcarifer), tank goby (Glossogobius giuris), grey mullet (Mugil cephalus), and Malabar trevally (Carangoides malabaricus). Methods and resultsRT-PCR amplification of the mRNAs extracted from these fishes yielded H2A-derived AMP genes with slight differences in the nucleotide sequences, nevertheless with similar translated amino acid sequences. The amino acid sequence was found to possess marked similarity at the N-terminal region and showed antimicrobial motif sequence characteristic of histone H2A-derived AMPs. Phylogenetic analysis showed an evolutionary relationship with invertebrate ones suggesting a common ancestral origin for the vertebrate and invertebrate histone H2A. The secondary structure of histone H2A-derived AMP showed the presence of α-helices and intermittent coils having a proline hinge connecting the N- and C-terminus. The peptide was also predicted to be a cell-penetrating one. ConclusionIn silico structural and functional characterization of histone H2A-derived AMP revealed its potential antibacterial property and point towards the possibility of being an efficient AMP with the potential of being made use of in therapeutics.

Effects of two fish-derived probiotics on growth performance, innate immune response, intestinal health, and disease resistance of Procambarus clarkii

Selection of the appropriate strain for particular aquatic animals is necessary for the desirable benefits of probiotics application in aquaculture, however, it is not very clear whether fish-derived probiotics could provide beneficial effects on invertebrates. In the present study, two fish-derived probiotics, Bacillus amyloliquefaciens, and Lactococcus lactis, were administrated as feed additives for crayfish (Procambarus clarkii) at concentrations of 106, 107, and 108 CFU/g for 28 days, respectively. Then their effects on the growth performance, innate immune enzyme activity, immune-related genes expression, haemocyte phagocytic activity, intestinal microbial diversity, and pathogen resistance of crayfish were evaluated. The results showed that the final weight, weight gain (WG), and specific growth rate (SGR) were significantly higher in 108 CFU/g B. coagulans and L. lactis additive groups. Compared to the control group, the activities of immune-related enzymes (acid phosphatase, alkaline phosphatase, glutathione peroxidase, and lysozyme) in the intestine were significantly increased in most of the probiotics added groups. In almost all experimental groups, the mRNA expression of two AMP genes (PcALF3 and PcAst) was enhanced in the intestinal of crayfish, and the expression level was proportional to the amounts of probiotics used. Moreover, the intestinal of crayfish in probiotics groups have better integrity and a thicker mucosal layer, and higher density granules in epithelial cells were also observed. According to the results of next-generation sequencing technologies, the diversity of intestinal microbiota in crayfish was also increased after supplementation with B. coagulans or L. lactis. Finally, the phagocytosis rate of haemocytes and pathogen resistance has also been improved. Taken together, our results suggested that fish-derived probiotics have beneficial effects on crayfish, and they can be treated as potential feed additives in practical applications.

Effect of amide protoporphyrin derivatives on immune response in Apis mellifera

The intracellular microsporidian parasite Nosema ceranae is known to compromise bee health by induction of energetic stress and downregulation of the immune system. Porphyrins are candidate therapeutic agents for controlling Nosema infection without adverse effects on honeybees. In the present work, the impact of two protoporphyrin IX derivatives, i.e. PP[Asp]2 and PP[Lys]2, on Apis mellifera humoral immune response has been investigated in laboratory conditions in non-infected and N. ceranae-infected honeybees. Fluorescence spectroscopy analysis of hemolymph showed for the first time that porphyrin molecules penetrate into the hemocoel of honeybees. Phenoloxidase (PO) activity and the expression of genes encoding antimicrobial peptides (AMPs: abaecin, defensin, and hymenoptaecin) were assessed. Porphyrins significantly increased the phenoloxidase activity in healthy honeybees but did not increase the expression of AMP genes. Compared with the control bees, the hemolymph of non-infected bees treated with porphyrins had an 11.3- and 6.1-fold higher level of PO activity after the 24- and 48-h porphyrin administration, respectively. Notably, there was a significant inverse correlation between the PO activity and the AMP gene expression level (r = − 0.61696, p = 0.0143). The PO activity profile in the infected bees was completely opposite to that in the healthy bees (r = − 0.5118, p = 0.000), which was related to the changing load of N. ceranae spores in the porphyrin treated-bees. On day 12 post-infection, the spore loads in the infected porphyrin-fed individuals significantly decreased by 74%, compared with the control bees. Our findings show involvement of the honeybee immune system in the porphyrin-based control of Nosema infection. This allows the infected bees to improve their lifespan considerably by choosing an optimal PO activity/AMP expression variant to cope with the varying level of N. ceranae infection.

Differential Expression of Major Royal Jelly Proteins in the Hypopharyngeal Glands of the Honeybee Apis mellifera upon Bacterial Ingestion.

Simple SummaryTransgenerational immune priming (TGIP) to elicit social immunity in the honeybee Apis mellifera has two axes: the first is the ingested pathogen fragments–vitellogenin (Vg)–queen’s ovary axis for the developing embryo, and the second is the ingested pathogen fragments–Vg–nurse’s hypopharyngeal gland axis for the queen and young larvae through royal jelly. However, the dynamics of the expression of the major royal jelly proteins (MRJPs) in the hypopharyngeal glands of A. mellifera nurse bees after bacterial ingestion must be determined to improve our understanding of the second axis of TGIP. In this study, we investigated the expression patterns of MRJPs 1–7 and defensin-1 in the hypopharyngeal glands and Vg in the fat body of nurse bees fed with live or heat-killed Paenibacillus larvae over 12 h or 24 h by using northern blot analysis. We found that the expression of MRJPs and defensin-1 in the hypopharyngeal glands and Vg in the fat body was significantly induced in nurse bees upon bacterial ingestion, indicating that the differential expression patterns of MRJPs, defensin-1, and Vg were dependent on the bacterial status and timing of bacterial ingestion. We also found that antimicrobial peptide (AMP) genes showed induced expression in young larvae upon bacterial ingestion. In summary, our findings indicate that MRJPs in the hypopharyngeal glands are upregulated along with Vg in the fat body of nurse bees upon bacterial ingestion, providing novel insights into the ingested pathogen fragments–Vg–nurse’s hypopharyngeal gland axis for TGIP.Honeybee vitellogenin (Vg) transports pathogen fragments from the gut to the hypopharyngeal glands and is also used by nurse bees to synthesize royal jelly (RJ), which serves as a vehicle for transferring pathogen fragments to the queen and young larvae. The proteomic profile of RJ from bacterial-challenged and control colonies was compared using mass spectrometry; however, the expression changes of major royal jelly proteins (MRJPs) in hypopharyngeal glands of the honeybee Apis mellifera in response to bacterial ingestion is not well-characterized. In this study, we investigated the expression patterns of Vg in the fat body and MRJPs 1–7 in the hypopharyngeal glands of nurse bees after feeding them live or heat-killed Paenibacillus larvae. The expression levels of MRJPs and defensin-1 in the hypopharyngeal glands were upregulated along with Vg in the fat body of nurse bees fed with live or heat-killed P. larvae over 12 h or 24 h. We observed that the expression patterns of MRJPs and defensin-1 in the hypopharyngeal glands and Vg in the fat body of nurse bees upon bacterial ingestion were differentially expressed depending on the bacterial status and the time since bacterial ingestion. In addition, the AMP genes had increased expression in young larvae fed heat-killed P. larvae. Thus, our findings indicate that bacterial ingestion upregulates the transcriptional expression of MRJPs in the hypopharyngeal glands as well as Vg in the fat body of A. mellifera nurse bees.

Microbiome of Pukzing Cave in India shows high antimicrobial activity against plant and animal pathogens

Pukzing cave, the largest cave of Mizoram, India was explored for bacterial diversity. Culture dependent method revealed 235 bacterial isolates using three different treatments. Identity of the microbial species was confirmed by 16S rDNA sequencing. The highest bacterial population was recovered from heat treatment (n = 97;41.2%) followed by normal (n = 79;33.6%) and cold treatment (n = 59;25.1%) indicating dominance of moderate thermophiles. Antimicrobial potential of isolates showed 20.4% isolates having antimicrobial ability against tested pathogens. Amplicon sequencing of PKSI, PKSII and NRP specific genes revealed presence of AMP genes in the microbial population. Six microbial pathogens were selected for screening as they are well known for different disease cause organism in various fields such as agriculture and human health. Cave environment harbors unique microbial flora and hypervariable region V4 is more informative. Higher activity of AMP assay against these microbes indicates that cave microbial communities could be potential source of future genomic resources.

FOXO regulates the expression of antimicrobial peptides and promotes phagocytosis of hemocytes in shrimp antibacterial immunity.

Invertebrates rely on innate immunity, including humoral and cellular immunity, to resist pathogenic infection. Previous studies showed that forkhead box transcription factor O (FOXO) participates in mucosal immune responses of mammals and the gut humoral immune regulation of invertebrates. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from shrimp (Marsupenaeus japonicus) and found that it was expressed at relatively basal levels in normal shrimp, but was upregulated significantly in shrimp challenged by Vibrio anguillarum. FOXO played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish, the transcription factor of the immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity. In the nucleus, activated FOXO directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C (Src), and two small GTPases, Rab5 and Rab7, which are related to phagosome trafficking to the lysosome in the cytoplasm. Taken together, our results indicated that FOXO exerts its effects on homeostasis of hemolymph and the enteric microbiota by activating the IMD pathway in normal shrimp, and directly or indirectly promoting AMP expression and enhancing phagocytosis of hemocytes against pathogens in bacteria-infected shrimp. This study revealed the different functions of FOXO in the mucosal (local) and systemic antibacterial immunity of invertebrates.

Multilocus typing of aster yellows phytoplasmas infecting lettuce withyellowing in Calabria, Italy

Lettuce is an important vegetable crop commercially produced in many countries and affected by several bacterial diseases. The presence of phytoplasmas in lettuce plants growing in open field in Calabria region (Southern Italy) and showing yellowing and leaf necrosis was detected by PCR assays with primers amplifying 16S rRNA gene sequence. The ‘Candidatus Phytoplasma asteris’ strain identified in the symptomatic samples was further amplified on amp, groEL, and leucine genes for its molecular characterization. This lettuce strain resulted scattered in the fields and was identified by RFLP analyses as 16SrI-B; a multigene characterization confirmed its ribosomal subgroup affiliation and the validity of these molecular markers for studying the geographic distribution of phytoplasma strains of this taxon.

Scavenger receptor B8 improves survivability by mediating innate immunity in silkworm, Bombyx mori

Scavenger receptor class B (SR-B) is an extracellular transmembrane glycoprotein that plays a vital role in innate immunity. Although SR-Bs have been widely studied in vertebrates, their functions remained to elucidate in insects. Here, we identified and characterized a scavenger receptor class B member from the silkworm, Bombyx mori (designated as BmSCRB8). BmSCRB8 is broadly expressed in various immune tissues/organs, including fat body, gut, and hemocyte. Its expression is dramatically enhanced after challenge with different types of bacteria or pathogen-associated molecular patterns (PAMPs). The recombinant BmSCRB8 protein can detect different types of bacteria by directly binding to PAMPs and significantly improve the bacterial clearance in vivo. After knockdown of BmSCRB8, the pathogenic bacterial clearance was strongly impaired, and several AMP genes were down-regulated following E. coli challenge. Moreover, pathogenic bacteria's treatment following the depletion of BmSCRB8 remarkably decreased silkworm larvae's survival rate. Taken together, these results demonstrate that BmSCRB8 acts as a pattern recognition protein and plays an essential role in silkworm innate immunity by enhancing bacterial clearance and contributing to the production of AMPs in vivo.

Bacterial but not fungal challenge up‐regulates the transcription of Coleoptericin genes in Tenebrio molitor

AbstractAntimicrobial peptides (AMPs) are effector candidates that elicit humoral immunity in invertebrates. AMPs facilitate bacterial clearance by either physically disrupting the microbial membranes or the intracellular targets. In the Coleopteran pest, Tenebrio molitor, transcriptional regulation of AMPs has been studied in the context of innate immune signaling cascades and antimicrobial immunity. Here, we report the transcriptional response of three AMP genes, Coleoptericin A, B, and C (TmCole A, B and C) in T. molitor in response to bacterial (Escherichia coli, Staphylococcus aureus), and fungal (Candida albicans) challenges. These genes were expressed essentially in the gut and hemocytes followed by the integument tissue of the T. molitor larva. Further, these genes were highly expressed in the late‐larval, pupal, and early adult stages. Furthermore, while all of these transcripts were highly upregulated in the fat body and Malpighian tubules after bacterial challenge, TmCole B and TmCole C were induced in the gut after E. coli challenge. Fungal stimulation was not required for the upregulation of the transcription of Coleoptericin genes in T. molitor.

Peptidoglycan recognition proteins regulate immune response of Antheraea pernyi in different ways

Peptidoglycan recognition proteins (PGRPs) are evolutionarily conserved molecules that act in innate immune responses against invading pathogens. Insect PGRPs activate the Toll and/or immune deficiency (IMD) signal transduction pathways. They induce cellular and humoral immune defense reactions that effectively protect insects from invasion of microorganisms. In this study, four cDNA clones encoding PGRPs (ApPGRP-A, ApPGRP-B, ApPGRP-C, ApPGRP-LE) were isolated from the Chinese oak silkworm, Antheraea pernyi. The deduced amino acid sequences of ApPGRP-B and -C share high identity with enzymatically active PGRP proteins and contain the amino acids required for amidase activity. The spatiotemporal expression patterns of ApPGRPs and their response to immune stimulations were determined, and suggest that PGRPs might act as a broad-spectrum pattern recognition protein and participate in the pathway activation system. Here, using an RNA-interference approach, we examined the function of PGRPs in response to immune stimulation. We observed that RNAi-mediated silencing of ApPGRP-A decreased the tested antimicrobial peptides (Attacin, Attacin 2, Ceropin, Gloverin, lysozyme and Lebocin) in response to Enterococcus pernyi challenge. However, reducing the ApPGRP-B, -C mRNA levels led to a strong increase of the AMP genes (except for Lebocin). These results suggest that ApPGRPs are necessary for pathway initiation complex formation and activate AMP generation. Our data also indicated that PGRP-B and -C down-regulate AMPs generation in the immune response.

Impact of Nosema ceranae and Nosema apis on individual worker bees of the two host species (Apis cerana and Apis mellifera) and regulation of host immune response

Nosema apis and Nosema ceranae are obligate intracellular microsporidian parasites infecting midgut epithelial cells of host adult honey bees, originally Apis mellifera and Apis cerana respectively. Each microsporidia cross-infects the other host and both microsporidia nowadays have a worldwide distribution. In this study, cross-infection experiments using both N. apis and N. ceranae in both A. mellifera and A. cerana were carried out to compare pathogen proliferation and impact on hosts, including host immune response. Infection by N. ceranae led to higher spore loads than by N. apis in both host species, and there was greater proliferation of microsporidia in A. mellifera compared to A. cerana. Both N. apis and N. ceranae were pathogenic in both host Apis species. N. ceranae induced subtly, though not significantly, higher mortality than N. apis in both host species, yet survival of A. cerana was no different to that of A. mellifera in response to N. apis or N. ceranae. Infections of both host species with N. apis and N. ceranae caused significant up-regulation of AMP genes and cellular mediated immune genes but did not greatly alter apoptosis-related gene expression. In this study, A. cerana enlisted a higher immune response and displayed lower loads of N. apis and N. ceranae spores than A. mellifera, suggesting it may be better able to defend itself against microsporidia infection. We caution against over-interpretation of our results, though, because differences between host and parasite species in survival were insignificant and because size differences between microsporidia species and between host Apis species may alternatively explain the differential proliferation of N. ceranae in A. mellifera.

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa - their role in the development of resistance.

The bacterial cell-wall that forms a protective layer over the inner membrane is called the murein sacculus - a tightly cross-linked peptidoglycan mesh unique to bacteria. Cell-wall synthesis and recycling are critical cellular processes essential for cell growth, elongation and division. Both de novo synthesis and recycling involve an array of enzymes across all cellular compartments, namely the outer membrane, periplasm, inner membrane and cytoplasm. Due to the exclusivity of peptidoglycan in the bacterial cell-wall, these players are the target of choice for many antibacterial agents. Our current understanding of cell-wall biochemistry and biogenesis in Gram-negative organisms stems mostly from studies of Escherichia coli. An incomplete knowledge on these processes exists for the opportunistic Gram-negative pathogen, Pseudomonas aeruginosa. In this review, cell-wall synthesis and recycling in the various cellular compartments are compared and contrasted between E. coli and P. aeruginosa. Despite the fact that there is a remarkable similarity of these processes between the two bacterial species, crucial differences alter their resistance to β-lactams, fluoroquinolones and aminoglycosides. One of the common mediators underlying resistance is the amp system whose mechanism of action is closely associated with the cell-wall recycling pathway. The activation of amp genes results in expression of AmpC β-lactamase through its cognate regulator AmpR which further regulates multi-drug resistance. In addition, other cell-wall recycling enzymes also contribute to antibiotic resistance. This comprehensive summary of the information should spawn new ideas on how to effectively target cell-wall processes to combat the growing resistance to existing antibiotics.

TmCactin plays an important role in Gram-negative and -positive bacterial infection by regulating expression of 7 AMP genes in Tenebrio molitor

Cactin was originally identified as an interactor of the Drosophila IκB factor Cactus and shown to play a role in controlling embryonic polarity and regulating the NF-κB signaling pathway. While subsequent studies have identified the roles for Cactin in the mammalian immune response, the immune function of Cactin in insects has not been described yet. Here, we identified a Cactin gene from the mealworm beetle, Tenebrio molitor (TmCactin) and characterized its functional role in innate immunity. TmCactin was highly expressed in prepupa to last instar stages, and its expression was high in the integument and Malpighian tubules of last instar larvae and adults. TmCactin was induced in larvae after infection with different pathogens and detectable within 3 hours of infection. The highest levels of TmCactin expression were detected at 9 hours post infection. TmCactin RNAi significantly decreased the survival rates of larvae after challenge with Escherichia coli and Staphylococcus aureus, but had no significant effect after challenge with Candida albicans. Furthermore, TmCactin RNAi significantly reduced the expression of seven antimicrobial peptide genes (AMPs) after bacterial challenge. Our results suggest that TmCactin may serve as an important regulator of innate immunity, mediating AMP responses against both Gram-positive and Gram-negative bacteria in T. molitor.

Biocontrol Potentials of Antimicrobial Peptide Producing Bacillus Species: Multifaceted Antagonists for the Management of Stem Rot of Carnation Caused by Sclerotinia sclerotiorum

Bacillus species are widely exploited as biocontrol agents because of their efficiency in impeding various plant pathogens with multifaceted approach. In this study, Bacillus species were isolated from rhizosphere of various plants viz., carnations, cotton, turmeric, and bananas in Tamil Nadu state of India. Their potential to control the mycelial growth of Sclerotinia sclerotiorum was assessed in vitro by dual plate and partition plate techniques. B. amyloliquefaciens strain VB7 was much effective in inhibiting mycelial growth (45% inhibition of over control) and sclerotial production (100%). PCR detection of AMP genes revealed that B. amyloliquefaciens (VB7) had a maximum of 10 diverse antibiotic biosynthesis genes, namely, ituD, ipa14, bacA, bacD, bamC, sfP, spaC, spaS, alba, and albF, that resulted in production of the antibiotics iturin, bacilysin, bacillomycin, surfactin, subtilin, and subtilosin. Further, metabolites from B. amyloliquefaciens strains VB2 and VB7, associated with inhibition of S. sclerotiorum, were identified as phenols and fatty acids by gas chromatography mass spectrometry (GC-MS). Delivery of bacterial suspension of the effective strains of Bacillus spp. as root dip was found promising for the management of stem rot of cultivated carnations. Minimal percent disease incidence (4.6%) and maximum plant growth promotion was observed in the plants treated with B. amyloliquefaciens (VB7).

The use of brief post-surgical low frequency electrical stimulation to enhance nerve regeneration in clinical practice.

Despite efforts to enhance peripheral nerve regeneration, there has been little progress in improving clinical outcomes. Recently, a method of brief post-surgical low frequency electrical stimulation of surgically repaired nerves has been developed. It was shown to accelerate axon outgrowth across the repair site and it hastened target reinnervation. In this brief review, we describe the mechanistic insights and functional impacts of the post-surgical electrical stimulation that have been gained through animal studies. Brain-derived neurotrophic factor, cyclic AMP and regeneration-associated genes play a vital role in expediting the outgrowth of axons across the injury site. The method of stimulation has also been shown to be effective in patients with severe compressive neuropathy as well as those with digital nerve laceration. Its clinical feasibility and positive impact open the door of further clinical translation in other peripheral nerve injuries.