Utilization of gene manipulation system for advancing the biotechnological potential of halophiles: A review.

Halophiles are salt-loving microorganisms known to have their natural resistance against media contamination even when cultivated in nonsterile and continuous bioprocess system, thus acting as promising cell factories for Next Generation of Industrial Biotechnology (NGIB). NGIB - a successor to the traditional industrial biotechnology, is a more sustainable and efficient bioprocess technology while saving energy and water in a more convenient way as well as reducing the investment cost and skilled workforce requirement. Numerous studies have achieved intriguing outcomes during synthesis of different metabolite using halophiles such as polyhydroxyalkanoates (PHA), ectoine, biosurfactants, and carotenoids. Present-day development in genetic maneuverings have shown optimistic effects on the industrial applications of halophiles. However, viable and competent genetic manipulation system and gene editing tools are critical to accelerate the process of halophile engineering. With the aid of such powerful gene manipulation systems, exclusive microbial chassis are being crafted with desirable features to breed another innovative area of research such as synthetic biology. This review provides an aerial perspective on how the expansion of adaptable gene manipulation toolkits in halophiles are contributing towards biotechnological advancement, and also focusses on their subsequent application for production improvement. This current methodical and comprehensive review will definitely help the scientific fraternity to bridge the gap between challenges and opportunities in halophile engineering.

Recent advancement in protected delivery methods for carotenoid: a smart choice in modern nutraceutical formulation concept.

Carotenoids are fat-soluble bio pigments often responsible for red, orange, pink and yellow coloration of fruits and vegetables. They are commonly referred as nutraceutical which is an alternative to pharmaceutical drugs claiming to have numerous physiological benefits. However their activity often get disoriented by photonic exposure, temperature and aeration rate thus leading to low bioavailability and bio accessibility. Most of the market value for carotenoids revolves around food and cosmetic industries as supplement where they have been continuously exposed to rigorous physico-chemical treatment. Though several encapsulation techniques are now in practice to improve stability of carotenoids, the factors like shelf life during storage and controlled release from the delivery vehicle always appeared to be a bottleneck in this field. In this situation, different technologies in nanoscale is showing promising result for carotenoid encapsulation and delivery as they provide greater mass per surface area and protects most of their bioactivities. However, safety concerns related to carrier material and process must be evaluated crucially. Thus, the aim of this review was to collect and correlate technical information concerning the parameters playing pivotal role in characterization and stabilization of designed vehicles for carotenoids delivery. This comprehensive study predominantly focused on experiments carried out in past decade explaining how researchers have fabricated bioprocess engineering in amalgamation with nano techniques to improve the bioavailability for carotenoids. Furthermore, it will help the readers to understand the cognisance of carotenoids in nutraceutical market for their trendy application in food, feed and cosmeceutical industries in contemporary era.

Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea.

Although the use of multiple replication origins for chromosome replication has been widely characterized in haloarchaea, whether it is possible to manipulate the chromosome copy number by their genetic engineering is not known, and how it would affect the cell functioning is poorly understood. Here, we demonstrate that deletion of the three active chromosomal origins in Haloferax mediterranei remarkably reduces its DNA amounts and ploidy numbers. Consequently, the mutant strain H. mediterranei Δ123 is more sensitive to UV and mitomycin C. Surprisingly, the cell size increases by 21.2%, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production in shake flask culture enhances from 7.23 to 8.11 g/L in ΔEPSΔ123, although there is also a decrease in cell growth. In this mutant, the chromosomal copy number decreases, whereas the pha-encoding pHM300 megaplasmid copy number increases. Moreover, our transcriptome analysis reveals that the genes involved in primary metabolisms are significantly downregulated in ΔEPSΔ123, whereas those responsible for starch utilization and precursor supplying for PHBV monomers are upregulated. This indicates that more energy and carbon flux is redirected from primary metabolism to PHBV synthesis, thereby enhancing its PHBV accumulation. These findings may therefore provide a rational design to enhance PHBV synthesis by simply tuning the replication origins to modulate the chromosome/megaplasmid copy number ratio and subsequently influence cellular metabolism and physiological functions. IMPORTANCE The haloarchaeon Haloferax mediterranei is a potential producer of PHBV (100% biodegradable plastic) from inexpensive carbon sources. We previously reported that H. mediterranei possessed three active chromosomal origins and, when these origins were deleted, a dormant origin was activated to initiate the replication of chromosome. In this context, in the present study, we first found a close connection between replication initiation and PHBV accumulation. We describe the potential industrial advantages of the strain H. mediterranei ΔEPSΔ123, which includes the enlargement of cell volume by 21.2% and enhancement of PHBV production by 11.2%. We further reveal the possible mechanism that contributes to the greater PHBV production in the ΔEPSΔ123 strain. Overall, we provide here a conceptual advance in the field of synthetic biology by modulating chromosome replication to improve the production of bio-based chemicals.

Halomonas spp., as chassis for low-cost production of chemicals.

Halomonas spp. are the well-studied platform organisms or chassis for next-generation industrial biotechnology (NGIB) due to their contamination-resistant nature combined with their fast growth property. Several Halomonas spp. have been studied regarding their genomic information and molecular engineering approaches. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), proteins including surfactants and enzymes, small molecular compounds including amino acids and derivates, as well as organic acids. This paper reviews all the progress reported in the last 10 years regarding this robust microbial cell factory. KEY POINTS: • Halomonas spp. are robust chassis for low-cost production of chemicals • Genomic information of some Halomonas spp. has been revealed • Molecular tools and approaches for Halomonas spp. have been developed • Halomonas spp. are becoming more and more important for biotechnology.

Deficiency of exopolysaccharides and O-antigen makes Halomonas bluephagenesis self-flocculating and amenable to electrotransformation.

Halomonas bluephagenesis, a haloalkaliphilic bacterium and native polyhydroxybutyrate (PHB) producer, is a non-traditional bioproduction chassis for the next generation industrial biotechnology (NGIB). A single-sgRNA CRISPR/Cas9 genome editing tool is optimized using dual-sgRNA strategy to delete large DNA genomic fragments (>50 kb) with efficiency of 12.5% for H. bluephagenesis. The non-essential or redundant gene clusters of H. bluephagenesis, including those encoding flagella, exopolysaccharides (EPSs) and O-antigen, are sequentially deleted using this improved genome editing strategy. Totally, ~3% of the genome is reduced with its rapid growth and high PHB-production ability unaffected. The deletion of EPSs and O-antigen gene clusters shows two excellent properties from industrial perspective. Firstly, the EPSs and O-antigen deleted mutant rapidly self-flocculates and precipitates within 20 min without centrifugation. Secondly, DNA transformation into the mutant using electroporation becomes feasible compared to the wild-type H. bluephagenesis. The genome-reduced H. bluephagenesis mutant reduces energy and carbon source requirement to synthesize PHB comparable to its wild type. The H. bluephagenesis chassis with a reduced genome serves as an improved version of a NGIB chassis for productions of polyhydroxyalkanoates (PHA) or other chemicals.

An updated overview on the regulatory circuits of polyhydroxyalkanoates synthesis.

Polyhydroxyalkanoates (PHA) are a promising and sustainable alternative to the petroleum-based synthetic plastics. Regulation of PHA synthesis is receiving considerable importance as engineering the regulatory factors might help developing strains with improved PHA-producing abilities. PHA synthesis is dedicatedly regulated by a number of regulatory networks. They tightly control the PHA content, granule size and their distribution in cells. Most PHA-accumulating microorganisms have multiple regulatory networks that impart a combined effect on PHA metabolism. Among them, several factors ranging from global to specific regulators, have been identified and characterized till now. This review is an attempt to categorically summarize the diverse regulatory circuits that operate in some important PHA-producing microorganisms. However, in several organisms, the detailed mechanisms involved in the regulation of PHA synthesis is not well-explored and hence further research is needed. The information presented in this review might help researcher to identify the prevailing research gaps in PHA regulation.

Current Advances towards 4-Hydroxybutyrate Containing Polyhydroxyalkanoates Production for Biomedical Applications.

Polyhydroxyalkanoates (PHA) are polyesters having high promise in biomedical applications. Among different types of PHA, poly-4-hydroxybutyrate (P4HB) is the only polymer that has received FDA approval for medical applications. However, most PHA producing microorganisms lack the ability to synthesize P4HB or PHA comprising 4-hydroxybutyrate (4HB) monomer due to their absence of a 4HB monomer supplying pathway. Thus, most microorganisms require supplementation of 4HB precursors to synthesize 4HB polymers. However, usage of 4HB precursors incurs additional production cost. Therefore, researchers have adopted strategies to reduce the cost, such as utilizing low-cost substrate as well as constructing 4HB monomer supplying pathways in microorganisms. We herein summarize the biomedical applications of P4HB, the natural producers of 4HB polymer, and the various strategies that have been applied in producing 4HB polymers in non-4HB producing microorganisms. It is expected that the readers would gain a vivid idea on the different strategic developments in the field of 4HB polymer production.

Optimising PHBV biopolymer production in haloarchaea via CRISPRi-mediated redirection of carbon flux.

The haloarchaeon Haloferax mediterranei is a potential strain for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production, yet the production yield and cost are the major obstacles hindering the use of this archaeal strain. Leveraging the endogenous type I-B CRISPR-Cas system in H. mediterranei, we develop a CRISPR-based interference (CRISPRi) approach that allows to regulate the metabolic pathways related to PHBV synthesis, thereby enhancing PHBV production. Our CRISPRi approach can downregulate the gene expression in a range of 25% to 98% depending upon the target region. Importantly, plasmid-mediated CRISPRi downregulation on the citrate synthase genes (citZ and gltA) improves the PHBV accumulation by 76.4% (from 1.78 to 3.14 g/L). When crRNA cassette integrated into chromosome, this further shortens the PHBV fermentation period and enhances PHA productivity by 165%. Our transcriptome analysis shows that repression of citrate synthase genes redirects metabolic flux from the central metabolic pathways to PHBV synthesis pathway. These findings demonstrate that the CRISPRi-based gene regulation is a transformative toolkit for fine-tuning the endogenous metabolic pathways in the archaeal system, which can be applied to not only the biopolymer production but also many other applications.

Deletion of the pps-like gene activates the cryptic phaC genes in Haloferax mediterranei.

Haloferax mediterranei, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) producing haloarchaeon, possesses four PHA synthase encoding genes, phaC, phaC1, phaC2, and phaC3. In the wild-type strain, except phaC, the other three genes are cryptic and not transcribed under PHA-accumulating conditions. The PhaC protein together with PhaE subunit forms the active PHA synthase and catalyzes PHBV polymerization. Previously, it was observed that the deletion of a gene named pps-like significantly enhanced PHBV accumulation probably resulted from the upregulation of pha cluster genes (phaR-phaP-phaE-phaC). The present study demonstrated the influence of pps-like gene deletion on the cryptic phaC genes. As revealed by qRT-PCR, the expression level of the three cryptic genes was upregulated in the ΔEPSΔpps-like geneΔphaC mutant. Sequential knockout of the cryptic phaC genes and fermentation experiments showed that PhaC1 followed by PhaC3 had the ability to synthesize PHBV in ΔEPSΔpps-like geneΔphaC mutant. Both PhaC1 and PhaC3 could complex with PhaE to form functionally active PHA synthase. However, the expression of phaC2 did not lead to PHBV synthesis. Moreover, PhaC, PhaC1, and PhaC3 exhibited distinct substrate specificity as the 3HV content in PHBV copolymers was different. The EMSA result showed that PPS-like protein might be a negative regulator of phaC1 gene by binding to its promoter region. Taken together, PhaC1 had the most pronounced effect on PHBV synthesis in ΔEPSΔpps-like geneΔphaC mutant and deletion of pps-like gene released the negative effect from phaC1 expression and thereby restored PHBV accumulating ability in ΔphaC mutant. KEY POINTS: • Cryptic phaC genes were activated by pps-like gene deletion. • PPS-like protein probably regulated phaC1 expression by binding to its promoter. • Both PhaC1 and PhaC3 formed active PHA synthase with PhaE.

Current developments on polyhydroxyalkanoates synthesis by using halophiles as a promising cell factory.

Plastic pollution is a severe threat to our environment which necessitates implementation of bioplastics to realize sustainable development for a green world. Polyhydroxyalkanoates (PHA) represent one of the potential candidates for these bioplastics. However, a major challenge faced by PHA is the high production cost which limits its commercial application. Halophiles are considered to be a promising cell factory for PHA synthesis due to its several unique characteristics including high salinity requirement preventing microbial contamination, high intracellular osmotic pressure allowing easy cell lysis for PHA recovery, and capability to utilize wide spectrum of low-cost substrates. Optimization of fermentation parameters has made it plausible to achieve large-scale production at low cost by using halophiles. Further deeper insights into halophiles have revealed the existence of diversified and even novel PHA synthetic pathways within different halophilic species that greatly affects PHA type. Thus, precise metabolic engineering of halophiles with the help of advanced tools and strategies have led to more efficient microbial cell factory for PHA production. This review is an endeavour to summarize the various research achievements in these areas which will help the readers to understand the current developments as well as the future efforts in PHA research.

Unusual Phosphoenolpyruvate (PEP) Synthetase-Like Protein Crucial to Enhancement of Polyhydroxyalkanoate Accumulation in Haloferax mediterranei Revealed by Dissection of PEP-Pyruvate Interconversion Mechanism.

Phosphoenolpyruvate (PEP)/pyruvate interconversion is a major metabolic point in glycolysis and gluconeogenesis and is catalyzed by various sets of enzymes in different Archaea groups. In this study, we report the key enzymes that catalyze the anabolic and catabolic directions of the PEP/pyruvate interconversion in Haloferax mediterranei The in silico analysis showed the presence of a potassium-dependent pyruvate kinase (PYKHm [HFX_0773]) and two phosphoenol pyruvate synthetase (PPS) candidates (PPSHm [HFX_0782] and a PPS homolog protein named PPS-like [HFX_2676]) in this strain. Expression of the pykHm gene and ppsHm was induced by glycerol and pyruvate, respectively; whereas the pps-like gene was not induced at all. Similarly, genetic analysis and enzyme activities of purified proteins showed that PYKHm catalyzed the conversion from PEP to pyruvate and that PPSHm catalyzed the reverse reaction, while PPS-like protein displayed no function in PEP/pyruvate interconversion. Interestingly, knockout of the pps-like gene led to a 70.46% increase in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production. The transcriptome sequencing (RNA-Seq) and quantitative reverse transcription-PCR (qRT-PCR) results showed that many genes responsible for PHBV monomer supply and for PHBV synthesis were upregulated in a pps-like gene deletion strain and thereby improved PHBV accumulation. Additionally, our phylogenetic evidence suggested that PPS-like protein diverged from PPS enzyme and evolved as a distinct protein with novel function in haloarchaea. Our findings attempt to fill the gaps in central metabolism of Archaea by providing comprehensive information about key enzymes involved in the haloarchaeal PEP/pyruvate interconversion, and we also report a high-yielding PHBV strain with great future potentials.IMPORTANCEArchaea, the third domain of life, have evolved diversified metabolic pathways to cope with their extreme habitats. Phosphoenol pyruvate (PEP)/pyruvate interconversion during carbohydrate metabolism is one such important metabolic process that is highly differentiated among Archaea However, this process is still uncharacterized in the haloarchaeal group. Haloferax mediterranei is a well-studied haloarchaeon that has the ability to produce polyhydroxyalkanoates (PHAs) under unbalanced nutritional conditions. In this study, we identified the key enzymes involved in this interconversion and discussed their differences with their counterparts from other members of the Archaea and Bacteria domains. Notably, we found a novel protein, phosphoenolpyruvate synthetase-like (PPS-like), which exhibited high homology to PPS enzyme. However, PPS-like protein has evolved some distinct sequence features and functions, and strikingly the corresponding gene deletion helped to enhance poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) synthesis significantly. Overall, we have filled the gap in knowledge about PEP/pyruvate interconversion in haloarchaea and reported an efficient strategy for improving PHBV production in H. mediterranei.

Growth profiling, kinetics and substrate utilization of low-cost dairy waste for production of ß-cryptoxanthin by Kocuria marina DAGII.

The dairy industry produces enormous amount of cheese whey containing the major milk nutrients, but this remains unutilized all over the globe. The present study investigates the production of ß-cryptoxanthin (ß-CRX) by Kocuria marina DAGII using cheese whey as substrate. Response surface methodology (RSM) and an artificial neural network (ANN) approach were implemented to obtain the maximum ß-CRX yield. Significant factors, i.e. yeast extract, peptone, cheese whey and initial pH, were the input variables in both the optimizing studies, and ß-CRX yield and biomass were taken as output variables. The ANN topology of 4-9-2 was found to be optimum when trained with a feed-forward back-propagation algorithm. Experimental values of ß-CRX yield (17.14 mg l-1) and biomass (5.35 g l-1) were compared and ANN predicted values (16.99 mg l-1 and 5.33 g l-1, respectively) were found to be more accurate compared with RSM predicted values (16.95 mg l-1 and 5.23 g l-1, respectively). Detailed kinetic analysis of cellular growth, substrate consumption and product formation revealed that growth inhibition took place at substrate concentrations higher than 12% (v/v) of cheese whey. The Han and Levenspiel model was the best fitted substrate inhibition model that described the cell growth in cheese whey with an R2 and MSE of 0.9982% and 0.00477%, respectively. The potential importance of this study lies in the development, optimization and modelling of a suitable cheese whey supplemented medium for increased ß-CRX production.

Modelling the growth kinetics of Kocuria marina DAGII as a function of single and binary substrate during batch production of ß-Cryptoxanthin.

In the present investigation, growth kinetics of Kocuria marina DAGII during batch production of ß-Cryptoxanthin (ß-CRX) was studied by considering the effect of glucose and maltose as a single and binary substrate. The importance of mixed substrate over single substrate has been emphasised in the present study. Different mathematical models namely, the Logistic model for cell growth, the Logistic mass balance equation for substrate consumption and the Luedeking-Piret model for ß-CRX production were successfully implemented. Model-based analyses for the single substrate experiments suggested that the concentrations of glucose and maltose higher than 7.5 and 10.0 g/L, respectively, inhibited the growth and ß-CRX production by K. marina DAGII. The Han and Levenspiel model and the Luong product inhibition model accurately described the cell growth in glucose and maltose substrate systems with a R 2 value of 0.9989 and 0.9998, respectively. The effect of glucose and maltose as binary substrate was further investigated. The binary substrate kinetics was well described using the sum-kinetics with interaction parameters model. The results of production kinetics revealed that the presence of binary substrate in the cultivation medium increased the biomass and ß-CRX yield significantly. This study is a first time detailed investigation on kinetic behaviours of K. marina DAGII during ß-CRX production. The parameters obtained in the study might be helpful for developing strategies for commercial production of ß-CRX by K. marina DAGII.

Reciprocal regulation of NF-kB (Relish) and Subolesin in the tick vector, Ixodes scapularis.

BACKGROUND: Tick Subolesin and its ortholog in insects and vertebrates, Akirin, have been suggested to play a role in the immune response through regulation of nuclear factor-kappa B (NF-kB)-dependent and independent gene expression via interaction with intermediate proteins that interact with NF-kB and other regulatory proteins, bind DNA or remodel chromatin to regulate gene expression. The objective of this study was to characterize the structure and regulation of subolesin in Ixodes scapularis. I. scapularis is a vector of emerging pathogens such as Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti that cause in humans Lyme disease, anaplasmosis and babesiosis, respectively. The genome of I. scapularis was recently sequenced, and this tick serves as a model organism for the study of vector-host-pathogen interactions. However, basic biological questions such as gene organization and regulation are largely unknown in ticks and other arthropod vectors. PRINCIPAL FINDINGS: The results presented here provide evidence that subolesin/akirin are evolutionarily conserved at several levels (primary sequence, gene organization and function), thus supporting their crucial biological function in metazoans. These results showed that NF-kB (Relish) is involved in the regulation of subolesin expression in ticks, suggesting that as in other organisms, different NF-kB integral subunits and/or unknown interacting proteins regulate the specificity of the NF-kB-mediated gene expression. These results suggested a regulatory network involving cross-regulation between NF-kB (Relish) and Subolesin and Subolesin auto-regulation with possible implications in tick immune response to bacterial infection. SIGNIFICANCE: These results advance our understanding of gene organization and regulation in I. scapularis and have important implications for arthropod vectors genetics and immunology highlighting the possible role of NF-kB and Subolesin/Akirin in vector-pathogen interactions and for designing new strategies for the control of vector infestations and pathogen transmission.

The human respiratory syncytial virus matrix protein is required for maturation of viral filaments.

An experimental system was developed to generate infectious human respiratory syncytial virus (HRSV) lacking matrix (M) protein expression (M-null virus) from cDNA. The role of the M protein in virus assembly was then examined by infecting HEp-2 and Vero cells with the M-null virus and assessing the impact on infectious virus production and viral protein trafficking. In the absence of M, the production of infectious progeny was strongly impaired. Immunofluorescence (IF) microscopy analysis using antibodies against the nucleoprotein (N), attachment protein (G), and fusion protein (F) failed to detect the characteristic virus-induced cell surface filaments, which are believed to represent infectious virions. In addition, a large proportion of the N protein was detected in viral replication factories termed inclusion bodies (IBs). High-resolution analysis of the surface of M-null virus-infected cells by field emission scanning electron microscopy (SEM) revealed the presence of large areas with densely packed, uniformly short filaments. Although unusually short, these filaments were otherwise similar to those induced by an M-containing control virus, including the presence of the viral G and F proteins. The abundance of the short, stunted filaments in the absence of M indicates that M is not required for the initial stages of filament formation but plays an important role in the maturation or elongation of these structures. In addition, the absence of mature viral filaments and the simultaneous increase in the level of the N protein within IBs suggest that the M protein is involved in the transport of viral ribonucleoprotein (RNP) complexes from cytoplasmic IBs to sites of budding.

The papillomavirus E1 helicase activates a cellular DNA damage response in viral replication foci.

The papillomavirus E1 and E2 proteins are essential for viral genome replication. E1 is a helicase that unwinds the viral origin and recruits host cellular factors to replicate the viral genome. E2 is a transcriptional regulator that helps recruit the E1 helicase to the origin and also plays a role in genome partitioning. We find that when coexpressed, the E1 and E2 proteins from several papillomavirus types localize to defined nuclear foci and result in growth suppression of the host cells. Growth suppression was due primarily to E1 protein function, and nuclear expression of E1 was accompanied by activation of a DNA damage response, resulting in phosphorylation of ATM, Chk2, and H2AX. Growth suppression and ATM activation required the ATPase and origin-specific binding functions of the E1 protein and resulted in active DNA repair, as evidenced by incorporation of nucleotide analogs and detection of free DNA ends. In the presence of the E2 protein, these activities became localized to nuclear foci. We postulate that these foci represent viral replication factories and that a cellular DNA damage response is activated to facilitate replication of viral DNA.

Subolesin expression in response to pathogen infection in ticks.

BACKGROUND: Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin expression was characterized in ticks experimentally infected with the cattle pathogen, Anaplasma marginale. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in Dermacentor variabilis males exposed to various pathogens by capillary feeding (CF). RESULTS: Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with A. marginale. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in D. variabilis infected with A. marginale and other tick-borne pathogens resulted in lower infection levels, while infection with Francisella tularensis increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF, subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was upregulated in D. variabilis exposed to Escherichia coli, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism. CONCLUSIONS: Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional support for the role of subolesin in other molecular pathways including those required for tissue development and function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control of global gene expression in ticks.

Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis.

BACKGROUND: The cattle pathogen, Anaplasma marginale, undergoes a developmental cycle in ticks that begins in gut cells. Transmission to cattle occurs from salivary glands during a second tick feeding. At each site of development two forms of A. marginale (reticulated and dense) occur within a parasitophorous vacuole in the host cell cytoplasm. However, the role of tick genes in pathogen development is unknown. Four genes, found in previous studies to be differentially expressed in Dermacentor variabilis ticks in response to infection with A. marginale, were silenced by RNA interference (RNAi) to determine the effect of silencing on the A. marginale developmental cycle. These four genes encoded for putative glutathione S-transferase (GST), salivary selenoprotein M (SelM), H+ transporting lysosomal vacuolar proton pump (vATPase) and subolesin. RESULTS: The impact of gene knockdown on A. marginale tick infections, both after acquiring infection and after a second transmission feeding, was determined and studied by light microscopy. Silencing of these genes had a different impact on A. marginale development in different tick tissues by affecting infection levels, the densities of colonies containing reticulated or dense forms and tissue morphology. Salivary gland infections were not seen in any of the gene-silenced ticks, raising the question of whether these ticks were able to transmit the pathogen. CONCLUSION: The results of this RNAi and light microscopic analyses of tick tissues infected with A. marginale after the silencing of genes functionally important for pathogen development suggest a role for these molecules during pathogen life cycle in ticks.

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection.

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.

The potato virus X TGBp2 protein association with the endoplasmic reticulum plays a role in but is not sufficient for viral cell-to-cell movement.

Potato virus X (PVX) TGBp1, TGBp2, TGBp3, and coat protein are required for virus cell-to-cell movement. Plasmids expressing GFP fused to TGBp2 were bombarded to leaf epidermal cells and GFP:TGBp2 moved cell to cell in Nicotiana benthamiana leaves but not in Nicotiana tabacum leaves. GFP:TGBp2 movement was observed in TGBp1-transgenic N. tabacum, indicating that TGBp2 requires TGBp1 to promote its movement in N. tabacum. In this study, GFP:TGBp2 was detected in a polygonal pattern that resembles the endoplasmic reticulum (ER) network. Amino acid sequence analysis revealed TGBp2 has two putative transmembrane domains. Two mutations separately introduced into the coding sequences encompassing the putative transmembrane domains within the GFP:TGBp2 plasmids and PVX genome, disrupted membrane binding of GFP:TGBp2, inhibited GFP:TGBp2 movement in N. benthamiana and TGBp1-expressing N. tabacum, and inhibited PVX movement. A third mutation, lying outside the transmembrane domains, had no effect on GFP:TGBp2 ER association or movement in N. benthamiana but inhibited GFP:TGBp2 movement in TGBp1-expressing N. tabacum and PVX movement in either Nicotiana species. Thus, ER association of TGBp2 may be required but not be sufficient for virus movement. TGBp2 likely provides an activity for PVX movement beyond ER association.