Chemical shift assignments and secondary structure prediction for Q4DY78, a conserved kinetoplastid-specific protein from <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Trypanosoma cruzi, Trypanosma brucei and Leishmania spp. are kinetoplastid protozoa causative agents of Chagas disease, sleeping sickness and leishmaniasis, respectively, neglected tropical diseases estimated to infect millions of people worldwide. Their genome sequencing has revealed approximately 50 % of genes encoding hypothetical proteins of unknown function, opening possibilities for novel target identification and drug discovery. Q4DY78 is a putative essential protein from T. cruzi conserved in the related kinetoplastids and divergent from mammalian host proteins. Here we report the ¹H, ¹⁵N, and ¹³C chemical shift assignments and secondary structure analysis of the Q4DY78 protein as basis for NMR structure determination, functional analysis and drug screening.     

In silico structural characterization of protein targets for drug development against <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Trypanosoma cruzi is the protozoan pathogen responsible for Chagas disease, which is a major public health problem in tropical and subtropical regions of developing countries and particularly in Brazil. Despite many studies, there is no efficient treatment against Chagas disease, and the search for new therapeutic targets specific to T. cruzi is critical for drug development. Here, we have revisited 41 protein sequences proposed by the analogous enzyme pipeline, and found that it is possible to provide structures for T. cruzi sequences with clear homologs or analogs in H. sapiens and likely associated with trypanothione reductase, cysteine synthase, and ATPase functions, and structures for sequences specific to T. cruzi and absent in H. sapiens associated with 2,4-dienoyl-CoA reductase, and leishmanolysin activities. The implications of our structures refined by atomistic molecular dynamics (monomer or dimer states) in their in vitro environments (aqueous solution or membrane bilayers) are discussed for drug development and suggest that all protein targets, except cysteine synthase, merit further investigation.     

Identification of drug target candidates of the swine pathogen <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> by construction of protein–protein interaction network

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae has led to severe economic losses in the pig industry worldwide. A. pleuropneumoniae displays various levels of antimicrobial resistance, leading to the dire need to identify new drug targets. Protein–protein interaction (PPI) network can aid the identification of drug targets by discovering essential proteins during the life of bacteria. The aim of this study is to identify drug target candidates of A. pleuropneumoniae from essential proteins in PPI network. The homologous protein mapping method (HPM) was utilized to construct A. pleuropneumoniae PPI network. Afterwards, the subnetwork centered with H-NS was selected to verify the PPI network using bacterial two-hybrid assays. Drug target candidates were identified from the hub proteins by analyzing the topology of the network using interaction degree and homologous comparison with the pig proteome. An A. pleuropneumoniae PPI network containing 2737 non-redundant interaction pairs among 533 proteins was constructed. These proteins were distributed in 21 COG functional categories and 28 KEGG metabolic pathways. The A. pleuropneumoniae PPI network was scale free and the similar topological tendencies were found when compared with other bacteria PPI network. Furthermore, 56.3% of the H-NS subnetwork interactions were validated. 57 highly connected proteins (hub proteins) were identified from the A. pleuropneumoniae PPI network. Finally, 9 potential drug targets were identified from the hub proteins, with no homologs in swine. This study provides drug target candidates, which are promising for further investigations to explore lead compounds against A. pleuropneumoniae.     

<Emphasis Type="Italic">In silico</Emphasis> identification of essential proteins in <Emphasis Type="Italic">Corynebacterium pseudotuberculosis</Emphasis> based on protein-protein interaction networks

Background

Corynebacterium pseudotuberculosis (Cp) is a gram-positive bacterium that is classified into equi and ovis serovars. The serovar ovis is the etiological agent of caseous lymphadenitis, a chronic infection affecting sheep and goats, causing economic losses due to carcass condemnation and decreased production of meat, wool, and milk. Current diagnosis or treatment protocols are not fully effective and, thus, require further research of Cp pathogenesis.

Results

Here, we mapped known protein-protein interactions (PPI) from various species to nine Cp strains to reconstruct parts of the potential Cp interactome and to identify potentially essential proteins serving as putative drug targets. On average, we predict 16,669 interactions for each of the nine strains (with 15,495 interactions shared among all strains). An in silico sanity check suggests that the potential networks were not formed by spurious interactions but have a strong biological bias. With the inferred Cp networks we identify 181 essential proteins, among which 41 are non-host homologous.

Conclusions

The list of candidate interactions of the Cp strains lay the basis for developing novel hypotheses and designing according wet-lab studies. The non-host homologous essential proteins are attractive targets for therapeutic and diagnostic proposes. They allow for searching of small molecule inhibitors of binding interactions enabling modern drug discovery. Overall, the predicted Cp PPI networks form a valuable and versatile tool for researchers interested in Corynebacterium pseudotuberculosis.

     

The characterization of anti-<Emphasis Type="Italic">T. cruzi</Emphasis> activity relationships between ferrocenyl,cyrhetrenyl complexes and ROS release

Trypanosoma cruzi (T. cruzi) is the parasite that causes Chagas disease. Nifurtimox is the most used drug against the T. cruzi, this drug increases intermediaries nitro group, being mainly responsible for the high toxicity component, for this reason it is important to study new organic compounds and thus improve therapeutic strategies against Chagas disease. The electronic effects of ferrocenyl and cyrhetrenyl fragments were investigated by DFT calculation. A close correlation was found between HOMO–LUMO gap of nitro radical NO ₂ ^? with the experimental reduction potential found for nitro group and IC₅₀ of two forms the T. cruzi (epimastigote and trypomastigote). The IC₅₀ on human hepatoma cells is higher for both compounds compared to IC₅₀ demonstrated in the two forms the T. cruzi, and additionally show reactive oxygen species release. The information obtained in this paper could generate two new drugs with anti-T. cruzi activity, but additional studies are needed.     

Phase variation of Opa proteins of <Emphasis Type="BoldItalic">Neisseria meningitidis</Emphasis> and the effects of bacterial transformation

Opa proteins are major proteins involved in meningococcal colonization of the nasopharynx and immune interactions. Opa proteins undergo phase variation (PV) due to the presence of the 5′-CTCTT-3′ coding repeat (CR) sequence. The dynamics of PV of meningococcal Opa proteins is unknown. Opa PV, including the effect of transformation on PV, was assessed using a panel of Opa-deficient strains of Neisseria meningitidis. Analysis of Opa expression from UK disease-causing isolates was undertaken. Different opa genes demonstrated variable rates of PV, between 6.4 ×10^–4 and 6.9 ×10^–3 per cell per generation. opa genes with a longer CR tract had a higher rate of PV (r ²=0.77, p=0.1212). Bacterial transformation resulted in a 180-fold increase in PV rate. The majority of opa genes in UK disease isolates (315/463, 68.0%) were in the ‘on’ phase, suggesting the importance of Opa proteins during invasive disease. These data provide valuable information for the first time regarding meningococcal Opa PV. The presence of Opa PV in meningococcal populations and high expression of Opa among invasive strains likely indicates the importance of this protein in bacterial colonization in the human nasopharynx. These findings have potential implications for development of vaccines derived from meningococcal outer membranes.     

In silico study of peptide inhibitors against BACE 1

Peptides are increasingly used as inhibitors of various disease specific targets. Several naturally occurring and synthetically developed peptides are undergoing clinical trials. Our work explores the possibility of reusing the non-expressing DNA sequences to predict potential drug-target specific peptides. Recently, we experimentally demonstrated the artificial synthesis of novel proteins from non-coding regions of Escherichia coli genome. In this study, a library of synthetic peptides (Synpeps) was constructed from 2500 intergenic E. coli sequences and screened against Beta-secretase 1 protein, a known drug target for Alzheimer’s disease (AD). Secondary and tertiary protein structure predictions followed by protein–protein docking studies were performed to identify the most promising enzyme inhibitors. Interacting residues and favorable binding poses of lead peptide inhibitors were studied. Though initial results are encouraging, experimental validation is required in future to develop efficient target specific inhibitors against AD.     

Genome-wide identification and expression analysis of sulphate transporter (<Emphasis Type="Italic">SULTR</Emphasis>) genes under sulfur deficiency in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>

Sulphur is an important mineral element for plant growth and development. It involves in a number of metabolic processes with crucial functions. This study has performed a genome-wide analysis of sulfate transporter (SULTR) genes in Brachypodium distachyon. Ten putative SULTR genes were identified in Brachypodium genome. BdSULTR genes included 6–17 exons encoding a protein of 647–693 residues with basic nature. BdSULTR proteins included both sulfate_transp (PF00916) and STAS (PF01740) domains. BdSULTRs were classified into 4 groups based on the phylogenetic distribution. Promoter regions of all BdSULTR genes, except for BdSULTR3;3 and 3;5 included the SURECOREATSULTR11 elements. A considerable structural overlap was identified between superimposed SULTR1;3 and 3;1 proteins, indicating that SULTR1 members may also involve in plant stress response/tolerance like SULTR3 members. Microarray and RNA-Seq analyses also revealed the differential expression of SULTR 1 and 3 genes under different biotic/abiotic stresses. Protein–protein interaction partners of BdSULTRs were mainly related with adenylyl-sulfate kinases, 5′-adenylylsulfate reductases, ATP sulfurylases, and acyl carrier proteins. Moreover, expression profiles of identified BdSULTR genes under S-deficiency were analyzed using RT-qPCR. It was revealed that BdSULTR1;1 and 3;1 are highly expressed in plant roots as ~tenfold and ~fivefold, respectively, while BdSULTR2 (~15-fold) and 3;1 (~twofold) are abundantly expressed in leaf tissues.     

Identification of Ribosomal Protein L1-Binding Sites in <Emphasis Type="Italic">Thermus thermophilus</Emphasis> and <Emphasis Type="Italic">Thermotoga maritima</Emphasis> mRNAs

The conserved two-domain ribosomal protein (r-protein) L1 is a structural part of the L1 stalk of the large ribosomal subunit and regulates the translation of the operon that comprises its own gene. The regulatory properties of the bacterial r-protein L1 have only been studied in detail for Escherichia coli; however, there were no such studies for other bacteria, in particular, Thermus thermophilus and Thermotoga maritima, which are more evolutionarily ancient. It is known that domain I of the r-protein L1 might have regulatory properties of the whole protein. The aim of this study was to identify regulatory sites on the mRNA of T. thermophilus and T. maritima that interact with r-proteins L1, as well as with their domains I from the same organisms. An analysis of the mRNA of the L11 operon T. thermophilus showed the presence of one potential binding site of the L1 r-protein, two such regions were found also in the mRNA sequence of the L11 operon of T. maritima. The dissociation constants for the L1 proteins from T. thermophilus and T. maritima and their domains I with mRNA fragments from the same organisms that contain the supposed L1-binding sites were determined by surface plasmon resonance. It has been shown that the ribosomal proteins L1 as their domains I bind specific fragments of mRNA from the same organisms that may suggest regulatory activity of the L1 protein in the T. thermophilus and T. maritima and conservatism of the principles of L1-RNA interactions.     

Proteome wide identification of iron binding proteins of <Emphasis Type="Italic">Xanthomonas translucens</Emphasis> pv. <Emphasis Type="Italic">undulosa</Emphasis>: focus on secretory virulent proteins

Xanthomonas translucens pv. undulosa (Xtu) causes Bacterial Leaf Streak disease in the staple food crops such as wheat and barley. The survival strategies of pathogen and host are determined by the complex interactions occurring between the host plants and the pathogenic microbes. Iron binding proteins are important in the plant–microbe interactions as they are indulged in enzyme catalysis, virulence, metabolic and transport activities. In the presented study, we have identified that ~9.8% of Xtu proteome possess iron binding sequence motifs. Further, the analysis of Xtu proteome for secretory iron binding virulent proteins (IBVPs) revealed the fact that iron co-regulate the function of secretory proteins in virulence. We have found 26 secretory IBVPs and observed that these proteins are diverse in their biological functions ranging from transport to antimicrobial resistance, Reactive oxygen species detoxification and carbohydrate catabolism. The inferences may instigate to design the new strategies to combat and control the microbial diseases of staple food crops.     

Characterization of volatile organic compounds emitted by kiwifruit plants infected with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">actinidiae</Emphasis> and their effects on host defences

Key message

Specific VOC emissions from infected plants allow their recognition and elicit defence responses in neighboring plants, which are, however, insufficient to induce resistance.

Abstract

A wide range of volatile organic compounds (VOCs) is released during plant–pathogen interactions both by the pathogens and the hosts. Some of these VOCs are specific for the different diseases and are known to play a role in the pathogenicity or in plant defence responses. Besides, disease-specific VOCs may serve as markers for diagnostic protocols, which allow a non-destructive and rapid screening of bulk samples of plant material. This work aimed to verify the feasibility of a VOC-based diagnosis and to investigate the possible biological role of VOCs released during the plant–pathogen interactions. The volatile emissions from Pseudomonas syringae pv. actinidiae in axenic cultures and from inoculated in vitro kiwifruit plants were characterized by gas chromatography–mass spectrometry (GC–MS) and proton transfer reaction–time-of-flight-mass spectrometry (PTR–ToF-MS). By GC–MS, several putative biochemical markers, such as 1-undecene, were identified. PTR–ToF-MS resulted highly effective in screening the plant material for latent infections. To develop a more user-friendly, portable and less expensive diagnosis system, two different electronic nose models were tested for the early diagnosis of P. syringae pv. actinidiae in asymptomatic plant material. Our experiments demonstrated the feasibility of the electronic nose-based screening of infected plant material. Concerning the biological role of the VOCs released during the plant–pathogen interactions, the exposure of healthy plants to VOCs from infected ones influences the plant growth and induces the stimulation of protective responses. However, after the infection, P. syringae pv. actinidiae is able to selectively inactivate the induced plant defences.

     

Study of the physiological mechanisms of two species of <Emphasis Type="Italic">Spiraea</Emphasis> during adaptation to drought treatment

The photosynthetic rate, light saturation point, light compensation point, changes in the MDA and SOD activities, and protein expression of two different drought-resistance species, Spiraea fritschiana and Spiraea trichocarpa, were assessed in this study. Furthermore, the drought-resistant physiological mechanisms of both species were analyzed at the protein level. The photosynthetic capacities of two Spiraea species decreased under drought stress, and the light saturation point and light compensation point decreased. However, their capacities to use weak light increased. Spiraea fritschiana, which demonstrated a stronger drought resistance, showed a better ability to adapt to weak light than S. trichocarpa. The content of MDA in S. fritschiana was notably lower than that in S. trichocarpa, indicating that the concentration of the membrane peroxidation products of S. fritschiana was less than those of S. trichocarpa. Compared with S. trichocarpa, S. fritschiana’s SOD activity was higher, and its ability to remove ROS was also better. Sixty-six proteins were identified with significantly different expression behavior and included regulatory, redox homeostasis, metabolism and energy, and cytoskeleton proteins. The results showed that the photosynthesis of S. trichocarpa was significantly affected by the drought stress. Enzymes in photosynthesis changed significantly; the expression of the RuBisCo large subunit decreased; and RuBisCo carboxylase, the chlorophyll a–b binding protein, ATP synthase, OEC 33 kD photosystem II protein and 23 kD OEC protein greatly increased. In addition, four antioxidant enzymes greatly increased, GroES chaperonin decreased, and eIF5A significantly increased under light stress. When S. fritschiana Schneid encountered serious drought stress, in addition to those enzymes that changed significantly under light drought stress in S. trichocarpa Nakai, NAD(P)H-quinone oxidoreductase and eIF5A were up-regulated. Specifically, three heat-shock proteins were induced. The expression of the enzymes of the two Spiraea that were related to photosynthesis, oxidation–reduction and regulation were all affected, but their species and expression patterns were different. In S. trichocarpa Nakai and S. fritschiana Schneid, there were significant changes in the proteins related to energy metabolism and the proteins related to energy transport, respectively. Thus, we considered that, in the case of protein involvement, the differences in the metabolic pathways and adjustment levels might contribute to S. trichocarpa having a weaker drought tolerance than S. fritschiana.     

Chemical shift assignments of Ribosomal protein S1 from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

RpsA, also known as ribosomal protein S1, is an essential protein required for translation initiation of mRNAs when their Shine-Dalgarno sequence is degenerated (Sorensen et al. 1998). In addition, RpsA of Mycobacterium tuberculosis (M. tb) is involved in trans-translation, which is an effective system mediated by tmRNA-SmpB to release stalled ribosomes from mRNA in the presence of rare codons (Keiler 2008). Shi et al. found that POA binds to RpsA of Mtb and disrupts the formation of RpsA–tmRNA complex (Shi et al. 2011) and mutations at the C-terminus of RpsA confer PZA resistance. The previous work reported the pyrazinoic acid-binding domain of RpsA (Yang et al. Mol Microbiol 95:791–803, 2015). However, the HSQC spectra of the isolated S1 domain does not overlap with that of MtRpsA280-438, suggesting that substantial interactions occur between the flexible C-terminus and the S1 domain in MtRpsA .To further study the PZA resistance and how substantial interactions influence/affect protein structure, using heteronuclear NMR spectroscopy, we have completed backbone and side-chain ¹H, ¹⁵N, ¹³C chemical shift assignments of MtRpsA280-438 which contains S1 domain and the flexible C-terminus. These NMR resonance assignments provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.     

Identification and characterization of a gene encoding a putative lysophosphatidyl acyltransferase from <Emphasis Type="Italic">Arachis hypogaea</Emphasis>

Lysophosphatidyl acyltransferase (LPAT) is the important enzyme responsible for the acylation of lysophosphatidic acid (LPA), leading to the generation of phosphatidic acid (PA) in plant. Its encoding gene is an essential candidate for oil crops to improve oil composition and increase seed oil content through genetic engineering. In this study, a full-length AhLPAT4 gene was isolated via cDNA library screening and rapid amplification of cDNA ends (RACE); our data demonstrated that AhLPAT4 had 1631 nucleotides, encoding a putative 43.8 kDa protein with 383 amino acid residues. The deduced protein included a conserved acyltransferase domain and four motifs (I–IV) with putative LPA and acyl-CoA catalytic and binding sites. Bioinformatic analysis indicated that AhLPAT4 contained four transmembrane domains (TMDs), localized to the endoplasmic reticulum (ER) membrane; detailed analysis indicated that motif I and motifs II–III in AhLPAT4 were separated by the third TMD, which located on cytosolic and ER luminal side respectively, and hydrophobic residues on the surface of AhLPAT4 protein fold to form a hydrophobic tunnel to accommodate the acyl chain. Subcellular localization analysis confirmed that AhLPAT4 was a cytoplasm protein. Phylogenetic analysis revealed that AhLPAT4 had a high homology (63.7–78.3%) with putative LPAT4 proteins from Glycine max, Arabidopsis thaliana and Ricinus communis. AhLPAT4 was ubiquitously expressed in diverse tissues except in flower, which is almost undetectable. The expression analysis in different developmental stages in peanut seeds indicated that AhLPAT4 did not coincide with oil accumulation.     

Hydrophilic Acylated Surface Protein A (HASPA) of <Emphasis Type="Italic">Leishmania donovani</Emphasis>: Expression,Purification and Biophysico-Chemical Characterization

Hydrophilic acylated surface proteins (HASPs) are acidic surface proteins which get localized on the surface of Leishmania parasite during infective stages through a “non-classical” pathway. In this study, we report the heterologous expression and purification of Leishmania donovani HASPA (r-LdHASPA) in E. coli system and its partial characterization. The structural aspects of the purified protein were analyzed using CD spectroscopy and modeling studies which indicate that r-LdHASPA consists of random coils. Studies in mouse macrophage RAW264.7 cell lines indicate that r-LdHASPA enhances reactive oxygen species (ROS) production. Co-immunoprecipitation (IP) studies indicate that r-LdHASPA interacts with certain macrophage proteins which however could not be identified unambiguously. The present study provides key insights into the structural and functional aspects of an important Leishmania protein, HASPA, which we believe could be useful for further research on vaccine/drug development.     

Comparative pan genome analysis of oral <Emphasis Type="Italic">Prevotella</Emphasis> species implicated in periodontitis

Prevotella is part of the oral bacterial community implicated in periodontitis. Pan genome analyses of eight oral Prevotella species, P. dentalis, P. enoeca, P. fusca, P. melaninogenica, P. denticola, P. intermedia 17, P. intermedia 17-2 and P. sp. oral taxon 299 are presented in this study. Analysis of the Prevotella pan genome revealed features such as secretion systems, resistance to oxidative stress and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems that enable the bacteria to adapt to the oral environment. We identified the presence of type VI secretion system (T6SS) in P. fusca and P. intermedia strains. For some VgrG and Hcp proteins which were not part of the core T6SS loci, we used gene neighborhood analysis and identified putative effector proteins and putative polyimmunity loci in P. fusca and polymorphic toxin systems in P. intermedia strains. Earlier studies have identified the presence of Por secretion system (PorSS) in P. gingivalis, P. melaninogenica and P. intermedia. We noted the presence of their homologs in six other oral Prevotella studied here. We suggest that in Prevotella, PorSS is used to secrete cysteine proteases such as interpain and C-terminal domain containing proteins with a “Por_secre_tail” domain. We identified subtype I-B CRISPR-Cas system in P. enoeca. Putative CRISPR-Cas system subtypes for 37 oral Prevotella and 30 non-oral Prevotella species were also predicted. Further, we performed a BLASTp search of the Prevotella proteins which are also conserved in the red-complex pathogens, against the human proteome to identify potential broad-spectrum drug targets. In summary, the use of a pan genome approach enabled identification of secretion systems and defense mechanisms in Prevotella that confer adaptation to the oral cavity.