Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

<p>Abstract</p> <p>Background</p> <p>Butanol is a second generation biofuel produced by <it>Clostridium acetobutylicum </it>through acetone-butanol-ethanol (ABE) fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of <it>C. acetobutylicum </it>from ABE fermentation using glucose and xylose to understand the functional mechanisms of <it>C. acetobutylicum </it>proteins involved in butanol production.</p> <p>Results</p> <p>We identified 894 different proteins in <it>C. acetobutylicum </it>from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylose substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates.</p> <p>Conclusion</p> <p>Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of <it>C. acetobutylicum </it>ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report.</p

The Schistosomiasis SpleenOME: Unveiling the Proteomic Landscape of Splenomegaly Using Label-Free Mass Spectrometry

Schistosomiasis is a neglected parasitic disease that affects millions of people worldwide and is caused by helminth parasites from the genus Schistosoma. When caused by S. mansoni, it is associated with the development of a hepatosplenic disease caused by an intense immune response to the important antigenic contribution of adult worms and to the presence of eggs trapped in liver tissue. Although the importance of the spleen for the establishment of immune pathology is widely accepted, it has received little attention in terms of the molecular mechanisms operating in response to the infection. Here, we interrogated the spleen proteome using a label-free shotgun approach for the potential discovery of molecular mechanisms associated to the peak of the acute phase of inflammation and the development of splenomegaly in the murine model. Over fifteen hundred proteins were identified in both infected and control individuals and 325 of those proteins were differentially expressed. Two hundred and forty-two proteins were found upregulated in infected individuals while 83 were downregulated. Functional enrichment analyses for differentially expressed proteins showed that most of them were categorized within pathways of innate and adaptive immunity, DNA replication, vesicle transport and catabolic metabolism. There was an important contribution of granulocyte proteins and antigen processing and presentation pathways were augmented, with the increased expression of MHC class II molecules but the negative regulation of cysteine and serine proteases. Several proteins related to RNA processing were upregulated, including splicing factors. We also found indications of metabolic reprogramming in spleen cells with downregulation of proteins related to mitochondrial metabolism. Ex-vivo imunophenotyping of spleen cells allowed us to attribute the higher abundance of MHC II detected by mass spectrometry to increased number of macrophages (F4/80+/MHC II+ cells) in the infected condition. We believe these findings add novel insights for the understanding of the immune mechanisms associated with the establishment of schistosomiasis and the processes of immune modulation implied in the host-parasite interactions

Integration of shot-gun proteomics and bioinformatics analysis to explore plant hormone responses

Abstract Background Multidimensional protein identification technology (MudPIT)-based shot-gun proteomics has been proven to be an effective platform for functional proteomics. In particular, the various sample preparation methods and bioinformatics tools can be integrated to improve the proteomics platform for applications like target organelle proteomics. We have recently integrated a rapid sample preparation method and bioinformatics classification system for comparative analysis of plant responses to two plant hormones, zeatin and brassinosteroid (BR). These hormones belong to two distinct classes of plant growth regulators, yet both can promote cell elongation and growth. An understanding of the differences and the cross-talk between the two types of hormone responses will allow us to better understand the molecular mechanisms and to identify new candidate genes for plant engineering. Results As compared to traditional organelle proteomics, the organelle-enrichment method both simplifies the sample preparation and increases the number of proteins identified in the targeted organelle as well as the entire sample. Both zeatin and BR induce dramatic changes in signaling and metabolism. Their shared-regulated protein components indicate that both hormones may down-regulate some key components in auxin responses. However, they have shown distinct induction and suppression of metabolic pathways in mitochondria and chloroplast. For zeatin, the metabolic pathways in sucrose and starch biosynthesis and utilization were significantly changed, yet the lipid biosynthesis remained unchanged. For BR, lipid biosynthesis and β-oxidation were both down-regulated, yet the changes in sucrose and starch metabolism were minor. Conclusions We present a rapid sample preparation method and bioinformatics classification for effective proteomics analysis of plant hormone responses. The study highlighted the largely differing response to zeatin and brassinosteroid by the metabolic pathways in chloroplast and mitochondria.</p

Across intra-mammalian stages of the liver fluke Fasciola hepatica: a proteomic study

Fasciola hepatica is the agent of fasciolosis, a foodborne zoonosis that affects livestock production and human health. Although flukicidal drugs are available, re-infection and expanding resistance to triclabendazole demand new control strategies. Understanding the molecular mechanisms underlying the complex interaction with the mammalian host could provide relevant clues, aiding the search for novel targets in diagnosis and control of fasciolosis. Parasite survival in the mammalian host is mediated by parasite compounds released during infection, known as excretory/secretory (E/S) products. E/S products are thought to protect parasites from host responses, allowing them to survive for a long period in the vertebrate host. This work provides in-depth proteomic analysis of F. hepatica intra-mammalian stages, and represents the largest number of proteins identified to date for this species. Functional classification revealed the presence of proteins involved in different biological processes, many of which represent original findings for this organism and are important for parasite survival within the host. These results could lead to a better comprehension of host-parasite relationships, and contribute to the development of drugs or vaccines against this parasite

Across intra-mammalian stages of the liver f luke Fasciola hepatica: a proteomic study

Fasciola hepatica is the agent of fasciolosis, a foodborne zoonosis that affects livestock production and human health. Although flukicidal drugs are available, re-infection and expanding resistance to triclabendazole demand new control strategies. Understanding the molecular mechanisms underlying the complex interaction with the mammalian host could provide relevant clues, aiding the search for novel targets in diagnosis and control of fasciolosis. Parasite survival in the mammalian host is mediated by parasite compounds released during infection, known as excretory/secretory (E/S) products. E/S products are thought to protect parasites from host responses, allowing them to survive for a long period in the vertebrate host. This work provides in-depth proteomic analysis of F. hepatica intra-mammalian stages, and represents the largest number of proteins identified to date for this species. Functional classification revealed the presence of proteins involved in different biological processes, many of which represent original findings for this organism and are important for parasite survival within the host. These results could lead to a better comprehension of host-parasite relationships, and contribute to the development of drugs or vaccines against this parasite

Proteomic insights into the modulation of foetal neurogenesis by the anti-retroviral efavirenz

Background: South African guidelines recommend that HIV-positive pregnant women immediately initiate antiretroviral therapy (efavirenz, emtricitabine, and tenofovir), regardless of trimester. Efavirenz causes central nervous system neuropathy and has been linked to birth defects such as encephalocoele. Cohort studies of HIV-uninfected children exposed to antiretroviral treatment in utero report minor learning delays but are inconclusive. Non-transformed human derived neuroepithelial stem (NES) represent a unique pre-clinical model in which to investigate the effects of efavirenz on the developing neural system. Efavirenz-induced global cellular molecular changes may be characterised using mass spectrometry (MS). Aims: To optimise an MS-based efavirenz extraction and detection assay, and to investigate efavirenzinduced NES proteomic responses. Methods: A TSQ Vantage triple quadrupole mass spectrometer was employed to optimise targeted detection of efavirenz extracted from cultured cells and supernatant. Cells were cultured for 72 hours, incorporating a 24-hourly efavirenz treatment. Efavirenz concentration dynamics were assessed over this period, and cells were harvested every 24 hours for discovery proteomic analysis using a Q-Exactive quadrupole-Orbitrap mass spectrometer. Results: Drug extraction with acetonitrile was selected as the optimal extraction and detection technique. In cell culture, efavirenz concentration increased after 24 hours and decreased after 48 hours. A total of 1663 protein groups were identified, with 26, 39, and 80 protein groups differentially expressed 24, 48, and 72 hours respectively post EFV treatment. The most significantly enriched deregulated pathways included cholesterol biosynthesis, mRNA splicing, and JAK/STAT and Wnt signalling. Conclusions: Efavirenz-altered protein expression reflects functional pathway perturbations, which may contribute to clinically-observed neurological effects. Orthoganal and in vivo confirmation is required

Proteomics Guided Gene Discovery for Plant Growth and Defense

Research carried out in this dissertation aims to discover and validate novel gene functions in plant defense and growth. Plants deploy many mechanisms to respond and overcome biological, physical, and environmental challenges. By taking advantage of proteomics, we can discover dynamic changes of the proteome during the plant response. Nonetheless, the complexity of the proteome and the effect of high abundant proteins hinder the discovery of proteins with vital functions at low abundance. I developed a new sample preparation method, dubbed Polyethyleneimine Assisted Rubisco Cleanup (PARC) to fractionate and deplete an abundant protein, namely Rubisco, from plant protein samples. The new approach was applied to investigate mechanisms for plant defense against herbivorous insects. My results indicated that PARC can effectively remove Rubisco and almost two times more differentially regulated proteins were identified. Over-expression of jacalin-like and cupin-like genes was carried out to validate their role in insect resistance in rice. The results further highlighted that PARC can serve as an effective strategy for gene discovery. Furthermore, I integrated a rapid sample preparation method and bioinformatics classification system for comparative analysis of plant responses to two plant hormones, zeatin and brassinosteroid (BR). My data showed the metabolic pathways in sucrose and starch biosynthesis and utilization were significantly changed in zeatin treated plants, yet the lipid biosynthesis remained unchanged. For brassinosteroid treated plants, lipid biosynthesis and β-oxidation were both down-regulated, yet the changes in sucrose and starch metabolism were minor. Finally, a prohibitin (PHB8) involved in plant growth regulation was discovered by bioinformatics and proteomics methods. The over-expression of PHB8 in Arabidopsis increased the plant height, stem diameter, branch number and seed yield significantly. Downstream proteomics revealed that due to PHB8 over-expression, an ATPase beta subunit is significantly up-regulated. Further genetic study showed that over-expression of ATPase led to a similar phenotype as PHB8 over-expression lines. Pull-down assays revealed that PHB8 interacts with PHB9 and PHB16 to regulate ATPase level and energy metabolism. Overall, my research shows that the latest proteomics platform and appropriate sample preparation methods can facilitate the discovery of genes involved in plant defense and growth regulation

Mass & secondary structure propensity of amino acids explain their mutability and evolutionary replacements

Why is an amino acid replacement in a protein accepted during evolution? The answer given by bioinformatics relies on the frequency of change of each amino acid by another one and the propensity of each to remain unchanged. We propose that these replacement rules are recoverable from the secondary structural trends of amino acids. A distance measure between high-resolution Ramachandran distributions reveals that structurally similar residues coincide with those found in substitution matrices such as BLOSUM: Asn Asp, Phe Tyr, Lys Arg, Gln Glu, Ile Val, Met → Leu; with Ala, Cys, His, Gly, Ser, Pro, and Thr, as structurally idiosyncratic residues. We also found a high average correlation (\overline{R} R = 0.85) between thirty amino acid mutability scales and the mutational inertia (I X ), which measures the energetic cost weighted by the number of observations at the most probable amino acid conformation. These results indicate that amino acid substitutions follow two optimally-efficient principles: (a) amino acids interchangeability privileges their secondary structural similarity, and (b) the amino acid mutability depends directly on its biosynthetic energy cost, and inversely with its frequency. These two principles are the underlying rules governing the observed amino acid substitutions. © 2017 The Author(s)

An in vitro investigation of Mycobacterium Tuberculosis biofilm formation and its effect on the host innate immune response

Mycobacterium tuberculosis is ostensibly an intracellular pathogen, which may form pellicle-like biofilms in the peripheries of tuberculosis cavities. Environment-induced cell wall modifications and extracellular polymeric substance production may alter host-pathogen interactions. Specifically, expectorated mycobacteria from cavities, which establish infection in new hosts, may have distinct phenotypic adaptations to impair early clearance by the innate immune system. M. tuberculosis H37Rv biofilm extracellular polymeric substance was identified using scanning electron microscopy. Biofilm phenotype non-covalently-bound extracts of cell wall lipids and carbohydrates were compared to planktonic phenotype and a relative reduction in the proportion of constituent glucose in biofilm carbohydrate extracts was discovered, indicative of a reduction in α-glucan prevalence. Comparison of carbohydrate extracts’ potency in stimulating cytokine and chemokine secretion in whole blood and complement activation elucidated reduced C3b/iC3b deposition onto biofilm carbohydrate extracts. Labelling live dispersed M. tuberculosis planktonic and biofilm samples with fluorescent antibodies showed C3b/iC3b, C5b-9, MBL and C1q deposition was reduced on biofilm phenotype cells, using flow cytometry. The relative contribution of each major pathway of complement activation was investigated and greater dependence on classical pathway activation by M. tuberculosis biofilm cells compared to planktonic cells was observed. Implications of these findings in M. tuberculosis pathogenesis are discussed