The development and application of protemics to the analysis of Chlamydia Trachomatis

The bacterial pathogen Chlamydia trachomatis causes Trachoma, the worlds leading cause of preventable blindness and is also responsible for the most common curable sexually transmitted disease in the UK and United States. C. trachomatis is an obligate intracellular organism characterised by a unique and complex growth cycle. Its study presents many challenges since it has historically been recalcitrant to genetic manipulation and growth in the absence of a host cell. Nevertheless, the sequencing of the C. trachomatis genome and its relatively small size by comparison to genomes from other bacterial pathogens, has paved the way for studies at the proteomic level.This thesis describes the development and application of proteomic approaches to study C. trachomatis L2. To survey the expressed chlamydial proteome, a combination of the qualitative approaches, 2-DGE, MudPIT and GeLC-MS/MS; and the quantitative approaches AQUA, iTRAQ and LC-MSE were used. Collectively, the approaches efficiently identified 648 expressed proteins, representing ~72% of the predicted proteome of C. trachomatis L2, from both the infectious (elementary body, EB) and replicating (reticulate body, RB) form of the pathogen. In the infectious EB, the entire set of predicted glycolytic enzymes were detected, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. Further, proteomic analysis of the RB form also uncovered biosynthetic enzymes for chlamydial cell wall synthesis, indicating that peptidoglycan is produced in some form during growth in host cells. Comparison of the quantitative approaches iTRAQ and LC-MSE demonstrated that LC-MSE quantitative data was significantly more robust and extensive relative to iTRAQ data. In addition to information on relative amounts of these proteins between the two forms, LC-MSE data also yielded the cellular concentration (molecules per cell) for 489 proteins.This extensive set of absolute quantitation data permits estimates of the energy invested in the synthesis of various classes of proteins. The results indicate that C. trachomatis devotes most of its energy into maintenance of the translational machinery. However, it also expends significant amounts of energy into making cell envelope components and a set of hitherto hypothetical proteins. These proteins, which account for the bulk of the energy invested by the intracellular RB form of the pathogen as it converts to the extracellular EB form, highlight the importance of absolute quantitation data for understanding the biological processing status of the cell. The datasets also revealed a large number of proteins that were differentially expressed between replicating RBs and infectious EBs, ranging from 8.4-fold down-regulation to 3.5-fold up-regulation. Consistent with transcriptomic studies (Belland et al., 2003), proteins involved in protein synthesis, ATP generation, central metabolism, secretion and nutrient uptake were predominant in the metabolically active RB at 15 h PI. Although many of the proteins in these functional categories were down-regulated in EBs, proteins required for glycolysis, central metabolism, protein synthesis, and type III secretion were present in significant amounts in EBs suggesting that the infectious EB is primed ‘ready-to-go’ upon contact with the host cell

Mining whole sample mass spectrometry proteomics data for biomarkers: an overview

In this paper we aim to provide a concise overview of designing and conducting an MS proteomics experiment in such a way as to allow statistical analysis that may lead to the discovery of novel biomarkers. We provide a summary of the various stages that make up such an experiment, highlighting the need for experimental goals to be decided upon in advance. We discuss issues in experimental design at the sample collection stage, and good practise for standardising protocols within the proteomics laboratory. We then describe approaches to the data mining stage of the experiment, including the processing steps that transform a raw mass spectrum into a useable form. We propose a permutation-based procedure for determining the significance of reported error rates. Finally, because of its general advantages in speed and cost, we suggest that MS proteomics may be a good candidate for an early primary screening approach to disease diagnosis, identifying areas of risk and making referrals for more specific tests without necessarily making a diagnosis in its own right. Our discussion is illustrated with examples drawn from experiments on bovine blood serum conducted in the Centre for Proteomic Research (CPR) at Southampton University

Analysis of the hippocampal proteome in ME7 prion disease reveals a predominant astrocytic signature and highlights the brain-restricted production of clusterin in chronic neurodegeneration

Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ(TM)/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration

Soluble axoplasm enriched from injured CNS axons reveals the early modulation of the actin cytoskeleton

Axon injury and degeneration is a common consequence of diverse neurological conditions including multiple sclerosis, traumatic brain injury and spinal cord injury. The molecular events underlying axon degeneration are poorly understood. We have developed a novel method to enrich for axoplasm from rodent optic nerve and characterised the early events in Wallerian degeneration using an unbiased proteomics screen. Our detergent-free method draws axoplasm into a dehydrated hydrogel of the polymer poly(2-hydroxyethyl methacrylate), which is then recovered using centrifugation. This technique is able to recover axonal proteins and significantly deplete glial contamination as confirmed by immunoblotting. We have used iTRAQ to compare axoplasm-enriched samples from naïve vs injured optic nerves, which has revealed a pronounced modulation of proteins associated with the actin cytoskeleton. To confirm the modulation of the actin cytoskeleton in injured axons we focused on the RhoA pathway. Western blotting revealed an augmentation of RhoA and phosphorylated cofilin in axoplasm-enriched samples from injured optic nerve. To investigate the localisation of these components of the RhoA pathway in injured axons we transected axons of primary hippocampal neurons in vitro. We observed an early modulation of filamentous actin with a concomitant redistribution of phosphorylated cofilin in injured axons. At later time-points, RhoA is found to accumulate in axonal swellings and also colocalises with filamentous actin. The actin cytoskeleton is a known sensor of cell viability across multiple eukaryotes, and our results suggest a similar role for the actin cytoskeleton following axon injury. In agreement with other reports, our data also highlights the role of the RhoA pathway in axon degeneration. These findings highlight a previously unexplored area of axon biology, which may open novel avenues to prevent axon degeneration. Our method for isolating CNS axoplasm also represents a new tool to study axon biology

Quantifying integrated proteomic responses to iron stress in the globally important marine diazotroph trichodesmium

Trichodesmium is a biogeochemically important marine cyanobacterium, responsible for a significant proportion of the annual ‘new’ nitrogen introduced into the global ocean. These non-heterocystous filamentous diazotrophs employ a potentially unique strategy of near-concurrent nitrogen fixation and oxygenic photosynthesis, potentially burdening Trichodesmium with a particularly high iron requirement due to the iron-binding proteins involved in these processes. Iron availability may therefore have a significant influence on the biogeography of Trichodesmium. Previous investigations of molecular responses to iron stress in this keystone marine microbe have largely been targeted. Here a holistic approach was taken using a label-free quantitative proteomics technique (MSE) to reveal a sophisticated multi-faceted proteomic response of Trichodesmium erythraeum IMS101 to iron stress. Increased abundances of proteins known to be involved in acclimation to iron stress and proteins known or predicted to be involved in iron uptake were observed, alongside decreases in the abundances of iron-binding proteins involved in photosynthesis and nitrogen fixation. Preferential loss of proteins with a high iron content contributed to overall reductions of 55–60% in estimated proteomic iron requirements. Changes in the abundances of iron-binding proteins also suggested the potential importance of alternate photosynthetic pathways as Trichodesmium reallocates the limiting resource under iron stress. Trichodesmium therefore displays a significant and integrated proteomic response to iron availability that likely contributes to the ecological success of this species in the ocean

Geologic setting and stratigraphy of the Ziegler Reservoir fossil site, Snowmass Village, Colorado

The geologic setting of the Ziegler Reservoir fossil site is somewhat unusual—the sediments containing the Pleistocene fossils were deposited in a lake on top of a ridge. The lake basin was formed near Snowmass Village, Colorado (USA) when a glacier flowing down Snowmass Creek Valley became thick enough to overtop a low point in the eastern valley wall and entered the head of Brush Creek Valley. When the glacier retreated at about 155–130 ka, near the end of Marine Oxygen Isotope Stage 6, the Brush Creek Valley lobe left behind a moraine that impounded a small alpine lake. The lake was initially ~10m deep and appears to have been highly productive during most of its existence, based on the abundant and exquisitely preserved organic material present in the sediments. Over time, the basin slowly filled with (mostly) eolian sediment such that by ~87 ka it contained a marsh or wetland rather than a true lake. Open-water conditions returned briefly between ~77 and 55 ka before the impoundment was finally breached to the east, establishing ties with the Brush Creek drainage system and creating an alpine meadow that persisted until historic times

Geologic setting and stratigraphy of the Ziegler Reservoir fossil site, Snowmass Village, Colorado

The geologic setting of the Ziegler Reservoir fossil site is somewhat unusual—the sediments containing the Pleistocene fossils were deposited in a lake on top of a ridge. The lake basin was formed near Snowmass Village, Colorado (USA) when a glacier flowing down Snowmass Creek Valley became thick enough to overtop a low point in the eastern valley wall and entered the head of Brush Creek Valley. When the glacier retreated at about 155–130 ka, near the end of Marine Oxygen Isotope Stage 6, the Brush Creek Valley lobe left behind a moraine that impounded a small alpine lake. The lake was initially ~10m deep and appears to have been highly productive during most of its existence, based on the abundant and exquisitely preserved organic material present in the sediments. Over time, the basin slowly filled with (mostly) eolian sediment such that by ~87 ka it contained a marsh or wetland rather than a true lake. Open-water conditions returned briefly between ~77 and 55 ka before the impoundment was finally breached to the east, establishing ties with the Brush Creek drainage system and creating an alpine meadow that persisted until historic times

Low concentrations of nitric oxide modulate Streptococcus pneumoniae biofilm metabolism and antibiotic tolerance

Streptococcus pneumoniae is one of the key pathogens responsible for otitis media (OM), the most common infection in children and the largest cause of childhood antibiotic prescription. Novel therapeutic strategies that reduce the overall antibiotic consumption due to OM are required because although widespread pneumococcal conjugate immunization has controlled invasive pneumococcal disease, overall OM incidence has not decreased. Biofilm formation represents an important phenotype contributing to the antibiotic tolerance and persistence of S. pneumoniae in chronic or recurrent OM. We investigated the treatment of pneumococcal biofilms with nitric oxide (NO), an endogenous signaling molecule and therapeutic agent that has been demonstrated to trigger biofilm dispersal in other bacterial species. We hypothesised that addition of low concentrations of NO to pneumococcal biofilms would improve antibiotic efficacy and higher concentrations exert direct antibacterial effects. Unlike in many other bacterial species, low concentrations of NO, did not result in S. pneumoniae biofilm dispersal. Instead, treatment of both in vitro biofilms and ex vivo adenoid tissue samples (a reservoir for S. pneumoniae biofilms) with low concentrations of NO enhanced pneumococcal killing when combined with amoxicillin-clavulanic acid, an antibiotic commonly used to treat chronic OM. Quantitative proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) identified 13 proteins that were differentially expressed following low-concentration NO treatment, 85% of which function in metabolism or translation. Treatment with low-concentration NO therefore appears to modulate pneumococcal metabolism and may represent a novel therapeutic approach to reduce antibiotic tolerance in pneumococcal biofilms

The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects