Helminth parasite proteomics: from experimental models to human infections

Schistosomiasis is a major human helminth infection endemic in developing countries. Urogenital schistosomiasis, caused by S. haematobium, is the most prevalent human schistosome disease in sub-Saharan Africa. Currently control of schistosome infection is by treatment of infected people with the anthelmintic drug praziquantel, but there are calls for continued efforts to develop a vaccine against the parasites. In order for successful vaccine development, it is necessary to understand the biology and molecular characteristics of the parasite. Ultimately, there is need to understand the nature and dynamics of the relationship between the parasite and the natural host. Thus, my studies have focused on molecular characterization of different parasite stages and integrating this information with quantitative approaches to investigate the nature and development of protective immunity against schistosomes in humans. Proteomics has proved a powerful tool in these studies allowing the proteins expressed by the parasite to be characterized at a molecular and immunological level. In this review, the application of proteomic approaches to understanding the human-schistosome relationship as well as testing specific hypotheses on the nature and development of schistosome-specific immune responses is discussed. The contribution of these approaches to informing schistosome vaccine development is highlighted.     

Chronic infection drives expression of the inhibitory receptor CD200R, and its ligand CD200, by mouse and human CD4 T cells

Certain parasites have evolved to evade the immune response and establish chronic infections that may persist for many years. T cell responses in these conditions become muted despite ongoing infection. Upregulation of surface receptors with inhibitory properties provides an immune cell-intrinsic mechanism that, under conditions of chronic infection, regulates immune responses and limits cellular activation and associated pathology. The negative regulator, CD200 receptor, and its ligand, CD200, have been shown to regulate macrophage activation and reduce pathology following infection. We show that CD4 T cells also increase expression of inhibitory CD200 receptors (CD200R) in response to chronic infection. CD200R was upregulated on murine effector T cells in response to infection with bacterial, Salmonella enterica, or helminth, Schistosoma mansoni, pathogens that respectively drive predominant Th1- or Th2-responses. In vitro chronic and prolonged stimuli were required for the sustained upregulation of CD200R, and its expression coincided with loss of multifunctional potential in T effector cells during infection. Importantly, we show an association between IL-4 production and CD200R expression on T effector cells from humans infected with Schistosoma haematobium that correlated effectively with egg burden and, thus infection intensity. Our results indicate a role of CD200R:CD200 in T cell responses to helminths which has diagnostic and prognostic relevance as a marker of infection for chronic schistosomiasis in mouse and man.     

Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny of the Drosophila repleta species group

We present a revised molecular phylogeny of the Drosophila repleta group including 62 repleta group taxa and nine outgroup species based on four mitochondrial and six nuclear DNA sequence fragments. With ca. 100 species endemic to the New World, the repleta species group represents one of the major species radiations in the genus Drosophila. Most repleta group species are associated with cacti in arid or semiarid regions. Contrary to previous results, maximum likelihood and Bayesian phylogenies of the 10-gene dataset strongly support the monophyly of the repleta group. Several previously described subdivisions in the group were also recovered, despite poorly resolved relationships between these clades. Divergence time estimates suggested that the repleta group split from its sister group about 21millionyears ago (Mya), although diversification of the crown group began ca. 16Mya. Character mapping of patterns of host plant use showed that flat leaf Opuntia use is common throughout the phylogeny and that shifts in host use from Opuntia to the more chemically complex columnar cacti occurred several times independently during the history of this group. Although some species retained the use of Opuntia after acquiring the use of columnar cacti, there were multiple, phylogenetically independent instances of columnar cactus specialization with loss of Opuntia as a host. Concordant with our proposed timing of host use shifts, these dates are consistent with the suggested times when the Opuntioideae originated in South America. We discuss the generally accepted South American origin of the repleta group.     

Differentially regulated proteins in Prevotella intermedia after oxidative stress analyzed by 2D electrophoresis and mass spectrometry

Prevotella intermedia is a rod-shaped, Gram-negative anaerobic bacterium found in human indigenous microbiota that plays an important role in opportunistic infections. The successful colonization depends on the ability of anaerobes to respond to oxidative stress (OS) in oxygenated tissues as well as to resist oxidative events from the host immune system until anaerobic conditions are present at the infection site. As knowledge of the mechanisms of protection against OS in Prevotella is limited, studies are needed to clarify aspects of molecular biology, physiology and ecology of this bacterium. The aim of this study was to access the proteins differentially regulated in P. intermedia after exposure to molecular oxygen by using two-dimensional gel electrophoresis (2DE) associated with the approach of MALDI-TOF/TOF Tandem Mass Spectrometry. The identity of the protein was evaluated by database search for homologous genomic sequences of P. intermedia strain 17 (TIGR). Twenty five out of 72 proteins found were identified as up-regulated (17) or down-regulated (9). These proteins were related to a variety of metabolic process, some of which could be associated to antioxidant and redox regulatory roles. Our data indicate that OS may stimulate an adaptive response in P. intermedia whose effect on its biology may be evidenced by the increase in aerotolerance and changes in protein abundance in the oxygen adapted cells.     

A Novel Melanocortin-4 Receptor Mutation MC4R-P272L Associated with Severe Obesity Has Increased Propensity To Be Ubiquitinated in the ER in the Face of Correct Folding

Heterozygous mutations in the melanocortin-4 receptor (MC4R) gene represent the most frequent cause of monogenic obesity in humans. MC4R mutation analysis in a cohort of 77 children with morbid obesity identified previously unreported heterozygous mutations (P272L, N74I) in two patients inherited from their obese mothers. A rare polymorphism (I251L, allelic frequency: 1/100) reported to protect against obesity was found in another obese patient. When expressed in neuronal cells, the cell surface abundance of wild-type MC4R and of the N74I and I251L variants and the cAMP generated by these receptors in response to exposure to the agonist, α-MSH, were not different. Conversely, MC4R P272L was retained in the endoplasmic reticulum and had reduced cell surface expression and signaling (by ≈3-fold). The chemical chaperone PBA, which promotes protein folding of wild-type MC4R, had minimal effects on the distribution and signaling of the P272L variant. In contrast, incubation with UBE-41, a specific inhibitor of ubiquitin activating enzyme E1, inhibited ubiquitination of MC4R P272L and increased its cell surface expression and signaling to similar levels as wild-type MC4R. UBE41 had much less profound effects on MC4R I316S, another obesity-linked MC4R variant trapped in the ER. These data suggest that P272L is retained in the ER by a propensity to be ubiquitinated in the face of correct folding, which is only minimally shared by MC4R I316S. Thus, studies that combine clinical screening of obese patients and investigation of the functional defects of the obesity-linked MC4R variants can identify specific ways to correct these defects and are the first steps towards personalized medicine.     

Cardiac protein changes in ischaemic and dilated cardiomyopathy: a proteomic study of human left ventricular tissue

The development of heart failure (HF) is characterized by progressive alteration of left ventricle structure and function. Previous works on proteomic analysis in cardiac tissue from patients with HF remain scant. The purpose of our study was to use a proteomic approach to investigate variations in protein expression of left ventricle tissue from patients with ischaemic (ICM) and dilated cardiomyopathy (DCM). Twenty-four explanted human hearts, 12 from patients with ICM and 12 with DCM undergoing cardiac transplantation and six non-diseased donor hearts (CNT) were analysed by 2DE. Proteins of interest were identified by mass spectrometry and validated by Western blotting and immunofluorescence. We encountered 35 differentially regulated spots in the comparison CNT versus ICM, 33 in CNT versus DCM, and 34 in ICM versus DCM. We identified glyceraldehyde 3-phophate dehydrogenase up-regulation in both ICM and DCM, and alpha-crystallin B down-regulation in both ICM and DCM. Heat shock 70 protein 1 was up-regulated only in ICM. Ten of the eleven differentially regulated proteins common to both aetiologies are interconnected as a part of a same network. In summary, we have shown by proteomics analysis that HF is associated with changes in proteins involved in the cellular stress response, respiratory chain and cardiac metabolism. Although we found altered expression of eleven proteins common to both ischaemic and dilated aetiology, we also observed different proteins altered in both groups. Furthermore, we obtained that seven of these eleven proteins are involved in cell death and apoptosis processes, and therefore in HF progression.     

C957T polymorphism of the dopamine D2 receptor gene is associated with motor learning and heart rate

Genetic variants that are related to the dopaminergic system have been frequently found to be associated with various neurological and mental disorders. Here, we studied the relationships between some of these genetic variants and some cognitive and psychophysiological processes that are implicated in such disorders. Two single nucleotide polymorphisms were chosen: one in the dopamine D2 receptor gene (rs6277-C957T) and one in the catechol-O-methyltransferase gene (rs4680-Val158Met), which is involved in the metabolic degradation of dopamine. The performance of participants on two long-term memory tasks was assessed: free recall (declarative memory) and mirror drawing (procedural motor learning). Heart rate (HR) was also monitored during the initial trials of the mirror-drawing task, which is considered to be a laboratory middle-stress generator (moderate stress), and during a rest period (low stress). Data were collected from 213 healthy Caucasian university students. The C957T C homozygous participants showed more rapid learning than the T allele carriers in the procedural motor learning task and smaller differences in HR between the moderate- and the low-stress conditions. These results provide useful information regarding phenotypic variance in both healthy individuals and patients.