Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.     

Quantifying the importance of MSP1-19 as a target of growth-inhibitory and protective antibodies against Plasmodium falciparum in humans

Background

Antibodies targeting blood stage antigens are important in protection against malaria, but the key targets and mechanisms of immunity are not well understood. Merozoite surface protein 1 (MSP1) is an abundant and essential protein. The C-terminal 19 kDa region (MSP1-19) is regarded as a promising vaccine candidate and may also be an important target of immunity.

Methodology/Findings

Growth inhibitory antibodies against asexual-stage parasites and IgG to recombinant MSP1-19 were measured in plasma samples from a longitudinal cohort of 206 children in Papua New Guinea. Differential inhibition by samples of mutant P. falciparum lines that expressed either the P. falciparum or P. chabaudi form of MSP1-19 were used to quantify MSP1-19 specific growth-inhibitory antibodies. The great majority of children had detectable IgG to MSP1-19, and high levels of IgG were significantly associated with a reduced risk of symptomatic P. falciparum malaria during the 6-month follow-up period. However, there was little evidence of PfMSP1-19 specific growth inhibition by plasma samples from children. Similar results were found when testing non-dialysed or dialysed plasma, or purified antibodies, or when measuring growth inhibition in flow cytometry or microscopy-based assays. Rabbit antisera generated by immunization with recombinant MSP1-19 demonstrated strong MSP1-19 specific growth-inhibitory activity, which appeared to be due to much higher antibody levels than human samples; antibody avidity was similar between rabbit antisera and human plasma.

Conclusions/Significance

These data suggest that MSP1-19 is not a major target of growth inhibitory antibodies and that the protective effects of antibodies to MSP1-19 are not due to growth inhibitory activity, but may instead be mediated by other mechanisms. Alternatively, antibodies to MSP1-19 may act as a marker of protective immunity.     

CD133 positive embryonal rhabdomyosarcoma stem-like cell population is enriched in rhabdospheres

Cancer stem cells (CSCs) have been identified in a number of solid tumors, but not yet in rhabdomyosarcoma (RMS), the most frequently occurring soft tissue tumor in childhood. Hence, the aim of this study was to identify and characterize a CSC population in RMS using a functional approach. We found that embryonal rhabdomyosarcoma (eRMS) cell lines can form rhabdomyosarcoma spheres (short rhabdospheres) in stem cell medium containing defined growth factors over several passages. Using an orthotopic xenograft model, we demonstrate that a 100 fold less sphere cells result in faster tumor growth compared to the adherent population suggesting that CSCs were enriched in the sphere population. Furthermore, stem cell genes such as oct4, nanog, c-myc, pax3 and sox2 are significantly upregulated in rhabdospheres which can be differentiated into multiple lineages such as adipocytes, myocytes and neuronal cells. Surprisingly, gene expression profiles indicate that rhabdospheres show more similarities with neuronal than with hematopoietic or mesenchymal stem cells. Analysis of these profiles identified the known CSC marker CD133 as one of the genes upregulated in rhabdospheres, both on RNA and protein levels. CD133(+) sorted cells were subsequently shown to be more tumorigenic and more resistant to commonly used chemotherapeutics. Using a tissue microarray (TMA) of eRMS patients, we found that high expression of CD133 correlates with poor overall survival. Hence, CD133 could be a prognostic marker for eRMS. These experiments indicate that a CD133(+) CSC population can be enriched from eRMS which might help to develop novel targeted therapies against this pediatric tumor.     

Features and prognosis of severe malaria caused by Plasmodium falciparum, Plasmodium vivax and mixed Plasmodium species in Papua New Guinean children

Background

Mortality from severe pediatric falciparum malaria appears low in Oceania but Plasmodium vivax is increasingly recognized as a cause of complications and death. The features and prognosis of mixed Plasmodium species infections are poorly characterized. Detailed prospective studies that include accurate malaria diagnosis and detection of co-morbidities are lacking.

Methods and Findings

We followed 340 Papua New Guinean (PNG) children with PCR-confirmed severe malaria (77.1% P. falciparum, 7.9% P. vivax, 14.7% P. falciparum/vivax) hospitalized over a 3-year period. Bacterial cultures were performed to identify co-incident sepsis. Clinical management was under national guidelines. Of 262 children with severe falciparum malaria, 30.9%, 24.8% and 23.2% had impaired consciousness, severe anemia, and metabolic acidosis/hyperlactatemia, respectively. Two (0.8%) presented with hypoglycemia, seven (2.7%) were discharged with neurologic impairment, and one child died (0.4%). The 27 severe vivax malaria cases presented with similar phenotypic features to the falciparum malaria cases but respiratory distress was five times more common (P = 0.001); one child died (3.7%). The 50 children with P. falciparum/vivax infections shared phenotypic features of mono-species infections, but were more likely to present in deep coma and had the highest mortality (8.0%; P = 0.003 vs falciparum malaria). Overall, bacterial cultures were positive in only two non-fatal cases. 83.6% of the children had alpha-thalassemia trait and seven with coma/impaired consciousness had South Asian ovalocytosis (SAO).

Conclusions

The low mortality from severe falciparum malaria in PNG children may reflect protective genetic factors other than alpha-thalassemia trait/SAO, good nutrition, and/or infrequent co-incident sepsis. Severe vivax malaria had similar features but severe P. falciparum/vivax infections were associated with the most severe phenotype and worst prognosis.     

Identification of a Gene Cluster for the Biosynthesis of a Long,Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase

Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in beneficial bacteria. We identified a gene cluster that encodes the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain Lactobacillus rhamnosus GG. Subsequent mutation of the welE gene, encoding the priming glycosyltransferase within this cluster, and comparative phenotypic analyses of wild-type versus mutant strains confirmed the specific function of this gene cluster in the biosynthesis of high-molecular-weight, galactose-rich heteropolymeric EPS molecules. The phenotypic analyses included monomer composition determination, estimation of the polymer length of the isolated EPS molecules, and single-molecule force spectroscopy of the surface polysaccharides. Further characterization of the welE mutant also showed that deprivation of these long, galactose-rich EPS molecules results in an increased adherence and biofilm formation capacity of L. rhamnosus GG, possibly because of less shielding of adhesins such as fimbria-like structures.Bacterial surface polysaccharides are considered to be key macromolecules in determining microbe-host interactions, as they display a high degree of variety and diversity among bacterial species in terms of composition, monomer linkages, branching degree, polymer size, production level, etc. (24, 46). Since most bacteria contain more than one type of surface polysaccharides, such as lipopolysaccharides (O antigens), capsular polysaccharides (CPS), exopolysaccharides (EPS), and/or glycan chains as part of glycoproteins, the elucidation of their exact role is complex. Nevertheless, surface polysaccharides are now known to exert important functions at several stages during pathogenesis, including tissue adherence, biofilm formation, and evasion of host defenses such as phagocytosis (9, 24, 33). In addition to their role in pathogens, an important biological role for CPS and glycoproteins has also recently been shown in colonization of the gut by bacteria of the genus Bacteroides (10, 34).Conversely, the role of surface polysaccharides in probiotic-host interactions has not yet been studied in great detail. A probiotic bacterium is defined as “a live microorganism that, when administered or ingested in adequate amounts, confers a health benefit on the host” (18). Members of the genus Lactobacillus are commonly studied for their health-promoting capacities (26, 31, 37). As polysaccharides display a high diversity among lactobacilli, they are thought to be involved in determining strain-specific properties important for probiotic action, such as adhesion, stress resistance, and interactions with specific receptors and effectors of the host defense system (13, 56). Moreover, these EPS molecules are of interest in the dairy industry for conferring textural and rheological properties to fermented products such as yogurt and soft cheese (56). Nevertheless, detailed genetic and functional studies of EPS molecules of lactobacilli are currently limited (26, 56).Lactobacillus rhamnosus GG (ATCC 53103) is one of the probiotic strains with the largest number of proven health benefits (15). Several clinical trials have reported that L. rhamnosus GG can prevent and relieve certain types of diarrhea (22) and atopic disease (25) and reduce inflammation in some milder states of inflammatory bowel diseases (60). However, the cell surface factors or specific characteristics of L. rhamnosus GG that underlie these health benefits are largely unknown.We recently showed by single-molecule force spectroscopy (SMFS) with specific lectin tips that the cell surface of L. rhamnosus GG wild-type cells contains two major types of cell wall-associated polysaccharides (CW-PS) (21). The longest and most abundant polysaccharides are galactose-rich and seem to correspond with the EPS molecules of L. rhamnosus GG, which were previously structurally identified by Landersjö et al. (27) using nuclear magnetic resonance spectroscopy. Additionally, shorter, yet-uncharacterized glucose-rich polysaccharides are present on the L. rhamnosus GG surface (21). In the current study, we describe the identification and annotation of the L. rhamnosus GG gene cluster that encodes the enzymes and transporter and regulatory proteins involved in the biosynthesis of long, galactose-rich EPS molecules. This was experimentally confirmed by the construction of a knockout mutant of the corresponding priming glycosyltransferase and subsequent characterization of the surface polysaccharides of wild-type and mutant strains. We also studied the specific role of these EPS molecules in adherence to mucus and gut epithelial cells and in biofilm formation by L. rhamnosus GG.