Individuals with Le(a+b-) blood group have increased susceptibility to symptomatic vibrio cholerae O1 infection

Background

Human genetic factors such as blood group antigens may affect the severity of infectious diseases. Presence of specific ABO and Lewis blood group antigens has been shown previously to be associated with the risk of different enteric infections. The aim of this study was to determine the relationship of the Lewis blood group antigens with susceptibility to cholera, as well as severity of disease and immune responses to infection.

Methodology

We determined Lewis and ABO blood groups of a cohort of patients infected by Vibrio cholerae O1, their household contacts, and healthy controls, and analyzed the risk of symptomatic infection, severity of disease if infected and immune response following infection.

Principal Findings

We found that more individuals with cholera expressed the Le(a+b−) phenotype than the asymptomatic household contacts (OR 1.91, 95% CI 1.03–3.56) or healthy controls (OR 1.90, 95% CI 1.13–3.21), as has been seen previously for the risk of symptomatic ETEC infection. Le(a–b+) individuals were less susceptible to cholera and if infected, required less intravenous fluid replacement in hospital, suggesting that this blood group may be associated with protection against V. cholerae O1. Individuals with Le(a–b−) blood group phenotype who had symptomatic cholera had a longer duration of diarrhea and required higher volumes of intravenous fluid replacement. In addition, individuals with Le(a–b−) phenotype also had lessened plasma IgA responses to V. cholerae O1 lipopolysaccharide on day 7 after infection compared to individuals in the other two Lewis blood group phenotypes.

Conclusion

Individuals with Lewis blood type Le(a+b−) are more susceptible and Le(a–b+) are less susceptible to V. cholerae O1 associated symptomatic disease. Presence of this histo-blood group antigen may be included in evaluating the risk for cholera in a population, as well as in vaccine efficacy studies, as is currently being done for the ABO blood group antigens.     

Lateral elements inside synaptonemal complex-like polycomplexes in ndt80 mutants of yeast bind DNA

The synaptonemal complex (SC) keeps the synapsed homologous chromosomes together during pachytene in meiotic prophase I. Structures that resemble stacks of SCs, polycomplexes, are sometimes found before or after pachytene. We have investigated ndt80 mutants of yeast, which arrest in pachytene. SCs appear normal in spread chromosome preparations, but are only occasionally found in intact nuclei examined in the electron microscope. Instead, large polycomplexes occur in almost every ndt80 mutant nucleus. Immunoelectron microscopy using DNA antibodies show strong preferential labeling to the lateral element parts of the polycomplexes. In situ hybridization using chromosome-specific probes confirms that the chromosomes in ndt80 mutants are paired and attached to the SCs. Our results suggest that polycomplexes can be involved in binding of chromosomes and possibly also in synapsis.     

Stimulation of betacyanin synthesis through exogenous methyl jasmonate and other elicitors in suspension-cultured cells of Portulaca

Betacyanin production in suspension-cultured cells of Portulaca was significantly enhanced by both abiotic and biotic elicitors. Betacyanin levels increased 1.3 and 1.5-fold over the controls in the presence of two abiotic elicitors (20 mumol/L CuSO4 and 100 mumol/L FeEDTA) and increased 1.8 and 1.6-fold in the presence of two biotic elicitors (0.5 mg/L beta-glucan and 0.5 mg/L chitosan). Maximum betacyanin synthesis with the two most effective elicitors was obtained when cultures were treated on day 1 and day 0 by beta-glucan and FeEDTA, respectively. A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ). MJ alone at 0.1 mumol/L caused a 2.6-fold increase in betacyanin synthesis when administered to the suspension culture on day 3. However, no additive effect on betacyanin accumulation was observed in treatments, which combined MJ and beta-glucan or FeEDTA. Treatment with ibuprofen (IB), an inhibitor of jasmonate biosynthesis, reduced the level of betacyanin in cells cultured in standard medium at all concentrations tested (25, 50, 100 mumol/L). The effect of IB on betacyanin synthesis in the cells treated with MJ or beta-glucan, however, differed with the IB concentration applied. The two higher concentrations (50 and 100 mumol/L) of IB significantly reduced the betacyanin content while the lower concentration (25 mumol/L) did not show an adverse effect on the betacyanin enhancement triggered by MJ or beta-glucan. Our findings suggest that, in suspension-cultured cells of Portulaca, an MJ-mediated signal transduction pathway prominently exists in betacyanin synthesis. This pathway seems to act antagonistically towards beta-glucan-mediated signaling. As far as we know this is the first report on the elevation of betacyanin level by jasmonate or other elicitors in cell suspension cultures.     

Metabolic engineering for betaine accumulation in microbes and plants

Plants accumulate a variety of osmoprotectants that improve their ability to combat abiotic stresses. Among them, betaine appears to play an important role in conferring resistance to stresses. Betaine is synthesized via either choline oxidation or glycine methylation. An increased betaine level in transgenic plants is one of the potential strategies to generate stress-tolerant crop plants. Here, we showed that an exogenous supply of serine or glycine to a halotolerant cyanobacterium Aphanothece halophytica, which synthesizes betaine from glycine by a three-step methylation, elevated intracellular accumulation of betaine under salt stress. The gene encoding 3-phosphoglycerate dehydrogenase (PGDH), which catalyzes the first step of the phosphorylated pathway of serine biosynthesis, was isolated from A. halophytica. Expression of the Aphanothece PGDH gene in Escherichia coli caused an increase in levels of betaine as well as glycine and serine. Expression of the Aphanothece PGDH gene in Arabidopsis plants, in which the betaine synthetic pathway was introduced via glycine methylation, further increased betaine levels and improved the stress tolerance. These results demonstrate that PGDH enhances the levels of betaine by providing the precursor serine for both choline oxidation and glycine methylation pathways.     

The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability

Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed.Following synthesis, most proteins undergo various N-terminal (Nt) protein modifications, including removal of the Nt Met and signal peptide, N-terminal α-acetylation (NAA), ubiquitination, and acylations. These Nt modifications play an important role in the regulation of cellular functions. The N terminus of proteins has also been shown to be a major determinant of protein stability in bacteria (Varshavsky, 2011), eukaryotes (Graciet et al., 2009), mitochondria, and perhaps in plastids/chloroplasts (Apel et al., 2010; Nishimura et al., 2013; van Wijk, 2015). The role of the N terminus in protein stability is conceptualized in the N-end rule, which states that certain amino acids, when exposed at the N terminus of a protein, act as triggers for degradation (Bachmair et al., 1986; Dougan et al., 2012; Tasaki et al., 2012; Gibbs et al., 2014).Most of the approximately 3,000 plastid proteins are nucleus encoded (n-encoded) and are targeted to the plastid through an Nt chloroplast transit peptide (cTP). After import, the cTP is cleaved by the stromal processing peptidase (SPP; Richter and Lamppa, 1998; Trösch and Jarvis, 2011). The consensus site of cTP cleavage by SPP is only loosely defined, and the rules, mechanisms, and enzymes for possible subsequent processing, stabilization, and other posttranslational modifications (PTMs) are not well characterized (for discussion, see van Wijk, 2015). The exact N terminus is unknown for many chloroplast proteins and cannot be accurately predicted, because SPP specificity is not sufficiently understood (Emanuelsson et al., 2000; Zybailov et al., 2008) and probably also because additional Nt processing occurs for many chloroplast proteins (Fig. 1A). The approximately 85 plastid-encoded (p-encoded) proteins typically first undergo cotranslational Nt deformylation, followed by N-terminal Met excision (NME; Giglione et al., 2009; Fig. 1B); both these PTMs are required for normal plastid/chloroplast development and protein stability (Dirk et al., 2001, 2002; Giglione et al., 2003; Meinnel et al., 2006). Both n-encoded and p-encoded proteins can undergo NAA inside the plastid (Zybailov et al., 2008; Fig. 1). Postulated functions of NAA in eukaryotes include the mediation of protein location, assembly, and stability (Jones and O’Connor, 2011; Starheim et al., 2012; Hoshiyasu et al., 2013; Xu et al., 2015), thereby affecting a variety of processes, including drought tolerance in Arabidopsis (Arabidopsis thaliana; Linster et al., 2015).Open in a separate windowFigure 1.Conceptual illustration of Nt maturation of n-encoded and p-encoded proteins. Ac, Acetylated; MAP, Met amino peptidase; NAT, N-acetyltransferase; N-term, N-terminal; PDF, peptide deformylase. A, Nt maturation of n-encoded plastid proteins including removal of cTP by SPP and potential subsequent Nt modifications. B, Nt maturation of p-encoded proteins. *, The removal depends on the penultimate residue, generally following the N-terminal Met Excision (NME) rule; **, N-terminal acetylation typically occurs only for selected residues; “Results”).Typical proteomics work flows generally yield only partial coverage of protein sequences, and it is often difficult to know which peptides represent the true N termini (Nti) or C termini. Systematic identification of Nti or C termini requires specific labeling and enrichment strategies, such as combined fractional diagonal chromatography, developed by Gevaert and colleagues (Staes et al., 2011), and terminal amine isotopic labeling of substrates (TAILS), developed by the group of Overall (Kleifeld et al., 2011; Lange and Overall, 2013). These strategies allow the identification of different Nt proteoforms and were recently also applied to plants (Tsiatsiani et al., 2013; Carrie et al., 2015; Kohler et al., 2015; Zhang et al., 2015) and diatoms (Huesgen et al., 2013). We previously reported on Nti of chloroplast proteins based on tandem mass spectrometry (MS/MS) analysis, but because no Nt enrichment/labeling technique was used, only those that underwent NAA could be considered bona fide Nti (Zybailov et al., 2008). Nt Edman degradation sequencing was systematically carried out for thylakoid lumen proteins (Peltier et al., 2000, 2002) but not for stromal proteins or chloroplast membrane proteins with their Nti exposed to the stroma. The Nti of thylakoid lumen proteins are mostly generated by lumenal peptidases (Hsu et al., 2011; Midorikawa et al., 2014), and the thylakoid lumen contains a different set of peptidases than the stroma; hence, rules for Nt maturation and stability are likely different than those for stroma-exposed proteins.The objective of this study was to systematically determine the Nti of stroma-exposed chloroplast proteins of Arabidopsis (the N-terminome) and to provide a baseline for understanding Nt protein maturation and protein stability in the chloroplast stroma. To that end, we applied the TAILS technique and determined the Nti of approximately 250 chloroplast proteins by mass spectrometry (MS). We observed that many n-encoded plastid proteins accumulated with two or even three different Nt residues, in many cases both with and without NAA. The extent of accumulation of different Nt proteoforms is surprising and will be discussed. The p-encoded proteins generally showed very similar Nt residues as compared with the n-encoded proteins, with the exception of Met. Our data show that small, apolar, or hydroxylated residues (Ala, Val, Ser, and Thr) are the most frequent Nt residues of stromal proteins, whereas other residues are strictly avoided or are only present for very specific proteins likely to aid in their function. Chloroplast protein degradation products were also detected, with enrichment for peptides generated by cleavage between Arg and Thr residues. We present testable hypotheses for understanding Nt processing and maturation, stability, and a possible N-end rule in chloroplast stroma.     

Evaluation of functional groups on amino acids in cyclic tetrapeptides in histone deacetylase inhibition

The naturally occurring cyclic tetrapeptide, chlamydocin, originally isolated from fungus Diheterospora chlamydosphoria, consists of α-aminoisobutyric acid, l-phenylalanine, d-proline and an unusual amino acid (S)-2-amino-8-((S)-oxiran-2-yl)-8-oxooctanoic acid (Aoe) and inhibits the histone deacetylases (HDACs), a class of regulatory enzymes. The epoxyketone moiety of Aoe is the key functional group for inhibition. The cyclic tetrapeptide scaffold is supposed to play important role for effective binding to the surface of enzymes. In place of the epoxyketone group, hydroxamic acid and sulfhydryl group have been applied to design inhibitor ligands to zinc atom in catalytic site of HDACs. In the research for more potent HDAC inhibitors, we replaced the epoxyketone moiety of Aoe with different functional groups and synthesized a series of chlamydocin analogs as HDAC inhibitors. Among the functional groups, methoxymethylketone moiety showed as potent inhibition as the hydroxamic acid. On the contrary, we confirmed that borate, trifruoromethylketone, and 2-aminoanilide are almost inactive in HDAC inhibition.