Regulatory rewiring confers serotype‐specific hyper‐virulence in the human pathogen group A Streptococcus

Phenotypic heterogeneity is commonly observed between isolates of a given pathogen. Epidemiological analyses have identified that some serotypes of the group A Streptococcus (GAS) are non‐randomly associated with particular disease manifestations. Here, we present evidence that a contributing factor to the association of serotype M3 GAS isolates with severe invasive infections is the presence of a null mutant allele for the orphan kinase RocA. Through use of RNAseq analysis, we identified that the natural rocA mutation present within M3 isolates leads to the enhanced expression of more than a dozen immunomodulatory virulence factors, enhancing phenotypes such as hemolysis and NAD⁺ hydrolysis. Consequently, an M3 GAS isolate survived human phagocytic killing at a level 13‐fold higher than a rocA complemented derivative, and was significantly more virulent in a murine bacteremia model of infection. Finally, we identified that RocA functions through the CovR/S two‐component system as levels of phosphorylated CovR increase in the presence of functional RocA, and RocA has no regulatory activity following covR or covS mutation. Our data are consistent with RocA interfacing with the CovR/S two‐component system, and that the absence of this activity in M3 GAS potentiates the severity of invasive infections caused by isolates of this serotype.     

Trends and Determinants of Antiretroviral Therapy Patient Monitoring Practices in Kenya and Uganda

Introduction

Patients receiving antiretroviral therapy (ART) require routine monitoring to track response to treatment and assess for treatment failure. This study aims to identify gaps in monitoring practices in Kenya and Uganda.

Methods

We conducted a systematic retrospective chart review of adults who initiated ART between 2007 and 2012. We assessed the availability of baseline measurements (CD4 count, weight, and WHO stage) and ongoing CD4 and weight monitoring according to national guidelines in place at the time. Mixed-effects logistic regression models were used to analyze facility and patient factors associated with meeting monitoring guidelines.

Results

From 2007 to 2012, at least 88% of patients per year in Uganda had a recorded weight at initiation, while in Kenya there was a notable increase from 69% to 90%. Patients with a documented baseline CD4 count increased from 69% to about 80% in both countries. In 2012, 83% and 86% of established patients received the recommended quarterly weight monitoring in Kenya and Uganda, respectively, while semiannual CD4 monitoring was less common (49% in Kenya and 38% in Uganda). Initiating at a more advanced WHO stage was associated with a lower odds of baseline CD4 testing. On-site CD4 analysis capacity was associated with increased odds of CD4 testing at baseline and in the future.

Discussion

Substantial gaps were noted in ongoing CD4 monitoring of patients on ART. Although guidelines have since changed, limited laboratory capacity is likely to remain a significant issue in monitoring patients on ART, with important implications for ensuring quality care.     

The Eucalyptus terpene synthase gene family

Background

Terpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts.

Results

The genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus.

Conclusions

Our data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1598-x) contains supplementary material, which is available to authorized users.     

Focus on Metabolism: Molecular Genetic Analysis of Glucan Branching Enzymes from Plants and Bacteria in Arabidopsis Reveals Marked Differences in Their Functions and Capacity to Mediate Starch Granule Formation

The major component of starch is the branched glucan amylopectin, the branching pattern of which is one of the key factors determining its ability to form semicrystalline starch granules. Here, we investigated the functions of different branching enzyme (BE) types by expressing proteins from maize (Zea mays BE2a), potato (Solanum tuberosum BE1), and Escherichia coli (glycogen BE [EcGLGB]) in Arabidopsis (Arabidopsis thaliana) mutant plants that are deficient in their endogenous BEs and therefore, cannot make starch. The expression of each of these three BE types restored starch biosynthesis to differing degrees. Full complementation was achieved using the class II BE ZmBE2a, which is most similar to the two endogenous Arabidopsis isoforms. Expression of the class I BE from potato, StBE1, resulted in partial complementation and high amylose starch. Expression of the glycogen BE EcGLGB restored only minimal amounts of starch production, which had unusual chain length distribution, branch point distribution, and granule morphology. Nevertheless, each type of BE together with the starch synthases and debranching enyzmes were able to create crystallization-competent amylopectin polymers. These data add to the knowledge of how the properties of the BE influence the final composition of starch and fine structure of amylopectin.Starch is composed of two glucan polymers: amylopectin and amylose. Amylopectin constitutes around 80% of the mass of most starches and is a large, branched polymer with a tree-like architecture. The positioning and frequency of branch points together with the distribution of chain lengths are thought to be critical factors allowing amylopectin to adopt a semicrystalline state. Within amylopectin molecules, clusters of unbranched chain segments align, and adjacent chains form double helices. These pack into crystalline lamellae that alternate with amorphous regions containing the branch points. Longer chain segments span from one cluster to the next (Zeeman et al., 2010).Amylopectin is synthesized by three enzyme activities. First, starch synthases (SSs) transfer the glucosyl part of ADP-Glc to the nonreducing end of existing glucan chains, forming new α-1,4 glucosidic bonds. Second, branching enzymes (BEs) cleave part of an α-1,4-linked chain and through an inter- or intramolecular transfer reaction, reattach it, creating α-1,6-branch points. This reaction creates additional nonreducing ends on which SSs can act. Third, debranching enzymes (DBEs) hydrolyze some of these branches, tailoring the structure of the polymer to promote its crystallization.Several SS and BE isoforms are involved in starch synthesis in plants. There are five conserved classes of SSs (granule-bound starch synthase [GBSS] and SS1–SS4) and two conserved classes of BEs (classes I and II; also referred to as classes B and A, respectively; Nougué et al., 2014). In addition, plants contain two classes of DBEs: isoamylases (ISAs) and limit dextrinases (LDAs; also called pullulanases). One ISA, a multimeric enzyme composed of either a mixture of ISA1 and ISA2 subunits or just ISA1 subunits, is primarily involved in amylopectin synthesis (James et al., 1995; Mouille et al., 1996; Nakamura et al., 1996; Delatte et al., 2005). The other DBEs (i.e. ISA3 and LDA) are primarily involved in starch degradation (Wattebled et al., 2005; Delatte et al., 2006).Based on the in vitro analysis of purified or recombinant proteins and the phenotypes of mutant plants, the different SS isoforms are proposed to have distinct, albeit overlapping, functions. SS1 is thought to preferentially elongate short chains produced by the branching reactions to between 8 and 12 Glc units (Delvallé et al., 2005; Fujita et al., 2006). SS2 is proposed to elongate such chains farther to about 20 Glc units, optimal for cluster formation (Edwards et al., 1999; Umemoto et al., 2002; Zhang et al., 2008). The precise role of SS3 is less clear, although it has been proposed to generate long, cluster-spanning chains (Fujita et al., 2007). SS4 has a distinct role in initiating and/or coordinating granule formation (Roldán et al., 2007; Crumpton-Taylor et al., 2013).The two different BE classes are also proposed to have distinct functions in amylopectin synthesis. In vitro analyses of maize (Zea mays), rice (Oryza sativa), and potato (Solanum tuberosum) enzymes suggest that the class I enzymes preferentially act on amylose and transfer longer chains, whereas class II enzymes preferentially act on branched substrates, such as amylopectin, and transfer shorter chains (Guan and Preiss, 1993; Rydberg et al., 2001; Nakamura et al., 2010). This knowledge derives largely from experiments where linear or branched substrates were provided to recombinant or purified enzymes and the increased degree of branching was monitored. Similar conclusions were gained by recombinant protein expression in Escherichia coli and yeast (Saccharomyces cerevisiae) strains deficient in their endogenous glycogen BEs (Guan et al., 1995; Seo et al., 2002), where chain elongation by glycogen synthases occurred concurrently with branching.Models have been proposed in which both BE classes help create the final cluster structure of amylopectin: class I BEs initiate branching by transferring long or branched chains, which are subsequently acted on by class II BEs to create more numerous shorter chains. These shorter chains are then elaborated by the SSs to create the clusters (Nakamura et al., 2010). After the branching reactions, a degree of debranching occurs, which is thought to control branch number and positioning and thereby, facilitate amylopectin crystallization (Myers et al., 2000; Zeeman et al., 2010). Several studies have shown that isa1-deficient mutants produce starch with an altered amylopectin, accumulate a related soluble polymer (phytoglycogen), or both (James et al., 1995; Mouille et al., 1996; Nakamura et al., 1996; Delatte et al., 2005).Despite the wide conservation of the two BE classes, major alterations in starch properties are only observed when genes encoding class II enzymes are mutated or repressed. Loss of class I BE activity in maize endosperm, rice endosperm, or potato tuber did not alter starch content and caused only minor differences in amylopectin structure (e.g. the distribution of chain lengths and branch points) and/or starch properties (e.g. gelatinization or digestibility; Safford et al., 1998; Blauth et al., 2002; Satoh et al., 2003; Xia et al., 2011). In contrast, loss of class II BE results in significant changes, such as decreased starch content and a high apparent amylose content. This has been observed in several species, including maize (Stinard et al., 1993), potato (Jobling et al., 1999), pea (Pisum sativum; Bhattacharyya et al., 1990), rice (Mizuno et al., 1993), barley (Hordeum vulgare; Regina et al., 2010), and wheat (Triticum aestivum; Regina et al., 2006). The high apparent amylose content was caused at least in part by the accumulation of less-frequently branched amylopectin that stains with a higher wavelength of maximal absorption (λ_max) than that of the wild type (Boyer et al., 1976). In potato, this phenotype was enhanced by the simultaneous suppression of BE1 (Schwall et al., 2000), a result also shown recently in barley (Carciofi et al., 2012).Arabidopsis (Arabidopsis thaliana) has three genes annotated as BEs, At3g20440 (BE1), At5g03650 (BE2), and At2g36390 (BE3), but it seems that only BE2 and BE3 are active. Both BE2 and BE3 are class II BEs, making Arabidopsis somewhat unusual in not possessing a class I BE. The gene annotated as BE1 encodes a related protein that falls into a separate clade to either class I or II BEs (Dumez et al., 2006; Han et al., 2007; Wang et al., 2010). It was initially suggested that plants with mutations in this gene had a wild-type phenotype (Dumez et al., 2006), but subsequent work indicated that homozygous be1 mutation causes embryo lethality (hence, its alternative name EMBRYO DEFECTIVE2729; Wang et al., 2010). Thus, the function of the protein encoded at At3g20440 is currently unknown but unlikely to be a functional BE.The be2 and be3 single mutants have phenotypes that closely resemble the wild type, indicating that there is a high degree of redundancy between the enzymes. However, be2be3 double mutants lack starch (Dumez et al., 2006). Instead, the plants accumulate large amounts of maltose and other linear malto-oligosaccharides (MOSs). This is presumably because linear chains produced by the SSs are cleaved by starch-degrading enzymes (α- and β-amylases; Dumez et al., 2006). The altered metabolism of these double-mutant plants impedes growth, and they are smaller and paler than the wild type. The precise reason for this is unclear.In addition to mutagenesis, there have been several studies where BEs were overexpressed in transgenic plants. Overexpression of the E. coli glycogen BE (EcGLGB) in potato tubers or rice endosperm resulted in an increased degree of branching of amylopectin (Shewmaker et al., 1994; Kortstee et al., 1996; Kim et al., 2005). Overexpression of endogenous plant BE2 genes has also been performed in both rice and potato, increasing the proportion of shorter amylopectin chains (Tanaka et al., 2004; Brummell et al., 2015), and rice, leading to the accumulation of highly branched, water-soluble polysaccharides (Tanaka et al., 2004). Transgenic expression of genes from different photosynthetic organisms has also shown the degree of functional conservation within the plant BE classes. Sawada et al. (2009) showed that class II BE from Chlorella kessleri could rescue the BE2b-deficient phenotype in rice endosperm.The aim of this work was to investigate the capacity of different types of BEs to mediate starch granule formation by assessing their ability to function in the context of an otherwise intact starch biosynthesis pathway. To do this, we used the Arabidopsis be2be3 double mutants as a line in which to express three types of BEs. We chose BE2a from maize (required for leaf starch synthesis and similar to the endogenous Arabidopsis proteins; Yandeau-Nelson et al., 2011), BE1 from potato (represents the plant class I BEs that Arabidopsis lacks; Safford et al., 1998), and GLGB (the BE from E. coli involved in glycogen biosynthesis). This approach differs from previous investigations, because the activity of each BE type (working in planta with the same set of SSs and DBEs) can be assessed, and the results can be directly compared. In addition, we sought to address whether a glycogen BE was sufficient for starch production—in other words, whether the remaining starch biosynthetic enzymes are capable of generating a crystallization competent polymer, even when partnered with a BE with a different specificity. In previously described transgenic plants expressing E. coli GLGB, the endogenous plant BEs were still present (Shewmaker et al., 1994; Kortstee et al., 1996; Kim et al., 2005).In the transgenic lines generated here, we analyzed glucan synthesis, starch structure, and composition. Our results show that all three BE types can mediate starch granule production but to differing degrees. In each case, the structure of amylopectin and the amylose content depend on the type of BE present, as does starch granule morphology. We discuss the reasons for these differences in relation to previously reported BE properties.     

Down's syndrome,Edwards' syndrome,Patau's syndrome —synthesis of glycosaminoglycans

Synthesis of glycosaminoglycans (GAGS) by fibroblasts derived from seven patients with Down's syndrome, five patients with Edwards' syndrome, and two patients with Patau's syndrome were studied in cell culture. The aneuploid strains were compared with diploid fibroblasts from age-matched controls. In terms of hyaluronic acid and sulfated GAG synthesis, the amount of synthesized hyaluronic acid was not significantly different between postnatal aneuploid strains and controls.     

Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion

Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.     

Hydrogen peroxide-mediated inactivation of microsomal cytochrome P450 during monooxygenase reactions

Cytochrome P450 can undergo inactivation following monooxygenase reactions in liver microsomes of untreated, phenobarbital and 3-methylcholanthrene-treated rats and rabbits. The acceleration of cytochrome P450 loss in the presence of catalase inhibitors (sodium azide, hydroxylamine) indicates that hydrogen peroxide is involved in hemoprotein degradation. It was revealed that cytochrome P450 is inactivated mainly by H₂O₂ formed through peroxy complex breakdown, whereas H₂O₂ formed via the dismutation of superoxide anions produces a slight inactivating effect. The hydrogen peroxide added outside or formed by a glucose-glucose oxidase system has less of an inactivating effect than H₂O₂ produced within the cytochrome P450 active center. Self-inactivation of cytochrome P450 during oxygenase reactions is highly specific. Other components of the monooxygenase system, such as cytochrome b₅, NADH- and NADPH-specific flavorproteins, undergo no inactivation. The alterations in phospholipid content and in the rate of lipid peroxidation were not observed as well. The inactivation of cytochrome P450 by H₂O₂ is the result of heme loss or destruction without cytochrome P420 formation. Such. a mechanism operates with different substrates and cytochrome P450 species catalyzing the partially coupled monooxygenase reactions.     

International Society for Reef Studies

International Society for Reef Studies