Integrated Translatomics with Proteomics to Identify Novel Iron–Transporting Proteins in Streptococcus pneumoniae

Streptococcus pneumoniae (S. pneumoniae) is a major human pathogen causing morbidity and mortality worldwide. Efficiently acquiring iron from the environment is critical for S. pneumoniae to sustain growth and cause infection. There are only three known iron-uptake systems in Streptococcal species responsible for iron acquisition from the host, including ABC transporters PiaABC, PiuABC and PitABC. Besides, no other iron-transporting system has been suggested. In this work, we employed our newly established translating mRNA analysis integrated with proteomics to evaluate the possible existence of novel iron transporters in the bacterium. We simultaneously deleted the iron-binding protein genes of the three iron-uptake systems to construct a piaA/piuA/pitA triple mutant (Tri-Mut) of S. pneumoniae D39, in which genes and proteins related to iron transport should be regulated in response to the deletion. With ribosome associated mRNA sequencing-based translatomics focusing on translating mRNA and iTRAQ quantitative proteomics based on the covalent labeling of peptides with tags of varying mass, we indeed observed a large number of genes and proteins representing various coordinated biological pathways with significantly altered expression levels in the Tri-Mut mutant. Highlighted in this observation is the identification of several new potential iron-uptake ABC transporters participating in iron metabolism of Streptococcus. In particular, putative protein SPD_1609 in operon 804 was verified to be a novel iron-binding protein with similar function to PitA in S. pneumoniae. These data derived from the integrative translatomics and proteomics analyses provided rich information and insightful clues for further investigations on iron-transporting mechanism in bacteria and the interplay between Streptococcal iron availability and the biological metabolic pathways

Metabolic engineering and comparative performance studies of Synechocystis sp. PCC 6803 strains for effective utilization of xylose.

Wood sugars such as xylose can be used as an inexpensive carbon source for biotechnological applications. The model cyanobacterium Synechocystis sp. PCC 6803 lacks the ability to catabolize wood sugars as an energy source. Here, we generated four Synechocystis strains that heterologously expressed XylAB enzymes, which mediate xylose catabolism, either in combination with or without one of three xylose transporters, namely XylE, GalP, or Glf. Except for glf, which is derived from the bacterium Zymomonas mobilis ZM4, the heterologous genes were sourced from Escherichia coli K-12. All of the recombinant strains were able to utilize xylose in the absence of catabolite repression. When xylose was the lone source of organic carbon, strains possessing the XylE and Glf transporters were most efficient in terms of dry biomass production and xylose consumption and the strain lacking a heterologous transporter was the least efficient. However, in the presence of a xylose-glucose mixed sugar source, the strains exhibited similar levels of growth and xylose consumption. This study demonstrates that various bacterial xylose transporters can boost xylose catabolism in transgenic Synechocystis strains, and paves the way for the sustainable production of bio-compounds and green fuels from lignocellulosic biomass

Polyamine regulates tolerance to water stress in leaves of white clover associated with antioxidant defense and dehydrin genes via involvement in calcium messenger system and hydrogen peroxide signaling

Endogenous polyamine (PA) may play a critical role in tolerance to water stress in plants acting as a signaling molecule activator. Water stress caused increases in endogenous PA content in leaves, including putrescine (Put), spermidine (Spd), and spermine (Spm). Exogenous application of Spd could induce the instantaneous H2O2 burst and accumulation of cytosolic free Ca2+, and activate NADPH oxidase and CDPK gene expression in cells. To a great extent, PA biosynthetic inhibitor reduced the water stress-induced H2O2 accumulation, free cytosolic Ca2+ release, antioxidant enzyme activities and genes expression leading to aggravate water stress-induced oxidative damage, while these suppressing effects were alleviated by the addition of exogenous Spd, indicating PA was involved in water stress-induced H2O2 and cytosolic free Ca2+ production as well as stress tolerance. Dehydrin genes (Y2SK, Y2K, and SK2) were showed to be highly responsive to exogenous Spd. PA-induced antioxidant defense and dehydrin genes expression could be blocked by the scavenger of H2O2 and the inhibitors of H2O2 generation or Ca2+ channels blockers, a calmodulin antagonist, as well as the inhibitor of CDPK. These findings suggested that PA regulated tolerance to water stress in white clover associated with antioxidant defenses and dehydrins via involvement in the calcium messenger system and H2O2 signaling pathways. PA-induced H2O2 production required Ca2+ release, while PA-induced Ca2+ release was also essential for H2O2 production, suggesting an interaction between PA-induced H2O2 and Ca2+ signaling

Serological response and diagnostic value of recombinant candida cell wall protein enolase, phosphoglycerate kinase and β- glucosidase

There are no specific signs and symtoms for invasive candidiasis (IC), which makes its diagnosis a challenge. Efforts have been made for decades to establish serological assays for rapid diagnosis of invasive candidiasis, but none of them have found widespread clinical use. Using a systemic candiasis murine model, serological response to recombinant proteins of enolase (rEno1), phosphoglycerate kinase (rPgk1) and β-glucosidase (rBgl2) were evaluated and rEno1 was found to possess the strongest immunoreactivity, followed by rPgk1 and rBgl2. Likewise, IgG antibody titers to rEno1, rPgk1 and rBgl2 in the positive sera of proven IC patients were determined by ELISA. Results show anti-rEno1 antibody possesses the highest titer, followed by rPgk1 and rBgl2. Antibodies against rEno1, rPgk1 and rBgl2 were detected by ELISA tests in a group of 52 proven IC patients or 50 healthy subjects, The sensitivity, specificity, positive and negative predictive values were 88.5%, 90.0%, 90.2%, and 88.2% for anti-rEno1 detection, 86.5%, 92.0%, 91.8% and 86.8% for anti-rPgk1 detection, and 80.8%, 90.0%, 89.4% and 81.8% for anti-rBgl2 detection, respectively. The data clearly demonstrate that the recombinant proteins of Eno1, Pgk1 and Bgl2 are promising candidates for IC serodiagnosis. There’s great possibility that the recombinant Eno1 will be more applicable in serodiagnosis and vaccine research on account of its strong serological response

Alterations in Fecal Lactobacillus and Bifidobacterium Species in Type 2 Diabetic Patients in Southern China Population

Background: The connection between gut microbiota and metabolism and its role in the pathogenesis of diabetes are increasingly recognized. The objective of this study was to quantitatively measure Bifidobacterium and Lactobacillus species, members of commensal bacteria found in human gut, in type 2 diabetic patients (T2D) patients from Southern China. Methods: Fifty patients with T2D and thirty control individuals of similar BMI were recruited from Southern China. T2D and control subjects were confirmed with both oral glucose tolerance test (OGTT) and HbA1c measurements. Bifidobacterium and Lactobacillus species in feces were measured by real-time quantitative PCR. Data were analyzed with STATA 11.0 statistical software.Results: In comparison to control subjects T2D patients had significantly more total Lactobacillus (+18%), L. bugaricus (+13%), L. rhamnosum (+37%) and L. acidophilus (+48%) (P <0.05). In contrast, T2D patients had less amounts of total Bifidobacteria (-7%) and B. adolescentis (-12%) (P <0.05). Cluster analysis showed that gut microbiota pattern of T2D patients is characterized by greater numbers of L. rhamnosus and L. acidophillus, together with lesser numbers of B. adolescentis (P <0.05). Conclusion: The gut microflora in T2D patients is characterized by greater numbers of Lactobacillus and lesser numbers of Bifidobacterium species

Characterization of Coxsackievirus A6- and Enterovirus 71-associated Hand Foot and Mouth disease in Beijing, China, from 2013 to 2015

Background: Etiology surveillance of Hand Foot and Mouth disease (HFMD) in Beijing showed that Coxsackievirus A6 (CVA6) became the major pathogen of HFMD in 2013 and 2015. In order to understand the epidemiological characteristics and clinical manifestations of CVA6-associated HFMD, a comparison study among CVA6-, EV71- (Enterovirus 71) and CVA16- (Coxsackievirus A16) associated HFMD was performed.Methods: Epidemiological characteristics and clinical manifestations among CVA6-, EV71- and CVA16-associated mild or severe cases were compared from 2013 to 2015. VP1 gene of CVA6 and EV71 from mild cases, severe cases were sequenced, aligned and compared with strains from 2009 to 2015 in Beijing and strains available in GenBank. Phylogenetic tree was constructed by neighbor-joining method.Results: CVA6 became the predominant causative agent of HFMD and accounted for 35.4% and 36.9% of total positive cases in 2013 and 2015, respectively. From 2013 to 2015, a total of 305 severe cases and 7 fatal cases were reported. CVA6 and EV71 were responsible for 57.5% of the severe cases. Five out six samples from fatal cases were identified as EV71. High fever, onychomadesis and decrustation were the typical symptoms of CVA6-associated mild HFMD. CVA6-associated severe cases were characterized by high fever with shorter duration and twitch compared with EV71-associated severe cases which were characterized by poor mental condition, abnormal pupil and vomiting. Poor mental condition, lung wet rales, abnormal pupil and tachycardia were the most common clinical features of fatal cases. The percentage of lymphocyte in CVA6-associated cases was significantly lower than that of EV71. High percentage of lymphocyte and low percentage of neutrophils were the typical characteristics of fatal cases. VP1 sequences between CVA6- or EV71-associated mild and severe cases were highly homologous.Conclusion: CVA6 became one of the major pathogens of HFMD in 2013 and 2015 in Beijing. Epidemiological characteristics, clinical manifestations of CVA6-, EV71- and CVA16-associated cases in this study enriched the definition of HFMD caused by different pathogens and shed light to accurate diagnosis, appropriate treatment and effective prevention of HFMD