The molecular basis of glycogen breakdown and transport in Streptococcus pneumoniae

The genome of Streptococcus pneumoniae strains, as typified by the TIGR4 strain, contain several genes encoding proteins putatively involved in α‐glucan degradation, modification and synthesis. The extracellular components comprise an ATP binding cassette‐transporter with its solute binding protein, MalX, and the hydrolytic enzyme SpuA. We show that of the commonly occurring exogenous α‐glucans, S. pneumoniae TIGR4 is only able to grow on glycogen in a MalX‐ and SpuA‐dependent manner. SpuA is able to degrade glycogen into a ladder of α‐1,4‐glucooligosaccharides while the high‐affinity interaction (K_a ～ 10⁶ M^?1) of MalX with maltooligosaccharides plays a key role in promoting the selective uptake of the glycogen degradation products that are produced by SpuA. The X‐ray crystallographic analyses of apo‐ and complexed MalX illuminate the protein's specificity for the degradation products of glycogen and its striking ability to recognize the helical structure of the ligand. Overall, the results of this work provide new structural and functional insight into streptococcal α‐glucan metabolism while supplying biochemical support for the hypothesis that the substrate of the S. pneumoniaeα‐glucan metabolizing machinery is glycogen, which in a human host is abundant in lung epithelial cells, a common target for invasive S. pneumoniae.     

The Role of ClpP in Protein Expression of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Previous reports suggest that ClpP proteolytic activity is important not only for cell physiology but also for regulation of virulence properties of Streptococcus pneumoniae (S. pneumoniae). In order to get a more comprehensive picture of the role of ClpP protease on protein expression in S. pneumoniae D39 and how it relates to physiology and virulence, a clpP mutant strain was constructed in S. pneumoniae D39, and global proteome expression was studied by 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. We report here that clpP deletion affects the expression of proteins which are involved in the general stress response, nucleotide metabolism, energy metabolism, and proteins metabolism. These provide clues for understanding the role of ClpP in the physiology and pathogenesis of pneumococcus.     

Characterization of hypothetical proteins Cpn0146, 0147, 0284 &; 0285 that are predicted to be in the <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> inclusion membrane

Background  

Although more than 100 Chlamydia pneumoniae hypothetical proteins have been predicted to be inclusion membrane proteins, only a few have been experimentally demonstrated to be in the inclusion membrane. Using antibodies raised with fusion proteins, we characterized four such hypothetical proteins encoded by two gene clusters (Cpn0146-147 and Cpn0284-285) in the C. pneumoniae genome.     

Streptococcus pneumoniae Invades Erythrocytes and Utilizes Them to Evade Human Innate Immunity

Streptococcus pneumoniae, a Gram-positive bacterium, is a major cause of invasive infection-related diseases such as pneumonia and sepsis. In blood, erythrocytes are considered to be an important factor for bacterial growth, as they contain abundant nutrients. However, the relationship between S. pneumoniae and erythrocytes remains unclear. We analyzed interactions between S. pneumoniae and erythrocytes, and found that iron ion present in human erythrocytes supported the growth of Staphylococcus aureus, another major Gram-positive sepsis pathogen, while it partially inhibited pneumococcal growth by generating free radicals. S. pneumoniae cells incubated with human erythrocytes or blood were subjected to scanning electron and confocal fluorescence microscopic analyses, which showed that the bacterial cells adhered to and invaded human erythrocytes. In addition, S. pneumoniae cells were found associated with human erythrocytes in cultures of blood from patients with an invasive pneumococcal infection. Erythrocyte invasion assays indicated that LPXTG motif-containing pneumococcal proteins, erythrocyte lipid rafts, and erythrocyte actin remodeling are all involved in the invasion mechanism. In a neutrophil killing assay, the viability of S. pneumoniae co-incubated with erythrocytes was higher than that without erythrocytes. Also, H₂O₂ killing of S. pneumoniae was nearly completely ineffective in the presence of erythrocytes. These results indicate that even when S. pneumoniae organisms are partially killed by iron ion-induced free radicals, they can still invade erythrocytes. Furthermore, in the presence of erythrocytes, S. pneumoniae can more effectively evade antibiotics, neutrophil phagocytosis, and H₂O₂ killing.     

Discovery of novel inhibitors of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> based on the virtual screening with the homology-modeled structure of histidine kinase (VicK)

Background  

Due to the widespread abusage of antibiotics, antibiotic-resistance in Streptococcus pneumoniae (S. pneumoniae) has been increasing quickly in recent years, and it is obviously urgent to develop new types of antibiotics. Two-component systems (TCSs) are the major signal transduction pathways in bacteria and have emerged as potential targets for antibacterial drugs. Among the 13 pairs of TCSs proteins presenting in S. pneumoniae, VicR/K is the unique one essential for bacterium growth, and block agents to which, if can be found, may be developed as effective antibiotics against S. pneumoniae infection.     

Streptococcus pneumoniae secretes a glyceraldehyde-3-phosphate dehydrogenase,which binds haemoglobin and haem

Streptococcus pneumoniae is a gram positive encapsulated bacterium responsible of septicaemia and upper respiratory infections in children. This pathogen requires iron to survive in the host, which it can obtain of haemoglobin (Hb) or haem. Only two Hb-binding membrane proteins have been identified up to now. However it is unknown whether this pathogen secretes proteins in order to scavenge iron from the Hb or haem. Therefore, in order to explore these possibilities, cellular growth of S. pneumoniae was tested with several alternative iron supplies. The bacterial growth was supported with iron, Hb and haem. Additionally, S. pneumoniae expressed and secreted a protein of 38 kDa which was purified and characterized as Hb and haem-binding protein. This protein was also identified by mass spectrometry as glyceraldehyde-3-phosphate dehydrogenase. Our overall results suggest that S. pneumoniae secretes a protein capable of binding two usefull iron sources for this bacterium (Hb and haem). This protein could be playing a dynamic role in the success of the invasive and infective processes of this pathogen.     

Analysis of Structure and Function of Putative Surface-Exposed Proteins Encoded in the Streptococcus pneumoniae Genome: A Bioinformatics-Based Approach to Vaccine and Drug Design

Streptococcus pneumoniae is the most common cause of fatal community-acquired pneumonia, middle ear infection, and meningitis. The prevention and treatment of this infection have become a top priority for the medical-scientific community. The present polysaccharide-based vaccine used to immunize susceptible hosts is only ～60% effective and is ineffective in children younger than 2 years of age. The new conjugate vaccine, based on the engineered diphtheria toxin coupled to polysaccharide antigens, is approved only for use in children under 2 years of age to treat invasive disease. While penicillin is the drug of choice to treat infections secondary to S. pneumoniae, increasing numbers of bacterial strains are resistant to penicillin as well as to broad spectrum antibiotics such as vancomycin. Thus, there is a need to identify new strategies to prevent and treat diseases caused by to S. pneumoniae.

In this article, we summarize the utilization of the recently available S. pneumoniae genomic information in order to identify and characterize novel proteins likely located on the surface of this Gram-positive pathogenic bacterium. Because only a limited number of surface proteins of S. pneumoniae have been characterized to date, this information provides new insights into the pathogenesis of this organism as well as highlights possible avenues for its treatment and/or prevention in the future. The review is divided into two sections.

First, we briefly summarize current information about known surface-exposed proteins of S. pneumoniae. This is followed by the illustration of procedures for the identification of new putative surface-exposed proteins. These have signal peptides required for their extra-cytoplasmic transport and/or additional signature sequences. Some of these will be S. pneumoniae virulence factors. The signature sequences we have chosen are those leading to protein binding to choline present on the bacterial surface, attachment to peptidoglycan of the cell wall, or anchoring to lipids of the cytoplasmic membrane. All these signatures are indicative of binding of proteins to the surface of this organism.

Secondly, we illustrate the application of bioinformatics and modeling tools to these selected proteins in order to provide information about their likely functions and preliminary three-dimensional structure models. The focal point of the analysis of these proteins, their sequences, and structures is the evaluation of their antigenic properties and possible roles in pathogenicity. The information obtained from the genome analysis will be instrumental in the development of a more effective prophylactic and/or therapeutic agents to prevent and to treat infections due to S. pneumoniae.     

Role of hydrogen generation by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in the oral cavity

Some gastrointestinal bacteria synthesize hydrogen (H₂) by fermentation. Despite the presence of bactericidal factors in human saliva, a large number of bacteria also live in the oral cavity. It has never been shown that oral bacteria also produce H₂ or what role H₂ might play in the oral cavity. It was found that a significant amount of H₂ is synthesized in the oral cavity of healthy human subjects, and that its generation is enhanced by the presence of glucose but inhibited by either teeth brushing or sterilization with povidone iodine. These observations suggest the presence of H₂-generating bacteria in the oral cavity. The screening of commensal bacteria in the oral cavity revealed that a variety of anaerobic bacteria generate H₂. Among them, Klebsiella pneumoniae (K. pneumoniae) generated significantly large amounts of H₂ in the presence of glucose. Biochemical analysis revealed that various proteins in K. pneumoniae are carbonylated under standard culture conditions, and that oxidative stress induced by the presence of Fe⁺⁺ and H₂O₂ increases the number of carbonylated proteins, particularly when their hydrogenase activity is inhibited by KCN. Inhibition of H₂ generation markedly suppresses the growth of K. pneumoniae. These observations suggest that H₂ generation and/or the reduction of oxidative stress is important for the survival and growth of K. pneumoniae in the oral cavity.     

Structural characterization of the PTS IIA and IIB proteins associated with pneumococcal fucose utilization

Streptococcus pneumoniae harbors a significant number of transporters, including phosphotransferase (PTS) systems, allowing the bacterium to utilize a number of different carbohydrates for metabolic and other purposes. The genes encoding for one PTS transport system in particular (EII^fuc) are found within a fucose utilization operon in S. pneumoniae TIGR4. Here, we report the three‐dimensional structures of IIA^fuc and IIB^fuc providing evidence that this PTS system belongs to the EII^man family. Additionally, the predicted metabolic pathway for this distinctive fucose utilization system suggests that EII^fuc transports the H‐disaccharide blood group antigen, which would represent a novel PTS transporter specificity. Proteins 2017; 85:963–968. © 2016 Wiley Periodicals, Inc.     

Acclimation of the photosynthetic apparatus and alterations in sugar metabolism in response to inoculation with endophytic fungi

The role of an endophytic Zygomycete Mucor sp. in growth promotion and adaptation of the photosynthetic apparatus to increased energy demands of its hosts Arabidopsis arenosa and Arabidopsis thaliana was evaluated. Inoculation with the fungus improved the water use efficiency of the plants and allowed for them to utilize incident light for photochemistry more effectively by upregulating the expression of several photosystem I‐ and II‐related genes and their respective proteins, proteins involved in light harvesting in PSII and PSI and carbon assimilation. This effect was independent of the ability of the plants to acquire nutrients from the soil. We hypothesize that the accelerated growth of the symbiotic plants resulted from an increase in their demand for carbohydrates and carbohydrate turnover (sink strength) that triggered a simultaneous upregulation of carbon assimilation. Arabidopsis plants inoculated with Mucor sp. exhibited upregulated expression in several genes encoding proteins involved in carbohydrate catabolism, sugar transport, and smaller starch grains that indicate a significant upregulation of carbohydrate metabolism.     

肺炎链球菌与宿主炎症小体的相互作用机制

肺炎链球菌(Streptococcus pneumoniae)是一种定植于上呼吸道的革兰阳性胞外菌,是导致侵袭性肺炎的主要原因,所致疾病具有较高的发病率和死亡率。炎症小体(inflammasome)是胞浆内重要的蛋白复合体,在先天免疫应答过程中起着重要作用。大量研究表明,肺炎链球菌感染可诱导宿主炎症小体的激活、半胱天冬酶1的活化和促炎性细胞因子的分泌。在长期选择压力的作用下,肺炎链球菌的部分突变菌株可以逃避炎症小体的识别。本文就肺炎链球菌感染过程中炎症小体的激活、炎症小体在抗肺炎链球菌过程中的作用以及肺炎链球菌逃避宿主炎症小体识别的机制三方面对肺炎链球菌与炎症小体之间相互作用的研究进展进行综述。     

A rhesus macaque model of Streptococcus pneumoniae carriage

Background Nasopharyngeal colonization by Streptococcus pneumoniae precedes pneumococcal disease. Elucidation of procedures to prevent or eradicate nasopharyngeal carriage in a model akin to the human would help to diminish the incidence of both pneumonia and invasive pneumococcal disease. Methods We conducted a survey of the nasopharynx of infant rhesus macaques from our breeding colony, in search of natural carriers of S. pneumoniae. We also attempted experimental induction of colonization, by nasopharyngeal instillation of a human S. pneumoniae strain (19F). Results None of 158 colony animals surveyed carried S. pneumoniae in the nasopharynx. Colonization was induced in eight of eight infant rhesus by nasopharyngeal instillation and lasted 2 weeks in 100% of the animals and 7 weeks in more than 60%. Conclusion Rhesus macaques are probably not natural carriers of S. pneumoniae. The high rate and duration of colonization obtained in our experiments indicates that the rhesus macaque will serve as a human‐like carriage model.     

MATE transporter GFD1 cooperates with sugar transporters,mediates carbohydrate partitioning and controls grain-filling duration,grain size and number in rice

More than half of the world's food is provided by cereals, as humans obtain >60% of daily calories from grains. Producing more carbohydrates is always the final target of crop cultivation. The carbohydrate partitioning pathway directly affects grain yield, but the molecular mechanisms and biological functions are poorly understood, including rice (Oryza sativa L.), one of the most important food sources. Here, we reported a prolonged grain filling duration mutant 1 (gfd1), exhibiting a long grain-filling duration, less grain number per panicle and bigger grain size without changing grain weight. Map-based cloning and molecular biological analyses revealed that GFD1 encoded a MATE transporter and expressed high in vascular tissues of the stem, spikelet hulls and rachilla, but low in the leaf, controlling carbohydrate partitioning in the stem and grain but not in the leaf. GFD1 protein was partially localized on the plasma membrane and in the Golgi apparatus, and was finally verified to interact with two sugar transporters, OsSWEET4 and OsSUT2. Genetic analyses showed that GFD1 might control grain-filling duration through OsSWEET4, adjust grain size with OsSUT2 and synergistically modulate grain number per panicle with both OsSUT2 and OsSWEET4. Together, our work proved that the three transporters, which are all initially classified in the major facilitator superfamily family, could control starch storage in both the primary sink (grain) and temporary sink (stem), and affect carbohydrate partitioning in the whole plant through physical interaction, giving a new vision of sugar transporter interactome and providing a tool for rice yield improvement.     

Wogonin attenuates the pathogenicity of Streptococcus pneumoniae by double-target inhibition of Pneumolysin and Sortase A

Streptococcus pneumoniae (S. pneumoniae) is a major causative agent of respiratory disease in patients and can cause respiratory distress and other symptoms in severe cases. Pneumolysin (PLY) is a pore-forming toxin that induces host tissue injury and inflammatory responses. Sortase A (SrtA), a catalytic enzyme that anchors surface-associated virulence factors, is critical for S. pneumoniae virulence. Here, we found that the active ingredient of the Chinese herb Scutellaria baicalensis, wogonin, simultaneously inhibited the haemolytic activity of PLY and SrtA activity. Consequently, wogonin decreased PLY-mediated cell damage and reduced SrtA-mediated biofilm formation by S. pneumoniae. Furthermore, our data indicated that wogonin did not affect PLY expression but directly altered its oligomerization, leading to reduced activity. Furthermore, the analysis of a mouse pneumonia model further revealed that wogonin reduced mortality in mice infected with S. pneumoniae laboratory strain D39 and S. pneumoniae clinical isolate E1, reduced the number of colony-forming units in infected mice and decreased the W/D ratio and levels of the inflammatory factors TNF-α, IL-6 and IL-1β in the lungs of infected mice. Thus, wogonin reduces S. pneumoniae pathogenicity by inhibiting the dual targets PLY and SrtA, providing a treatment option for S. pneumoniae infection.     

Protein expression analysis ofChlamydia pneumoniae persistence by combined surface-enhanced laser desorption ionization time-of-flight mass spectrometry and two-dimensional polyacrylamide gel electrophoresis

The aim of this study was to examine the protein expression profiles of persistentChlamydia pneumoniae by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Although 2D PAGE is still the method of choice for separating and detecting components of complex protein mixtures, it has several distinct disadvantages; i.e., being labor-intensive and having a bias toward proteins within the dynamic range of the gel condition. Hence, SELDI-TOF-MS technology was used to complement 2D PAGE.C. pneumoniae-infected HEp2 cells were treated with or without IFN-γ, and protein expression profiles were determined at 48 h postinfection (hpi). Unfractionated monolayers were also used for protein profiling by SELDI-TOF, using two different chip surface types: weak cation exchanger and hydrophobic surface. Under IFN-γ-induced persistence,C. pneumoniae expresses an altered protein expression profile. Twenty chlamydial proteins showed differential regulatory patterns by SELDI-TOF-MS, two of which, HSP-70 cofactor, and a hypothetical protein, were identified by 2D PAGE and mass spectrometry. Two additional proteins, phosphatidylserine decarboxylase and 30S ribosomal protein S17, were exclusively identified by SELDI TOF-MS analysis, as these were not present in sufficient quantity for detection by 2D PAGE. We propose that a combination of 2D-PAGE and SELDI-TOF-MS may complement the disadvantages of each technique alone and may provide a rapid and precise screening technique.     

In silico synteny based comparative genomics approach for identification and characterization of novel therapeutic targets in Chlamydophila pneumoniae

Chlamydophila pneumoniae is one of the most important and well studied gram negative bacterial strain with respect to community acquired pneumonia and other respiratory diseases like Chronic obstructive pulmonary disease (COPD), Chronic asthma, Alzheimer''s disease, Atherosclerosis and Multisclerosis which have a great potential to infect humans and many other mammals. According to WHO prediction, COPD is to become the third leading cause of death by 2030. Unfortunately, the molecular mechanisms leading to chronic infections are poorly understood and the difficulty in culturing C pneumoniae in experimental conditions and lack of entirely satisfactory serological methods for diagnosis is also a hurdle for drug discovery and development. We have performed an insilico synteny based comparative genomics analysis of C pneumoniae and other eight Chlamydial organisms to know the potential of C pneumoniae which cause COPD but other Chlamydial organisms lack in potential to cause COPD though some are involved in human pathogenesis. We have identified total 354 protein sequences as non-orthologous to other Chlamydial organisms, except hypothetical proteins 70 were found functional out of which 60 are non homologous to Homo sapiens proteome and among them 18 protein sequences are found to be essential for survival of the C pneumoniae based on BLASTP search against DEG database of essential genes. CELLO analysis results showed that about 80% proteins are found to be cytoplasmic, Among which 5 were found as bacterial exotoxins and 2 as bacterial endotoxins, remaining 11 proteins were found to be involved in DNA binding, RNA binding, catalytic activity, ATP binding, oxidoreductase activity, hydrolase activity and proteolysis activity. It is expected that our data will facilitate selection of C pneumoniae proteins for successful entry into drug design pipelines.