Cell surface engineering of microorganisms towards adsorption of heavy metals

Abstract

Heavy metal contamination has become a worldwide environmental concern due to its toxicity, non-degradability and food-chain bioaccumulation. Conventional physical and chemical treatment methods for heavy metal removal have disadvantages such as cost-intensiveness, incomplete removal, secondary pollution and the lack of metal specificity. Microbial biomass-based biosorption is one of the approaches gaining increasing attention because it is effective, cheap, and environmental friendly and can work well at low concentrations. To enhance the adsorption properties of microbial cells to heavy metal ions, the cell surface display of various metal-binding proteins/peptides have been performed using a cell surface engineering approach. The surface engineering of Gram-negative bacteria, Gram-positive bacteria and yeast towards the adsorption of heavy metals are reviewed in this article. The problems and future perspectives of this technology are discussed.     

Role for the fibrinogen-binding proteins Coagulase and Efb in the Staphylococcus aureus–Candida interaction

Staphylococcus aureus and Candida species are increasingly coisolated from implant-associated polymicrobial infections creating an incremental health care problem. Synergistic effects between both genera seem to facilitate the formation of mixed S. aureus–Candida biofilms, which is thought to play a critical role in coinfections with these microorganisms. To identify and characterize S. aureus factors involved in the interaction with Candida species, we affinity-panned an S. aureus phage display library against Candida biofilms in the presence or absence of fibrinogen. Repeatedly isolated clones contained DNA fragments encoding portions of the S. aureus fibrinogen-binding proteins coagulase or Efb. The coagulase binds to prothrombin in a 1:1 ratio thereby inducing a conformational change and non-proteolytic activation of prothrombin, which in turn cleaves fibrinogen to fibrin. Efb has been known to inhibit opsonization. To study the role of coagulase and Efb in the S. aureus–Candida cross-kingdom interaction, we performed flow-cytometric phagocytosis assays. Preincubation with coagulase reduced the phagocytosis of Candida yeasts by granulocytes significantly and dose-dependently. By using confocal laser scanning microscopy, we demonstrated that the coagulase mediated the formation of fibrin surrounding the candidal cells. Furthermore, the addition of Efb significantly protected the yeasts against phagocytosis by granulocytes in a dose-dependent and saturable fashion. In conclusion, the inhibition of phagocytosis of Candida cells by coagulase and Efb via two distinct mechanisms suggests that S. aureus might be beneficial for Candida to persist as it helps Candida to circumvent the host immune system.     

同伴冲突情境下学龄儿童的情绪表达

目的考察同伴冲突情境下学龄儿童情绪表达策略的特点及发展规律。方法从两所普通小学抽取三四五年级共886名被试,采用故事情境法进行考察。结果①同伴冲突情境下,学龄儿童夸大策略的使用显著高于其他3种策略(t≥3.17,P0.01),弱化策略使用最少(t≤-7.91,P0.001);②学龄儿童情绪表达策略总分的年级主效应显著(F=3.72,P0.05),性别主效应显著(F=5.80,P0.05);③在轻微冲突情境中,更多使用夸大策略(F=2.94,P=0.06);在模糊伤害情境中,更多使用平静化策略,且女生使用次数显著多于男生(F=3.46,P0.05);在故意伤害情境中,更多地使用弱化策略,男生使用次数多于女生(F=3.95,P0.05)。结论同伴冲突情境具有情境特异性,在该情境下,学龄儿童不同情绪表达策略存在性别、年级和冲突情境类型差异。     

Genetic analysis of structural proteins in the adsorption apparatus of bacteriophage epsilon 15

AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15 (E15) using genetic and biochemical methodologyMETHODS: Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under non-permissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins. These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of ³⁵S-methionine and co-purified along with E15wt phage on CsCl block gradients.RESULTS: Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive CsCl buoyant density centrifugation.CONCLUSION: The portal ring (gp4) of E15 is bound to tail spikes (gp20) and the tail tube (gp15 and gp17); gp17’s attachment requires both gp15 and gp16.     

Phage-Displayed Peptides for Targeting Tyrosine Kinase Membrane Receptors in Cancer Therapy

Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides can target in vitro and in vivo tumor cells and represent a novel therapeutic approach for cancer therapy. Further, they are more convenient compared to antibodies, being less expensive and non-immunogenic. In this review, we describe the state-of-the-art of phage display for development of peptide ligands of tyrosine kinase membrane receptors and discuss their potential applications for tumor-targeted therapy.     

Antigenicity and immunogenicity of phage library-selected peptide mimics of the major surface proteophosphoglycan antigens of Entamoeba histolytica

Entamoeba histolytica is the protozoan parasite responsible for intestinal amoebiasis and amoebic liver abscess, which cause significant morbidity and mortality in many countries of the world. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) represent dominant surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against these components protected SCID mice from amoebic liver abscess, so PPGs might be regarded as vaccine candidates; however, their structure is very complex and only known in part. They are glycosylphosphatidylinositol-linked polypeptides of unknown sequence carrying glycan side-chains linked to serine residues via phosphodiester bonds. In order to identify peptide mimics of the E. histolytica PPG antigens, we screened six different phage-displayed random peptide libraries with the antibody EH5. Various peptide mimics of different length were identified and, in all the peptides, a distinct consensus sequence Gly-Thr-His-Pro-X-Leu could be identified. The phages strongly bound to the antibody, and the natural antigen inhibited binding of the phages to antibody EH5. In addition, several of the phages induced a significant immunoglobulin G response against amoebic antigens in immunized mice.     

A Rheumatoid Factor Specific Mimotope Identified by a Peptide Display Library

We screened a 10 amino acid peptide display library in filamentous phage with B'20, a monoclonal high affinity IgM rheumatoid factor (RF) expressing the Vk111a-dependent 4C9 idiotype. Using direct and indirect selection techniques, 12 B'20 reactive peptides were identified, 9 of which belonged to one of two motifs. Binding of B'20 to phage-bearing peptides was inhibited by both IgG and 4C9 antiidiotype. Synthetic peptides corresponding to the two motifs inhibited the Fc binding of a low avidity IgA B'20 construct. Purified IgM from 6/8 RF-positive RA patients and 8/11 monoclonal RFs with Vk111-encoded light chains bound to the phage, whereas none of the four monoclonal RFs with Vk1 or Vk11 encoded light chains bound. Phage binding appeared to be RF specific. Three 4C9 positive/RF negative cell lines from RA patients did not bind to phage nor did three B'20 mutants that had lost RF specificity, whereas two mutants that retained RF specificity also retained phage binding. We propose that there is a common epitope(s) recognized by Vk11 I encoded RFs that is mimicked by the structure of these peptides. Such mimotopes might be exploited to design novel agents that interfere with autoantibody binding.     

Hierarchy and extremes in selections from pools of randomized proteins

Variation and selection are the core principles of Darwinian evolution, but quantitatively relating the diversity of a population to its capacity to respond to selection is challenging. Here, we examine this problem at a molecular level in the context of populations of partially randomized proteins selected for binding to well-defined targets. We built several minimal protein libraries, screened them in vitro by phage display, and analyzed their response to selection by high-throughput sequencing. A statistical analysis of the results reveals two main findings. First, libraries with the same sequence diversity but built around different “frameworks” typically have vastly different responses; second, the distribution of responses of the best binders in a library follows a simple scaling law. We show how an elementary probabilistic model based on extreme value theory rationalizes the latter finding. Our results have implications for designing synthetic protein libraries, estimating the density of functional biomolecules in sequence space, characterizing diversity in natural populations, and experimentally investigating evolvability (i.e., the potential for future evolution).Diversity is the fuel of evolution by natural selection, but translating this concept into quantitative measurements is not straightforward (1). A simple count of the number of different individuals in a population, for instance, fails to account for the very different responses to selection that two populations with the same number of different individuals may elicit. The problem is even acute at the molecular scale, where it also takes a very practical form: libraries of diverse proteins are routinely screened as a way to identify biomolecules of interest (binders, catalysts, etc.), and a proper “diversity” is critical for success (2, 3). However, beyond a general agreement that maximizing the number of different elements is desirable, there is no general rule for engineering and comparing diversity in these libraries.A common design of many protein libraries is to concentrate variations at one or a few variable parts located around a fixed “framework,” which is shared by all members of the library (2, 3). The natural design of antibody repertoires, the pools of immune proteins with potential to recognize nearly every molecular target, follows this pattern. Most of the sequence variations in antibodies are, indeed, concentrated at a few loops extending from a common structural scaffold (4). This architecture has inspired the conception of artificial protein libraries built on frameworks other than the Ig fold (5).Here, we present an approach to quantitatively characterize the selective potential of molecular libraries. To develop this approach, we designed and screened 24 synthetic protein libraries with identical sequence variations but different frameworks and analyzed their response to well-defined selective pressures by high-throughput sequencing. Between libraries, we find that selective potentials vary widely and define a hierarchy of frameworks. Within libraries, we find that selective potentials exhibit a simple scaling law, characterized by few parameters. The essence of these results is captured by an elementary probabilistic model based on extreme value theory (EVT).Previous work has quantified the functional potential of totally or partially random biomolecules by counting the number of positive hits resulting from successive rounds of selections and amplifications of a large sample of these biomolecules (6–11). Our results lead us to propose a different approach to characterize the selective potential of a population. Compared with previous analyses, this approach does not depend strongly on the sensitivity of the experimental assay or the number of copies in which each distinct biomolecule is present in the initial population.     

Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells. Baculovirus expressed rVP28 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that rVP28 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired rVP28 from the insect cell membrane via the budding process. Using this baculovirus displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and 73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.