Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development

By combining X-ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.     

TiO2 nanoparticle assisted mass spectrometry as biosensor of Staphylococcus aureus, key pathogen in nosocomial infections from air, skin surface and human nasal passage

For the first time, we have demonstrated the use of mass spectrometry as a biosensor for detecting a clinically important bacterium: Staphylococcus aureus in air, nasal passage and skin samples using culture-free, rapid, direct analysis via TiO(2) nanoparticles (NPs) assisted MALDI-MS. When this bacterium is predominating, the nasal passage of an individual is observed to lead to wound infections especially when the individual has a surgery or some wounds. This study indicates that even at very low concentrations of an individual bacterium can be directly detected from a mixture of bacteria using the MALDI-MS analysis without the requirement of any culturing steps or any other sample pretreatment techniques. The current approach is extremely simple, rapid, straightforward and sensitive which could be widely applied for the detection of this deadly pathogen in clinical as well as environmental samples.     

The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H(2)O(2) versus L-012 and cytochrome c for O(2)(·-). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H(2)O(2) to O(2)(·-) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O(2)()-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O(2)(·-). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H(2)O(2) but increased O(2)(·-) formation by Nox4 without affecting Nox1-dependent O(2)(·-) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H(2)O(2). Mutation of this but not of four other conserved histidines also switched Nox4 from H(2)O(2) to O(2)(·-) formation. Thus, H(2)O(2) formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O(2)(·-) egress and/or provide a source for protons, allowing dismutation to form H(2)O(2).     

Activated protein C attenuates systemic lupus erythematosus and lupus nephritis in MRL-Fas(lpr) mice

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease leading to inflammatory tissue damage in multiple organs (e.g., lupus nephritis). Current treatments including steroids, antimalarials, and immunosuppressive drugs have significant side effects. Activated protein C is a natural protein with anticoagulant and immunomodulatory effects, and its recombinant version has been approved by the U.S. Food and Drug Administration to treat severe sepsis. Given the similarities between overshooting immune activation in sepsis and autoimmunity, we hypothesized that recombinant activated protein C would also suppress SLE and lupus nephritis. To test this concept, autoimmune female MRL-Fas(lpr) mice were injected with either vehicle or recombinant human activated protein C from week 14-18 of age. Activated protein C treatment significantly suppressed lupus nephritis as evidenced by decrease in activity index, glomerular IgG and complement C3 deposits, macrophage counts, as well as intrarenal IL-12 expression. Further, activated protein C attenuated cutaneous lupus and lung disease as compared with vehicle-treated MRL-Fas(lpr) mice. In addition, parameters of systemic autoimmunity, such as plasma cytokine levels of IL-12p40, IL-6, and CCL2/MCP-1, and numbers of B cells and plasma cells in spleen were suppressed by activated protein C. The latter was associated with lower total plasma IgM and IgG levels as well as lower titers of anti-dsDNA IgG and rheumatoid factor. Together, recombinant activated protein C suppresses the abnormal systemic immune activation in SLE of MRL-Fas(lpr) mice, which prevents subsequent kidney, lung, and skin disease. These results implicate that recombinant activated protein C might be useful for the treatment of human SLE.     

A model for group B Streptococcus pilus type 1: the structure of a 35-kDa C-terminal fragment of the major pilin GBS80

The Gram-positive pathogen Streptococcus agalactiae, known as group B Streptococcus (GBS), is the leading cause of bacterial septicemia, pneumonia, and meningitis among neonates. GBS assembles two types of pili—pilus islands (PIs) 1 and 2—on its surface to adhere to host cells and to initiate colonization for pathogenesis. The GBS PI-1 pilus is made of one major pilin, GBS80, which forms the pilus shaft, and two secondary pilins, GBS104 and GBS52, which are incorporated into the pilus at various places. We report here the crystal structure of the 35-kDa C-terminal fragment from GBS80, which is composed of two IgG-like domains (N2-N3). The structure was solved by single-wavelength anomalous dispersion using sodium-iodide-soaked crystals and diffraction data collected at the home source. The N2 domain exhibits a cnaA/DEv-IgG fold with two calcium-binding sites, while the N3 domain displays a cnaB/IgG-rev fold. We have built a model for full-length GBS80 (N1, N2, and N3) with the help of available homologous major pilin structures, and we propose a model for the GBS PI-1 pilus shaft. The N2 and N3 domains are arranged in tandem along the pilus shaft, whereas the respective N1 domain is tilted by approximately 20° away from the pilus axis. We have also identified a pilin-like motif in the minor pilin GBS52, which might aid its incorporation at the pilus base.     

Characterization of Arabidopsis thaliana SufE2 and SufE3: functions in chloroplast iron-sulfur cluster assembly and Nad synthesis

In this study we characterize two novel chloroplast SufE-like proteins from Arabidopsis thaliana. Other SufE-like proteins, including the previously described A. thaliana CpSufE, participate in sulfur mobilization for Fe-S biosynthesis through activation of cysteine desulfurization by NifS-like proteins. In addition to CpSufE, the Arabidopsis genome encodes two other proteins with SufE domains, SufE2 and SufE3. SufE2 has plastid targeting information. Purified recombinant SufE2 could activate the cysteine desulfurase activity of CpNifS 40-fold. SufE2 expression was flower-specific and high in pollen; we therefore hypothesize that SufE2 has a specific function in pollen Fe-S cluster biosynthesis. SufE3, also a plastid targeted protein, was expressed at low levels in all major plant organs. The mature SufE3 contains two domains, one SufE-like and one with similarity to the bacterial quinolinate synthase, NadA. Indeed SufE3 displayed both SufE activity (stimulating CpNifS cysteine desulfurase activity 70-fold) and quinolinate synthase activity. The full-length protein was shown to carry a highly oxygen-sensitive (4Fe-4S) cluster at its NadA domain, which could be reconstituted by its own SufE domain in the presence of CpNifS, cysteine and ferrous iron. Knock-out of SufE3 in Arabidopsis is embryolethal. We conclude that SufE3 is the NadA enzyme of A. thaliana, involved in a critical step during NAD biosynthesis.     

<Emphasis Type="Italic">compact shoot and leafy head 1</Emphasis>, a mutation affects leaf initiation and developmental transition in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The shoot apical meristem (SAM) produces lateral organs in a regular spacing (phyllotaxy) and at a regular interval (phyllochron) during the vegetative phase. In a Dissociation (Ds) insertion rice population, we identified a mutant, compact shoot and leafy head 1 (csl1), which produced massive number of leaves (∼70) during the vegetative phase. In csl1, the transition from the vegetative to the reproductive phase was delayed by about 2 months under long-day conditions. With a reduced leaf size and severe dwarfism, csl1 failed to produce a normal panicle after the transition to reproductive growth. Instead, it produced a leafy panicle, in which all primary rachis-branches were converted to vegetative shoots. Phenotypically csl1 resembled pla mutants in short plastochron but was more severe in the conversion of the reproductive organs to vegetative organs. In addition, neither the expression nor the coding region of PLA1 or PLA2 was affected in csl1. csl1 is most likely a dominant mutation because no mutant segregant was observed in progeny of 67 siblings of the csl1 mutant. CSL1 may represent a novel gene, which functions downstream of PLA1 and/or PLA2, or alternatively functions in a separate pathway, involved in the regulation of leaf initiation and developmental transition via plant hormones or other mobile signals.     

Development of silverleaf assay, protein and nucleic acid-based diagnostic techniques for the quick and reliable detection and monitoring of biotype B of the whitefly, Bemisia tabaci (Gennadius)

The aim of this study was to develop and optimize silverleaf bioassay, esterase analysis and PCR-based techniques to distinguish quickly and reliably biotype B of the whitefly, Bemisia tabaci (Gennadius), from Indian indigenous biotypes. Zucchini and squash readily develop silverleaf symptoms upon feeding by the B biotype, but they are not readily available in Indian markets. A local pumpkin variety 'Big' was, therefore, used in silverleaf assay, which developed symptoms similar to those on zucchini and squash and can be used reliably to detect B biotype. Analysis of non-specific esterases of B and the indigenous biotypes indicated both quantitative and qualitative differences in esterase patterns. Two high molecular weight bands were unique to B biotype and they occurred in abundance. These esterases were used to develop quick and field-based novel detection methods for differentiating B from the indigenous biotypes. Development of these simple and cost-effective protocols has wider application as they can be potentially used to identify other agricultural pests. Mitochondrial cytochrome oxidase I gene sequences and randomly amplified polymorphic DNA (RAPD) polymorphisms, generated using the primer OpB11, were also found useful for detecting B. tabaci biotypes. A B biotype-specific RAPD band of 800 bp was sequenced, which was used to a develop sequence characterized amplified region (SCAR) marker. The SCAR marker involved the development of B biotype-specific primers that amplified 550 bp PCR products only from B biotype genomic DNA. Silverleaf assay, esterases, RAPDs or a SCAR marker were used in combination to analyse whitefly samples collected from selected locations in India, and it was found that any of these techniques can be used singly or in combination to detect B biotype reliably. The B biotype was found in southern parts of India but not in the north in 2004-06.     

PPS: A computing engine to find Palindromes in all Protein sequences

The primary structure of a protein molecule comprises a linear chain of amino acid residues. Certain parts of this linear chain are unique in nature and function. They can be classified under different categories and their roles studied in detail. Two such unique categories are the palindromic sequences and the Single Amino Acid Repeats (SAARs), which plays a major role in the structure, function and evolution of the protein molecule. In spite of their presence in various protein sequences, palindromes have not yet been investigated in detail. Thus, to enable a comprehensive understanding of these sequences, a computing engine, PPS, has been developed. The users can search the occurrences of palindromes and SAARs in all the protein sequences available in various databases and can view the three-dimensional structures (in case it is available in the known three-dimensional protein structures deposited to the Protein Data Bank) using the graphics plug-in Jmol. The proposed server is the first of its kind and can be freely accessed through the World Wide Web.

Availability

URL http://pranag.physics.iisc.ernet.in/pps/