Krishnan Sankaran

A Database of Bacterial Lipoproteins (DOLOP) with Functional Assignments to Predicted Lipoproteins

Lipid modification of the N-terminal Cys residue (N-acyl-S-diacylglyceryl-Cys) has been found to be an essential, ubiquitous, and unique bacterial posttranslational modification. Such a modification allows anchoring of even highly hydrophilic proteins to the membrane which carry out a variety of functions important for bacteria, including pathogenesis. Hence, being able to identify such proteins is of great value. To this end, we have created a comprehensive database of bacterial lipoproteins, called DOLOP, which contains information and links to molecular details for about 278 distinct lipoproteins and predicted lipoproteins from 234 completely sequenced bacterial genomes. The website also features a tool that applies a predictive algorithm to identify the presence or absence of the lipoprotein signal sequence in a user-given sequence. The experimentally verified lipoproteins have been classified into different functional classes and more importantly functional domain assignments using hidden Markov models from the SUPERFAMILY database that have been provided for the predicted lipoproteins. Other features include the following: primary sequence analysis, signal sequence analysis, and search facility and information exchange facility to allow researchers to exchange results on newly characterized lipoproteins. The website, along with additional information on the biosynthetic pathway, statistics on predicted lipoproteins, and related figures, is available at http://www.mrc-lmb.cam.ac.uk/genomes/dolop/.

DOLOP-database of bacterial lipoproteins

UNLABELLED Bacterial lipoproteins and lipid modification are gaining importance owing to their essential nature, roles in pathogenesis and interesting commercial applications. We have created an exclusive knowledge base for bacterial lipoproteins by processing information from 510 entries to provide a list of 199 distinct lipoproteins with relevant links to molecular details. Features include functional classification, predictive algorithm for query sequences, primary sequence analysis and lists of predicted lipoproteins from 43 completed bacterial genomes along with interactive information exchange facility. AVAILABILITY The website called Database Of bacterial LipOProteins (DOLOP) is available at http://www.mrc-lmb.cam.ac.uk/genomes/dolop along with additional information on the biosynthetic pathway, supplementary material and other related figures.

[49] Modification of bacterial lipoproteins

Publisher Summary This chapter summarizes current knowledge of the lipid modification enzymes in the pathway for the maturation of bacterial lipoproteins. Lipid modification of bacterial lipoproteins is catalyzed by three enzymes: diacylglyceryltransferase, signal peptidase II, and N -acyltransferase. Major phospholipids in the bacterial cell envelope serve as both the diacylglyceryl and acyl donors. Phosphatidylglycerol (PG) is the major diacylglyceryl donor for diacylglyceryltransferase, whereas N -acyltransferase does not exhibit a preference for any particular acyl donor. The ability of Tricine- sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) to separate the various intermediates in the biosynthesis of murein lipoprotein is exploited to develop assays for diacylglyceryl modification of prolipoprotein and for N -acylation of apolipoprotein. A simpler and quicker peptide-based assay for diacylglyceryltransferase is available and a similar one for N -acyltransferase is required for its purification. The availability of the genes for the enzymes should enable hyper expression and the development of simple purification protocols. The specificity toward PG in vivo is demonstrated in vitro by delipidating the inverted vesicles with aqueous acetone extraction followed by incubation of the delipidated enzyme preparations with individual phospholipid species.

Structure-Function Relationship Studies on the Frog Skin Antimicrobial Peptide Tigerinin 1: Design of Analogs with Improved Activity and Their Action on Clinical Bacterial Isolates

ABSTRACT Structure-function relationships in antimicrobial peptides have been extensively investigated in order to obtain improved analogs. Most of these studies have targeted either α-helical peptides or β-sheet peptides with multiple disulfide bridges. Tigerinins are short, nonhelical antimicrobial peptides with a single disulfide bridge. In this study, we have synthesized several analogs of tigerinin 1 with an aim to understand the structural basis of activity as well as improve its activity. The studies demonstrate that the loop structure of tigerinin 1 is essential for its optimal activity. However, linearization with increased cationic charges can compensate for loss of loop structure to some extent. Morphology of the cells after treatment with the active analogs shows extensive leakage of cytoplasmic contents. Tigerinin 1 and two of its analogs exhibit impressive activity against a variety of clinical bacterial isolates.

TAT-Pathway-Dependent Lipoproteins as a Niche-Based Adaptation in Prokaryotes

Bacterial lipoproteins, characterized by the N-terminal N-acyl S-diacylglyceryl Cysteine, are key membrane proteins in bacterial homeostasis. It is generally thought that during the modification lipoprotein precursors are translocated via the Sec-machinery in an unfolded state. The recent discovery of twin-arginine translocation (TAT) machinery, meant for exporting folded-proteins, and the presence of TAT-type signal sequences in co-factor-containing (hence already folded) lipoproteins, prompted us to investigate its role and significance in lipoprotein biosynthesis. We systematically analyzed 696 prokaryotic genomes using an algorithm based on DOLOP and TatP rules to predict TAT-pathway-dependent lipoprotein substrates. Occurrence of the deduced TAT-pathway-dependent lipoprotein substrates in relation to genome size, presence or absence of TAT machinery, and extent of its usage for lipoprotein export and habitat types revealed that unlike the host-obligates, the free-living prokaryotes in complex hostile environments (e.g., soil) depend more on TAT-exported lipoproteins. Functional classification of the predicted TAT-dependent lipoproteins revealed enrichment in hydrolases and oxido-reductases, which are fast-folding and co-factor-containing proteins. The role of the TAT pathway in the export of folded-lipoproteins and in niche-specific adaptation for survival has important implications not only in lipoprotein biosynthesis, but also for protein and metabolic engineering applications.

Localization and characterization of prolipoprotein diacylglyceryl transferase (Lgt) critical in bacterial lipoprotein biosynthesis.

Lipid modification of proteins is an essential post-translational event that can be targeted for protein engineering and pharmaceutical applications. In this regard, the unique and ubiquitous bacterial N-terminal lipid modification (N-acyl S-diacylglyceryl modification of N-terminal cysteine) is particularly attractive. It is initiated by phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) and therefore its properties, which remain uninvestigated, largely define the specifics of the modification. A synthetic peptide-substrate (MKATKSAVGSTLAGCSSHHHHHH) with a short hydrophilic h-region, unlike that of the prototypical substrate used so far, demonstrated lack of enzymes substrate preference based on hydrophobicity, perhaps accounting for a significant number of lipoproteins possessing hydrophilic signal peptides. Solubilization experiments revealed a peripheral and possibly reversible hydrophobic association of Lgt with the inner-membrane on the cytosolic side contradicting its deduced transmembrane topology. Except for heat stability, the soluble enzyme was indistinguishable from the membrane-bound enzyme in kinetic behaviour, indicating that the committed first step of bacterial lipid modification may be aqueous compatible. The direct, more accurate, precise and easier paper electrophoretic assay, designed anew, and Lgts ready extraction with water or low ionic strength solutions from inverted vesicles could aid better understanding and exploitation of the enzyme and bacterial lipid modification.

Shigella apyrase ^ a novel variant of bacterial acid phosphatases?

A virulence‐associated ATP diphosphohydrolase activity in the periplasm of Shigella, identified as apyrase, was found to be markedly similar to bacterial non‐specific acid phosphatases in primary structure. When the Shigella apyrase sequence was threaded in to the recently published 3D structure of the highly similar (73%) Escherichia blattae acid phosphatase it was found to have a highly overlapping 3D structure. Our analysis, which included assays for phosphatase, haloperoxidase and catalase activities, led us to hypothesize that Shigella apyrase might belong to a new class of pyrophosphatase originating as one more variant in the family of bacterial non‐specific acid phosphatases. It revealed interesting structure–function relationships and probable roles relevant to pathogenesis.

Cytopathic effects of outer-membrane preparations of enteropathogenic Escherichia coli and co-expression of maltoporin with secretory virulence factor, EspB

Enteropathogenic Escherichia coli (EPEC) is an important aetiological agent of persistent infantile diarrhoea. EPEC pathogenicity is not mediated through known toxins and the role played by outer-membrane proteins (OMPs) in the initial adherence of the bacterium, intimate attachment to epithelial cells and ultimately in the effacement of the intestinal epithelium is being pursued vigorously. In this study of the different cellular fractions of the bacterium investigated, only the outer-membrane fraction was able to disrupt HEp-2 cells. The outer-membrane fraction was also found to be cytotoxic and caused actin accumulation around the periphery of the host cells. To understand the role of OMPs in pathogenesis, protein profiles of outer-membrane preparations of wild-type and attenuated mutants lacking either the EPEC adherence factor (EAF) mega-plasmid or EPEC attaching and effacing gene A (eaeA) coding for a 94-kDa OMP, intimin or EPEC secretory protein gene B (espB) coding for a 34-kDa translocated signal transducing protein were compared and correlated with their cytopathic effects. A 43-kDa protein seen along with intimin in the outer membrane of EPEC was identified as maltoporin, an E. coli outer-membrane porin normally expressed only in response to maltose in the growth medium. In the case of EPEC, not only was this regulation lost, but also the expression of maltoporin was found to be tightly coupled to the expression of the secretory virulence factor EspB. Maltoporin per se is not toxic, as evidenced by the treatment of HEp-2 cells with the outer-membrane preparation of E. coli DH5a grown in the presence of maltose and the significance of this pathogenic adaptation is not clear. However, when maltoporin and possibly other unidentified proteins were not present as a component of the outer-membrane preparation, as in the outer-membrane preparation of an espB-negative strain, cellular disruption as well as actin accumulation proceeded at a very slow rate even though the cytotoxic effects were comparable to those of the wild-type EPEC strains.

An electrochemical immunosensor for efficient detection of uropathogenic E. coli based on thionine dye immobilized chitosan/functionalized-MWCNT modified electrode

Uropathogenic Escherichia coli (UPEC) is the major cause of 150 million Urinary Tract Infections (UTI) reported annually world-wide. High prevalence of multi-drug-resistance makes it dangerous and difficult to cure. Therefore simple, quick and early diagnostic tools are essential for effective treatment and control. We report an electrochemical immunosensor based on thionine dye (Th) immobilized on functionalized-multiwalled carbon nanotube+chitosan composite coated on glassy carbon electrode (GCE/f-MWCNT-Chit@Th) for quick and sensitive detection of UPEC in aqueous solution. This immunosensor was constructed by sequential immobilization of UPEC, bovine serum albumin, primary antibody and Horse Radish Peroxidase (HRP) tagged secondary antibody on the surface of GCE/f-MWCNT-Chit@Th. When analyzed using 2.5mM of hydrogen peroxide reduction reaction using cyclic voltammetry in phosphate buffer, pH 7.0, the immunosensor showed excellent linearity in a range of 10(2)-10(9)cfu of UPEC mL(-1) with a current sensitivity of 7.162μA {log(cfumL(-1))}(-1). The specificity of this immunosensor was tested using other UTI and non-UTI bacteria, Staphylococcus, Klebsiella, Proteus and Shigella. The clinical applicability of the immunosensor was also successfully tested directly in UPEC spiked urine samples (simulated sample).

Salmonid alphavirus replicon is functional in fish, mammalian and insect cells and in vivo in shrimps (Litopenaeus vannamei)

The Salmonid alphavirus (SAV) is the etiological agent of pancreas disease in Atlantic salmon (Salmo salar) and Sleeping disease in rainbow trout (Oncorhynchus mykiss). SAV differs from alphaviruses infecting terrestrial animals in that it infects salmonid fish at low temperatures and does not use an arthropod vector for transmission. In this study we have shown that a SAVbased replicon could express proteins when driven by the subgenomic promoter in vitro in cells from fish, mammals and insects, as well as in vivo in shrimps (Litopanaeus vannamei). The SAV-replicon was found to be functional at temperatures ranging from 4 to 37°C. Protein expression was slow and moderate compared to that reported from terrestrial alphavirus replicons or from vectors where protein expression was under control of the immediate early CMV-promoter. No cytopathic effect was visually observable in cells transfected with SAV-replicon vectors. Double stranded RNA was present for several days after transfection of the SAV-replicon in fish cell lines and its presence was indicated also in shrimp. The combination of prolonged dsRNA production, low toxicity, and wide temperature range for expression, may potentially be advantageous for the use of the SAV replicon to induce immune responses in aquaculture of fish and shrimp.