Pan-serotype diagnostic for foot-and-mouth disease using the consensus antigen of nonstructural protein 3B

An amino acid consensus sequence for the seven serotypes of foot-and-mouth disease virus (FMDV) nonstructural protein 3B, including all three contiguous repeats, and its use in the development of a pan-serotype diagnostic test for all seven FMDV serotypes are described. The amino acid consensus sequence of the 3B protein was determined from a multiple-sequence alignment of 125 sequences of 3B. The consensus 3B (c3B) protein was expressed as a soluble recombinant fusion protein with maltose-binding protein (MBP) using a bacterial expression system and was affinity purified using amylose resin. The MBP-c3B protein was used as the antigen in the development of a competition enzyme-linked immunosorbent assay (cELISA) for detection of anti-3B antibodies in bovine sera. The comparative diagnostic sensitivity and specificity at 47% inhibition were estimated to be 87.22% and 93.15%, respectively. Reactivity of c3B with bovine sera representing the seven FMDV serotypes demonstrated the pan-serotype diagnostic capability of this bioreagent. The consensus antigen and competition ELISA are described here as candidates for a pan-serotype diagnostic test for FMDV infection.

Bacterial lessons on organelle division

Mitochondria and chloroplasts arose from bacterial endosymbionts about a billion years ago. This ancestry is now showing us how these organelles divide in modem cells.

Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts – organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms – one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.

Evidence for a common role for the serine-type Plasmodium falciparum serine repeat antigen proteases : implications for vaccine and drug design

Serine repeat antigens (SERAs) are a family of secreted “cysteine-like” proteases of Plasmodium parasites. Several SERAs possess an atypical active-site serine residue in place of the canonical cysteine. The human malaria parasite Plasmodium falciparum possesses six “serine-type” (SERA1 to SERA5 and SERA9) and three “cysteine-type” (SERA6 to SERA8) SERAs. Here, we investigate the importance of the serine-type SERAs to blood-stage parasite development and examine the extent of functional redundancy among this group. We attempted to knock out the four P. falciparum serine-type SERA genes that have not been disrupted previously. SERA1, SERA4, and SERA9 knockout lines were generated, while only SERA5, the most strongly expressed member of the SERA family, remained refractory to genetic deletion. Interestingly, we discovered that while SERA4-null parasites completed the blood-stage cycle normally, they exhibited a twofold increase in the level of SERA5 mRNA. The inability to disrupt SERA5 and the apparent compensatory increase in SERA5 expression in response to the deletion of SERA4 provides evidence for an important blood-stage function for the serine-type SERAs and supports the notion of functional redundancy among this group. Such redundancy is consistent with our phylogenetic analysis, which reveals a monophyletic grouping of the serine-type SERAs across the genus Plasmodium and a predominance of postspeciation expansion. While SERA5 is to some extent further validated as a target for vaccine and drug development, our data suggest that the expression level of other serine-type SERAs is the only barrier to escape from anti-SERA5-specific interventions.

Nucleic acid vaccines: tasks and tactics

There are no adequate vaccines against some of the new or reemerged infectious scourges such as HIV and TB. They may require strong and enduring cell-mediated immunity to be elicited. This is quite a task, as the only known basis of protection by current commercial vaccines is antibody. As DNA or RNA vaccines may induce both cell-mediated and humoral immunity, great interest has been shown in them. However, doubt remains whether their efficacy will suffice for their clinical realization. We look at the various tactics to increase the potency of nucleic acid vaccines and divided them broadly under those affecting delivery and those affecting immune induction. For delivery, we have considered ways of improving uptake and the use of bacterial, replicon or viral vectors. For immune induction, we considered aspects of immunostimulatory CpG motifs, coinjection of cytokines or costimulators and alterations of the antigen, its cellular localization and its anatomical localization including the use of ligand-targeting to lymphoid tissue. We also thought that mucosal application of DNA deserved a separate section. In this review, we have taken the liberty to discuss these enhancement methods, whenever possible, in the context of the underlying mechanisms that might argue for or against these strategies.

Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ

The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.

Changes in bacterial concentration in the liver correlate with that in the hepaticojejunostomy after bile duct reconstruction: implication in the pathogenesis of postoperative cholangitis

Postoperative cholangitis is a frequent and unpredictable complication of unknown etiology following bile duct reconstruction (BDR), particularly for biliary atresia. This study was undertaken to correlate the growth of bacteria in the hepaticojejunostomy with that in the liver after BDR. Quantitative bacterial culture was done on the specimens taken from the liver and from the hepaticojejunostomy at 1 week (group 1, n = 7), 1 month (group 2, n = 7), and 2 months (group 3, n = 7) following BDR with Roux-en-Y hepaticojejunostomy in piglets after 2 weeks of common bile duct ligation. The histological examination of the liver and the hepaticojejunostomy, as well as serial monitoring of hemogram and liver function tests, were performed to correlate the findings with the bacterial concentration of the liver and the hepaticojejunostomy following BDR. The bacterial concentration of the hepaticojejunostomy, expressed as log10 colony-forming units per gram (log10 CFU/g) of the hepaticojejunostomy, showed a progressive decrease from 8.38 ± 1.36 in group 1, 7.07 ± 2.54 in group 2, to 3.56 ± 1.31 in group 3 (p = 0.001). The log10 CFU/g of the liver also showed a progressive decrease from 5.02 ± 1.59 in group 1, 3.16 ± 1.56 in group 2, to 2.19 ± 1.09 in group 3 (p = 0.006). There was a significant positive correlation of the log10 CFU/g of the liver (n = 21) with that of the hepaticojejunostomy (n = 21) following BDR (r = 0.600, p = 0.004). Most of the infectious pathogens isolated from the liver were also isolated from the hepaticojejunostomy. The changes in hemoglobin, bilirubin, albumin, and ammonia significantly correlated with the changes of the bacterial concentration of the liver. The results of the study suggests that hepatic bacterial proliferation after BDR is significantly affected by microbial overgrowth in the bilioenteric anastomosis and is associated with deteriorated liver function and hemogram.

Suppression by abscisic acid of lignin production and monolignol pathway gene expression in interactions of Arabidopsis with oomycete and bacterial pathogens

The phytohormone, abscisic acid (ABA) has been shown to influence the outcome of the interactions between various hosts with biotrophic and hemibiotrophic pathogens. Susceptibility to avirulent isolates can be induced in plants by addition of low physiological concentrations of ABA. In contrast, addition of ABA biosynthesis inhibitors induced resistance following challenge of plants by virulent isolates. ABA deficient mutants of Arabidopsis, such as aba1-1, were resistant to virulent isolates of Peronospora parasitica. In interactions of Arabidopsis with avirulent isolates of Pseudomonas syringae pv. tomato, susceptibility was induced following addition of ABA or imposition of drought stress. These results indicate a pivotal, albiet undefined, role for ABA in determining either susceptibility or resistance to pathogen attack. We have found that the production of the cell wall strengthening compound, lignin, is increased during resistant interactions of aba1-1 but suppressed in ABA-induced susceptible interactions. Using RT-PCR and microarray analysis we have found down-regulation by ABA of key genes of the phenylpropanoid pathway especially of those genes involved directly in lignin biosynthesis. ABA also down-regulates a number of genes in other functional classes including those involved in defence and cell signalling.

Water quality, bacterial flora and production in shrimp farms in Sri Lanka

This study investigated the possibilities of improvement in the brackish water shrimp culture industry in Sri Lanka. Feeding rates could be further reduced without negative effect on shrimp growth while improving effluent water quality. Improvements of feed quality and pond management practices were also suggested.

A new rodent model to assess blood stage immunity to the plasmodium falciparum antigen merozoite surface protein 119 reveals a protective role for invasion inhibitory antibodies

Antibodies capable of inhibiting the invasion of Plasmodium merozoites into erythrocytes are present in individuals that are clinically immune to the malaria parasite. Those targeting the 19-kD COOH-terminal domain of the major merozoite surface protein (MSP)-119 are a major component of this inhibitory activity. However, it has been difficult to assess the overall relevance of such antibodies to antiparasite immunity. Here we use an allelic replacement approach to generate a rodent malaria parasite (Plasmodium berghei) that expresses a human malaria (Plasmodium falciparum) form of MSP-119. We show that mice made semi-immune to this parasite line generate high levels of merozoite inhibitory antibodies that are specific for P. falciparum MSP-119. Importantly, protection from homologous blood stage challenge in these mice correlated with levels of P. falciparum MSP-119–specific inhibitory antibodies, but not with titres of total MSP-119–specific immunoglobulins. We conclude that merozoite inhibitory antibodies generated in response to infection can play a significant role in suppressing parasitemia in vivo. This study provides a strong impetus for the development of blood stage vaccines designed to generate invasion inhibitory antibodies and offers a new animal model to trial P. falciparum MSP-119 vaccines.

Systemic activation of dendritic cells by toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity

The mechanisms responsible for the immunosuppression associated with sepsis or some chronic blood infections remain poorly understood. Here we show that infection with a malaria parasite (Plasmodium berghei) or simple systemic exposure to bacterial or viral Toll-like receptor ligands inhibited cross-priming. Reduced cross-priming was a consequence of downregulation of cross-presentation by activated dendritic cells due to systemic activation that did not otherwise globally inhibit T cell proliferation. Although activated dendritic cells retained their capacity to present viral antigens via the endogenous major histocompatibility complex class I processing pathway, antiviral responses were greatly impaired in mice exposed to Toll-like receptor ligands. This is consistent with a key function for cross-presentation in antiviral immunity and helps explain the immunosuppressive effects of systemic infection. Moreover, inhibition of cross-presentation was overcome by injection of dendritic cells bearing antigen, which provides a new strategy for generating immunity during immunosuppressive blood infections.

Immobilization of bacterial recombinant proteins

Recombinant α-L Rhamnosidase has several potential applications in citrus fruit juice processing industries. Immobilized recombinant α-L Rhamnosidase further provides an added advantage to this industrially important enzyme. Various techniques have been used to immobilize native rhamnosidase from fungal origin and applications were explored in great details by several workers. (Puri et al., 1996, 2000, 2001)

A recombinant rhamnosidase from a bacterial source was expressed in E.coli has been immobilized in calcium alginate beads (entrapment method). A batch bioreactor was created for the hydrolysis of naringin using immobilized recombinant α-L Rhamnosidase under shaking and stationary conditions and it was found to hydrolyze naringin effectively. The system was efficient to hydrolyze narigin under shaking conditions and was operationally stable up to 9 days. A high percent hydrolysis of naringin was achieved at pH 7.5 and 60˚C by immobilized rhamnosidase. Entrapped rhamnosidase was able to hydrolyze naringin content in kinnow juice repeatedly and this feature makes this technique economically suitable for debittering of fruit juices.

Insights into bacterial genome composition through variable target GC content profiling

This study presents a new computational method for guanine (G) and cytosine (C), or GC, content profiling based on the idea of multiple resolution sampling (MRS). The benefit of our new approach over existing techniques follows from its ability to locate significant regions without prior knowledge of the sequence, nor the features being sought. The use of MRS has provided novel insights into bacterial genome composition. Key findings include those that are related to the core composition of bacterial genomes, to the identification of large genomic islands (in Enterobacterial genomes), and to the identification of surface protein determinants in human pathogenic organisms (e.g., Staphylococcus genomes). We observed that bacterial surface binding proteins maintain abnormal GC content, potentially pointing to a viral origin. This study has demonstrated that GC content holds a high informational worth and hints at many underlying evolutionary processes. For online Supplementary Material, see www.liebertonline.com.

Bacterial genomic G + C composition-eliciting environmental adaptation

Bacterial genomes reflect their adaptation strategies through nucleotide usage trends found in their chromosome composition. Bacteria, unlike eukaryotes contain a wide range of genomic G + C. This wide variability may be viewed as a response to environmental adaptation. Two overarching trends are observed across bacterial genomes, the first, correlates genomic G + C to environmental niches and lifestyle, while the other utilizees intra-genomic G + C incongruence to delineate horizontally transferred material. In this review, we focus on the influence of several properties including biochemical, genetic flows, selection biases, and the biochemical-energetic properties shaping genome composition. Outcomes indicate a trend toward high G + C and larger genomes in free-living organisms, as a result of more complex and varied environments (higher chance for horizontal gene transfer). Conversely, nutrient limiting and nutrient poor environments dictate smaller genomes of low GC in attempts to conserve replication expense. Varied processes including translesion repair mechanisms, phage insertion and cytosine degradation has been shown to introduce higher AT in genomic sequences. We conclude the review with an analysis of current bioinformatics tools seeking to elicit compositional variances and highlight the practical implications when using such techniques.

DIOXOLANE NORBORNANE / NORBORNENE COMPOUNDS SUITABLE AS ANTIMICROBIAL AGENTS TO TREAT BACTERIAL INFECTIONS

The invention provides a compound including : A core having a first face and a second face; A binding portion attached to the first face of the core, wherein the binding portion is capable of binding to an anionic group present in a cell membrane of a microorganism; and A hydrophobic portion attached to the second face of the core, wherein the hydrophobic portion is capable of interacting with the cell membrane of the microorganism; and The core comprises a dioxolane norbornane / norbornene of formula (II): Or a salt or ion thereof, wherein R' is a moiety forming part of a hydrophobic portion; R2 is a first binding portion; and R3 is a seconding binding portion. The invention also provides compositions including at least one such compound. The invention also provides methods and uses for treatment or prophylaxis of infection of a mammal by a microorganism, and methods and uses for treating or preventing contamination of a substrate by a microorganism, using the compounds and compositions.