Isolation and characterization of enteroaggregative,enterotoxigenic, diffusely adherent <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella</Emphasis> Worthington from human diarrhoeic faecal samples in Kashmir and Secunderabad,India

Seven hundred and thirty-five diarrhoeic faecal samples from children were investigated for presence of enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), diffusely adherent E. coli (DAEC) and Salmonella spp. by polymerase chain reaction (PCR) and bacterial culture. Out of 675 samples from Kashmir, 55 isolates were obtained, which carried at least one virulence gene studied. Out of the 55 isolates, 36 (65.45%) were EAEC, 18 (32.72%) were ETEC while only one isolate (1.81%) was DAEC. All the EAEC isolates were found to be typical as they possessed aggR gene. Six (16.66%) EAEC isolates carried the astA gene. Out of the 18 ETEC isolates, 13 carried the elt gene alone, four possessed both the elt and est genes and the remaining one harboured the est gene alone. Five ETEC isolates also possessed astA gene. Nineteen EAEC isolates belonged to 10 different serogroups. Serogroup O153 was most frequent. The ETEC isolates also belonged to 10 different serogroups of which O159 was most predominant. Out of 224 E. coli isolates from 60 samples of Secunderabad, 27 isolates carried at least one virulence gene. Out of 27 isolates 22 (81.48%) were typical EAEC, three (11.11%) were ETEC and two (7.4%) were DAEC. Fifteen EAEC isolates belonged to seven different serogroups with O86 as most frequent. Four EAEC isolates also possessed the astA gene. All the three ETEC isolates harboured elt gene only and belonged to three different serogroups. Two isolates of Salmonella Worthington were obtained from only two samples in Kashmir.     

Staphylococcus aureus Panton-Valentine Leukocidin Is a Very Potent Cytotoxic Factor for Human Neutrophils

The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.     

Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae

The EfeM protein is a component of the putative EfeUOBM iron-transporter of Pseudomonas syringae pathovar syringae and is thought to act as a periplasmic, ferrous-iron binding protein. It contains a signal peptide of 34 amino acid residues and a C-terminal ‘Peptidase_M75’ domain of 251 residues. The C-terminal domain contains a highly conserved ‘HXXE’ motif thought to act as part of a divalent cation-binding site. In this work, the gene (efeM or ‘Psyr_3370’) encoding EfeM was cloned and over-expressed in Escherichia coli, and the mature protein was purified from the periplasm. Mass spectrometry confirmed the identity of the protein (M_W 27,772 Da). Circular dichroism spectroscopy of EfeM indicated a mainly α-helical structure, consistent with bioinformatic predictions. Purified EfeM was crystallised by hanging-drop vapor diffusion to give needle-shaped crystals that diffracted to a resolution of 1.6 Å. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role.     

Glucan, water dikinase activity stimulates breakdown of starch granules by plastidial beta-amylases

Glucan phosphorylating enzymes are required for normal mobilization of starch in leaves of Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), but mechanisms underlying this dependency are unknown. Using two different activity assays, we aimed to identify starch degrading enzymes from Arabidopsis, whose activity is affected by glucan phosphorylation. Breakdown of granular starch by a protein fraction purified from leaf extracts increased approximately 2-fold if the granules were simultaneously phosphorylated by recombinant potato glucan, water dikinase (GWD). Using matrix-assisted laser-desorption ionization mass spectrometry several putative starch-related enzymes were identified in this fraction, among them beta-AMYLASE1 (BAM1; At3g23920) and ISOAMYLASE3 (ISA3; At4g09020). Experiments using purified recombinant enzymes showed that BAM1 activity with granules similarly increased under conditions of simultaneous starch phosphorylation. Purified recombinant potato ISA3 (StISA3) did not attack the granular starch significantly with or without glucan phosphorylation. However, starch breakdown by a mixture of BAM1 and StISA3 was 2 times higher than that by BAM1 alone and was further enhanced in the presence of GWD and ATP. Similar to BAM1, maltose release from granular starch by purified recombinant BAM3 (At4g17090), another plastid-localized beta-amylase isoform, increased 2- to 3-fold if the granules were simultaneously phosphorylated by GWD. BAM activity in turn strongly stimulated the GWD-catalyzed phosphorylation. The interdependence between the activities of GWD and BAMs offers an explanation for the severe starch excess phenotype of GWD-deficient mutants.     

Rapid genetic analysis in congenital hyperinsulinism

BACKGROUND: In severe, medically unresponsive congenital hyperinsulinism (CHI), the histological differentiation of focal versus diffuse disease is vital, since the surgical management is completely different. Genetic analysis may help in the differential diagnosis, as focal CHI is associated with a paternal germline ABCC8 or KCNJ11 mutation and a focal loss of maternal chromosome 11p15, whereas a maternal mutation, or homozygous/compound heterozygous ABCC8 and KCNJ11 mutations predict diffuse-type disease. However, genotyping usually takes too long to be helpful in the absence of a founder mutation. METHODS: In 4 patients, a rapid genetic analysis of the ABBC8 and KCNJ11 genes was performed within 2 weeks on request prior to the decision of pancreatic surgery. RESULTS: Two patients had no mutations, rendering the genetic analysis non-informative. Peroperative multiple biopsies showed diffuse disease. One patient had a paternal KCNJ11 mutation and focal disease confirmed by positron emission tomography scan and biopsies. One patient had a de novo heterozygous ABBC8 mutation and unexplained diffuse disease confirmed by positron emission tomography scan and biopsies. CONCLUSION: A rapid analysis of the entire ABBC8 and KCNJ11 genes should not stand alone in the preoperative assessment of patients with CHI, except for the case of maternal, or homozygous/compound heterozygous disease-causing mutations.     

Insights into Fanconi Anaemia from the structure of human FANCE

Fanconi Anaemia (FA) is a cancer predisposition disorder characterized by spontaneous chromosome breakage and high cellular sensitivity to genotoxic agents. In response to DNA damage, a multi-subunit assembly of FA proteins, the FA core complex, monoubiquitinates the downstream FANCD2 protein. The FANCE protein plays an essential role in the FA process of DNA repair as the FANCD2-binding component of the FA core complex. Here we report a crystallographic and biological study of human FANCE. The first structure of a FA protein reveals the presence of a repeated helical motif that provides a template for the structural rationalization of other proteins defective in Fanconi Anaemia. The portion of FANCE defined by our crystallographic analysis is sufficient for interaction with FANCD2, yielding structural information into the mode of FANCD2 recruitment to the FA core complex. Disease-associated mutations disrupt the FANCE–FANCD2 interaction, providing structural insight into the molecular mechanisms of FA pathogenesis.     

Biodegradation of α- and β-endosulfan by soil bacteria

Extensive applications of persistent organochlorine pesticides like endosulfan on cotton have led to the contamination of soil and water environments at several sites in Pakistan. Microbial degradation offers an effective approach to remove such toxicants from the environment. This study reports the isolation of highly efficient endosulfan degrading bacterial strains from soil. A total of 29 bacterial strains were isolated through enrichment technique from 15 specific sites using endosulfan as sole sulfur source. The strains differed substantially in their potential to degrade endosulfan in vitro ranging from 40 to 93% of the spiked amount (100 mg l⁻¹). During the initial 3 days of incubation, there was very little degradation but it got accelerated as the incubation period proceeded. Biodegradation of endosulfan by these bacteria also resulted in substantial decrease in pH of the broth from 8.2 to 3.7 within 14 days of incubation. The utilization of endosulfan was accompanied by increased optical densities (OD₅₉₅) of the broth ranging from 0.511 to 0.890. High performance liquid chromatography analyses revealed that endosulfan diol and endosulfan ether were among the products of endosulfan metabolism by these bacterial strains while endosulfan sulfate, a persistent and toxic metabolite of endosulfan, was not detected in any case. The presence of endosulfan diol and endosulfan ether in the bacterial metabolites was further confirmed by GC-MS. Abiotic degradation contributed up to 21% of the spiked amount. The three bacterial strains, Pseudomonas spinosa, P. aeruginosa, and Burkholderia cepacia, were the most efficient degraders of both α- and β-endosulfan as they consumed more than 90% of the spiked amount (100 mg l⁻¹) in the broth within 14 days of incubation. Maximum biodegradation by these three selected efficient bacterial strains was observed at an initial pH of 8.0 and at an incubation temperature of 30°C. The results of this study may imply that these bacterial strains could be employed for bioremediation of endosulfan polluted soil and water environments.