Division of the Salmonella-Containing Vacuole and Depletion of Acidic Lysosomes in Salmonella-Infected Host Cells Are Novel Strategies of Salmonella enterica To Avoid Lysosomes▿

Salmonella has evolved several strategies to counteract intracellular microbicidal agents like reactive oxygen and nitrogen species. However, it is not yet clear how Salmonella escapes lysosomal degradation. Some studies have demonstrated that Salmonella can inhibit phagolysosomal fusion, whereas other reports have shown that the Salmonella-containing vacuole (SCV) fuses/interacts with lysosomes. Here, we have addressed this issue from a different perspective by investigating if the infected host cell has a sufficient quantity of lysosomes to target Salmonella. Our results suggest that SCVs divide along with Salmonella, resulting in a single bacterium per SCV. As a consequence, the SCV load per cell increases with the division of Salmonella inside the host cell. This demands more investment from the host cell to counteract Salmonella. Interestingly, we observed that Salmonella infection decreases the number of acidic lysosomes inside the host cell both in vitro and in vivo. These events potentially result in a condition in which an infected cell is left with insufficient acidic lysosomes to target the increasing number of SCVs, which favors the survival and proliferation of Salmonella inside the host cell

δ-Aminolevulinate dehydrase from Plasmodium falciparum: indigenous versus imported

The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host δ-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit M<SUB>r</SUB> of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20-30% upon addition of Mg<SUP>2+</SUP>. The enzyme is inhibited by Zn<SUP>2+</SUP> and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg<SUP>2+</SUP> independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite

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Not AvailableIn foot-and-mouth disease (FMD) control programme, liquid-phase blocking ELISA (LPBE) is widely used to assay vaccine-induced seroconversion. Currently, the assay utilizes inactivated FMD virus antigen for the detection of antibodies in serum samples. To develop a non-infectious substitute for the antigen in LPBE, we expressed the structural polypeptide of FMDV (serotype A) using a baculovirus expression system, and show that inclusion of viral 3C with reduced protease activity resulted in a higher yield of structural proteins. Structural proteins expressed in insect cells assembled into empty virus-like particles (VLPs) and showed antigenicity comparable to chemically inactivated FMDV. Screening of serum samples from FMD-vaccinated cattle showed that the test performance of VLP-LPBE had a correlation of 0.89 with conventional inactivated virus antigen LPBE. The VLP-LPBE developed here demonstrates the diagnostic application of recombinant FMDV VLPs in monitoring seroconversion following FMD vaccination.Not Availabl

Cerebrospinal Fluid from Patients with Sporadic Amyotrophic Lateral Sclerosis Induces Degeneration of Motor Neurons Derived from Human Embryonic Stem Cells

Disease modeling has become challenging in the context of amyotrophic lateral sclerosis (ALS), as obtaining viable spinal motor neurons from postmortem patient tissue is an unlikely possibility. Limitations in the animal models due to their phylogenetic distance from human species hamper the success of translating possible findings into therapeutic options. Accordingly, there is a need for developing humanized models as a lead towards identifying successful therapeutic possibilities. In this study, human embryonic stem cellsBJNHem20were differentiated into motor neurons expressing HB9, Islet1, and choline acetyl transferase using retinoic acid and purmorphamine. These motor neurons discharged spontaneous action potentials with two different frequencies (5Hz), and majority of them were principal neurons firing with</p