Rickettsia typhi Possesses Phospholipase A2 Enzymes that Are Involved in Infection of Host Cells

The long-standing proposal that phospholipase A₂ (PLA₂) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA₂ activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA₂ activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA₂ activity were reduced by PLA₂ inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.     

Redescription of Robustodorus megadorus with Molecular Characterization and Analysis of Its Phylogenetic Position within the Family Aphelenchoididae

Based on a new record of the rare species Robustodorus megadorus from Utah, the generic diagnosis was amended to include the following characters: a labial disc surrounded by six pore-like sensilla; the absence of a cephalic disc; a lobed cephalic region devoid of annulation; a hexagonal inner cuticular structure of the pouch surrounding the stylet cone; large stylet knobs, rounded in outline and somewhat flattened on their lateral margins; a large spermatheca with an occluded lumen and lacking sperm; the excretory pore located between the median bulb and nerve ring. The stylet orifice consists of an open, ventral, elongate slit or groove. These characters distinguish the genus from the closely related genus Aphelenchoides. A lectotype and paralectotypes were designated. Results of phylogenetic analyses of the 18S and D2-D3 of 28S rRNA gene sequences revealed that R. megadorus occupies a basal position within one of the two main clades of the subfamily Aphelenchoidinae and shares close relationships with a species group of the genus Aphelenchoides that includes A. blastophthorus, A. fragariae, A. saprophilus, A. xylocopae, and A. subtenuis. Several specimens in our collection of R. megadorus were infected with Pasteuria sp. as were some of the paralectotypes.     

Nitric oxide regulates tissue transglutaminase localization and function in the vasculature

The multifunctional enzyme tissue transglutaminase (TG2) contributes to the development and progression of several cardiovascular diseases. Extracellular rather than intracellular TG2 is enzymatically active, however, the mechanism by which it is exported out of the cell remains unknown. Nitric oxide (NO) is shown to constrain TG2 externalization in endothelial and fibroblast cells. Here, we examined the role of both exogenous and endogenous (endothelial cell-derived) NO in regulating TG2 localization in vascular cells and tissue. NO synthase inhibition in endothelial cells (ECs) using N-nitro l-arginine methyl ester (l-NAME) led to a time-dependent decrease in S-nitrosation and increase in externalization of TG2. Laminar shear stress led to decreased extracellular TG2 in ECs. S-nitrosoglutathione treatment led to decreased activity and externalization of TG2 in human aortic smooth muscle and fibroblast (IMR90) cells. Co-culture of these cells with ECs resulted in increased S-nitrosation and decreased externalization and activity of TG2, which was reversed by l-NAME. Aged Fischer 344 rats had higher tissue scaffold-associated TG2 compared to young. NO regulates intracellular versus extracellular TG2 localization in vascular cells and tissue, likely via S-nitrosation. This in part, explains increased TG2 externalization and activity in aging aorta.     

A method to extract intact and pure RNA from mycobacteria

We describe a high-yielding, simple, and aerosol-free protocol for the isolation of RNA from mycobacteria that does not require sophisticated instruments. The method yielded 50 μg of RNA from 10⁷ cells, 50 times more than a recently reported method. Our method can extract total RNA from aerobically grown bacteria and from in vitro hypoxia-induced dormant bacilli and mycobacteria residing within infected macrophages.     

Mechanism of hypochlorous acid-mediated heme destruction and free iron release

Here, we show that hypochlorous acid (HOCl), a potent neutrophil-generated oxidant, can mediate destruction of free heme (Ht) and the heme precursor, protoporphyrin IX (PPIX). Ht displays a broad Soret absorbance peak centered at 365 and 394 nm, indicative of the presence of monomer and μ-oxo-dimer. Oxidation of Ht by HOCl was accompanied by a marked decrease in the Soret absorption peak and release of free iron. Kinetic measurements showed that the Ht-HOCl reaction was triphasic. The first two phases were HOCl concentration dependent and attributable to HOCl binding to the monomeric and dimeric forms. The third phase was HOCl concentration independent and attributed to Ht destruction with the release of free iron. HPLC and LC-ESI-MS analyses of the Ht-HOCl reaction revealed the formation of a number of degradation products, resulting from the cleavage or modification of one or more carbon-methene bridges of the porphyrin ring. Similar studies with PPIX showed that HOCl also mediated tetrapyrrole ring destruction. Collectively, this work demonstrates the ability of HOCl to modulate destruction of heme, through a process that occurs independent of the iron molecule that resides in the porphyrin center. This phenomenon may play a role in HOCl-mediated oxidative injury in pathological conditions.     

Evaluation of amoebicidal potential of Paneth cell cryptdin-2 against Entamoeba histolytica

Background

Amoebiasis is a major public health problem in tropical and subtropical countries. Currently, metronidazole is the gold choice medication for the treatment of this disease. However, reports have indicated towards the possibility of development of metronidazole-resistance in Entamoeba strains in near future. In view of the emergence of this possibility, in addition to the associated side effects and mutagenic ability of the currently available anti-amoebic drugs, there is a need to explore newer therapeutics against this disease. In this context, the present study evaluated the amoebicidal potential of cryptdin-2 against E. histolytica.

Methods/Principal Findings

In the present study, cryptdin-2 exhibited potent in-vitro amoebicidal activity against E. histolytica in a concentration dependent manner at a minimum amoebicidal concentration (MAC) of 4 mg/L. Scanning electron microscopy as well as phase contrast microscopic investigations of cryptdin-2 treated trophozoites revealed that the peptide was able to induce significant morphological alterations in terms of membrane wrinkling, leakage of the cytoplasmic contents and damaged plasma membrane suggesting a possible membrane dependent amoebicidal activity. N-phenyl napthylamine (NPN) uptake assay in presence of sulethal, lethal as well as twice the lethal concentrations further confirmed the membrane-dependent mode of action of cryptdin-2 and suggested that the peptide could permeabilize the plasma membrane of E. histolytica. It was also found that cryptdin-2 interfered with DNA, RNA as well as protein synthesis of E. histolytica exerting the highest effect against DNA synthesis. Thus, the macromolecular synthesis studies correlated well with the observations of membrane permeabilization studies.

Significance/Conclusions

The amoebicidal efficacy of cryptdin-2 suggests that it may be exploited as a promising option to combat amoebiasis or, at least, may act as an adjunct to metronidazole and/or other available anti-amoebic drugs.     

Immobilization of Pholiota adiposa xylanase onto SiO2 nanoparticles and its application for production of xylooligosaccharides

Enhanced yields of different lignocellulases were obtained under statistically-optimized parameters using Pholiota adiposa. The k (cat) value (4,261 s(-1)) of purified xylanase under standard assay conditions was the highest value ever reported. On covalent immobilization of the crude xylanase preparation onto functionalized silicon oxide nanoparticles, 66 % of the loaded enzyme was retained on the particle. Immobilized enzyme gave 45 % higher concentrations of xylooligosaccharides compared to the free enzyme. After 17 cycles, the immobilized enzyme retained 97 % of the original activity, demonstrating its prospects for the synthesis of xylooligosaccharides in industrial applications.     

Evaluation of Insecticides Susceptibility and Malaria Vector Potential of Anopheles annularis s.l. and Anopheles vagus in Assam,India

During the recent past, development of DDT resistance and reduction to pyrethroid susceptibility among the malaria vectors has posed a serious challenge in many Southeast Asian countries including India. Current study presents the insecticide susceptibility and knock-down data of field collected Anopheles annularis sensu lato and An. vagus mosquito species from endemic areas of Assam in northeast India. Anopheles annularis s.l. and An. vagus adult females were collected from four randomly selected sentinel sites in Orang primary health centre (OPHC) and Balipara primary health centre (BPHC) areas, and used for testing susceptibility to DDT, malathion, deltamethrin and lambda-cyhalothrin. After insecticide susceptibility tests, mosquitoes were subjected to VectorTest^™ assay kits to detect the presence of malaria sporozoite in the mosquitoes. An. annularis s.l. was completely susceptible to deltamethrin, lambda-cyhalothrin and malathion in both the study areas. An. vagus was highly susceptible to deltamethrin in both the areas, but exhibited reduced susceptibility to lambda-cyhalothrin in BPHC. Both the species were resistant to DDT and showed very high KDT₅₀ and KDT₉₉ values for DDT. Probit model used to calculate the KDT₅₀ and KDT₉₉ values did not display normal distribution of percent knock-down with time for malathion in both the mosquito species in OPHC (p<0.05) and An. vagus in BPHC (χ² = 25.3; p = 0.0), and also for deltamethrin to An. vagus in BPHC area (χ² = 15.4; p = 0.004). Minimum infection rate (MIR) of Plasmodium sporozoite for An. vagus was 0.56 in OPHC and 0.13 in BPHC, while for An. annularis MIR was found to be 0.22 in OPHC. Resistance management strategies should be identified to delay the expansion of resistance. Testing of field caught Anopheles vectors from different endemic areas for the presence of malaria sporozoite may be useful to ensure their role in malaria transmission.     

New bithiazolyl hydrazones: Novel synthesis,characterization and antitubercular evaluation

New bithiazolyl hydrazones (6a–l) have been first time synthesized by carrying novel one pot cyclocondensation of 5-acyl thiazoles (1a–b), thiosemicarbazide (2) and substituted phenacyl chlorides (4a–f) in freshly prepared ionic liquid, diisopropyl ethyl ammonium acetate (DIPEAc) at room temperature. The newly synthesized compounds have been evaluated for their antitubercular activity and the compounds 3b, 6a, 6b, 6d, 6e, 6f, 6g, and 6l have displayed noticeable antitubercular activity compared to Rifampicin with tolerable cytotoxicity. All these compounds were also screened for their antibacterial activity and found that, compounds 6j and 6k have exhibited a very good antibacterial activity. Molecular docking study has shown better harmony with the evaluation trend shown by these compounds under in vitro antitubercular screening.