In-vivo protein nitration facilitates Vibrio cholerae cell survival under anaerobic, nutrient deprived conditions.

Protein tyrosine nitration (PTN), a highly selective post translational modification, occurs in both prokaryotic and eukaryotic cells under nitrosative stress. However, its physiological function is not yet clear. Like many gut pathogens, Vibrio cholerae also faces nitrosative stress, which makes its proteome more vulnerable to PTN. Here, we report for the first time in-vivo PTN in V. cholerae by immunoblotting and LC-ESI-MS/MS proteomic analysis. Our results indicated that in-vivo PTN in V. cholerae was culture media independent. Surprisingly, in-vivo PTN was reduced in V. cholerae proteome under anaerobic or hypoxic condition in a nutrient deprived state. Interestingly, intracellular nitrate content was more than the nitrite content in V. cholerae under anaerobic conditions. Additionally, biochemical measurement of GSH/GSSG ratio, activities of catalase and SOD, ROS and RNS imaging by confocal microscopy confirmed a relative intracellular oxidizing environment in V. cholerae under anaerobic conditions. This altered redox environment favors the oxidation of nitrite which may be generated from protein denitration enriching the intracellular nitrate pool. The cell survival of V. cholerae can finally be facilitated by nitrate reductase (NapA) utilizing that nitrate pool. Our cell viability study using wild type and ΔnapA strain of V. cholerae also supported the role of NapA mediated cell survival under nutrient deprived anaerobic conditions. In spite of having nitrate reductase (NapA), V. cholerae lacks any nitrite reductase (NiR). Hence, in-vivo nitration may provide an avenue for toxic nitrite storage and also may help in nitrosative stress tolerance mechanism preventing further unnecessary protein nitration in V. cholerae proteome.

Analysis of blood stream infections, antibiograms and clinical outcomes in haematological patients with febrile neutropenia: data from a tertiary care haematology institute in India.

Timely administration of appropriate empirical antibiotics in febrile neutropenia is crucial for favourable patient outcomes. There are guidelines in place recommending such antibiotics. However, regional variations and local epidemiological data must be evaluated to tailor the antibiotics for best possible and rational use. In this study, we audited the clinical and microbiological data of febrile neutropenic episodes occurring at a tertiary care haematology institution. Three hundred and ninety-three febrile neutropenic episodes occurring in 123 patients over a 1-year period were analysed for microbial profile, sensitivity and resistance patterns, and finally clinical outcomes. Gram-negative bacilli (GNB) blood stream infections (46.9%) were more prevalent as compared to gram-positive infections (41.9%). Overall mortality due to complicated neutropenic sepsis was 19.5% (24/123 patients). Increased resistance to carbapenems, beta-lactam beta-lactamase inhibitor combinations, aminoglycosides, fluoroquinolones, and cephalosporins were observed. Cefepime and tigecycline resistance were seen in 20% and 15% GNB isolates, respectively. Chest was the most frequent focus of infection, and acute myeloid leukaemia (AML) was the most common underlying disorder which correlated with the likelihood of death (p < 0.01). Multidrug-resistant GNB (esp. Klebsiella sp.) are still most worrisome isolates in neutropenic patients. Single-agent cefepime or piperacillin-tazobactam/tigecycline combination may be considered empirical agents. Chest infections and AML were independent predictors of poor clinical outcome in neutropenic patients. Regular audit of infections and antibiotic susceptibility data is needed to improve clinical outcomes in patients with febrile neutropenia.

Secretome analysis identified extracellular superoxide dismutase and catalase of Macrophomina phaseolina.

Macrophomina phaseolina, a necrotrophic fungal pathogen is known to cause charcoal rot disease in food crops, pulse crops, oil crops and cotton and fibre crops. Necrotrophic fungi survive on dead plant tissue. It is well known that reactive oxygen species (ROS) are produced by the host plant during plant-pathogen interaction. However, it is still unclear how M. phaseolina can overcome the ROS-induced cellular damage. To mimic the invasion of M. phaseolina inside the plant cell wall, we developed solid substrate fermentation where M. phaseolina spore suspension was inoculated on a wheat bran bed and incubated for vegetative growth. To analyse the secretome of M. phaseolina after different day interval, its secretory material was collected and concentrated. Both superoxide dismutase (SOD) and catalase were detected in the secretome by zymogram. The presence of SOD and catalase was further confirmed by liquid chromatography based mass spectrometry. The physicochemical properties of M. phaseolina catalase in terms of stability towards pH, temperature, metal ions and chaotropic agent and inhibitors indicated its fitness at different environmental conditions. Apart from the production of catalase in SSF, the studies on this particular microorganism may also have significance in necrotrophic fungal pathogen and their susceptible host plant interaction.

Reactive nitrogen species induced catalases promote a novel nitrosative stress tolerance mechanism in Vibrio cholerae.

Vibrio cholerae faces nitrosative stress during successful colonization in intestine. Very little information is available on the nitrosative stress protective mechanisms of V. cholerae. Reports show that NorR regulon control two genes hmpA and nnrS responsible for nitric oxide (NO) detoxification in V. cholerae. In the present study we first time report a novel role of V. cholerae catalases under nitrosative stress. Using zymogram analysis of catalase we showed that KatB and KatG activity were induced within 30 min in V. cholerae in the presence of sodium nitroprusside (SNP), a NO donor compound. Surprisingly, V. cholerae cell survival was found to be decreased under nitrosative stress if catalase activities were blocked by ATz, a catalase inhibitor. Flow cytometry study was conducted to detect reactive oxygen species (ROS) and reactive nitrogen species (RNS) using DHE and DHR123, fluorescent probes respectively. Short exposure of SNP to V. cholerae did not generate ROS but RNS was detectable within 30 min. Total glutathione content was increased in V. cholerae cells under nitrosative stress. Furthermore, Superoxide dismutase (SOD) and Glutathione reductase (GR) activities remained unchanged under nitrosative stress in V. cholerae indicated antioxidant role of NO which could produce peroxynitrite. To investigate the role of catalase induction under nitrosative stress in V. cholerae, we conducted peroxynitrite reductase assay using cell lysates. Interestingly, SNP treated V. cholerae cell lysates showed lowest DHR123 oxidation compared to the control set. The extent of DHR123 oxidation was more in V. cholerae cell lysate when catalases were blocked by ATz.

Secretome Analysis of Macrophomina phaseolina Identifies an Array of Putative Virulence Factors Responsible for Charcoal Rot Disease in Plants.

Macrophomina phaseolina is a global devastating necrotrophic fungal pathogen. It causes charcoal rot disease in more than 500 host plants including major food crops, pulse crops, fiber crops, and oil crops. Despite having the whole-genome sequence of M. phaseolina, understanding the M. phaseolina genome-based plant-pathogen interactions is limited in the absence of direct experimental proof of secretion. Thus, it is essential to understand the host-microbe interaction and the disease pathogenesis, which can ensure global agricultural crop production and security. An in silico-predicted secretome of M. phaseolina is unable to represent the actual secretome. We could identify 117 proteins present in the secretome of M. phaseolina using liquid chromatography-electrospray ionization-tandem mass spectrometry. Data are available via ProteomeXchange with identifier PXD032749. An array of putative virulence factors of M. phaseolina were identified in the present study using solid-state culture. Similar virulence factors have been reported in other plant pathogenic fungi also. Among the secretory fungal proteins with positive economic impacts, lignocellulolytic enzymes are of prime importance. Further, we validated our results by detecting the cell wall-degrading enzymes xylanase, endoglucanase, and amylase in the secretome of M. phaseolina. The present study may provide a better understanding about the necrotrophic fungi M. phaseolina, which modulate the host plant defense barriers using secretory proteins.

Derivative 6 as an additional chromosomal abnormality along with t(15;17): A case report.

Acute promyelocytic leukemia (APL) is a type of acute myeloid leukemia (AML) characterized by the presence of t(15;17)(q22;q21) translocation leading to fusion between PML and RARa gene. Treatment combining all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has dramatically improved the prognosis of APL. We report a rare finding of primary clone of t(15;17) followed by a sequential clonal evolution of additional derivative chromosome 6 formation by a two hit mechanism. Our case showed a good clinical response with a four years and nine months event free survival after ATRA and ATO combination therapy in spite of existence of three chromosomal abnormalities stating that targeted therapy overcomes the adverse effects of additional genetic markers. However, close monitoring with assessment for long term prognostic behavior is required.