Role of cAMP Homeostasis in Intra-Macrophage Survival and Infectivity of Unicellular Parasites like <em>Leishmania</em>

Unicellular eukaryotic pathogen Leishmania donovani, an intra-macrophage protozoan parasite, on exposure to phagolysosome conditions (PC) of mammalian macrophages, show increased cAMP level and cAMP-dependent protein kinase A (PKA) resulting in resistance to macrophage oxidative burst. In order to have a comprehensive understanding of cAMP signaling and their contribution to infectivity, studies were carried out on all the enzymes associated with cAMP metabolism such as adenylate cyclase, phosphodiesterase, pyrophosphatase and the regulatory and catalytic subunits of PKA. This chapter deals in detail the contribution of these components of cAMP signaling in cAMP homeostasis of the parasite as well as their role on successful host-parasite interaction leading to intracellular parasite survival and establishment of infection. Finally, a discussion is made about how these observations might be exploited for developing drug candidates targeting parasite specific features

A Socio cultural perspective on obesity: A study in kolkata city

Obesity as the cause of all sorts of chronic non communicable disease has been of great concern recently. The present study attempts to understand people’s experiences of being obese, their efforts to manage their body weight, and socio-cultural barriers they encounter. For this qualitative study, data was collected through in-depth interviews of middle class and upper middle class informants in Kolkata, the capital city of West Bengal state. The field of the study includes residents, patients in private hospitals and customers in fitness centers

The Tale of Mastering Macrophage Environment through the Control of Inflammasome-Mediated Macrophage Activation and cAMP Homeostasis by the Protozoan Parasite <em>Leishmania</em>

Leishmania, being an intelligent protozoan parasite, modulates the defensive arsenals of the host to create a favorable niche for their survival. When the intracellular parasite is encountered by the host, multimeric complexes of inflammasomes get assembled and activated, thereby leading to genesis of inflammatory response. In order to subvert host defensive strategies, Leishmania utilizes their cyclic adenosine monophosphate (cAMP) and cAMP-induced response to neutralize macrophage oxidative damage. In this chapter, we summarize our current understanding of the mechanisms of inflammasome activation in macrophages and cAMP homeostasis of the parasite, leading to parasite viability within the macrophages and establishment of infection. Furthermore, we took into account, recent progresses in translating these research areas into therapeutic strategies, aimed at combating macrophage associated diseases

Expanding Molecular Coverage in Mass Spectrometry Imaging of Microbial Systems Using Metal-Assisted Laser Desorption/Ionization

Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial ( approximately 67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels

Chemical and Physical Determinants of Bacterial Biofilm Development

Bacterial biofilms cause persistent and deadly infections in medical settings, which are resistant to conventional antibiotic doses. Medical biofilms are often multi-species communities of bacteria which are maintained through chemical signaling and metabolite exchange. An improved understanding of the interactions between bacteria governing community behavior will facilitate the discovery of drug targets for biofilm prevention and eradication. In this thesis, I describe coculture interactions between different species of bacteria and a method to track metabolic states of bacteria in different environments. One example of such coculture interactions is illustrated in the competition for iron in an in vitro infection model. Salmonellaenterica serovar Typhimurium (STm) causes acute infections in the gut, but STm infections are abolished in coculture with a probiotic bacterium, Escherichia coli Nissle (EcN). EcN outcompetes STm in the gut via a class of bactericidal compounds, called microcins, which are conjugated to iron scavenging siderophore molecules. In vitro biofilm models show that STm uses EcN siderophores to acquire iron, and that siderophore conjugation is an anti-cheating strategy employed by EcN to outcompete STm for nutrients.In another example of novel coculture interactions, E. coli and Pseudomonas aeruginosa have competitive interactions governing biofilm establishment and dispersal. The E. coli biofilm dispersal is triggered by P. aeruginosa quorum sensing (QS) compounds. However, E. coli biofilms grown on periodic microcturctures, resembling the stomach micro-villi, are shown to modulate this pathway by metabolite accumulation in engineered microenvironments. Substrate structures induce changes in E. coli biofilm morphology, which in turn increase the concentration of indole, a constitutively produced metabolite within the biofilm. Moreover, the monoculture biofilms grown on microstructured substrates are significantly more susceptible to antibiotics than monocultures on flat substrates. FLIM is a label free, non-invasive technique and has immense potential for use as a means to probe interactions in microbial communities. Fluorescence lifetime imaging microscopy (FLIM), demonstrates that this increased antibiotic susceptibility is due to changes in cellular metabolism induced by an altered microenvironment. The research in this thesis demonstrates more long term and permanent strategies for biofilm infections and will provide guidelines and inspiration for improved diagnostics, and treatments for biofilm infections

Metabolic fingerprinting of bacteria by fluorescence lifetime imaging microscopy.

Bacterial populations exhibit a range of metabolic states influenced by their environment, intra- and interspecies interactions. The identification of bacterial metabolic states and transitions between them in their native environment promises to elucidate community behavior and stochastic processes, such as antibiotic resistance acquisition. In this work, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to create a metabolic fingerprint of individual bacteria and populations. FLIM of autofluorescent reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, has been previously exploited for label-free metabolic imaging of mammalian cells. However, NAD(P)H FLIM has not been established as a metabolic proxy in bacteria. Applying the phasor approach, we create FLIM-phasor maps of Escherichia coli, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus epidermidis at the single cell and population levels. The bacterial phasor is sensitive to environmental conditions such as antibiotic exposure and growth phase, suggesting that observed shifts in the phasor are representative of metabolic changes within the cells. The FLIM-phasor approach represents a powerful, non-invasive imaging technique to study bacterial metabolism in situ and could provide unique insights into bacterial community behavior, pathology and antibiotic resistance with sub-cellular resolution

Metabolic fingerprinting of bacteria by fluorescence lifetime imaging microscopy.

Bacterial populations exhibit a range of metabolic states influenced by their environment, intra- and interspecies interactions. The identification of bacterial metabolic states and transitions between them in their native environment promises to elucidate community behavior and stochastic processes, such as antibiotic resistance acquisition. In this work, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to create a metabolic fingerprint of individual bacteria and populations. FLIM of autofluorescent reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, has been previously exploited for label-free metabolic imaging of mammalian cells. However, NAD(P)H FLIM has not been established as a metabolic proxy in bacteria. Applying the phasor approach, we create FLIM-phasor maps of Escherichia coli, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus epidermidis at the single cell and population levels. The bacterial phasor is sensitive to environmental conditions such as antibiotic exposure and growth phase, suggesting that observed shifts in the phasor are representative of metabolic changes within the cells. The FLIM-phasor approach represents a powerful, non-invasive imaging technique to study bacterial metabolism in situ and could provide unique insights into bacterial community behavior, pathology and antibiotic resistance with sub-cellular resolution

DECOLORIZATION OF SELECTIVE TEXTILE DYES USING WATERBORNE PATHOGENIC BACTERIAL STRAINS

Dyes are serious pollutants, causing environmental and health problems to human being and aquatic animals. Textile wastewater with dye contaminants presents a severe environmental peril because of persistent nature and allied toxicity along with bioaccumulation propensity. Therefore, treatment of dye effluents has become utmost important criterion prior discharged into the environment. In present study three different textile dyes namely, methylene blue, rhodamine B and reactive green 19, were selected for decolorization study using three types of water borne pathogens explicitly Bacillus subtilis, Staphylococcus aureus and Pseudomonas aeruginosa). Effect of major operational process parameters like, initial dye concentration, temperature and pH were investigated for decolorization of textile dyes and present study confirms the ability of Pseudomonas aeruginosa and Bacillus subtilis as potential decolorizing bacterial with efficiency of 97.28% for methylene blue and 98.73% for Reactive Green 19. Significantly high decolorization level and simplistic process conditions illustrate the potential for these bacterial strains to be used in the biological treatment of dyeing mill effluents

Mass spectrometry imaging of natural carbonyl products directly from agar-based microbial interactions using 4-APEBA derivatization

ABSTRACTAliphatic carboxylic acids, aldehydes, and ketones play diverse roles in microbial adaptation to their microenvironment, from excretion as toxins to adaptive metabolites for membrane fluidity. However, the spatial distribution of these molecules throughout biofilms and how microbes in these environments exchange these molecules remain elusive for many of these bioactive species due to inefficient molecular imaging strategies. Herein, we apply on-tissue chemical derivatization (OTCD) using 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) on a co-culture of a soil bacterium (Bacillus subtilis NCIB 3610) and fungus (Fusarium sp. DS 682) grown on agar as our model system. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we spatially resolved more than 300 different metabolites containing carbonyl groups within this model system. Various spatial patterns are observable in these species, which indicate possible extracellular or intercellular processes of the metabolites and their up- or downregulation during microbial interaction. The unique chemistry of our approach allowed us to bring additional confidence in accurate carbonyl identification, especially when multiple isomeric candidates were possible, and this provided the ability to generate hypotheses about the potential role of some aliphatic carbonyls in this B. subtilis/Fusarium sp. interaction. The results shown here demonstrate the utility of 4-ABEBA-based OTCD MALDI-MSI in probing interkingdom interactions directly from microbial co-cultures, and these methods will enable future microbial interaction studies with expanded metabolic coverage.IMPORTANCEThe metabolic profiles within microbial biofilms and interkingdom interactions are extremely complex and serve a variety of functions, which include promoting colonization, growth, and survival within competitive and symbiotic environments. However, measuring and differentiating many of these molecules, especially in an in situ fashion, remains a significant analytical challenge. We demonstrate a chemical derivatization strategy that enabled highly sensitive, multiplexed mass spectrometry imaging of over 300 metabolites from a model microbial co-culture. Notably, this approach afforded us to visualize over two dozen classes of ketone-, aldehyde-, and carboxyl-containing molecules, which were previously undetectable from colonies grown on agar. We also demonstrate that this chemical derivatization strategy can enable the discrimination of isobaric and isomeric metabolites without the need for orthogonal separation (e.g., online chromatography or ion mobility). We anticipate that this approach will further enhance our knowledge of metabolic regulation within microbiomes and microbial systems used in bioengineering applications