Enteropathogenic E. coli infection co-elicits lysosomal exocytosis and lytic host cell death.

IMPORTANCE: Enteropathogenic Escherichia coli (EPEC) infection is a significant cause of gastroenteritis, mainly in children. Therefore, studying the mechanisms of EPEC infection is an important research theme. EPEC modulates its host cell life by injecting via a type III secretion machinery cell death modulating effector proteins. For instance, while EspF and Map promote mitochondrial cell death, EspZ antagonizes cell death. We show that these effectors also control lysosomal exocytosis, i.e., the trafficking of lysosomes to the host cell plasma membrane. Interestingly, the capacity of these effectors to induce or protect against cell death correlates completely with their ability to induce LE, suggesting that the two processes are interconnected. Modulating host cell death is critical for establishing bacterial attachment to the host and subsequent dissemination. Therefore, exploring the modes of LE involvement in host cell death is crucial for elucidating the mechanisms underlying EPEC infection and disease.

Mitogen-Activated Protein Kinases (MAPKs) and Enteric Bacterial Pathogens: A Complex Interplay.

Diverse extracellular and intracellular cues activate mammalian mitogen-activated protein kinases (MAPKs). Canonically, the activation starts at cell surface receptors and continues via intracellular MAPK components, acting in the host cell nucleus as activators of transcriptional programs to regulate various cellular activities, including proinflammatory responses against bacterial pathogens. For instance, binding host pattern recognition receptors (PRRs) on the surface of intestinal epithelial cells to bacterial pathogen external components trigger the MAPK/NF-κB signaling cascade, eliciting cytokine production. This results in an innate immune response that can eliminate the bacterial pathogen. However, enteric bacterial pathogens evolved sophisticated mechanisms that interfere with such a response by delivering virulent proteins, termed effectors, and toxins into the host cells. These proteins act in numerous ways to inactivate or activate critical components of the MAPK signaling cascades and innate immunity. The consequence of such activities could lead to successful bacterial colonization, dissemination, and pathogenicity. This article will review enteric bacterial pathogens' strategies to modulate MAPKs and host responses. It will also discuss findings attempting to develop anti-microbial treatments by targeting MAPKs.

Topology and function of translocated EspZ.

EspZ and Tir are essential virulence effectors of enteropathogenic Escherichia coli (EPEC). EspZ, the second translocated effector, has been suggested to antagonize host cell death induced by the first translocated effector, Tir (translocated intimin receptor). Another characteristic of EspZ is its localization to host mitochondria. However, studies that explored the mitochondrial localization of EspZ have examined the ectopically expressed effector and not the more physiologically relevant translocated effector. Here, we confirmed the membrane topology of translocated EspZ at infection sites and the involvement of Tir in confining its localization to these sites. Unlike the ectopically expressed EspZ, the translocated EspZ did not colocalize with mitochondrial markers. Moreover, no correlation has been found between the capacity of ectopically expressed EspZ to target mitochondria and the ability of translocated EspZ to protect against cell death. Translocated EspZ may have to some extent diminished F-actin pedestal formation induced by Tir but has a marked effect on protecting against host cell death and on promoting host colonization by the bacteria. Taken together, our results suggest that EspZ plays an essential role in facilitating bacterial colonization, likely by antagonizing cell death mediated by Tir at the onset of bacterial infection. This activity of EspZ, which occurs by targeting host membrane components at infection sites, and not mitochondria, may contribute to successful bacterial colonization of the infected intestine. IMPORTANCE EPEC is an important human pathogen that causes acute infantile diarrhea. EspZ is an essential virulence effector protein translocated from the bacterium into the host cells. Detailed knowledge of its mechanisms of action is, therefore, critical for better understanding the EPEC disease. We show that Tir, the first translocated effector, confines the localization of EspZ, the second translocated effector, to infection sites. This activity is important for antagonizing the pro-cell death activity conferred by Tir. Moreover, we show that translocated EspZ leads to effective bacterial colonization of the host. Hence, our data suggest that translocated EspZ is essential because it confers host cell survival to allow bacterial colonization at an early stage of bacterial infection. It performs these activities by targeting host membrane components at infection sites. Identifying these targets is critical for elucidating the molecular mechanism underlying the EspZ activity and the EPEC disease.

EspH interacts with the host active Bcr related (ABR) protein to suppress RhoGTPases.

Enteropathogenic Escherichia coli are bacterial pathogens that colonize the gut and cause severe diarrhea in humans. Upon intimate attachment to the intestinal epithelium, these pathogens translocate via a type III secretion system virulent proteins, termed effectors, into the host cells. These effectors manipulate diverse host cell organelles and functions for the pathogen's benefit. However, the precise mechanisms underlying their activities are not fully understood despite intensive research. EspH, a critical effector protein, has been previously reported to disrupt the host cell actin cytoskeleton by suppressing RhoGTPase guanine exchange factors. However, native host proteins targeted by EspH to mediate these activities remained unknown. Here, we identified the active Bcr related (ABR), a protein previously characterized to possess dual Rho guanine nucleotide exchange factor and GTPase activating protein (GAP) domains, as a native EspH interacting partner. These interactions are mediated by the effector protein's C-terminal 38 amino acid segment. The effector primarily targets the GAP domain of ABR to suppress Rac1 and Cdc42, host cell cytotoxicity, bacterial invasion, and filopodium formation at infection sites. Knockdown of ABR expression abolished the ability of EspH to suppress Rac1, Cdc42. Our studies unravel a novel mechanism by which host RhoGTPases are hijacked by bacterial effectors.

Type III secreted effectors that target mitochondria.

A type III secretion system (T3SS) is used by Gram-negative bacterial pathogens to secrete and translocate a battery of proteins, termed effectors, from the bacteria directly into the host cells. These effectors, which are thought to play a key role in bacterial virulence, hijack and modify the activity of diverse host cell organelles, including mitochondria. Mitochondria-the energy powerhouse of the cell-are important cell organelles that play role in numerous critical cellular processes, including the initiation of apoptosis and the induction of innate immunity. Therefore, it is not surprising that pathogenic bacteria use mitochondrially targeted effectors to control host cell death and immunity pathways. Surprisingly, however, we found that despite their importance, only a limited number of type III secreted effectors have been characterised to target host mitochondria, and the mechanisms underlying their mitochondrial activity have not been sufficiently analysed. These include effectors secreted by the enteric attaching and effacing (A/E), Salmonella and Shigella bacterial pathogens. Here we give an overview of key findings, present gaps in knowledge and hypotheses concerning the mode by which these type III secreted effectors control the host and the bacterial cell life (and death) through targeting mitochondria.

Effective data convergence, mapping, and pollution categorization of ghats at Ganga River Front in Varanasi.

Rivers support life of Indian population but water pollution threatens human health. There is no consensus data for water quality (WQ) of rivers in India including River Ganga. For robust stakeholder participation and community involvement and governance, a consolidation of WQ parameters for River Ganga from different data sources is essentially needed. The priority to combat environmental, economic, and social dislocations due to river pollution also necessitates WQ data convergence, its availability in public domain for policy makers, citizens, researchers, etc, and mapping with respect to the flowing river. Lack of real-time data limits civic involvement in river management. This paper is a novel attempt to consolidate the WQ data available in literature for River Ganga at Varanasi during 1992-2016. Results indicate water of River Ganga to have high coliform and BOD levels due to direct discharge of sewage waste from Varuna and that from Varanasi city. Categorization of ghats based on WQ parameters reveal pollution status of ghats to vary from low to highly polluted (coliform data excluded as it changes the category of all the ghats to highly polluted). More urban population with lack of readiness of authorities to follow the law appear to be major contributors towards failure of river management strategies. The consolidated information mapped with demographic data can be used as data management tools for sustenance of River Ganga. A need for change in policy framework and publicizing the real-time data seem key solutions for improving water quality of River Ganga at Varanasi.

Expression, purification and functional characterization of recombinant hypervariable region (HVR) of Chikungunya virus nsP3 protein.

One of the most important rapidly emerging mosquito-borne alphavirus is Chikungunya virus (CHIKV). There is a necessity to develop anti-CHIKV therapeutics, as neither antiviral drug nor vaccines have been licensed yet. Several CHIKV proteins are being studied worldwide, but non-structural protein 3 (nsP3) has been less explored. This protein consists of three domains: macrodomain, alphavirus unique domain (AUD) and hypervariable region (HVR). The proline-rich regions of HVR contain SRC homology 3 (SH3)-binding domain which is essential for its functionality. Interaction of these motifs with host amphiphysin protein is crucial for viral RNA replication. Restricting the interactions of HVR could lead to inhibition of viral life cycle. Therefore, the present study focuses on purification of HVR protein and its structural and functional assay for therapeutic intervention in future use. In order to obtain purified protein, HVR region was amplified from TOPO clones of nsP3 of IND-06-Guj strain and cloned into expression vector. Expression and solubilization of the protein were optimized at various conditions of salt, detergent and imidazole before purification. The soluble recombinant HVR (His-HVR) protein was purified using affinity chromatography. Purified protein was analyzed for structural studies and functional assays. Circular dichroism of His-HVR protein was performed for structural study, and it was observed that it consists of mostly random coils. For functional assay, co-pull down of His-HVR protein was performed with endogenous amphiphysin-I protein of N2a cells and was analyzed using Western blotting. This purified protein obtained could be used as a potential target reagent for novel therapeutic interventions in the future.

Assessing the Accuracy of Citizen Scientist Reported Measurements for Agrichemical Contaminants.

Citizen science is a research tool capable of addressing major environmental challenges, including contamination of water resources by agrichemicals, such as nutrients and pesticides. The objectives of this study were (1) to identify the proportion of accurate observations by citizen scientists using rapid assessment water quality tools, and (2) to characterize how a user's prior experience with water quality tools was associated with the accuracy of citizen scientists. To achieve these objectives, we conducted group testing with over 136 citizen scientists and compared their results from water quality testing of water samples to results obtained using laboratory analytical methods. Following brief training, we observed that accuracy of reported results varies based on the user's experience level where experienced and expert users shared consistent and reliable measurements. Where erroneous measures were reported, citizen scientists tend to overestimate contaminant concentrations when using colorimetric water quality tools. Additionally, we identified differences in accuracy related to the types of water quality assessment tools used by citizen scientists from each experience group. This study demonstrates the importance of evaluating participant background experience in designing citizen science campaigns.

Microbial diversity of a Himalayan forest and characterization of rare actinomycetes for antimicrobial compounds.

The slow pace of discovery of new effective drugs against multi-drug resistant pathogens and largely unsuccessful combinatorial chemistry has resulted in shifting the focus back to natural products as sources of lead molecules for antimicrobial drugs, mainly due to their structural diversity. Investigation of under-explored habitats for potentially novel microorganisms provides for wider chemodiversity. In this study, four actinomycetes, namely UK-274, UK-281, UK-282 and UK-285, which showed broad-spectrum antibacterial and antifungal activities, were isolated from Timli forest range of the biodiversity-rich Himalayan region. 16S rRNA gene sequence analysis showed that the nearest neighbours of the isolates were Actinomadura nitrigenes, Streptomyces niveiscabiei, and Kitasatospora psammotica with similarity values ranging between 97 and 98% suggesting their potential as new isolates. Further morphological and phenotypic characterization strengthened this assumption. Isolate UK-282, of the rare actinomycetes Kitasatospora group, was found to produce antimicrobial activity. Metabolite fingerprinting of ethyl acetate fraction of isolate UK-282 by GC-MS and 1H NMR analysis showed the presence of three novel compounds. The study underlines that a combination approach of bioprospecting of under-studied habitats and focus on rare actinomycetes may result in the identification of novel chemodiversity.

Water sustainability: reforming water management in new global era of climate change.

The National Seminar on Sustainable Water Resource Management in Era of Changing Climate (NSWRM-2014) on 10-11 January 2014 organised by the Institute of Environment and Sustainable Development and Environmental Science and Technology, Banaras Hindu University, witnessed the presence of experts from environmentalists, industrialists and experts on water resources and its management. The deliberations and scientific discussions led to the conclusion that it is not just the resource but the natural capacity to sustain it that requires monitoring, understanding and stewardship. The focus of governance in India needs to move at a faster pace from conventional methods of sector-based water management to more integrated approach for sustainable water resource management. It is more of the people participation that is the future key towards sustainable water resource management in India.