Narendra Kumar, MSc, PhD
Nominated From: University of Hawaii
Research Site: India
Research Area: Infectious Disease & HIV/AIDS
Primary Mentor: Lishomwa Ndhlovu
Research Project
Microbial Translocation during HIV infection using the Caenorhabditis elegans model
Gastrointestinal (GI)-related morbidities are major clinical concern in HIV-infected patients that persists despite suppressive antiretroviral therapy (ART). HIV-infected patients are burdened with chronic diarrhea, alteration of gut-microbiota and elevated microbial translocation that cause increased inflammation. Elevated level of inflammation in treated HIV-infected patients appears to be the major cause of non-AIDS morbidity and mortality. Increased microbial translocation is associated with the up-regulation of inflammatory cytokines in peripheral blood and rapid disease progression. An altered intestinal microbiota during chronic HIV infection is also associated with mucosal dysfunction, inflammation and disease progression. While several attempts were made to mitigate the chronic inflammation in treated HIV-infected patients (SM Dillon et al), there has not been any promising therapy in normalizing the increased inflammation. Therefore, comprehensive study of gut microbiota, and the dynamics of real-time microbial translocation in will help us unravel the functional characteristics of gut micriobiota.
Gut microbiota describes the overall health of a person (Caitriona M et al) and fecal microbiota transfer (FMT) is a unique interventional approach currently being used for different inflammatory diseases including Inflammatory Bowel Disease (IBD). In order to understand the mechanism of gut microbiota-induced microbial translocation and inflammation, and the role of FMT to reverse the microbial translocation, it is imperative to develop an appropriate animal model. C. elegans is an important animal model system that has been the platform for many discoveries in biology, including key findings in the fields of neurobiology, development and small RNAs (Fire et al., 1998). C. elegans is commonly found in rotting fruits and stems, and appears to feed on a variety of different microbes, which can be nutritious or pathogenic. We propose to study C. elegans as a model to study the gut microbiota- associated microbial translocation and reversal of microbial translocation by FMT. C. elegans has a simple transparent body plan, which has facilitated many studies in the worm. The hypodermis of C. elegans tissues maintain regular contact with microbes, while a single layer, multi-nucleate syncytium intestinal epithelium surrounds the animal that secretes collagen to form a tough outer cuticle (Chisholm and Hardin, 2005). C. elegans feeds on microbes and its intestine is comprised of 20 epithelial cells that are mostly in pairs of cells that form a tube that runs the length of the animal (McGhee, 2007). C. elegans intestinal cells share several morphological similarities with human intestinal epithelial cells, including actin-rich microvilli on the apical side of cells that absorb nutrients from the lumen. These microvilli are anchored into a cytoskeletal structure called the terminal web, which spans the cell and connects into apical cell–cell junctions. The ability to visualize these structures and processes in a live animal provides a powerful system in which to examine pathogen infection of the intestine (Keir M. Balla and Emily R. Troemel).
Research Significance
Fecal microbiota transfer (FMT) therapy has emerged as one of the promising therapeutic intervention to cure several inflammatory diseases. In HIV infection, alteration in gut microbiota results in the increased microbial translocation and inflammation leading to HIV disease progression. FMT therapy could be used as a potential therapy together with antiretroviral therapy (ART) to reverse the inflammation and microbial translocation to the normal level (which in general persists despite therapy). This may lower the incidence of non-AIDS morbidity among ART-treated HIV subjects – in India and the United States.
Specific Aims
Aim 1. To establish C. elegans as a model to study the interaction between gut microbiota of HIV-infected (treated and untreated) patients and host epithelial cells.
AIM 2. To study the effect of fecal microbiota transplantation (FMT) on microbial translocation and associated GI inflammation.
Approach
To develop C. elegans as a model to study the interaction between gut microbiota of HIV-infected subjects and host epithelial cells. C. elegans will be cultured on Nematode Growth Medium. To assess the effect of HIV-infected patients-derived gut microbiota on gut epithelial cells, 20 ART-suppressed and 20 treatment naïve HIV positive patients will be recruited from ART center at AIIMS. For control, 10 HIV negative healthy subjects will be recruited. Fecal sample will be cultured and stained with Alexa Fluor-647 dye and fed to C. elegans. Microbial translocation will be studied under fluorescence microscope by measuring the intensity of translocated dye. As negative control, C. elegans will be fed on E. coli stained with the dye. In our preliminary study, we have established protocol for fecal feeding by C. elegans. In addition to feeding fecal samples to C. elegans, 2-3 gram of fecal samples from 5 representative subjects from each category will be subjected to high throughout sequencing using Illumina platform. Aerobic bacteria will be cultured separately on LB –Agar plate and individual colony will be screened their ability to induce inflammatory response on human epithelial colorectal cell line, Caco-2 cell, which is a model for gut epithelial barrier function. Briefly, Bacteria will be cultured on LB medium and 2 MOI bacterial cells will be used to stimulate 1 million Caco-2 cells. RNA will be extracted 24 h post infection and IL-6, TNF-a, IFN-gamma will be relatively qualified using SYBR Green based ΔΔCT method. To assess the overall level of inflammation in HIV-infected subjects, we will investigate inflammatory biomarkers like IFN-γ, TNF-a, IL-1β, IL- 8, IL-6 using Luminex based Multiplex assay in the plasma of treatment naïve and ART-suppressed HIV infected subjects and compare them with that of age matched HIV negative healthy controls. We will correlate these parameters with the level of microbial translocation in C.elegans. For Aim 2, C. elegans will be fed on the fecal sample from healthy individual following exposure with fecal samples from HIV-infected subjects. The overall objective for the aim-2 is to compare the differential expression of tight junction proteins, e.g. Occludin and ZO-1, in the gut epithelial cells of C. elegans after they are fed on fecal sample from HIV-infected subjects followed by FMT from healthy individuals. We seek to investigate the potential role of FMT in the restoration of tight junction proteins, which will correlate with the decrease in microbial translocation. The intestinal part of C. elegans will be removed using 27G needles under the microscope. The excised part of the intestine will be used for RNA and DNA extraction using Qiagen kit. The relative quantification of tight junction protein will be performed by ΔΔCt method.
Publications
Mentors
- Dr. Lishomwa Ndhlovu, John A. Burns School of Medicine, Department of Tropical Medicine, Medical Microbiology and Pharmacology, University of Hawaii at Manoa, USA
- Dr. Rupesh Chaturvedi, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India
- Dr. Ravi Tandon, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India
- Dr. Kalpana Luthra, Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
- Dr. V. Sreenivas, All India Institute of Medical Sciences, New Delhi, India
- Dr. Saguna Verma, John A. Burns School of Medicine, Department of Tropical Medicine, Medical Microbiology and Pharmacology, University of Hawaii at Manoa, USA
