Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

Abstract

MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

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Citation

Published Version (Please cite this version)

10.1371/journal.pone.0106434

Publication Info

Furuse, Yuki, Ryan Finethy, Hector A Saka, Ana M Xet-Mull, Dana M Sisk, Kristen L Jurcic Smith, Sunhee Lee, Jörn Coers, et al. (2014). Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells. PLoS One, 9(9). p. e106434. 10.1371/journal.pone.0106434 Retrieved from https://hdl.handle.net/10161/11186.

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Scholars@Duke

Coers

Jorn Coers

Associate Professor in Molecular Genetics and Microbiology

Bacterial infections remain one of the leading causes of morbidity and mortality worldwide. The Coers lab seeks to understand fundamental aspects of the innate immune response to bacterial pathogens as well as the corresponding immune evasion strategies evolved by human pathogens undermining immunity in order to establish infections. Defining innate immunity and microbial counter-immunity pathways on a molecular level will provide roadmaps for the rational design of novel antimicrobial therapies and improved vaccine strategies against pathogens such as the enteric pathogen Shigella or the sexually transmitted pathogen Chlamydia.

In addition to making major inroads in the fields of innate immunity, inflammation and bacterial pathogenesis, our second, but equally important goal, is to train the next generation of scientists in an environment that prioritizes excellence, research integrity, teamwork and inclusiveness. We strive to create an environment of mutual respect, openness, collegiality, integrity and, last but not least, fun, which promotes and awards curiosity and fosters collaborations. We strongly believe that diversity promotes excellence.

Valdivia

Raphael H. Valdivia

Nanaline H. Duke Distinguished Professor of Molecular Genetics and Microbiology

My laboratory is interested in microbes that influence human health, both in the context of host-pathogen and host-commensal interactions. For many pathogens, and certainly for most commensal microbes, we have an incomplete molecular understanding of how host and microbial factors contribute to health and disease. My research group focuses on two experimental systems:

Chlamydia trachomatis infections are responsible for the bulk of sexually transmitted bacterial diseases and are the leading cause of infectious blindness (trachoma) in the world. Chlamydia  resides within a membrane bound compartment (“inclusion”). From this location, the pathogen manipulates the cytoskeleton, inhibits lysosomal recognition of the inclusion, activates signaling pathways, re-routes lipid transport, and prevents the onset of programmed cell death. Our laboratory focuses on identifying and characterizing the bacterial factors that are secreted into the host cell cytoplasm to manipulate eukaryotic cellular functions. We use a combination of cell biology, biochemistry, genetics, genomics, proteomics and molecular biology to determining the function of virulence factors that reveal novel facets of the host-pathogen interaction. Our goal is to understand how these obligate intracellular bacterial pathogens manipulate host cellular functions to replicate, disseminate and cause disease, and in the process develop strategies to ameliorate the damage caused by these infections to the female reproductive organs.

Akkermansia muciniphila is prevalent member of the gut microbiota that proliferates in the mucus layers of our lower gastrointestinal tract and contribute to nutrient homeostasis and human immunological health. My research group developed genetic tools to characterize these microbes to define the mechanisms used to colonize the human gut and identify the molecular and cellular pathways that underscore Akkermansia's impact on immune homeostasis.  In the process, we seek to engineer strains of Akkermansia that enhance their probiotic potential.

Tobin

David M. Tobin

Professor of Molecular Genetics and Microbiology

Tuberculosis: Mycobacterial Pathogenesis and Host Susceptibility

Tuberculosis kills 1.5 million people annually. Our laboratory aims to understand the intricate interplay between mycobacteria and their hosts using a combination of model organism genetics, human genetics, pharmacology and high-resolution microscopy. By identifying key pathways utilized by the infecting bacteria and the host innate immune system, we hope to discover new therapeutic targets and interventions to combat this enduringly destructive disease.

Using a Mycobacterium/zebrafish model, we have identified new host susceptibility loci for tuberculosis. Zebrafish are natural hosts to Mycobacterium marinum, the closest relative of the Mycobacterium tuberculosis complex. Because zebrafish embryos and larvae are optically transparent, we are able to visualize the complex details of mycobacterial pathogenesis in whole, live animals. The facile genetics of the zebrafish allow us to map and positionally clone affected host susceptibility genes. In addition, zebrafish larvae are remarkably permeable to small molecules, providing a platform for whole-animal pharmacological manipulation of specific host immune responses.

We have identified novel pathways that modulate susceptibility to tuberculosis. We have shown that genes identified in the zebrafish model are also important in human tuberculosis. We find robust associations of human variants in a specific eicosanoid pathway with susceptibility to both tuberculosis and leprosy.

We have active collaborations in both Vietnam and Guatemala. In Guatemala, we are working with the Clínica Familiar Luis Angel García and the Asociación de Salud Integral to support projects involving HIV-infected patients and to understand the dynamics of TB transmission in Central America.


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