Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis.
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2016-03-22
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Abstract
Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, includingChlamydia trachomatis Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogenC. trachomatislacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against aC. trachomatisgenomic library using a conditional-lethallpxHmutantEscherichia colistrain, we have identified an open reading frame (Ct461, renamedlpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function inE. coliand catalyzes the conversion of UDP-DAGn to lipid Xin vitro LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity ofC. trachomatis, validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools forC. trachomatisand should be broadly applicable for the functional characterization of other essentialC. trachomatisgenes.IMPORTANCEChlamydia trachomatisis a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of manyChlamydiagenes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against theC. trachomatisgenomic library using a conditional-lethal mutant ofE. coliand identified the missing essential gene in the lipid A biosynthetic pathway, which we designatedlpxG We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activityin vitro Overexpression of LpxG inC. trachomatisleads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating thein vivofunction of LpxG and highlighting the importance of regulated lipid A biosynthesis inC. trachomatis.
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Young, Hayley E, Jinshi Zhao, Jeffrey R Barker, Ziqiang Guan, Raphael H Valdivia and Pei Zhou (2016). Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis. MBio, 7(2). p. e00090. 10.1128/mBio.00090-16 Retrieved from https://hdl.handle.net/10161/11783.
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Ziqiang Guan
We develop and apply mass spectrometry techniques to address biochemical and biomedical questions that are lipid-related. Research projects include:
1) Structural lipidomics
o Develop and apply high resolution tandem mass spectrometry-based lipidomics for the discovery, structural elucidation and functional study of novel lipids.
2) Elucidation of novel pathways/enzymes of lipid biosynthesis and metabolism
o Genetic, biochemical and MS approaches are employed to identify the substrates and pathways involved in lipid biosynthesis and metabolism
3) Identification of lipid biomarkers of genetic diseases and cancers
o Provide molecular insights into the disease mechanisms, as well as to serve as the diagnostic and prognostic tools of diseases.
Raphael H. Valdivia
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.
Pei Zhou
The Zhou lab focuses on the elucidation of the structure and dynamics of protein–protein and protein–ligand interactions and their functions in various cellular processes. Our current efforts are directed at enzymes and protein complexes involved in bacterial membrane biosynthesis, translesion DNA synthesis, co-transcriptional regulation, and host-pathogen interactions. Our investigations of these important cellular machineries have led to the development of novel antibiotics and cancer therapeutics, as well as the establishment of new biotechnology adventures.
The Zhou lab integrates a variety of biochemical and biophysical tools, including NMR, X-ray crystallography, cryo-EM, and enzymology. The lab has played a major role in the development and application of innovative NMR technologies, including high-resolution, high-dimensional spectral reconstruction techniques.
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