Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in <named-content content-type="genus-species">Chlamydia trachomatis</named-content>

ABSTRACT Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, including Chlamydia trachomatis. Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cl...

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Autores principales:	Hayley E. Young, Jinshi Zhao, Jeffrey R. Barker, Ziqiang Guan, Raphael H. Valdivia, Pei Zhou
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/482e2b09ddd54b878e07c4c7aa0035b7
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spelling	oai:doaj.org-article:482e2b09ddd54b878e07c4c7aa0035b72021-11-15T15:41:41ZDiscovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in <named-content content-type="genus-species">Chlamydia trachomatis</named-content>10.1128/mBio.00090-162150-7511https://doaj.org/article/482e2b09ddd54b878e07c4c7aa0035b72016-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00090-16https://doaj.org/toc/2150-7511ABSTRACT Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, including Chlamydia 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 pathogen C. trachomatis lacks 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 a C. trachomatis genomic library using a conditional-lethal lpxH mutant Escherichia coli strain, we have identified an open reading frame (Ct461, renamed lpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function in E. coli and catalyzes the conversion of UDP-DAGn to lipid X in 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 of C. 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 for C. trachomatis and should be broadly applicable for the functional characterization of other essential C. trachomatis genes. IMPORTANCE Chlamydia trachomatis is a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of many Chlamydia genes 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 the C. trachomatis genomic library using a conditional-lethal mutant of E. coli and identified the missing essential gene in the lipid A biosynthetic pathway, which we designated lpxG. We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activity in vitro. Overexpression of LpxG in C. trachomatis leads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating the in vivo function of LpxG and highlighting the importance of regulated lipid A biosynthesis in C. trachomatis.Hayley E. YoungJinshi ZhaoJeffrey R. BarkerZiqiang GuanRaphael H. ValdiviaPei ZhouAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 2 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Hayley E. Young Jinshi Zhao Jeffrey R. Barker Ziqiang Guan Raphael H. Valdivia Pei Zhou Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in <named-content content-type="genus-species">Chlamydia trachomatis</named-content>
description	ABSTRACT Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, including Chlamydia 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 pathogen C. trachomatis lacks 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 a C. trachomatis genomic library using a conditional-lethal lpxH mutant Escherichia coli strain, we have identified an open reading frame (Ct461, renamed lpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function in E. coli and catalyzes the conversion of UDP-DAGn to lipid X in 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 of C. 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 for C. trachomatis and should be broadly applicable for the functional characterization of other essential C. trachomatis genes. IMPORTANCE Chlamydia trachomatis is a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of many Chlamydia genes 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 the C. trachomatis genomic library using a conditional-lethal mutant of E. coli and identified the missing essential gene in the lipid A biosynthetic pathway, which we designated lpxG. We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activity in vitro. Overexpression of LpxG in C. trachomatis leads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating the in vivo function of LpxG and highlighting the importance of regulated lipid A biosynthesis in C. trachomatis.
format	article
author	Hayley E. Young Jinshi Zhao Jeffrey R. Barker Ziqiang Guan Raphael H. Valdivia Pei Zhou
author_facet	Hayley E. Young Jinshi Zhao Jeffrey R. Barker Ziqiang Guan Raphael H. Valdivia Pei Zhou
author_sort	Hayley E. Young
title	Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in <named-content content-type="genus-species">Chlamydia trachomatis</named-content>
title_short	Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in <named-content content-type="genus-species">Chlamydia trachomatis</named-content>
title_full	Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in <named-content content-type="genus-species">Chlamydia trachomatis</named-content>
title_fullStr	Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in <named-content content-type="genus-species">Chlamydia trachomatis</named-content>
title_full_unstemmed	Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in <named-content content-type="genus-species">Chlamydia trachomatis</named-content>
title_sort	discovery of the elusive udp-diacylglucosamine hydrolase in the lipid a biosynthetic pathway in <named-content content-type="genus-species">chlamydia trachomatis</named-content>
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/482e2b09ddd54b878e07c4c7aa0035b7
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Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in <named-content content-type="genus-species">Chlamydia trachomatis</named-content>

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