Functional conservation of the lipid II biosynthesis pathway in the cell wall‐less bacteria <i>Chlamydia</i> and <i>Wolbachia</i>: why is lipid II needed?

Henrichfreise, Beate; Schiefer, Andrea; Schneider, Tanja; Nzukou, Edith; Poellinger, Christina; Hoffmann, Theo-Julian; Johnston, Kelly L.; Moelleken, Katja; Wiedemann, Imke; Pfarr, Kenneth; Hoerauf, Achim; Sahl, Hans Georg

doi:10.1111/j.1365-2958.2009.06815.x

Cited by 67 publications

(99 citation statements)

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“…The presence of functional MraY enzymes in the reduced proteome of the cell wall-less Chlamydia cells was for a long time a matter of debate and only recently the presence and function of the enzyme was demonstrated (16). It is speculated that the function of MraY in C. pneumoniae might be necessary for cell division, rather than for biosynthesis of peptidoglycan in its classical function as a osmotic stabilizing cell wall envelope.…”

Section: Discussionmentioning

confidence: 99%

“…Genes for H. pylori MraY (Hp-MraY), B. pertussis MraY (Bp-MraY), and B. burgdorferi (Bb-MraY) were synthesized by Geneart optimized for E. coli codon usage and cloned into vector pET21a. C. pneumonia MraY (Cp-MraY) was amplified as described previously (16) and cloned into vector pET52b. Expression vectors and purification tags of the expressed proteins are specified in Table 1.…”

Section: Methodsmentioning

confidence: 99%

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Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Henrich

Engels

et al. 2016

Journal of Biological Chemistry

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Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cellfree expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were cotranslationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY⅐nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.Proteins involved in bacterial cell wall biosynthesis are highly conserved and often essential. In stage I, the cytoplasmic proteins MurA-F are responsible for the formation of the soluble peptidoglycan precursor (Park's nucleotide). In stage II, the integral membrane protein MraY catalyzes the ligation of Park's nucleotide with the membrane-bound undecaprenylphosphate (C 55 -P) 4 resulting into lipid I. The membrane-associated globular protein MurG catalyzes subsequent glycosyl transfer forming lipid II (Fig. 1). As the final monomeric building block in bacterial cell wall biosynthesis, lipid II will be transported through the membrane into the periplasmic space for further polymerization in stage III of cell wall biosynthesis (1).The bacterial cell wall biosynthetic pathway is an important and traditional target for antibiotics and numerous drugs directed against key catalysts for clinical applications have been developed. However, most known drugs such as the penicillin derivatives affect the periplasmic or extracellular steps of peptidoglycan synthesis catalyzed by soluble enzymes. In contrast, almost no clinical drugs have been developed that are targeted against the integral membrane proteins involve...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Henrich

Engels

et al. 2016

Journal of Biological Chemistry

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“…Interestingly, all the members of the heme biosynthetic pathway, except for the ferrochelatase (Wbm0719) and the protoporphyrinogen oxidase, which is absent from the Wolbachia genome, were detected in the present proteomic analysis. Although wBm is devoid of a cell wall, the functional cellular machinery for the synthesis of lipid-II exists (45). Proteomic analysis identified each of the components involved in lipid-II synthesis.…”

Section: Resultsmentioning

confidence: 99%

Stage-specific proteomic expression patterns of the human filarial parasiteBrugia malayiand its endosymbiontWolbachia

Bennuru

Meng

Ribeiro

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

123

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Global proteomic analyses of pathogens have thus far been limited to unicellular organisms (e.g., protozoa and bacteria). Proteomic analyses of most eukaryotic pathogens (e.g., helminths) have been restricted to specific organs, specific stages, or secretomes. We report here a large-scale proteomic characterization of almost all the major mammalian stages of Brugia malayi, a causative agent of lymphatic filariasis, resulting in the identification of more than 62% of the products predicted from the Bm draft genome. The analysis also yielded much of the proteome of Wolbachia, the obligate endosymbiont of Bm that also expressed proteins in a stage-specific manner. Of the 11,610 predicted Bm gene products, 7,103 were definitively identified from adult male, adult female, blood-borne and uterine microfilariae, and infective L3 larvae. Among the 4,956 gene products (42.5%) inferred from the genome as "hypothetical," the present study was able to confirm 2,336 (47.1%) as bona fide proteins. Analysis of protein families and domains coupled with stage-specific expression highlight the important pathways that benefit the parasite during its development in the host. Gene set enrichment analysis identified extracellular matrix proteins and those with immunologic effects as enriched in the microfilarial and L3 stages. Parasite sex-and stage-specific protein expression identified those pathways related to parasite differentiation and demonstrates stage-specific expression by the Bm endosymbiont Wolbachia as well.filaria | nematode D isease associated with infection by Brugia malayi (Bm) and Wuchereria bancrofti, the two major causative organisms of human lymphatic filariasis, is the second leading cause of morbidity/disability worldwide, in large part because of the parasites' ability to alter the structural and functional integrity of the lymphatics, leading to lymphedema and elephantiasis. Invasion, establishment of infection within the host and development are essential processes within the complex parasite life cycle (SI Appendix, Fig. S1), with many of the parasitic stages being targets for therapeutic intervention or vaccines. Each of the filarial life cycle stages has characteristics that are shared and others that are stage-specific.Filarial infections are often characterized by a series of discrete host responses directed at the parasite and its endosymbiont Wolbachia that evolve during the course of infection. Because proteins are usually the effectors of most biological functions, proteomic data enable a more direct understanding of these important processes compared with those inferred from genomic studies. Absolute quantification of genome-wide expressed proteins is not yet within our reach for most eukaryotes. However, spectral counts of massive MS-based data (e.g., observed frequencies of each peptide) allow for relative quantification. Proteomic data also allow for clearer genomic curation by improving annotation and the identification of translational sites, stop codon read-throughs, frame shifts, and predict...

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“…and wBm (Henrichfreise et al 2009), with the important exception of the bifunctional N-succinyldiaminopimelate-aminotransferase/ acetylornithine transaminase gene, argD, which is pseudogenized in wOo (Supplemental Table S5). …”

Section: Resultsmentioning

confidence: 99%

Analysis of gene expression from theWolbachiagenome of a filarial nematode supports both metabolic and defensive roles within the symbiosis

et al. 2012

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The a-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.

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Functional conservation of the lipid II biosynthesis pathway in the cell wall‐less bacteria Chlamydia and Wolbachia: why is lipid II needed?

Cited by 67 publications

References 37 publications

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Stage-specific proteomic expression patterns of the human filarial parasiteBrugia malayiand its endosymbiontWolbachia

Analysis of gene expression from theWolbachiagenome of a filarial nematode supports both metabolic and defensive roles within the symbiosis

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