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Quantitative proteomics of the infectious and replicative forms of Chlamydia trachomatis

Quantitative proteomics of the infectious and replicative forms of Chlamydia trachomatis
Quantitative proteomics of the infectious and replicative forms of Chlamydia trachomatis
The obligate intracellular developmental cycle of Chlamydia trachomatis presents significant challenges in defining its proteome. In this study we have applied quantitative proteomics to both the intracellular reticulate body (RB) and the extracellular elementary body (EB) from C. trachomatis. We used C. trachomatis L2 as a model chlamydial isolate for our study since it has a high infectivity:particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantified by chromosomal and plasmid copy number using PCR, from which the concentrations of chlamydial proteins per bacterial cell/genome were determined. RBs harvested at 15h post infection (PI) were purified by three successive rounds of gradient centrifugation. This is the earliest possible time to obtain purified RBs, free from host cell components in quantity, within the constraints of the technology. EBs were purified at 48h PI. We then used two-dimensional reverse phase UPLC to fractionate RB or EB peptides before mass spectroscopic analysis, providing absolute amount estimates of chlamydial proteins. The ability to express the data as molecules per cell gave ranking in both abundance and energy requirements for synthesis, allowing meaningful identification of rate-limiting components. The study assigned 562 proteins with high confidence and provided absolute estimates of protein concentration for 489 proteins. Interestingly, the data showed an increase in TTS capacity at 15h PI. Most of the enzymes involved in peptidoglycan biosynthesis were detected along with high levels of muramidase (in EBs) suggesting breakdown of peptidoglycan occurs in the non-dividing form of the microorganism. All the genome-encoded enzymes for glycolysis, pentose phosphate pathway and tricarboxylic acid cycle were identified and quantified; these data supported the observation that the EB is metabolically active. The availability of detailed, accurate quantitative proteomic data will be invaluable for investigations into gene regulation and function.
1932-6203
1-17
Skipp, Paul J.S.
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Hughes, Chris
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McKenna, Thérèse
4bb2217b-44f5-4ee4-bae0-d9b685efa023
Edwards, Richard
9d25e74f-dc0d-455a-832c-5f363d864c43
Langridge, James
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Thomson, Nicholas R.
5497a110-069d-4156-bccb-c77db572b1c2
Clarke, Ian N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Skipp, Paul J.S.
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5
Hughes, Chris
f7d4e979-a7f9-4b39-9423-b53da62b6f24
McKenna, Thérèse
4bb2217b-44f5-4ee4-bae0-d9b685efa023
Edwards, Richard
9d25e74f-dc0d-455a-832c-5f363d864c43
Langridge, James
d795a34c-5487-45de-b7b9-c282365b095a
Thomson, Nicholas R.
5497a110-069d-4156-bccb-c77db572b1c2
Clarke, Ian N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b

Skipp, Paul J.S., Hughes, Chris, McKenna, Thérèse, Edwards, Richard, Langridge, James, Thomson, Nicholas R. and Clarke, Ian N. (2016) Quantitative proteomics of the infectious and replicative forms of Chlamydia trachomatis. PLoS ONE, 11 (2), 1-17. (doi:10.1371/journal.pone.0149011). (PMID:26871455)

Record type: Article

Abstract

The obligate intracellular developmental cycle of Chlamydia trachomatis presents significant challenges in defining its proteome. In this study we have applied quantitative proteomics to both the intracellular reticulate body (RB) and the extracellular elementary body (EB) from C. trachomatis. We used C. trachomatis L2 as a model chlamydial isolate for our study since it has a high infectivity:particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantified by chromosomal and plasmid copy number using PCR, from which the concentrations of chlamydial proteins per bacterial cell/genome were determined. RBs harvested at 15h post infection (PI) were purified by three successive rounds of gradient centrifugation. This is the earliest possible time to obtain purified RBs, free from host cell components in quantity, within the constraints of the technology. EBs were purified at 48h PI. We then used two-dimensional reverse phase UPLC to fractionate RB or EB peptides before mass spectroscopic analysis, providing absolute amount estimates of chlamydial proteins. The ability to express the data as molecules per cell gave ranking in both abundance and energy requirements for synthesis, allowing meaningful identification of rate-limiting components. The study assigned 562 proteins with high confidence and provided absolute estimates of protein concentration for 489 proteins. Interestingly, the data showed an increase in TTS capacity at 15h PI. Most of the enzymes involved in peptidoglycan biosynthesis were detected along with high levels of muramidase (in EBs) suggesting breakdown of peptidoglycan occurs in the non-dividing form of the microorganism. All the genome-encoded enzymes for glycolysis, pentose phosphate pathway and tricarboxylic acid cycle were identified and quantified; these data supported the observation that the EB is metabolically active. The availability of detailed, accurate quantitative proteomic data will be invaluable for investigations into gene regulation and function.

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Accepted/In Press date: 26 January 2016
e-pub ahead of print date: 12 February 2016
Published date: 12 February 2016
Organisations: Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 388544
URI: https://eprints.soton.ac.uk/id/eprint/388544
ISSN: 1932-6203
PURE UUID: 2c4baea7-c5c7-4300-9ec9-2ca5c09fb405
ORCID for Paul J.S. Skipp: ORCID iD orcid.org/0000-0002-2995-2959
ORCID for Ian N. Clarke: ORCID iD orcid.org/0000-0002-4938-1620

Catalogue record

Date deposited: 29 Feb 2016 11:15
Last modified: 10 Dec 2019 01:59

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