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Subproteomic Fraction Approach Results in 'Most Comprehensive Quantification' of S. Aureus

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This story originally ran on Dec. 16.

By Tony Fong

By integrating data from four different subproteome fractions, German and Dutch researchers have created a proteomic map of Staphylococcus aureas that they called the "most comprehensive quantitative study of a bacterial genome" to date.

The work, described in a study published Dec. 4 in PLoS One, enabled the team to identify 1,703 proteins and 80 percent of the expressed genes for S. aureas, paving the way "toward a new level in understanding of cell physiology and pathophysiology" of the bacterium and related pathogenic bacteria, the researchers said.

For their work, they combined four subproteomic fractions — cytosolic proteins, membrane-bound proteins, cell surface-associated proteins, and extracellular proteins — in order to obtain comprehensive coverage of the bacterium.

The study, which the scientists said integrates data "ranging from gene expression to subcellular localization" in growing and non-growing cells, demonstrates a proof-of-concept for their "whole-cell physiological proteomics" approach that can "now be extended to address physiological questions in infection-relevant settings."

Dörte Becher, the first author of the study, told ProteoMonitor that she and her colleagues chose the four categories of proteins because they wanted to find out not only whether certain proteins were expressed and under what conditions, but also where the proteins were located.

"If a protein is situated on the surface, [for example], it may play an important role in the interaction between the host and the pathogen," said Becher, who is a PhD candidate in microbiology at the Institute for Microbiology at Ernst-Mortiz-Arndt University in Germany. "If certain proteins … are expressed on the surface, when they are in touch with the host, you can really see if these proteins are expressed only on the surface."

While she said that the approach used by her and her colleagues was "a lot of work," and more labor-intensive than if they had investigated a single subproteome, it resulted in deeper coverage and possibly opens the door to finding new ways to attack the bacterium, which has become a major concern in the healthcare arena as some strains of S. aureus have become antibiotic-resistant.

The bacterium is associated with a wide range of infectious diseases — from minor skin conditions, such as pimples, to more life-threatening ailments such as pneumonia and toxic shock syndrome.

In recent years, the number of cases involving a particularly difficult-to-treat strain, methicillin-resistant S. aureus, or MRSA, has also been on the increase, resulting in thousands of deaths. According to a 2007 article in the Journal of the American Medical Association, more than 94,000 serious cases of MRSA were estimated to have occurred in the US in 2005, leading to nearly 19,000 deaths.

While the authors of the PLoS One study have performed extensive proteomics research into S. aureus, this current work represents their most extensive effort, made possible only by recent improvements to the sensitivity of mass spectrometers, Becher said. Much of the prior work was gel-based.

"Now that we have the tools … we decided to do it now on S. aureus because it is truly important for medical research," she said.

The study was not aimed at developing new therapeutics against S. aureus-related infections, but Becher said that as the research opens new knowledge about the organism and its pathogenicity, new treatments may follow.

"Since therapeutic strategies directed against S. aureus could either target extracellular and secreted proteins or the pathogen's metabolism, we integrated the analysis of the different subproteomes to provide evidence that an integrated proteomic view of cell physiology and virulence will help come to a new quality in the understanding of general cellular processes in S. aureus," the researchers wrote.

Multiple Subproteomes, Multiple Views

For their work, which analyzed the proteome pattern in growing and non-growing cells, the researchers first investigated cytosolic proteins using gel-based and LC-MS/MS approaches. They identified about 1,190 proteins, including 500 new ones with an approach combining 1D gel with LC-MS/MS that they dubbed GeLC-MS/MS. These included mostly low-abundance proteins, "but also very acidic or alkaline, as well as very large or small proteins," they wrote.

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They also identify some proteins that they suggest may be "vulnerable to proteolytic attack" — including ribosomal proteins, translational factors, and enzymes for amino acid biosynthesis — though their molecular mechanisms still need to be studied.

They then went on to membrane proteins, using a method that combined GeLC-MS/MS and a "shaving approach." This resulted in the identification of "a large fraction of cytosolic proteins," as well proteins with one to 21 transmembrane proteins. The researchers noted that the shaving approach was the only method that allowed them to identify "the most hydrophobic species of membrane-spanning peptides, usually lacking lysine- and arginine-residues as targets for tryptic digestion."

The work enabled them to quantify for the first time the membrane proteome of S. aureus, they added. Glycerol uptake facilitator, GlpF, was up-regulated, as were two transporters importing C3 carbon sources, phosphoglycerate, and glycerol -3-phosphate.

Four cytosolic enzymes "acting downstream of C3 catabolite uptake were also detected in increased quantity in the membrane fraction,” the researchers reported, “indicating that "the glycerol consuming pathway forms enzyme complexes associated with the membrane-bound uptake facilitator."

Previous studies have tied glycerol consumption with virulence in two close relatives of S. aureus Listeria monocytogenes and Mycoplasma pneumonia — and further research in glycerol use in S. aureus and its potential involvement in pathogenicity is needed, they added.

Lastly, they investigated proteins bound to the cell surface, and those secreted into the extracellular space. These proteins, Becher and her colleagues said, "form the most crucial class of proteins in S. aureus" from an infection-related point of view "because most of the virulence factors can be found in these two subproteomic fractions."

They identified 146 cell surface-associated proteins, the highest coverage of such proteins to have been identified and quantified to date. This included 48 membrane proteins, four proteins covalently bonded to peptidoglycan, 37 lipoproteins, and 57 cell wall-associated proteins containing a single peptide.

The researchers also identified 57 extracellular proteins, but said not all proteins "characterized by an N-terminal signal sequence and signal peptidase I cleavage site will be secreted to the extracellular space."

Such Sec-type proteins — or those that stay attached to the membrane despite the presence of a signal peptide that governs protein secretion — have recently been discovered, though "it is still difficult to predict the proteins that belong to this class," the researchers wrote.

"Importantly, the present panorama view of the S. aureus secretome can now be used to address crucial issues, such as the assignment of secreted proteins to virulence regulons, or assessment of the virulence potential of well-defined clinical isolates," they said.

As they continue their research, Becher said she and her co-authors plan to carry out "timeline experiments" with multiple timepoints for events "to get much more detailed information about the dynamics [of] the proteome."

They also are working on improving the sensitivity of their approach to get to the low-abundance proteins. Calling this a "difficult" task, Becher said one strategy they are exploring includes methods that perform more fractionation on the proteins.

As a whole, the data generated from the research can now be the basis for a more targeted study, she said. "If we want to analyze what is going on with this or that protein, which role a [certain protein] plays, and a certain process in infection or host-cell interaction , or even by forming of biofilms …we can now [do] that targeted analysis of that protein," she said.

She and her colleagues added in the study that "the future challenge will now be to transfer this huge proteome data to new hypotheses, which will finally generate new physiological knowledge." Protein expression profiling in response to environmental stimuli can now be done with high proteomic coverage "to identify almost all proteins that make the cell viable," they said, and "the virulence protein inventory of a cell can now be captured, analyzed and quantified."

In addition, by using a combination of gel-based and gel-free proteomics, "the fate and final destination of each single protein can be followed at a proteome-wide scale," they added, suggesting that the dynamics of the membrane proteome, phosphoproteome and its role in signal transduction can be studied, "thereby opening a new era in infection-related research."

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