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A New S. aureus Virulence Factor

 

By: Carol A. Rouzer, VICB Communications
Published:  April 6, 2018

 

Activity-based protein profiling reveals previously uncharacterized hydrolases - one of which promotes infection of heart and liver.

 

Although Staphylococcus aureus (Figure 1) occupies a niche in the skin microbiome of ~30% of healthy people, if it penetrates the skin's protective barrier, it can cause severe, often life-threatening infections. Hydrolytic enzymes play an important metabolic role in the ability of all pathogenic bacteria to colonize a host. Among these are the α,β-hydrolase class of serine hydrolases, characterized by a structural domain comprising eight β-strands connected by six α-helices. These enzymes also contain a catalytic triad composed of an acid residue, a basic residue, and a nucleophilic residue, which in the case of serine hydrolases, is serine. Despite the growing realization of the importance of these enzymes in mammalian metabolism, we know almost nothing about them in S. aureus. To address this problem, Vanderbilt Institute of Chemical Biology (VICB) member Eric Skaar and his postdoc Jessica Sheldon paired with Matthew Bogyo (Stanford University School of Medicine) and his postdoc Christian Lentz to identify α,β-hydrolases and explore their function in S. aureus infection [C. S. Lentz, J.R. Sheldon, et al. (2018) Nat. Chem. Biol, published online May 16, doi: 10.1038/s41589-018-0060-1].

 

 

FIGURE 1. Electron micrograph of S. aureus bacteria (gold) escaping from a white blood cell (blue). Figure is reproduced from NIAID and is in the public domain.

 

 

A characteristic of S. aureus is its ability to form biofilms on surfaces of internal organs or prosthetic devices. These films, which are made up of bacteria embedded in a complex matrix, convey antibiotic resistance and enable S. aureus to withstand attack by the immune system. Due to the importance of biofilm formation to bacterial virulence, the researchers decided to identify serine hydrolases expressed by the bacteria grown under conditions that promote biofilm formation. To accomplish their goal, they took advantage of the fact that serine hydrolases have been studied for decades, leading to the development of numerous reactive electrophilic compounds that can be used to label enzymes in this class. The investigators decided to use such a probe, fluorophosphonate tetramethylrhodamine (FP-TMR), employing a technique known as activity-based protein profiling (ABPP) to identify serine hydrolases in S. aureus (Figure 2a).

 

FIGURE 2. Diagrammatic representation of the ABPP approach. (a) Bacterial cells are incubated with FP-TMR (red stars), which covalently labels the active site serine residues (-OH) on serine hydrolase enzymes. The cells are lysed, and proteins are separated by gel electrophoresis. Labeled proteins are visualized by fluorescence. (b) Diagrammatic representation of the screen for compounds that bind to bacterial serine hydrolases. Bacteria are preincubated with compounds that covalently bind to serine hydrolases (colored hexagons). If a test compound binds to a hydrolase, it prevents labeling by FP-TMR, which is added next. Any protein to which a compound has bound will not be detected on subsequent gel electrophoresis, which is revealed by comparison to a gel obtained from bacteria incubated with FP-TMR in the absence of any added compounds. Figure reproduced by permission from Nature America and Springer Nature from C. S. Lentz, J.R. Sheldon, et al. (2018) Nat. Chem. Biol, published online May 16, doi: 10.1038/s41589-018-0060-1. Copyright 2018.

 

 

 

They initiated their studies by incubating the bacteria with FP-TMR, and then using the probe's fluorescence to visualize all of the labeled proteins by gel electrophoresis. Next, they screened a library of ~500 compounds that were designed to covalently bind to serine hydrolases, in a search for molecules that blocked FP-TMR labeling (Figure 2b). From this search, they identified two compounds, JCP251 and JCP678 (Figure 3), which very potently blocked labeling of a 36 kDa protein and a 28 kDa protein, respectively.

 


FIGURE 3. Structures of JCP251 and JCP678. Figure reproduced by permission from Nature America and Springer Nature from C. S. Lentz, J.R. Sheldon, et al. (2018) Nat. Chem. Biol, published online May 16, doi: 10.1038/s41589-018-0060-1. Copyright 2018.

 

 

To further characterize their labeled proteins, the investigators used a biotin-tagged fluorophosphonate probe, FP-biotin, to label all serine hydrolases in S. aureus. In this case, the biotin tag enabled the investigators to capture the proteins using streptavidin-coated beads. They then identified the proteins by tandem liquid-chromatography-mass spectrometry (LC/LC-MS/MS). Incubation of the bacteria with an inhibitor identified in their earlier screen prior to treatment with FP-Biotin eliminated labeling of the target protein, enabling the researchers to confirm its identity (Figure 4). The results yielded 12 proteins that were predicted to contain an α,β-fold. Two of these, SAL1 and SAL2, were known S. aureus lipases. The remaining ten had not been previously characterized, so the investigators named them fluorophosphonate-binding serine hydrolases (Fph) A through J, in order of decreasing molecular weight. FphB and FphF were the targets of JCP251 and JCP678, respectively.

 

FIGURE 4. Method used to identify α,β-hydrolase inhibitor targets in S. aureus. Cells were incubated with or without a hydrolase inhibitor (green hexagon) and then labeled with FP-biotin (blue star). Proteins bearing the biotin tag were then isolated by adherence to streptavidin beads and identified by LC/LC-MS/MS. The α,β-hydrolase targets were distinguished by their presence in lysates from cells not preincubated with the inhibitor but absent in lysates from cells that had been preincubated with the inhibitor. Figure reproduced by permission from Nature America and Springer Nature from C. S. Lentz, J.R. Sheldon, et al. (2018) Nat. Chem. Biol, published online May 16, doi: 10.1038/s41589-018-0060-1. Copyright 2018.

 

To further confirm the identity of their hydrolases, the researchers used a transposon mutant library of S. aureus, comprising bacterial strains in which the genes encoding the individual proteins had been mutated. For 8 of the 12 proteins, they were able to confirm that the bacteria bearing a mutation in the encoding gene lacked an appropriate band on gel electrophoresis.
     

Among the proteins identified, FphB stood out because of its high activity and the availability of the potent and selective inhibitor, JCP251. These considerations led the investigators to focus their attention on this enzyme. Transcriptomic analyses demonstrated that expression of FphB was upregulated in response to cell-wall-targeted antibiotics and antimicrobial peptides in addition to other environmental stressors. Tests of its activity with a range of substrates revealed that the enzyme preferred short chain (C4>C7>C8) fatty acid esters, displaying no activity for peptides, glycosides, phosphates, or phosphonates. The researchers synthesized a fluorescent probe based on the structure of JCP251 (JCP261-bT) and demonstrated that the molecule bound to FphB with high selectivity. They used the probe to explore the patterns of FphB expression in S. aureus. Results showed that enzyme expression increased in the presence of RAW264.7 murine macrophage-like cells in addition to other human cell lines and Fetalplex Animal Serum Complex. Further studies suggested that the trigger inducing expression under these conditions was a heat-stable cell surface component, possibly a lipid. A somewhat surprising finding was that only about 10% of cells in an S. aureus population strongly expressed the protein (Figure 5), and that a subset of dividing cells exhibited particularly strong expression in the septal wall, a location of cell wall biogenesis (Figure 6).

 

 

FIGURE 5. S. aureus expressing green fluorescent protein (green) were also labeled with JCP251-bT (violet) to identify cells expressing FphB. Note the number of cells that do not show strong expression of FphB as indicated by the lack of violet staining. Figure reproduced by permission from Nature America and Springer Nature from C. S. Lentz, J.R. Sheldon, et al. (2018) Nat. Chem. Biol, published online May 16, doi: 10.1038/s41589-018-0060-1. Copyright 2018.

 

 

 

FIGURE 6. S. aureus expressing green fluorescent protein (green, center) were also labeled with JCP251-bT (violet, left) to identify cells expressing FphB. The merged image (right) shows the concentration of FphB at the septal wall of dividing cells. Figure reproduced by permission from Nature America and Springer Nature from C. S. Lentz, J.R. Sheldon, et al. (2018) Nat. Chem. Biol, published online May 16, doi: 10.1038/s41589-018-0060-1. Copyright 2018.

 

 

Bacteria bearing an inactivating mutation of the gene encoding FphB grew normally in culture. However, they exhibited a reduced ability to colonize the heart and liver in a mouse model of S. aureus infection. This deficit was not global, however, as colonization of the kidneys was not affected by loss of FphB expression. The exact role of FphB in S. aureus metabolism is not yet known, but it is likely that it is involved in some aspect of cell membrane or cell wall metabolism. Research into the function of FphB is ongoing.
     

Together, the results provide important new information on a range of α,β-hydrolases in S. aureus. More specifically, the findings highlight the power of ABPP to identify and characterize serine hydrolases, such as FphB, that are important in S. aureus infection. This study helps to lay the groundwork for the future development of probes and inhibitors of serine hydrolases that can be used in the identification, monitoring and treatment of bacterial infections. This remains a very interesting topic for future work.


 

View Nature Chemical Biology article: Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP)

 

 

 

 

 

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