Immucillin-H, a purine nucleoside phosphorylase transition state analog, causes non-lethal attenuation of growth in Staphylococcus aureus

Abstract

Purine nucleoside phosphorylase (PNP; EC: 2.4.2.1) is a key enzyme involved in the purine salvage pathway. A recent bioinformatic study by Yadav, P. K. et al. (Bioinformation 2012, 8(14), 664–672) reports PNP as an essential enzyme and potential drug target in community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). We conducted an analysis using the methodology outlined by the authors, but were unable to identify PNP as an essential gene product in CA-MRSA. In addition, the treatment of Staphylococcus aureus cultures with immucillin-H, a powerful inhibitor of PNP, resulted in the non-lethal attenuation of growth, suggesting that PNP activity is not essential for cell viability.

Background

A recent study by Yadav, P. K. et al. used a bioinformatic approach to identify potential drug targets in communityacquired methicillin-resistant Staphylococcus aureus (CA-MRSA) [1]. In this work, the BLASTp program was used to search metabolically relevant gene products in CA-MRSA against the proteome of Homo sapiens. A collection of non-homologous gene products was generated and further refined by the identification of essential genes in the DEG 6.8 database [2]. Using this approach, Yadav, P. K. et al. identified purine nucleoside phosphorylase (PNP; EC: 2.4.2.1) as an essential enzyme and potential drug target in CA-MRSA [1].

PNP catalyzes the reversible phosphorolysis of 6-oxypurine ribonucleosides to the corresponding purine nucleobases and ribose 1-phosphate (Figure 1A) [3]. PNP is a key enzyme involved in the purine salvage pathway and is essential in humans for the formation of uric acid [3]. Our laboratory has previously investigated the catalytic mechanism and transition state structure of PNP (Figure 1B) using kinetic isotope effect (KIE) analysis [4, 5]. Transition state analysis was used in the design of transition state analogs for PNP, including immucillin-H (ImmH; Figure 1C), which is a potent inhibitor of bovine PNP (K_i = 23 pM) [6], human PNP (K_i = 58 pM) [5], and bacterial PNP (K_i = 28 pM; M. tuberculosis) [7]. ImmH is currently in clinical trials for the treatment of leukemia [8, 9].

Figure 1.

PNP-catalyzed Phosphorolysis of Inosine and PNP Transition State Structure. (A) PNP catalyzes the reversible phosphorolysis of purine nucleosides to form ribose 1- phosphate and a purine nucleobase. Here, the PNP-catalyzed phosphorolysis of inosine is shown. (B) Transition state structure of the PNP-catalyzed phosphorolysis of inosine. (C) Immucillin H (ImmH) is a transition state analog and powerful inhibitor of PNP.

Yadav, P. K. et al. used non-homology between gene products in CA-MRSA and Homo sapiens as a key parameter to select potential drug targets. We were interested to find the authors identified PNP as a candidate despite the presence of PNP homologs in both CA-MRSA (NCBI Gene ID: 1004861) and Homo sapiens (NCBI Gene ID: 4860). An independent analysis of the DEG 6.8 database following the methodology outlined by Yadav, P. K. et al. was unable to identify PNP as an essential enzyme in CA-MRSA. Moreover, we conducted a series of cellular growth assays using a laboratory strain of Staphylococcus aureus and ImmH, a potent inhibitor of PNP developed by our laboratory. These assays indicate that ImmH causes non-lethal attenuation of growth in Staphylococcus aureus, suggesting that PNP activity is not essential to the organism.

Methodology

Database searches:

The DEG 6.8 database was accessed at URL: http://www.essentialgene.org and searches were executed for DEG accession number: DEG10020139.

Staphylococcus aureus growth assays with ImmH:

The effect of ImmH on Staphylococcus aureus subsp. aureus Rosenbach growth was assessed by comparison of bacterial cultures grown on solid media (LB agar) in the presence and absence of inhibitor. Control plates (no inhibitor) were prepared using an LB agar mix (Fisher Scientific) with no additional additives; plates containing inhibitor were prepared by adding ImmH to molten LB agar (final concentration of ImmH = 100 µM) just prior to pouring. Each plate of solid media was inoculated with Staphylococcus aureus using 30 µL of a culture in liquid LB. The inoculated plates were incubated at 37 °C for 18 h and then removed from the incubator for analysis (Figure 2A, B). The plates were returned to the incubator for an additional 24 h (42 h total) and then removed for analysis (Figure 2C, D). All experiments on solid media were performed in triplicate and cell culture work was carried out under aseptic conditions in a laminar flow hood.

Figure 2.

Effect of ImmH on Staphylococcus aureus Growth. (A) Growth of Staphylococcus aureus after 18 h incubation at 37 °C (Control / no ImmH). (B) Growth of Staphylococcus aureus in the presence of 100 µM ImmH after 18 h incubation at 37 °C. (C) Growth of Staphylococcus aureus after 42 h incubation at 37 °C (Control/no ImmH). (D) Growth of Staphylococcus aureus in the presence of 100 µM ImmH after 42 h incubation at 37 °C.

Discussion

Yadav, P. K. et al. report PNP (EC: 2.4.2.1) is an essential enzyme in Staphylococcus aureus N315 based on identification of the gene pnp (DEG10020139) in the DEG 6.8 database [1]. A search of the DEG database for accession number DEG10020139 returned an entry for the gene pnpA (NCBI Gene ID: 1123948), which codes for polyribonucleotide nucleotidyltransferase (EC: 2.7.7.8), an enzyme that exhibits 3´-to-5´ exonuclease activity. A subsequent search of the DEG database for essential genes in Staphylococcus aureus N315 returned 302 entries (see supplementary material). Examination of these genes failed to identify an entry for PNP (EC: 2.4.2.1). Based on these findings, we believe PNP (EC: 2.4.2.1) was incorrectly reported as an essential gene product in Staphylococcus aureus N315.

To investigate further the essentiality of PNP, we used ImmH to probe the effect of PNP inhibition on Staphylococcus aureus growth. It was found that the growth of Staphylococcus aureus cultures after 18 h at 37 °C was attenuated (relative to controls) in the presence of 100 µM ImmH (Figure 2A, B). This observation is consistent with ImmH acting to inhibit PNP and block the purine salvage pathway. When the same cultures were incubated for an additional 24 h at 37 °C (42 h total), bacterial growth recovered and appeared indistinguishable from the controls (Figure 2C, D). These data suggest the inhibition of PNP is non-lethal to Staphylococcus aureus, but rather only slows bacterial growth. Based on these results, we propose that PNP is not an essential enzyme in Staphylococcus aureus and that the inhibition of PNP activity can be compensated for by de novo nucleoside biosynthesis.

Conclusion

In conclusion, we believe PNP (EC: 2.4.2.1) was incorrectly cited as an essential enzyme in CA-MRSA by Yadav, P. K. et al. A follow-up analysis using the methodology outlined by the authors was unable to identify PNP as an essential enzyme in CA-MRSA, but instead found the correct citation should be for polyribonucleotide nucleotidyltransferase (EC: 2.7.7.8). In addition, cellular growth assays using ImmH, a potent inhibitor of PNP, suggest that inhibition of PNP activity is not lethal to Staphylococcus aureus. These data further suggest that PNP is not an essential enzyme in Staphylococcus aureus and thus, may be of limited use as a therapeutic target in CA-MRSA.