CcpA regulates arginine biosynthesis in Staphylococcus aureus through repression of proline catabolism.

Staphylococcus aureus is a leading cause of community-associated and nosocomial infections. Imperative to the success of S. aureus is the ability to adapt and utilize nutrients that are readily available. Genomic sequencing suggests that S. aureus has the genes required for synthesis of all twenty amino acids. However, in vitro experimentation demonstrates that staphylococci have multiple amino acid auxotrophies, including arginine. Although S. aureus possesses the highly conserved anabolic pathway that synthesizes arginine via glutamate, we demonstrate here that inactivation of ccpA facilitates the synthesis of arginine via the urea cycle utilizing proline as a substrate. Mutations within putA, rocD, arcB1, argG and argH abolished the ability of S. aureus JE2 ccpA::tetL to grow in the absence of arginine, whereas an interruption in argJBCF, arcB2, or proC had no effect. Furthermore, nuclear magnetic resonance demonstrated that JE2 ccpA::ermB produced (13)C(5) labeled arginine when grown with (13)C(5) proline. Taken together, these data support the conclusion that S. aureus synthesizes arginine from proline during growth on secondary carbon sources. Furthermore, although highly conserved in all sequenced S. aureus genomes, the arginine anabolic pathway (ArgJBCDFGH) is not functional under in vitro growth conditions. Finally, a mutation in argH attenuated virulence in a mouse kidney abscess model in comparison to wild type JE2 demonstrating the importance of arginine biosynthesis in vivo via the urea cycle. However, mutations in argB, argF, and putA did not attenuate virulence suggesting both the glutamate and proline pathways are active and they, or their pathway intermediates, can complement each other in vivo

NMR Metabolomics Protocols for Drug Discovery

Drug discovery is an extremely difficult and challenging endeavor with a very high failure rate. The task of identifying a drug that is safe, selective and effective is a daunting proposition because disease biology is complex and highly variable across patients. Metabolomics enables the discovery of disease biomarkers, which provides insights into the molecular and metabolic basis of disease and may be used to assess treatment prognosis and outcome. In this regard, metabolomics has evolved to become an important component of the drug discovery process to resolve efficacy and toxicity issues, and as a tool for precision medicine. A detailed description of an experimental protocol is presented that outlines the application of NMR metabolomics to the drug discovery pipeline. This includes: (1) target identification by understanding the metabolic dysregulation in diseases, (2) predicting the mechanism of action of newly discovered or existing drug therapies, (3) and using metabolomics to screen a chemical lead to assess biological activity. Unlike other OMICS approaches, the metabolome is “fragile”, and may be negatively impacted by improper sample collection, storage and extraction procedures. Similarly, biologically-irrelevant conclusions may result from incorrect data collection, pre-processing or processing procedures, or the erroneous use of univariate and multivariate statistical methods. These critical concerns are also addressed in the protocol

CcpA Regulates Arginine Biosynthesis in \u3ci\u3eStaphylococcus aureus\u3c/i\u3e through Repression of Proline Catabolism

Staphylococcus aureus is a leading cause of community-associated and nosocomial infections. Imperative to the success of S. aureus is the ability to adapt and utilize nutrients that are readily available. Genomic sequencing suggests that S. aureus has the genes required for synthesis of all twenty amino acids. However, in vitro experimentation demonstrates that staphylococci have multiple amino acid auxotrophies, including arginine. Although S. aureus possesses the highly conserved anabolic pathway that synthesizes arginine via glutamate, we demonstrate here that inactivation of ccpA facilitates the synthesis of arginine via the urea cycle utilizing proline as a substrate. Mutations within putA, rocD, arcB1, argG and argH abolished the ability of S. aureus JE2 ccpA::tetL to grow in the absence of arginine, whereas an interruption in argJBCF, arcB2, or proC had no effect. Furthermore, nuclear magnetic resonance demonstrated that JE2 ccpA::ermB produced 13C5 labeled arginine when grown with 13C5 proline. Taken together, these data support the conclusion that S. aureus synthesizes arginine from proline during growth on secondary carbon sources. Furthermore, although highly conserved in all sequenced S. aureus genomes, the arginine anabolic pathway (ArgJBCDFGH) is not functional under in vitro growth conditions. Finally, a mutation in argH attenuated virulence in a mouse kidney abscess model in comparison to wild type JE2 demonstrating the importance of arginine biosynthesis in vivo via the urea cycle. However, mutations in argB, argF, and putA did not attenuate virulence suggesting both the glutamate and proline pathways are active and they, or their pathway intermediates, can complement each other in vivo

CcpA Regulates Arginine Biosynthesis in <em>Staphylococcus aureus</em> through Repression of Proline Catabolism

<div><p><em>Staphylococcus aureus</em> is a leading cause of community-associated and nosocomial infections. Imperative to the success of <em>S. aureus</em> is the ability to adapt and utilize nutrients that are readily available. Genomic sequencing suggests that <em>S. aureus</em> has the genes required for synthesis of all twenty amino acids. However, <em>in vitro</em> experimentation demonstrates that staphylococci have multiple amino acid auxotrophies, including arginine. Although <em>S. aureus</em> possesses the highly conserved anabolic pathway that synthesizes arginine via glutamate, we demonstrate here that inactivation of <em>ccpA</em> facilitates the synthesis of arginine via the urea cycle utilizing proline as a substrate. Mutations within <em>putA</em>, <em>rocD</em>, <em>arcB1</em>, <em>argG</em> and <em>argH</em> abolished the ability of <em>S. aureus</em> JE2 <em>ccpA::tetL</em> to grow in the absence of arginine, whereas an interruption in <em>argJBCF</em>, <em>arcB2</em>, or <em>proC</em> had no effect. Furthermore, nuclear magnetic resonance demonstrated that JE2 <em>ccpA::ermB</em> produced ¹³C₅ labeled arginine when grown with ¹³C₅ proline. Taken together, these data support the conclusion that <em>S. aureus</em> synthesizes arginine from proline during growth on secondary carbon sources. Furthermore, although highly conserved in all sequenced <em>S. aureus</em> genomes, the arginine anabolic pathway (ArgJBCDFGH) is not functional under <em>in vitro</em> growth conditions. Finally, a mutation in <em>argH</em> attenuated virulence in a mouse kidney abscess model in comparison to wild type JE2 demonstrating the importance of arginine biosynthesis <em>in vivo</em> via the urea cycle. However, mutations in <em>argB</em>, <em>argF</em>, and <em>putA</em> did not attenuate virulence suggesting both the glutamate and proline pathways are active and they, or their pathway intermediates, can complement each other <em>in vivo</em>.</p> </div

Mouse kidney abscess model.

<p>C57BL/6 mice were infected with 10⁶ CFU of JE2 (n = 16 mice), JE2 <i>argF::</i>φΝΣ (n = 14 mice), JE2 <i>argH::</i>φΝΣ (n = 15 mice), JE2 <i>argC::</i>φΝΣ (n = 13 mice), or JE2 <i>putA::</i>φΝΣ (n = 18 mice). Kidneys were homogenized after 20 days and bacterial burden determined through viable count (CFU/gram tissue). Horizontal line represents median log₁₀ CFU/gram; significant differences in bacterial burden were noted between JE2 and JE2 <i>argH::</i>φΝΣ (**<i>p</i><.01). Data were analyzed using two-way ANOVA.</p

Two-dimensional (2D) ¹H-¹³C heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) analysis of JE2 and JE2 <i>ccpA::ermB</i>.

<p>JE2 and JE2 <i>ccpA::ermB</i> were grown in the presence of ¹³C₅-glutamate or ¹³C₅-proline in CDM and CDM-R, respectively, and assayed using 2D ¹³C HSQC NMR. The differences in ¹³C-arginine relative intensity were determined by subtracting the average intensities between JE2 and JE2 <i>ccpA::ermB</i>, and a student's t-test was utilized to determine significance. A positive relative intensity value is indicative of a greater intensity of ¹³C-arginine in JE2 <i>ccpA::ermB</i> in comparison to JE2. JE2 <i>ccpA::ermB</i> accumulated significantly greater amounts of ¹³C-arginine when grown in CDM containing ¹³C₅-proline in comparison to JE2. Note that there was no significant difference in ¹³C-arginine accumulation between JE2 and JE2 <i>ccpA::ermB</i> when grown in CDM and CDM-R, respectively, containing ¹³C₅-glutamate.</p

Determination of arginine biosynthesis-dependent genes in <i>S. aureus</i> Newman and RN4220.

<p>Defined <i>bursa aurealis</i> transposon mutants in <i>argF</i> and <i>argH</i> were transduced into Newman <i>ccpA::tetL</i> and RN4220 <i>ccpA::tetL</i> and assessed for growth in CDM-R for 18 hours. Data represent means ± SEM of three independent experiments.</p

Northern analysis of arginine biosynthetic pathway in <i>S. aureus</i> JE2 and JE2 <i>ccpA::ermB</i>.

<p>JE2 and JE2 <i>ccpA::ermB</i> total RNA was isolated in mid-exponential phase of growth in CDM and CDM-R, respectively. DNA probes specific for <i>argJ</i>, <i>argB</i>, <i>argC</i>, <i>argD</i>, <i>argF</i>, <i>argG</i>, and <i>argH</i> were labeled with digoxygenin and detected using anti-digoxigenin-AP Fab fragments (Panel A). Panel B shows 16 s and 23 s rRNA depicting equal RNA loading.</p

Arginine biosynthetic pathway via glutamate and proline.

<p>Figure depicts highly conserved arginine biosynthetic pathway via glutamate and the proposed pathway from proline via PutA, RocD, ArcB1, ArgG and ArgH. Note the previously established reverse pathway from arginine to proline via RocF, RocD and ProC.</p