NMR Analysis of a Stress Response Metabolic Signaling Network

We previously hypothesized that Staphylococcus epidermidis senses a diverse set of environmental and nutritional factors associated with biofilm formation through a modulation in the activity of the tricarboxylic acid (TCA) cycle. Herein, we report our further investigation of the impact of additional environmental stress factors on TCA cycle activity and provide a detailed description of our NMR methodology. S. epidermidis wild-type strain 1457 was treated with stressors that are associated with biofilm formation, a sub-lethal dose of tetracycline, 5% NaCl, 2% glucose and autoinducer-2 (AI-2). As controls and to integrate our current data with our previous study, 4% ethanol stress and iron-limitation were also used. Consistent with our prior observations, the effect of many environmental stress factors on the S. epidermidis metabolome was essentially identical to the effect of TCA cycle inactivation in the aconitase mutant strain 1457-acnA::tetM. A detailed quantitative analysis of metabolite concentration changes using 2D 1H-13C HSQC and 1H-1H TOCSY spectra identified a network of 37 metabolites uniformly affected by the stressors and TCA cycle inactivation. We postulate that the TCA cycle acts as the central pathway in a metabolic signaling network

Inactivation of the Pta-AckA Pathway Causes Cell Death in \u3ci\u3eStaphylococcus aureus\u3c/i\u3e

During growth under conditions of glucose and oxygen excess, Staphylococcus aureus predominantly accumulates acetate in the culture medium, suggesting that the phosphotransacetylase-acetate kinase (Pta-AckA) pathway plays a crucial role in bacterial fitness. Previous studies demonstrated that these conditions also induce the S. aureus CidR regulon involved in the control of cell death. Interestingly, the CidR regulon is comprised of only two operons, both encoding pyruvate catabolic enzymes, suggesting an intimate relationship between pyruvate metabolism and cell death. To examine this relationship, we introduced ackA and pta mutations in S. aureus and tested their effects on bacterial growth, carbon and energy metabolism, cid expression, and cell death. Inactivation of the Pta-AckA pathway showed a drastic inhibitory effect on growth and caused accumulation of dead cells in both pta and ackA mutants. Surprisingly, inactivation of the Pta-AckA pathway did not lead to a decrease in the energy status of bacteria, as the intracellular concentrations of ATP, NAD+, and NADH were higher in the mutants. However, inactivation of this pathway increased the rate of glucose consumption, led to a metabolic block at the pyruvate node, and enhanced carbon flux through both glycolysis and the tricarboxylic acid (TCA) cycle. Intriguingly, disruption of the Pta-AckA pathway also induced the CidR regulon, suggesting that activation of alternative pyruvate catabolic pathways could be an important survival strategy for the mutants. Collectively, the results of this study demonstrate the indispensable role of the Pta-AckA pathway in S. aureus for maintaining energy and metabolic homeostasis during overflow metabolism

Tricarboxylic Acid Cycle-Dependent Regulation of \u3ci\u3eStaphylococcus epidermidis\u3c/i\u3e Polysaccharide Intercellular Adhesin Synthesis

Staphylococcus epidermidis is a major nosocomial pathogen primarily infecting immunocompromised individuals or those with implanted biomaterials (e.g., catheters). Biomaterial-associated infections often involve the formation of a biofilm on the surface of the medical device. In S. epidermidis, polysaccharide intercellular adhesin (PIA) is an important mediator of biofilm formation and pathogenesis. Synthesis of PIA is regulated by at least three DNA binding proteins (IcaR, SarA, and σB) and several environmental and nutritional conditions. Previously, we observed the environmental conditions that increased PIA synthesis decreased tricarboxylic acid (TCA) cycle activity. In this study, S. epidermidis TCA cycle mutants were constructed, and the function of central metabolism in PIA biosynthesis was examined. TCA cycle inactivation altered the metabolic status of S. epidermidis, resulting in a massive derepression of PIA biosynthetic genes and a redirection of carbon from growth into PIA biosynthesis. These data demonstrate that the bacterial metabolic status is a critical regulatory determinant of PIA synthesis. In addition, these data lead us to propose that the TCA cycle acts as a signal transduction pathway to translate external environmental cues into intracellular metabolic signals that modulate the activity of transcriptional regulators

Amino Acid Catabolism in \u3ci\u3eStaphylococcus aureus\u3c/i\u3e and the Function of Carbon Catabolite Repression

Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting that S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids, including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in the gudB gene, coding for glutamate dehydrogenase, which generates 2-oxoglutarate from glutamate, significantly reduced growth in CDM, suggesting that glutamate and those amino acids generating glutamate, particularly proline, serve as the major carbon source in this medium. Nuclear magnetic resonance (NMR) studies confirmed this supposition. Furthermore, a mutation in the ackA gene, coding for acetate kinase, also abrogated growth of JE2 in CDM, suggesting that ATP production from pyruvate-producing amino acids is also critical for growth. In addition, although a functional respiratory chain was absolutely required for growth, the oxygen consumption rate and intracellular ATP concentration were significantly lower during growth in CDM than during growth in glucose-containing media. Finally, transcriptional analyses demonstrated that expression levels of genes coding for the enzymes that synthesize glutamate from proline, arginine, and histidine are repressed by CcpA and carbon catabolite repression. These data show that pathways important for glutamate catabolism or ATP generation via Pta/AckA are important for growth in niches where glucose is not abundant, such as abscesses within skin and soft tissue infections

Ratiometric Fluorescent Sensor Array as a Versatile Tool for Bacterial Pathogen Identification and Analysis

Rapid and reliable identification of pathogenic microorganisms is of great importance for human and animal health. Most conventional approaches are time-consuming and require expensive reagents, sophisticated equipment, trained personnel, and special storage and handling conditions. Sensor arrays based on small molecules offer a chemically stable and cost-effective alternative. Here we present a ratiometric fluorescent sensor array based on the derivatives of 2-(4′-<i>N</i>,<i>N</i>-dimethylamino)-3-hydroxyflavone and investigate its ability to provide a dual-channel ratiometric response. We demonstrate that, by using discriminant analysis of the sensor array responses, it is possible to effectively distinguish between eight bacterial species and recognize their Gram status. Thus, multiple parameters can be derived from the same data set. Moreover, the predictive potential of this sensor array is discussed, and its ability to analyze unknown samples beyond the list of species used for the training matrix is demonstrated. The proposed sensor array and analysis strategies open new avenues for the development of advanced ratiometric sensors for multiparametric analysis

Stochastic Expression of Sae-Dependent Virulence Genes during Staphylococcus aureus Biofilm Development Is Dependent on SaeS

Staphylococcus aureus is an important human pathogen capable of colonizing diverse tissue types and inducing severe disease in both immunocompromised and otherwise healthy individuals. Biofilm infections caused by this bacterial species are of particular concern because of their persistence, even in the face of intensive therapeutic intervention. The results of the current study demonstrate the stochastic nature of Sae-mediated virulence gene expression in S. aureus and indicate that this regulatory system may function as a “bistable switch” in a manner similar to that seen with regulators controlling competence gene expression in Bacillus subtilis and persister cell formation in Escherichia coli. The results of this study provide a new perspective on the complex mechanisms utilized by S. aureus during the establishment of infections.The intricate process of biofilm formation in the human pathogen Staphylococcus aureus involves distinct stages during which a complex mixture of matrix molecules is produced and modified throughout the developmental cycle. Early in biofilm development, a subpopulation of cells detaches from its substrate in an event termed “exodus” that is mediated by SaePQRS-dependent stochastic expression of a secreted staphylococcal nuclease, which degrades extracellular DNA within the matrix, causing the release of cells and subsequently allowing for the formation of metabolically heterogenous microcolonies. Since the SaePQRS regulatory system is involved in the transcriptional control of multiple S. aureus virulence factors, the expression of several additional virulence genes was examined within a developing biofilm by introducing fluorescent gene reporter plasmids into wild-type S. aureus and isogenic regulatory mutants and growing these strains in a microfluidic system that supplies the bacteria with a constant flow of media while simultaneously imaging developing biofilms in 5-min intervals. This study demonstrated that multiple virulence genes, including nuc, were expressed stochastically within a specialized subpopulation of cells in nascent biofilms. We demonstrated that virulence genes regulated by SaePQRS were stochastically expressed in nearly all strains examined whereas Agr-regulated genes were expressed more homogenously within maturing microcolonies. The commonly used Newman strain contains a variant of SaeS (SaeSP) that confers constitutive kinase activity to the protein and caused this strain to lack the stochastic expression pattern observed in other strain backgrounds. Importantly, repair of the SaeSP allele resulting in reversion to the well-conserved SaeSL allele found in other strains restored stochastic expression in this strain