A Manganese-independent Aldolase Enables Staphylococcus aureus To Resist Host-imposed Metal Starvation.

The preferred carbon source of Staphylococcus aureus and many other pathogens is glucose, and its consumption is critical during infection. However, glucose utilization increases the cellular demand for manganese, a nutrient sequestered by the host as a defense against invading pathogens. Therefore, bacteria must balance glucose metabolism with the increasing demand that metal-dependent processes, such as glycolysis, impose upon the cell. A critical regulator that enables S. aureus to resist nutritional immunity is the ArlRS two-component system. This work revealed that ArlRS regulates the expression of FdaB, a metal-independent fructose 1,6-bisphosphate aldolase. Further investigation revealed that when S. aureus is metal-starved by the host, FdaB functionally replaces the metal-dependent isozyme FbaA, thereby allowing S. aureus to resist host-imposed metal starvation in culture. Although metal-dependent aldolases are canonically zinc-dependent, this work uncovered that FbaA requires manganese for activity and that FdaB protects S. aureus from manganese starvation. Both FbaA and FdaB contribute to the ability of S. aureus to cause invasive disease in wild-type mice. However, the virulence defect of a strain lacking FdaB was reversed in calprotectin-deficient mice, which have defects in manganese sequestration, indicating that this isozyme contributes to the ability of this pathogen to overcome manganese limitation during infection. Cumulatively, these observations suggest that the expression of the metal-independent aldolase FdaB allows S. aureus to alleviate the increased demand for manganese that glucose consumption imposes, and highlights the cofactor flexibility of even established metalloenzyme families. IMPORTANCE Staphylococcus aureus and other pathogens consume glucose during infection. Glucose utilization increases the demand for transition metals, such as manganese, a nutrient that the host limits as a defense mechanism against invading pathogens. Therefore, pathogenic bacteria must balance glucose and manganese requirements during infection. The two-component system ArlRS is an important regulator that allows S. aureus to adapt to both glucose and manganese starvation. Among the genes regulated by ArlRS is the metal-independent fructose 1,6-bisphosphate aldolase fdaB, which functionally substitutes for the metal-dependent isoenzyme FbaA and enables S. aureus to survive host-imposed manganese starvation. Unexpectedly, and differing from most characterized metal-dependent aldolases, FbaA requires manganese for activity. Cumulatively, these findings reveal a new mechanism for overcoming nutritional immunity as well as the cofactor plasticity of even well-characterized metalloenzyme families.

The Sensor Histidine Kinase ArlS Is Necessary for Staphylococcus aureus To Activate ArlR in Response to Nutrient Availability.

Staphylococcus aureus is a versatile opportunistic pathogen whose success is driven by its ability to adapt to diverse environments and host-imposed stresses. Two-component signal transduction systems, such as ArlRS, often mediate these adaptations. Loss of ArlRS or the response regulator ArlR alone impairs the ability of S. aureus to respond to host-imposed manganese starvation and glucose limitation. As sensor histidine kinases and response regulators frequently work as pairs, it has been assumed that ArlS senses and activates ArlR in response to these stimuli. However, recent work suggests that the sensor histidine kinase GraS can also activate ArlR, calling the contribution of ArlS in responding to manganese and glucose availability into question. The results of current studies reveal that ArlS is necessary to activate ArlR in response to manganese sequestration by the host immune effector calprotectin and glucose limitation. Although the loss of ArlS does not completely eliminate ArlR activity, this response regulator is no longer responsive to manganese or glucose availability in the absence of its cognate histidine kinase. Despite the residual activity of ArlR in the absence of ArlS, ArlR phosphorylation by ArlS is required for S. aureus to resist calprotectin-imposed metal starvation. Cumulatively, these findings contribute to the understanding of S. aureus signal transduction in response to nutritional immunity and support the previous observation indicating that ArlRS is activated by a common signal derived from host-imposed manganese and glucose limitation. IMPORTANCE The ability of pathogens, including Staphylococcus aureus, to sense and adapt to diverse environments partially relies on two-component systems, such as ArlRS. Recent work revealed that the response regulator ArlR can be cross-activated by the sensor histidine kinase GraS, rendering the role of its cognate partner, ArlS, in response to manganese and glucose limitation uncertain. The results of this study reveal that ArlS is necessary for the activation of ArlR in response to calprotectin and glucose limitation. Although a low level of ArlR activity remains in the absence of ArlS, ArlS phosphotransfer to ArlR is required for S. aureus to overcome calprotectin-induced nutritional stress. Collectively, this study provides fundamental information to understand how ArlRS mediates staphylococcal adaptation during infection.

Disruption of Phosphate Homeostasis Sensitizes Staphylococcus aureus to Nutritional Immunity.

To control infection, mammals actively withhold essential nutrients, including the transition metal manganese, by a process termed nutritional immunity. A critical component of this host response is the manganese-chelating protein calprotectin. While many bacterial mechanisms for overcoming nutritional immunity have been identified, the intersection between metal starvation and other essential inorganic nutrients has not been investigated. Here, we report that overexpression of an operon encoding a highly conserved inorganic phosphate importer, PstSCAB, increases the sensitivity of Staphylococcus aureus to calprotectin-mediated manganese sequestration. Further analysis revealed that overexpression of pstSCAB does not disrupt manganese acquisition or result in overaccumulation of phosphate by S. aureus However, it does reduce the ability of S. aureus to grow in phosphate-replete defined medium. Overexpression of pstSCAB does not aberrantly activate the phosphate-responsive two-component system PhoPR, nor was this two-component system required for sensitivity to manganese starvation. In a mouse model of systemic staphylococcal disease, a pstSCAB-overexpressing strain is significantly attenuated compared to wild-type S. aureus This defect is partially reversed in a calprotectin-deficient mouse, in which manganese is more readily available. Given that expression of pstSCAB is regulated by PhoPR, these findings suggest that overactivation of PhoPR would diminish the ability of S. aureus to resist nutritional immunity and cause infection. As PhoPR is also necessary for bacterial virulence, these findings imply that phosphate homeostasis represents a critical regulatory node whose activity must be precisely controlled in order for S. aureus and other pathogens to cause infection.

Role of respiratory NADH oxidation in the regulation of Staphylococcus aureus virulence.

The success of Staphylococcus aureus as a pathogen is due to its capability of fine-tuning its cellular physiology to meet the challenges presented by diverse environments, which allows it to colonize multiple niches within a single vertebrate host. Elucidating the roles of energy-yielding metabolic pathways could uncover attractive therapeutic strategies and targets. In this work, we seek to determine the effects of disabling NADH-dependent aerobic respiration on the physiology of S. aureus. Differing from many pathogens, S. aureus has two type-2 respiratory NADH dehydrogenases (NDH-2s) but lacks the respiratory ion-pumping NDHs. Here, we show that the NDH-2s, individually or together, are not essential either for respiration or growth. Nevertheless, their absence eliminates biofilm formation, production of α-toxin, and reduces the ability to colonize specific organs in a mouse model of systemic infection. Moreover, we demonstrate that the reason behind these phenotypes is the alteration of the fatty acid metabolism. Importantly, the SaeRS two-component system, which responds to fatty acids regulation, is responsible for the link between NADH-dependent respiration and virulence in S. aureus.

Disruption of Glycolysis by Nutritional Immunity Activates a Two-Component System That Coordinates a Metabolic and Antihost Response by Staphylococcus aureus.

During infection, bacteria use two-component signal transduction systems to sense and adapt to the dynamic host environment. Despite critically contributing to infection, the activating signals of most of these regulators remain unknown. This also applies to the Staphylococcus aureus ArlRS two-component system, which contributes to virulence by coordinating the production of toxins, adhesins, and a metabolic response that enables the bacterium to overcome host-imposed manganese starvation. Restricting the availability of essential transition metals, a strategy known as nutritional immunity, constitutes a critical defense against infection. In this work, expression analysis revealed that manganese starvation imposed by the immune effector calprotectin or by the absence of glycolytic substrates activates ArlRS. Manganese starvation imposed by calprotectin also activated the ArlRS system even when glycolytic substrates were present. A combination of metabolomics, mutational analysis, and metabolic feeding experiments revealed that ArlRS is activated by alterations in metabolic flux occurring in the latter half of the glycolytic pathway. Moreover, calprotectin was found to induce expression of staphylococcal leukocidins in an ArlRS-dependent manner. These studies indicated that ArlRS is a metabolic sensor that allows S. aureus to integrate multiple environmental stresses that alter glycolytic flux to coordinate an antihost response and to adapt to manganese starvation. They also established that the latter half of glycolysis represents a checkpoint to monitor metabolic state in S. aureus Altogether, these findings contribute to understanding how invading pathogens, such as S. aureus, adapt to the host during infection and suggest the existence of similar mechanisms in other bacterial species.IMPORTANCE Two-component regulatory systems enable bacteria to adapt to changes in their environment during infection by altering gene expression and coordinating antihost responses. Despite the critical role of two-component systems in bacterial survival and pathogenesis, the activating signals for most of these regulators remain unidentified. This is exemplified by ArlRS, a Staphylococcus aureus global regulator that contributes to virulence and to resisting host-mediated restriction of essential nutrients, such as manganese. In this report, we demonstrate that manganese starvation and the absence of glycolytic substrates activate ArlRS. Further investigations revealed that ArlRS is activated when the latter half of glycolysis is disrupted, suggesting that S. aureus monitors flux through the second half of this pathway. Host-imposed manganese starvation also induced the expression of pore-forming toxins in an ArlRS-dependent manner. Cumulatively, this work reveals that ArlRS acts as a sensor that links nutritional status, cellular metabolism, and virulence regulation.

Acquisition of the Phosphate Transporter NptA Enhances Staphylococcus aureus Pathogenesis by Improving Phosphate Uptake in Divergent Environments.

During infection, pathogens must obtain all inorganic nutrients, such as phosphate, from the host. Despite the essentiality of phosphate for all forms of life, how Staphylococcus aureus obtains this nutrient during infection is unknown. Differing from Escherichia coli, the paradigm for bacterial phosphate acquisition, which has two inorganic phosphate (Pi) importers, genomic analysis suggested that S. aureus possesses three distinct Pi transporters: PstSCAB, PitA, and NptA. While pitA and nptA are expressed in phosphate-replete media, expression of all three transporters is induced by phosphate limitation. The loss of a single transporter did not affect S. aureus However, disruption of any two systems significantly reduced Pi accumulation and growth in divergent environments. These findings indicate that PstSCAB, PitA, and NptA have overlapping but nonredundant functions, thus expanding the environments in which S. aureus can successfully obtain Pi Consistent with this idea, in a systemic mouse model of disease, loss of any one transporter did not decrease staphylococcal virulence. However, loss of NptA in conjunction with either PstSCAB or PitA significantly reduced the ability of S. aureus to cause infection. These observations suggest that Pi acquisition via NptA is particularly important for the pathogenesis of S. aureus While our analysis suggests that NptA homologs are widely distributed among bacteria, closely related less pathogenic staphylococcal species do not possess this importer. Altogether, these observations indicate that Pi uptake by S. aureus differs from established models and that acquisition of a third transporter enhances the ability of the bacterium to cause infection.

The Metallophore Staphylopine Enables Staphylococcus aureus To Compete with the Host for Zinc and Overcome Nutritional Immunity.

During infection, the host sequesters essential nutrients, such as zinc, to combat invading microbes. Despite the ability of the immune effector protein calprotectin to bind zinc with subpicomolar affinity, Staphylococcus aureus is able to successfully compete with the host for zinc. However, the zinc importers expressed by S. aureus remain unknown. Our investigations have revealed that S. aureus possesses two importers, AdcABC and CntABCDF, which are induced in response to zinc limitation. While AdcABC is similar to known zinc importers in other bacteria, CntABCDF has not previously been associated with zinc acquisition. Concurrent loss of the two systems severely impairs the ability of S. aureus to obtain zinc and grow in zinc-limited environments. Further investigations revealed that the Cnt system is responsible for the ability of S. aureus to compete with calprotectin for zinc in culture and contributes to acquisition of zinc during infection. The cnt locus also enables S. aureus to produce the broad-spectrum metallophore staphylopine. Similarly to the Cnt transporter, loss of staphylopine severely impairs the ability of S. aureus to resist host-imposed zinc starvation, both in culture and during infection. Further investigations revealed that together staphylopine and the Cnt importer function analogously to siderophore-based iron acquisition systems in order to facilitate zinc acquisition by S. aureus Analogous systems are found in a broad range of Gram-positive and Gram-negative bacterial pathogens, suggesting that this new type of zinc importer broadly contributes to the ability of bacteria to cause infection.IMPORTANCE A critical host defense against infection is the restriction of zinc availability. Despite the subpicomolar affinity of the immune effector calprotectin for zinc, Staphylococcus aureus can successfully compete for this essential metal. Here, we describe two zinc importers, AdcABC and CntABCDF, possessed by S. aureus, the latter of which has not previously been associated with zinc acquisition. The ability of S. aureus to compete with the host for zinc is dependent on CntABCDF and the metallophore staphylopine, both in culture and during infection. These results expand the mechanisms utilized by bacteria to obtain zinc, beyond Adc-like systems, and demonstrate that pathogens utilize strategies similar to siderophore-based iron acquisition to obtain other essential metals during infection. The staphylopine synthesis machinery is present in a diverse collection of bacteria, suggesting that this new family of zinc importers broadly contributes to the ability of numerous pathogens to cause infection.

The Two-Component System ArlRS and Alterations in Metabolism Enable Staphylococcus aureus to Resist Calprotectin-Induced Manganese Starvation.

During infection the host imposes manganese and zinc starvation on invading pathogens. Despite this, Staphylococcus aureus and other successful pathogens remain capable of causing devastating disease. However, how these invaders adapt to host-imposed metal starvation and overcome nutritional immunity remains unknown. We report that ArlRS, a global staphylococcal virulence regulator, enhances the ability of S. aureus to grow in the presence of the manganese-and zinc-binding innate immune effector calprotectin. Utilization of calprotectin variants with altered metal binding properties revealed that strains lacking ArlRS are specifically more sensitive to manganese starvation. Loss of ArlRS did not alter the expression of manganese importers or prevent S. aureus from acquiring metals. It did, however, alter staphylococcal metabolism and impair the ability of S. aureus to grow on amino acids. Further studies suggested that relative to consuming glucose, the preferred carbon source of S. aureus, utilizing amino acids reduced the cellular demand for manganese. When forced to use glucose as the sole carbon source S. aureus became more sensitive to calprotectin compared to when amino acids are provided. Infection experiments utilizing wild type and calprotectin-deficient mice, which have defects in manganese sequestration, revealed that ArlRS is important for disease when manganese availability is restricted but not when this essential nutrient is freely available. In total, these results indicate that altering cellular metabolism contributes to the ability of pathogens to resist manganese starvation and that ArlRS enables S. aureus to overcome nutritional immunity by facilitating this adaptation.