The bacterial pathogenStaphylococcus aureusis capable of infecting a broad spectrum of host tissues, in part due to flexibility of metabolic programs.S. aureus, like all organisms, requires essential biosynthetic intermediates to synthesize macromolecules. We therefore sought to determine the metabolic pathways contributing to synthesis of essential precursors during invasiveS. aureusinfection. We focused specifically on staphylococcal infection of bone, one of the most common sites of invasiveS. aureusinfection and a unique environment characterized by dynamic substrate accessibility, infection-induced hypoxia, and a metabolic profile skewed toward aerobic glycolysis. Using a murine model of osteomyelitis, we examined survival ofS. aureusmutants deficient in central metabolic pathways, including glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and amino acid synthesis/catabolism. Despite the high glycolytic demand of skeletal cells, we discovered thatS. aureusrequires glycolysis for survival in bone. Furthermore, the TCA cycle is dispensable for survival during osteomyelitis, andS. aureusinstead has a critical need for anaplerosis. Bacterial synthesis of aspartate in particular is absolutely essential for staphylococcal survival in bone, despite the presence of an aspartate transporter, which we identified as GltT and confirmed biochemically. This dependence on endogenous aspartate synthesis derives from the presence of excess glutamate in infected tissue, which inhibits aspartate acquisition byS. aureus. Together, these data elucidate the metabolic pathways required for staphylococcal infection within bone and demonstrate that the host nutrient milieu can determine essentiality of bacterial nutrient biosynthesis pathways despite the presence of dedicated transporters.

Staphylococcus aureus is a Gram-positive pathogen capable of infecting nearly every vertebrate organ. Among these tissues, invasive infection of bone (osteomyelitis) is particularly common and induces high morbidity. Treatment of osteomyelitis is notoriously difficult and often requires debridement of diseased bone in conjunction with prolonged antibiotic treatment to resolve infection. During osteomyelitis, S. aureus forms characteristic multicellular microcolonies in distinct niches within bone. Virulence and metabolic responses within these multicellular microcolonies are coordinated, in part, by quorum sensing via the accessory gene regulator (agr) locus, which allows staphylococcal populations to produce toxins and adapt in response to bacterial density. During osteomyelitis, the Agr system significantly contributes to dysregulation of skeletal homeostasis and disease severity but may also paradoxically inhibit persistence in the host. Moreover, the Agr system is subject to complex crosstalk with other S. aureus regulatory systems, including SaeRS and SrrAB, which can significantly impact the progression of osteomyelitis. The objective of this review is to highlight Agr regulation, its implications on toxin production, factors that affect Agr activation, and the potential paradoxical influences of Agr regulation on disease progression during osteomyelitis.

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis and osteomyelitis. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) facilitate the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of osseous tissue for MALDI IMS has been difficult due to its mineralized composition and heterogeneous morphology, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs, allowing the distribution of endogenous lipids to be linked to tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. High spatial resolution (10 μm) MALDI IMS was employed to characterize lipid distributions, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to the bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.

Staphylococcus aureus is the major causative agent of bacterial osteomyelitis, an invasive infection of bone. Inflammation generated by the immune response to S. aureus contributes to bone damage by altering bone homeostasis.

Hyperglycemia, or elevated blood glucose, renders individuals more prone to developing severe Staphylococcus aureus infections. S. aureus is the most common etiological agent of musculoskeletal infection, which is a common manifestation of disease in hyperglycemic patients. However, the mechanisms by which S. aureus causes severe musculoskeletal infection during hyperglycemia are incompletely characterized.

Hyperglycemia, or elevated blood glucose, renders individuals more prone to developing severeStaphylococcus aureusinfections.S. aureusis the most common etiological agent of musculoskeletal infection, which is a common manifestation of disease in hyperglycemic patients. However, the mechanisms by whichS. aureuscauses severe musculoskeletal infection during hyperglycemia are incompletely characterized. To examine the influence of hyperglycemia onS. aureusvirulence during invasive infection, we used a murine model of osteomyelitis and induced hyperglycemia with streptozotocin. We discovered that hyperglycemic mice exhibited increased bacterial burdens in bone and enhanced dissemination compared to control mice. Furthermore, infected hyperglycemic mice sustained increased bone destruction relative to euglycemic controls, suggesting that hyperglycemia exacerbates infection-associated bone loss. To identify genes contributing toS. aureuspathogenesis during osteomyelitis in hyperglycemic animals relative to euglycemic controls, we used transposon sequencing (TnSeq). We identified 71 genes uniquely essential forS. aureussurvival in osteomyelitis in hyperglycemic mice and another 61 mutants with compromised fitness. Among the genes essential forS. aureussurvival in hyperglycemic mice was superoxide dismutase A (sodA), one of twoS. aureussuperoxide dismutases involved in detoxifying reactive oxygen species (ROS). We determined that asodAmutant exhibits attenuated growthin vitroin high glucose andin vivoduring osteomyelitis in hyperglycemic mice. SodA therefore serves an important role during growth in high glucose and promotesS. aureussurvival in bone. Collectively, these studies demonstrate that hyperglycemia increases the severity of osteomyelitis and identify genes contributing toS. aureussurvival during hyperglycemic infection.

Background Staphylococcus aureus is a leading cause of antibiotic-resistant bacterial infections and can infect nearly every organ of the human body. One common manifestation of S. aureus disease is invasive bone infection, or osteomyelitis. Osteomyelitis is one of the most difficult to treat infections, often necessitating prolonged antibiotic treatment and surgical interventions. Hyperglycemia, or elevated blood glucose concentration, increases the risk for developing osteomyelitis. We hypothesized that S. aureus adapts specifically to the altered host environment during hyperglycemic osteomyelitis, thereby contributing to the increased infection severity. Methods To model hyperglycemic osteomyelitis, we treated mice with streptozotocin prior to infection with S. aureus in a post-traumatic osteomyelitis model. We analyzed bacterial burdens in homogenized tissues and bone parameters with microcomputed tomography. To identify changes in the spatial molecular architecture of infected, hyperglycemic femurs, we utilized imaging mass spectrometry. Finally, to obtain a comprehensive understanding of S. aureus metabolic and virulence changes and identify genes essential for staphylococcal growth in vivo in hyperglycemic mice, we performed a transposon sequencing experiment. Results We discovered that hyperglycemic mice sustained increased bacterial burdens within infected femurs and greater dissemination to other organs. We also found that hyperglycemic infected mice experienced increased rates of bone destruction and loss of trabecular bone volume, suggesting that hyperglycemia exacerbates infection-associated bone loss. Finally, we identified 71 genes as uniquely essential for S. aureus growth during hyperglycemic infection compared to euglycemic infection. Conclusion Hyperglycemia results in increased osteomyelitis infection severity. Multiple S. aureus genes involved in metabolism with roles in bacterial fitness in acidic environments were uniquely essential for bacterial fitness during comorbid infection. By identifying genes contributing to S. aureus survival during hyperglycemic osteomyelitis, we have the potential to inform targeted therapeutic development for treatment of exacerbated infections in patients with comorbid hyperglycemia. Disclosures All Authors: No reported disclosures.

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to pathological alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto ITO coated glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracks, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 μm) MALDI IMS was leveraged to characterize lipid distributions in fresh-frozen bone, and complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Furthermore, we discovered that [sphingomyelin(42:1) + H]+ was abundant in megakaryocytes, whereas [sphingomyelin(42:2) + H]+ was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Therefore, this application of multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage in a preserved native bone microenvironment.