Considerable progress has been made toward elucidating the mechanism of Staphylococcus aureus aggregation in synovial fluid. In this study, aggregate morphology was assessed following incubation under several simulated postsurgical joint conditions. Using fluorescently labeled synovial fluid polymers, we show that aggregation occurs through two distinct mechanisms: (i) direct bridging between S. aureus cells and host fibrinogen and (ii) an entropy-driven depletion mechanism facilitated by hyaluronic acid and albumin. By screening surface adhesin-deficient mutants (clfA, clfB, fnbB, and fnbA), we identified the primary genetic determinant of aggregation in synovial fluid to be clumping factor A. To characterize this bridging interaction, we employed an atomic force microscopy-based approach to quantify the binding affinity of either wild-type S. aureus or the adhesin mutant to immobilized fibrinogen. Surprisingly, we found there to be cell-to-cell variability in the binding strength of the bacteria for immobilized fibrinogen. Superhigh-resolution microscopy imaging revealed that fibrinogen binding to the cell wall is heterogeneously distributed at both the single cell and population levels. Finally, we assessed the antibiotic tolerance of various aggregate morphologies arising from newly deciphered mechanisms of polymer-mediated synovial fluid-induced aggregation. The formation of macroscopic aggregates under shear was highly tolerant of gentamicin, while smaller aggregates, formed under static conditions, were susceptible. We hypothesize that aggregate formation in the joint cavity, in combination with shear, is mediated by both polymer-mediated aggregation mechanisms, with depletion forces enhancing the stability of essential bridging interactions. IMPORTANCE The formation of a bacterial biofilm in the postsurgical joint environment significantly complicates the resolution of an infection. To form a resilient biofilm, incoming bacteria must first survive the initial invasion of the joint space. We previously found that synovial fluid induces the formation of Staphylococcus aureus aggregates, which may provide rapid protection during the early stages of infection. The state of the host joint environment, including the presence of fluid flow and fluctuating abundance of synovial fluid polymers, determines the rate and size of aggregate formation. By expanding on our knowledge of the mechanism and pathogenic implications of synovial fluid-induced aggregation, we hope to contribute insights for the development of novel methods of prevention and therapeutic intervention.

Staphylococcus aureus is an important pathogen responsible for nosocomial and community-acquired infections in humans, and methicillin-resistant S. aureus (MRSA) infections have continued to increase despite widespread preventative measures. S. aureus can colonize the female vaginal tract, and reports have suggested an increase in MRSA infections in pregnant and postpartum women as well as outbreaks in newborn nurseries. Currently, little is known about specific factors that promote MRSA vaginal colonization and subsequent infection. To study S. aureus colonization of the female reproductive tract in a mammalian system, we developed a mouse model of S. aureus vaginal carriage and demonstrated that both hospital-associated and community-associated MRSA isolates can colonize the murine vaginal tract. Immunohistochemical analysis revealed an increase in neutrophils in the vaginal lumen during MRSA colonization. Additionally, we observed that a mutant lacking fibrinogen binding adhesins exhibited decreased persistence within the mouse vagina. To further identify novel factors that promote vaginal colonization, we performed RNA sequencing to determine the transcriptome of MRSA growing in vivo during vaginal carriage at 5 h, 1 day, and 3 days postinoculation. Over 25% of the bacterial genes were differentially regulated at all time points during colonization compared to laboratory cultures. The most highly induced genes were those involved in iron acquisition, including the Isd system and siderophore transport systems. Mutants deficient in these pathways did not persist as well during in vivo colonization. These results reveal that fibrinogen binding and the capacity to overcome host nutritional limitation are important determinants of MRSA vaginal colonization.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen able to cause a wide variety of infections in humans. Recent reports have suggested an increasing prevalence of MRSA in pregnant and postpartum women, coinciding with the increased incidence of MRSA infections in neonatal intensive care units (NICUs) and newborn nurseries. Vertical transmission from mothers to infants at delivery is a likely route of MRSA acquisition by the newborn; however, essentially nothing is known about host and bacterial factors that influence MRSA carriage in the vagina. Here, we established a mouse model of vaginal colonization and observed that multiple MRSA strains can persist in the vaginal tract. Additionally, we determined that MRSA interactions with fibrinogen and iron uptake can promote vaginal persistence. This study is the first to identify molecular mechanisms which govern vaginal colonization by MRSA, the critical initial step preceding infection and neonatal transmission.

Staphylococcus aureus is a leading cause of endovascular infections. This bacterial pathogen uses a diverse array of surface adhesins to clump in blood and adhere to vessel walls, leading to endothelial damage, development of intravascular vegetations and secondary infectious foci, and overall disease progression. In this work, we describe a novel strategy used by S. aureus to control adhesion and clumping through activity of the ArlRS two-component regulatory system, and its downstream effector MgrA. Utilizing a combination of in vitro cellular assays, and single-cell atomic force microscopy, we demonstrated that inactivation of this ArlRS-MgrA cascade inhibits S. aureus adhesion to a vast array of relevant host molecules (fibrinogen, fibronectin, von Willebrand factor, collagen), its clumping with fibrinogen, and its attachment to human endothelial cells and vascular structures. This impact on S. aureus adhesion was apparent in low shear environments, and in physiological levels of shear stress, as well as in vivo in mouse models. These effects were likely mediated by the de-repression of giant surface proteins Ebh, SraP, and SasG, caused by inactivation of the ArlRS-MgrA cascade. In our in vitro assays, these giant proteins collectively shielded the function of other surface adhesins and impaired their binding to cognate ligands. Finally, we demonstrated that the ArlRS-MgrA regulatory cascade is a druggable target through the identification of a small-molecule inhibitor of ArlRS signaling. Our findings suggest a novel approach for the pharmacological treatment and prevention of S. aureus endovascular infections through targeting the ArlRS-MgrA regulatory system.

Staphylococcus aureus is a human commensal and opportunistic pathogen that causes devastating infections in a wide range of locations within the body. One of the defining characteristics of S. aureus is its ability to form clumps in the presence of soluble fibrinogen, which likely has a protective benefit and facilitates adhesion to host tissue. We have previously shown that the ArlRS two-component regulatory system controls clumping, in part by repressing production of the large surface protein Ebh. In this work we show that ArlRS does not directly regulate Ebh, but instead ArlRS activates expression of the global regulator MgrA. Strains lacking mgrA fail to clump in the presence of fibrinogen, and clumping can be restored to an arlRS mutant by overexpressing either arlRS or mgrA, indicating that ArlRS and MgrA constitute a regulatory pathway. We used RNA-seq to show that MgrA represses ebh, as well as seven cell wall-associated proteins (SraP, Spa, FnbB, SasG, SasC, FmtB, and SdrD). EMSA analysis showed that MgrA directly represses expression of ebh and sraP. Clumping can be restored to an mgrA mutant by deleting the genes for Ebh, SraP and SasG, suggesting that increased expression of these proteins blocks clumping by steric hindrance. We show that mgrA mutants are less virulent in a rabbit model of endocarditis, and virulence can be partially restored by deleting the genes for the surface proteins ebh, sraP, and sasG. While mgrA mutants are unable to clump, they are known to have enhanced biofilm capacity. We demonstrate that this increase in biofilm formation is partially due to up-regulation of SasG, a surface protein known to promote intercellular interactions. These results confirm that ArlRS and MgrA constitute a regulatory cascade, and that they control expression of a number of genes important for virulence, including those for eight large surface proteins.

Periprosthetic joint infections (PJIs) are a devastating complication that occurs in 2% of patients following joint replacement. These infections are costly and difficult to treat, often requiring multiple corrective surgeries and prolonged antimicrobial treatments. The Gram-positive bacterium Staphylococcus aureus is one of the most common causes of PJIs, and it is often resistant to a number of commonly used antimicrobials. This tolerance can be partially attributed to the ability of S. aureus to form biofilms. Biofilms associated with the surface of indwelling medical devices have been observed on components removed during chronic infection, however, the development and localization of biofilms during PJIs remains unclear. Prior studies have demonstrated that synovial fluid, in the joint cavity, promotes the development of bacterial aggregates with many biofilm-like properties, including antibiotic resistance. We anticipate these aggregates have an important role in biofilm formation and antibiotic tolerance during PJIs. Therefore, we sought to determine specifically how synovial fluid promotes aggregate formation and the impact of this process on surface attachment. Using flow cytometry and microscopy, we quantified the aggregation of various clinical S. aureus strains following exposure to purified synovial fluid components. We determined that fibrinogen and fibronectin promoted bacterial aggregation, while cell free DNA, serum albumin, and hyaluronic acid had minimal effect. To determine how synovial fluid mediated aggregation affects surface attachment, we utilized microscopy to measure bacterial attachment. Surprisingly, we found that synovial fluid significantly impeded bacterial surface attachment to a variety of materials. We conclude from this study that fibrinogen and fibronectin in synovial fluid have a crucial role in promoting bacterial aggregation and inhibiting surface adhesion during PJI. Collectively, we propose that synovial fluid may have conflicting protective roles for the host by preventing adhesion to surfaces, but by promoting bacterial aggregation is also contributing to the development of antibiotic tolerance.

Twenty to forty one percent of the world's population is either transiently or permanently colonized by the Gram-positive bacterium, Staphylococcus aureus. In 2017, the CDC designated methicillin-resistant S. aureus (MRSA) as a serious threat, reporting ∼300 000 cases of MRSA-associated hospitalizations annually, resulting in over 19 000 deaths, surpassing that of HIV in the USA. S. aureus is a proficient biofilm-forming organism that rapidly acquires resistance to antibiotics, most commonly methicillin (MRSA). This review focuses on a large group of (>30) S. aureus adhesins, either surface-associated or secreted that are designed to specifically bind to 15 or more of the proteins that form key components of the human extracellular matrix (hECM). Importantly, this includes hECM proteins that are pivotal to the homeostasis of almost every tissue environment [collagen (skin), proteoglycans (lung), hemoglobin (blood), elastin, laminin, fibrinogen, fibronectin, and fibrin (multiple organs)]. These adhesins offer S. aureus the potential to establish an infection in every sterile tissue niche. These infections often endure repeated immune onslaught, developing into chronic, biofilm-associated conditions that are tolerant to ∼1000 times the clinically prescribed dose of antibiotics. Depending on the infection and the immune response, this allows S. aureus to seamlessly transition from colonizer to pathogen by subtly manipulating the host against itself while providing the time and stealth that it requires to establish and persist as a biofilm. This is a comprehensive discussion of the interaction between S. aureus biofilms and the hECM. We provide particular focus on the role of these interactions in pathogenesis and, consequently, the clinical implications for the prevention and treatment of S. aureus biofilm infections.

Early bacterial survival in the postsurgical joint is still a mystery. Recently, synovial fluid-induced aggregation was proposed as a potential mechanism of bacterial protection upon entry into the joint. As synovial fluid is secreted back into the joint cavity following surgery, rapid fluctuations in synovial fluid concentrations, composition, and viscosity occur. These changes, along with fluid movement resulting from postoperative joint motion, modify the environment and potentially affect the kinetics of aggregate formation. Through this work, we sought to evaluate the influence of exposure time, synovial fluid concentration, viscosity, and fluid dynamics on aggregation. Furthermore, we aimed to elucidate the primary mechanism of aggregate formation by assessing the interaction of bacterial adhesins with the synovial fluid polymer fibrinogen. Following incubation under each simulated postoperative joint condition, the aggregates were imaged using confocal microscopy. Our analysis revealed the formation of two distinct aggregate phenotypes, depending on whether the incubation was conducted under static or dynamic conditions. Using a surface adhesin mutant, we have narrowed down the genetic determinants for synovial fluid aggregate formation and identified essential host polymers. We report here that synovial fluid-induced aggregation is influenced by various changes specific to the postsurgical joint environment. While we now have evidence that select synovial fluid polymers facilitate bridging aggregation through essential bacterial adhesins, we suspect that their utility is limited by the increasing viscosity under static conditions. Furthermore, dynamic fluid movement recovers the ability of the bacteria with surface proteins present to aggregate under high-viscosity conditions, yielding large, globular aggregates. IMPORTANCE Infection is a major complication of knee and hip joint replacement surgery, which is used to treat arthritis or joint damage. We have shown that Staphylococcus aureus, a common bacterial pathogen, aggregates upon contact with synovial fluid. Within seconds, the bacterial cells interact with synovial fluid polymers in the joint fluid through their cell wall adhesins. The rapid formation of these aggregates likely aids in early bacterial survival in the joint, potentially contributing to the likelihood of developing an infection. By strengthening our basic understanding of the mechanics of synovial fluid aggregate formation under clinically relevant conditions, we hope to expand the knowledge of how to prevent or disrupt aggregation and reduce and more successfully treat these joint infections.