Search

Implant-associated Staphylococcus aureus infections are difficult to treat because of biofilm formation. Bacteria in a biofilm are often insensitive to antibiotics and host immunity. Monoclonal antibodies (mAbs) could provide an alternative approach to improve the diagnosis and potential treatment of biofilm-related infections. Here, we show that mAbs targeting common surface components of S. aureus can recognize clinically relevant biofilm types. The mAbs were also shown to bind a collection of clinical isolates derived from different biofilm-associated infections (endocarditis, prosthetic joint, catheter). We identify two groups of antibodies: one group that uniquely binds S. aureus in biofilm state and one that recognizes S. aureus in both biofilm and planktonic state. Furthermore, we show that a mAb recognizing wall teichoic acid (clone 4497) specifically localizes to a subcutaneously implanted pre-colonized catheter in mice. In conclusion, we demonstrate the capacity of several human mAbs to detect S. aureus biofilms in vitro and in vivo.

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.

Summary: Skin is one of the most common sites of host immune response against Staphylococcus aureus infection. Here, through a combination of in vitro assays, mouse models, and intravital imaging, we find that S. aureus immune evasion in skin is controlled by a cascade composed of the ArlRS two-component regulatory system and its downstream effector, MgrA. S. aureus lacking either ArlRS or MgrA is less virulent and unable to form correct abscess structure due to de-repression of a giant surface protein, Ebh. These S. aureus mutants also have decreased expression of immune evasion factors (leukocidins, chemotaxis-inhibitory protein of S. aureus [CHIPS], staphylococcal complement inhibitor [SCIN], and nuclease) and are unable to kill neutrophils, block their chemotaxis, degrade neutrophil extracellular traps, and survive direct neutrophil attack. The combination of disrupted abscess structure and reduced immune evasion factors makes S. aureus susceptible to host defenses. ArlRS and MgrA are therefore the main regulators of S. aureus immune evasion and promising treatment targets.

ABSTRACT 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. IMPORTANCE Staphylococcus 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.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) infections impact all patient populations both in the community and in health care settings. Despite advances in our knowledge of MRSA virulence, little is known about the regulatory mechanisms of USA100 health care-associated MRSA isolates, which are the second most frequently identified MRSA isolates found in all infections. This work focused on the contribution of the USA100 agr type II quorum-sensing system to virulence and antibiotic resistance. From a MRSA strain collection, we selected 16 representative USA100 isolates, constructed mutants with Δagr mutations, and characterized selected strain pairs for virulence factor expression, murine skin infection, and antibiotic resistance. For each strain pair, hemolysis and extracellular protease expression were significantly greater in the wild-type (WT) strains than in the Δagr mutants. Similarly, mice challenged with the WT strains had larger areas of dermonecrosis and greater weight loss than those challenged with the Δagr mutants, demonstrating that the USA100 agr system regulates virulence. Although USA100 isolates exhibit a high level of antibiotic resistance, the WT and Δagr strain pairs showed no difference in MICs by MIC testing. However, in the presence of a sub-MIC of vancomycin, most of the USA100 Δagr mutants exhibited slower growth than the WT isolates, and a couple of the Δagr mutants also grew more slowly in the presence of a sub-MIC of cefoxitin. Altogether, our findings demonstrate that the USA100 agr system is a critical regulator of virulence, and it may have a contribution to the optimal survival of these MRSA strains in the presence of antibiotics. IMPORTANCE USA100 health care-associated MRSA isolates are highly antibiotic resistant and can cause invasive disease across all patient populations. Even though USA100 strains are some of the most frequently identified causes of infections, little is known about virulence regulation in these isolates. Our study demonstrates that the USA100 agr quorum-sensing system is important for the control of toxin and exoenzyme production and that the agr system has a key role in skin infection. In some USA100 isolates, the agr system is important for growth in the presence of low levels of antibiotics. Altogether, our findings demonstrate that the USA100 agr system is a critical regulator of virulence and that it may make a contribution to the optimal survival of these MRSA strains in the presence of antibiotics.

ABSTRACTStaphylococcus aureus is one of the leading causes of hospital-acquired infections, many of which begin following attachment and accumulation on indwelling medical devices or diseased tissue. These infections are often linked to the establishment of biofilms, but another often overlooked key characteristic allowing S. aureus to establish persistent infection is the formation of planktonic aggregates. Such aggregates are physiologically similar to biofilms and protect pathogens from innate immune clearance and increase antibiotic tolerance. The cell-wall-associated protein SasG has been implicated in biofilm formation via mechanisms of intercellular aggregation but the mechanism in the context of disease is largely unknown. We have previously shown that the expression of cell-wall-anchored proteins involved in biofilm formation is controlled by the ArlRS-MgrA regulatory cascade. In this work, we demonstrate that the ArlRS two-component system controls aggregation, by repressing the expression of sasG by activation of the global regulator MgrA. We also demonstrate that SasG must be proteolytically processed by a non-staphylococcal protease to induce aggregation and that strains expressing functional full-length sasG aggregate significantly upon proteolysis by a mucosal-derived host protease found in human saliva. We used fractionation and N-terminal sequencing to demonstrate that human trypsin within saliva cleaves within the A domain of SasG to expose the B domain and induce aggregation. Finally, we demonstrated that SasG is involved in virulence during mouse lung infection. Together, our data point to SasG, its processing by host proteases, and SasG-driven aggregation as important elements of S. aureus adaptation to the host environment.IMPORTANCEHere, we demonstrate that the Staphylococcus aureus surface protein SasG is important for cell-cell aggregation in the presence of host proteases. We show that the ArlRS two-component regulatory system controls SasG levels through the cytoplasmic regulator MgrA. We identified human trypsin as the dominant protease triggering SasG-dependent aggregation and demonstrated that SasG is important for S. aureus lung infection. The discovery that host proteases can induce S. aureus aggregation contributes to our understanding of how this pathogen establishes persistent infections. The observations in this study demonstrate the need to strengthen our knowledge of S. aureus surface adhesin function and processing, regulation of adhesin expression, and the mechanisms that promote biofilm formation to develop strategies for preventing chronic infections.