Walton, N. R. | Search bioRxiv

bioRxiv2025

Multivalent Nanobodies for Potent and Broad Neutralization of Staphylococcus aureus Toxins

Staphylococcus aureus is a leading cause of lethal bacteremia and pneumonia, which are driven by potent virulence factors such as T-cell superantigens and alpha hemolysin. S. aureus has among the highest rates of antibiotic resistance, yet no vaccines or alternative therapies are available despite decades of research. Here, we developed a repertoire of potent, high affinity nanobodies (Nbs) targeting key toxins in S. aureus infection, including superantigens (SAgs) SEB, SEC, TSST-1, and Hla. Comprehensive cryo-EM and AlphaFold3 analyses of these Nbs, which were elicited with clinical cocktail vaccines, revealed diverse neutralizing epitopes and mechanisms that provide strategic insights for immunotherapy and vaccine design. Guided by these findings, we engineered highly stable, multivalent, and multifunctional Nb constructs. These constructs included an aerosolizable trimeric Nb with enhanced neuralization activity against Hla and SEC, and an ultrapotent decameric Nb-IgG-Fc fusion construct against a wide range of major toxins in S. aureus sepsis (SEB, SEC, TSST-1, and Hla). These multifunctional Nbs demonstrated promising protective activity in murine models of pneumonia and sepsis, underscoring their potential as versatile immunotherapies that address the complex virulence profiles of S. aureus. Our work lays a foundation for precision immunotherapies beyond current treatment options to combat complex bacterial infections with multiple virulence mechanisms. Significance statementS. aureus is among the most common, antibiotic-resistant, and deadly causes of bacterial infections. We developed nanobodies against clinically significant virulence factors in S. aureus sepsis and pneumonia, including superantigens (SAgs) SEB, SEC, and TSST-1 as well as pore forming toxin Hla. These nanobodies displayed complete and potent neutralization of each toxin, exploiting a wide variety neutralizing mechanisms. Structural investigation of these diverse neutralizing nanobodies, which were elicited in llamas using clinically investigated cocktail vaccines, highlighted the importance of disrupting SAg interaction with TCR or MHCII and potential flaws in targeting poorly neutralizing conserved SAg epitopes using vaccine cocktails. Nb leads against each toxin were combined in different multivalent configurations, including an aerosolizable trimeric Nb and a half-life extended decameric Nb IgG Fc fusion construct. This work highlights multivalent nanobodies as a comprehensive yet therapeutically precise drug platform that addresses the complex virulence profiles of bacterial infectious diseases.

microbiology↗

bioRxiv2025

Elucidating the Staphylococcus aureus TSST-1 regulatory network as a response to vaginal pH

Menstrual toxic shock syndrome (mTSS) is a life-threatening disease caused by the Staphylococcus aureus superantigen TSST-1. At menstruation, the typical acidic vaginal environment rises to near neutral pH, which allows for optimal TSST-1 production. However, the regulation network which alters toxin production in response to pH is largely unknown, despite the importance of this cue in the vaginal environment. To mimic the vaginal environment, we used Vaginally Defined Medium to assess TSST-1 promoter (tst) activity in the mTSS strain S. aureus MN8 and discovered a significant upregulation of tst expression occurring at pH 4.5 in low glucose environment, referred to as the acidic virulence surge. This increase was also observed in all the regulatory mutant backgrounds tested, including in the absence of saeS, which has previously been thought to be required for TSST-1 production. Recent studies in non-mTSS S. aureus strains have identified GraXRS as a pH sensor, in addition to its function in cationic antimicrobial peptide sensing. We therefore hypothesized that GraXRS alters TSST-1 expression at low pH. Deletion of the sensor-kinase graS resulted in the loss of TSST-1 surge at pH 4.5, indicating that GraXRS is required for the acidic virulence surge. We also found expression from the SaeRS P1 and SarA promoters to be significantly attenuated in the {Delta}graS background. At low pH, the absence of GraS resulted in the least amount of T cell activation from S. aureus supernatants when compared to other regulatory mutants, suggesting that GraXRS is the dominant activator at pH 4.5. Finally, we developed an in vivo murine model to measure tst expression using luciferase expression. Our results demonstrate a complex sequence of events that occur in response to changes in pH and further suggests that GraXRS is the main activator of TSST-1 at low pH in S. aureus. AUTHOR SUMMARYMenstrual toxic shock syndrome is a life-threatening hyperinflammatory disease, resulting from the production of a toxin named TSST-1 by Staphylococcus aureus. Environmental cues within the vagina are sensed by S. aureus, resulting in changes in the production of TSST-1. A key environmental cue present within the vagina is acidic pH, which has previously been thought to limit TSST-1 production. Here, we use a luminescent reporter assay to determine how pH affects expression of TSST-1 in a vaginal mimicking medium. We found that expression of the toxin drastically increases at a pH of 4.5, and key TSST-1 regulators are not responsible for this phenotype. We found that deletion of graS abolishes the toxins production at pH 4.5 and limits the activation of primary T cells. We also established an in vivo model of murine vaginal colonization to examine the expression of TSST-1. Our results highlight the ability of TSST-1 to be produced outside of conventional menstrual conditions and provides insight on the necessity of modeling the environment when studying bacterial virulence factors.

microbiology↗