Biology subjects

McCormick, J. K.

Publications and source records attributed to McCormick, J. K..

10 recordsLinked to original sources

bioRxiv2026

The RND family efflux pump FemT contributes to lipid homeostasis in Staphylococcus aureus

The RND efflux pump FemT encoded by SAUSA300_2213 of Staphylococcus aureus USA300 is co-transcribed with femX which has an essential role in synthesizing the Lipid II precursor of peptidoglycan. Anticipating that this arrangement reflects a critical accessory role for femT, we constructed USA300{Delta}femT to assess its function. Although growth of USA300{Delta}femT in tryptic soy broth (TSB) was not impaired, transcriptomic data revealed a mild cellular stress response, accompanied by reduced expression of ohyA and crt genes involved in fatty acid metabolism and carotenoid lipid synthesis respectively. Accordingly, USA300{Delta}femT exhibited impaired growth on exposure to saturated and unsaturated fatty acids, and exposure to subinhibitory 50 {micro}M palmitic acid promoted accumulation of reactive oxygen species, reduced respiratory activity, and altered membrane function and morphology. The transcriptome of cells grown under this condition revealed strongly attenuated expression of ohyA and crt, and several genes required for oxidative and anaerobic respiration, concomitant with strongly enhanced expression of several stress response pathways. Cellular metabolites were also profoundly altered. Finally, lipidomic analysis of USA300{Delta}femT exposed to oleic acid revealed increased incorporation of oleic acid into phosphatidylglycerol, accompanied by a significant reduction in undecaprenol C55 lipid carrier, and respiratory quinones MK-7 and MK-8. Our data are consistent with a role for FemT in maintaining cellular lipid homeostasis by promoting efflux of isoprenoid and carotenoid lipids that are prone to oxidative damage, including C55 and menaquinones that undergo cyclic reactions in peptidoglycan synthesis and electron transport.

microbiology↗

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↗

bioRxiv2024

The Streptococcus pyogenes mannose phosphotransferase system (Man-PTS) influences antimicrobial activity and niche-specific nasopharyngeal infection

Streptococcus pyogenes is a human-adapted pathogen that causes a variety of infections including pharyngitis and skin infections, and although this bacterium produces many virulence and host colonization factors, how S. pyogenes competes with the host microbiota is not well understood. Here we detected antimicrobial activity produced from S. pyogenes MGAS8232 that was able to prevent the growth of Micrococcus luteus. This activity was produced when cells were grown in 5% CO2 and in M17 media supplemented with galactose; however, evaluation of the phenotype with the addition of alternative sugars coupled with genome sequencing experiments revealed the antimicrobial phenotype was not related to classical bacteriocins. To further determine genes involved in the production of this activity, a transposon mutant library in S. pyogenes MGAS8232 was generated. The transposon screen identified the mannose phosphotransferase system (Man-PTS), a major sugar transporter in S. pyogenes, as important for the antimicrobial phenotype. Additional loss-of-function transposon mutants linked to the antimicrobial activity were identified to also be involved in alternative sugar utilization and additionally, the Man-PTS was also further identified from a secondary mutation in a bacteriocin operon mutant. Sugar utilization profiles in all the Man-PTS mutants demonstrated that galactose, mannose, and N-acetylglucosamine utilization was impaired in different Man-PTS mutants. In vitro RNA-seq experiments in high and low glucose concentrations further identified the Man-PTS as a glucose transporter; however, there was no transcriptional regulators or virulence factors affected with the loss of the Man-PTS. A clean deletion in the Man-PTS demonstrated defects in a mouse model of nasopharyngeal infection. Overall, the ability of S. pyogenes to utilize alternative sugars presented by glycans seems to play a role in acute infection and interactions with the endogenous microbial population existing in the nasopharynx. IMPORTANCEStreptococcus pyogenes causes a wide range of infections and is responsible for over 500,000 deaths per year due to invasive infections and post-infection sequelae. The most common clinical manifestation of S. pyogenes however are acute infections such as pharyngitis or impetigo. S. pyogenes can adapt to its environment through alternative sugar metabolism and in this study, we identified an antimicrobial phenotype that was not bacteriocin-related but a by-product of alternative sugar metabolism. Evidently, the mannose phosphotransferase system, a well-studied sugar transporter, was involved in production of the antimicrobial, and was also important for S. pyogenes to utilize alternative sugars and establish nasopharyngeal infection, but not skin infection. Overall, this study identified potential strategies used by S. pyogenes for interactions with the endogenous microbiota and further elucidated the importance of sugar metabolism in acute infection.

microbiology↗

bioRxiv2024

TSST-1 promotes colonization of Staphylococcus aureus within the vaginal tract by activation of CD8+T cells

Toxic shock syndrome toxin-1 (TSST-1) is a superantigen produced by Staphylococcus aureus and is the determinant of menstrual toxic shock syndrome (mTSS); however, the impact of TSST-1 on the vaginal environment beyond mTSS is not understood. Herein, we assessed how TSST-1 affects vaginal colonization by S. aureus, host inflammatory responses, and changes in microbial communities within the murine vagina. We demonstrated that TSST-1 induced a CD8+ T cell-dependent inflammatory response by 24 hours that correlated with an increased bacteria burden within the vaginal tract. This increase was due to superantigen-dependent T cell activation that triggered a change in microbial composition within the vaginal tract. Altogether, this study demonstrates that within the vaginal tract, TSST-1 modulates the vaginal microbiota to favor the survival of S. aureus in the absence of mTSS. ImportanceToxic shock syndrome toxin-1 (TSST-1) is a superantigen toxin produced from Staphylococcus aureus that causes the menstrual form of toxic shock syndrome. This research demonstrates that TSST-1 also has a wider function within the vaginal tract than previously expected. We show that TSST-1, by activating CD8+ T cells, induces an inflammatory environment that modifies the vaginal microbiota to favor colonization by S. aureus. These are important findings as S. aureus can colonize the human vaginal tract efficiently and subsequently trigger dysbiosis within the microbial communities leading to several adverse outcomes such as decreased fertility, increased risks for sexually transmitted diseases and issues related to pregnancy and birth.

microbiology↗

bioRxiv2024

Discovery of an anti-virulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin 1 (TSST-1) production

Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets virulence of S. aureus, but it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS.

microbiology↗

bioRxiv2023

Vaginal Community State Types (CSTs) Alter Environmental Cues and Production of the Staphylococcus aureus Toxic Shock Syndrome Toxin-1 (TSST-1)

Menstrual toxic shock syndrome (mTSS) is a rare but life-threatening disease associated with use of high-absorbency tampons. The production of the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) is involved in nearly all cases of mTSS and is tightly controlled by regulators responding to the environment. In the prototypic mTSS strain S. aureus MN8, the major repressor of TSST-1 is the carbon catabolite protein A (CcpA), which responds to glucose concentrations in the vaginal tract. Healthy vaginal Lactobacillus species also depend on glucose for both growth and acidification of the vaginal environment through lactic acid production. We hypothesized that interactions between the vaginal microbiota (herein referred to as Community State Types, or CSTs) and MN8 depend on environmental cues, and that these interactions subsequently affect TSST-1 production. Using MN8 {Delta}1ccpA at various glucose levels, we demonstrate that the supernatants from different CSTs grown in vaginally defined media (VDM) significantly decrease tst expression. When co-culturing CST species with MN8 {Delta}ccpA, we show that L. jensenii completely inhibits TSST-1 production in conditions mimicking healthy menstruation or mTSS. Finally, we show that growing S. aureus in "unhealthy" or "transitional" CST supernatants results in higher IL-2 production from T cells. These findings suggest that dysbiotic CSTs may encourage TSST-1 production in the vaginal tract, and further indicates that the CSTs are likely important for the development of mTSS. IMPORTANCEIn this study, we investigate the impact of the vaginal microbiota against S. aureus in conditions mimicking the vaginal environment at various stages of the menstrual cycle. We demonstrate that L. jensenii can inhibit TSST-1 production, suggesting the potential for probiotic activity in treating mTSS. On the other side of the spectrum, "unhealthy" or "transient" bacteria such as G. vaginalis and L. iners support more TSST-1 production by S. aureus, suggesting that CSTs are important in the development of mTSS. This study sets forward a model for examining contact-independent interactions between pathogenic bacteria and the vaginal microbiota. It also demonstrates the necessity of replicating the environment when studying one as dynamic as the vagina.

microbiology↗

bioRxiv2022

The Streptococcus pyogenes hyaluronic acid capsule promotes experimental nasal and skin infection by preventing neutrophil-mediated clearance

Streptococcus pyogenes is a globally prominent human-specific pathogen responsible for an enormous burden of human illnesses, including >600 million pharyngeal and >100 million skin infections each year. Despite intensive efforts that focus on invasive indications, much remains unknown about this bacterium in its natural state during colonization of the nasopharynx and skin. Using acute experimental infection models in HLA-transgenic mice, we evaluated how the hyaluronic acid (HA) capsule contributes to S. pyogenes MGAS8232 infection within these limited biological niches. Herein, we demonstrate that HA capsule expression promotes bacterial burden in murine nasal turbinates and skin lesions by resisting neutrophil-mediated killing. HA capsule production is encoded by the hasABC operon and compared to wildtype S. pyogenes infections, mice infected with a {Delta}hasA mutant exhibited over a 1000-fold CFU reduction at 48-hours post{-}nasal challenge, and a 10,000-fold CFU reduction from skin lesions 72-hours post-skin challenge. HA capsule expression contributed substantially to skin lesion size development following subdermal inoculations. In the absence of capsule expression, S. pyogenes revealed drastically impeded growth in whole human blood and increased susceptibility to killing by isolated neutrophils ex vivo, highlighting its important role in resisting phagocytosis. Furthermore, we establish that neutrophil depletion in mice, but not macrophage depletion, recovered the reduced burden by the {Delta}hasA mutant in both the nasopharynx and skin. Together, this work confirms that the HA capsule is a key virulence determinant during acute infections by S. pyogenes and demonstrates that its predominant function is to protect S. pyogenes against neutrophil-mediated killing. AUTHOR SUMMARYStreptococcus pyogenes is a globally disseminated and human-adapted bacterial pathogen that has evolved an arsenal of evasion strategies to overcome and escape host immune clearing mechanisms. Many strains of S. pyogenes are covered by a polysaccharide capsule composed of hyaluronic acid (HA) that is widely recognized to promote severe infections. In this study, we demonstrate using the encapsulated S. pyogenes MGAS8232 strain that the HA capsule is a key virulence factor that facilitates non-invasive infections of the nasopharynx and skin. Although bacterial adhesion and entry into host cells was impeded by HA capsule expression, we show that the key function for both nasal and skin infections is to protect S. pyogenes from neutrophil-mediated killing. Depletion of neutrophils, but not macrophages, recovered the low bacterial burden by unencapsulated S. pyogenes at both sites of infection. Our findings outline an important interaction between the HA capsule and neutrophils in the establishment of acute upper respiratory and skin infections by S. pyogenes.

microbiology↗

bioRxiv2021

Superantigens promote Staphylococcus aureus bloodstream infection by eliciting pathogenic interferon-gamma (IFNγ) production that subverts macrophage function

Staphylococcus aureus is a foremost bacterial pathogen responsible for a vast array of human diseases. Staphylococcal superantigens (SAgs) constitute a family of potent exotoxins secreted by S. aureus, and SAg genes are found ubiquitously in human isolates. SAgs bind directly to MHC class II molecules and T cell receptors, driving extensive T cell activation and cytokine release. Although these toxins have been implicated in serious disease including toxic shock syndrome, we aimed to further elucidate the mechanisms by which SAgs contribute to staphylococcal pathogenesis during septic bloodstream infections. As most conventional mouse strains respond poorly to staphylococcal SAgs, we utilized transgenic mice encoding humanized MHC class II molecules (HLA-DR4) as these animals are much more susceptible to SAg activity. Herein, we demonstrate that SAgs contribute to the severity of S. aureus bacteremia by increasing bacterial burden, most notably in the liver. We established that S. aureus bloodstream infection severity is mediated by CD4+ T cells and interferon-gamma (IFN{gamma}) is produced to very high levels during infection in a SAg-dependent manner. Bacterial burden and disease severity were reduced by antibody blocking of IFN{gamma}, phenocopying isogenic SAg deletion mutant strains. Additionally, cytokine analysis demonstrated that the immune system was skewed towards a proinflammatory response that was reduced by IFN{gamma} blocking. Infection kinetics and flow cytometry analyses suggested this was a macrophage driven mechanism, which was confirmed through macrophage depletion experiments. Further validation with human leukocytes indicated that excessive IFN{gamma} allowed S. aureus to replicate at a higher rate within macrophages. Together, this suggests that SAgs promote S. aureus survival by manipulating immune responses that would otherwise be effective at clearing S. aureus. This work implicates SAg toxins as critical targets for preventing persistent or severe S. aureus disease.

microbiology↗

bioRxiv2020

Prophage exotoxins enhance colonization fitness in epidemic scarlet fever-causing Streptococcus pyogenes

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage {Phi}HKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here we report the comprehensive molecular characterization of {Phi}HKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for optimal growth in human blood, confers resistance to neutrophil killing, and degrades neutrophil extracellular traps (NETs). Investigating single, double and triple isogenic knockout mutants of the {Phi}HKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the etiology and pathogenesis of scarlet fever-causing GAS mediated by phage {Phi}HKU.vir exotoxins.

microbiology↗