scholarly journals Identification of Anti-Alpha Toxin Monoclonal Antibodies That Reduce the Severity of Staphylococcus aureus Dermonecrosis and Exhibit a Correlation between Affinity and Potency

2012 ◽  
Vol 19 (3) ◽  
pp. 377-385 ◽  
Author(s):  
C. Tkaczyk ◽  
L. Hua ◽  
R. Varkey ◽  
Y. Shi ◽  
L. Dettinger ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibodies ◽  
Dissociation Constants ◽  
Lesion Size ◽  
Lung Epithelial Cells ◽  
Clinical Isolates ◽  
Dependent Manner ◽  
Alpha Toxin ◽  
Binding Studies ◽  
Content Type

ABSTRACTStaphylococcus aureusalpha toxin (AT) is an important virulence determinant and may be a valid target for immunoprophylaxis against staphylococcal disease. Here we report the identification of potent inhibitory anti-AT monoclonal antibodies (MAbs) derived using B-cell hybridoma technology from VelocImmune mice engineered to produce IgG with a human variable domain. A small panel of inhibitory MAbs blocked AT-mediated lysis of rabbit red blood cells, A549 human lung epithelial cells, and THP-1 human monocytic cells, in a dose-dependent manner. Binding studies indicated that these MAbs recognize a similar epitope on AT and exhibit dissociation constants (KD) ranging from 0.50 to 15 nM. In anS. aureusdermonecrosis model, mice passively immunized with anti-AT inhibitory MAbs exhibited significant reductions of lesion size relative to mice treated with an irrelevant IgG control. Interestingly, there was a correlation between MAb affinity for a single epitope, the 50% inhibitory concentration (IC50) in the AT hemolytic assay, and lesion size reduction in the dermonecrosis model. A representative high-affinity MAb, 2A3.1, was demonstrated to significantly reduce lesion size following infection with three different clinical isolates (USA300, CC30, and CC5). Taken together, these results indicate thatin vitropotency of anti-AT MAbs predictsin vivopotency in this model, supporting their continued preclinical evaluation as molecules for immunoprophylaxis against staphylococcal skin and soft tissue infections caused by diverse clinical isolates.

scholarly journals Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils

2014 ◽  
Vol 82 (4) ◽  
pp. 1559-1571 ◽  
Author(s):  
Mark J. White ◽  
Jeffrey M. Boyd ◽  
Alexander R. Horswill ◽  
William M. Nauseef
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Phospholipase C ◽  
Whole Blood ◽  
Dependent Manner ◽  
Binding Studies ◽  
Content Type ◽  
Positive Regulators

ABSTRACTStaphylococcus aureusis an important human pathogen that employs a large repertoire of secreted virulence factors to promote disease pathogenesis. Many strains ofS. aureuspossess aplcgene that encodes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) capable of hydrolyzing PI and cleaving glycosyl-PI (GPI)-linked proteins from cell surfaces. Despite being secreted by virulent staphylococci, the contribution of PI-PLC to the capacity ofS. aureusto cause disease remains undefined. Our goal in these studies was to understand PI-PLC in the context ofS. aureusbiology. Among a collection of genetically diverse clinical isolates ofS. aureus, community-associated methicillin-resistantS. aureus(CA-MRSA) USA300 secreted the most PI-PLC. Screening a collection of two-component system (TCS) mutants ofS. aureus, we identified both theagrquorum-sensing system and the SrrAB TCS to be positive regulators ofplcgene expression. Real-time PCR and PI-PLC enzyme assays of the TCS mutants, coupled with SrrA promoter binding studies, demonstrated that SrrAB was the predominant transcriptional activator ofplc. Furthermore,plcregulation was linked to oxidative stress bothin vitroandin vivoin a SrrAB-dependent manner. A Δplcmutant in a CA-MRSA USA300 background exhibited a survival defect in human whole blood and in isolated neutrophils. However, the same mutant strain displayed no survival defect in murine models of infection or murine whole blood. Overall, these data identify potential links between bacterial responses to the host innate immune system and to oxidative stress and suggest how PI-PLC could contribute to the pathogenesis ofS. aureusinfections.

scholarly journals Staphylococcus aureus Nuclease Is an SaeRS-Dependent Virulence Factor

2013 ◽  
Vol 81 (4) ◽  
pp. 1316-1324 ◽  
Author(s):  
Michael E. Olson ◽  
Tyler K. Nygaard ◽  
Laynez Ackermann ◽  
Robert L. Watkins ◽  
Oliwia W. Zurek ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factor ◽  
Dependent Manner ◽  
Binding Studies ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Activity Measurements ◽  
Two Component

ABSTRACTSeveral prominent bacterial pathogens secrete nuclease (Nuc) enzymes that have an important role in combating the host immune response. Early studies ofStaphylococcus aureusNuc attributed its regulation to theagrquorum-sensing system. However, recent microarray data have indicated thatnucis under the control of the SaeRS two-component system, which is a major regulator ofS. aureusvirulence determinants. Here we report that thenucgene is directly controlled by the SaeRS two-component system through reporter fusion, immunoblotting, Nuc activity measurements, promoter mapping, and binding studies, and additionally, we were unable identify a notable regulatory link to theagrsystem. The observed SaeRS-dependent regulation was conserved across a wide spectrum of representativeS. aureusisolates. Moreover, with community-associated methicillin-resistantS. aureus(CA MRSA) in a mouse model of peritonitis, we observedin vivoexpression of Nuc activity in an SaeRS-dependent manner and determined that Nuc is a virulence factor that is important forin vivosurvival, confirming the enzyme's role as a contributor to invasive disease. Finally, natural polymorphisms were identified in the SaeRS proteins, one of which was linked to Nuc regulation in a CA MRSA USA300 endocarditis isolate. Altogether, our findings demonstrate that Nuc is an importantS. aureusvirulence factor and part of the SaeRS regulon.

scholarly journals Efficacy of a Multimechanistic Monoclonal Antibody Combination against Staphylococcus aureus Surgical Site Infections in Mice

2019 ◽  
Vol 63 (8) ◽  
Author(s):  
Roger V. Ortines ◽  
Yu Wang ◽  
Haiyun Liu ◽  
Dustin A. Dikeman ◽  
Bret L. Pinsker ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Mouse Models ◽  
Virulence Factors ◽  
Surgical Site Infections ◽  
Alpha Toxin ◽  
Content Type

ABSTRACT Surgical site infections (SSIs) are commonly caused by Staphylococcus aureus. We report that a combination of three monoclonal antibodies (MEDI6389) that neutralize S. aureus alpha-toxin, clumping factor A, and four leukocidins (LukSF, LukED, HlgAB, and HlgCB) plus vancomycin had enhanced efficacy compared with control antibody plus vancomycin in two mouse models of S. aureus SSI. Therefore, monoclonal antibody-based neutralization of multiple S. aureus virulence factors may provide an adjunctive perioperative approach to combat S. aureus SSIs.

scholarly journals Linezolid Attenuates Lethal Lung Damage during Postinfluenza Methicillin-Resistant Staphylococcus aureus Pneumonia

2019 ◽  
Vol 87 (10) ◽  
Author(s):  
Atul K. Verma ◽  
Christopher Bauer ◽  
Vijaya Kumar Yajjala ◽  
Shruti Bansal ◽  
Keer Sun
Keyword(s):  
Staphylococcus Aureus ◽  
Therapeutic Effect ◽  
Critical Role ◽  
Lung Damage ◽  
Alpha Toxin ◽  
Methicillin Resistant ◽  
Content Type ◽  
Linezolid Therapy ◽  
Staphylococcus Aureus Pneumonia ◽  
The Impact

ABSTRACT Postinfluenza methicillin-resistant Staphylococcus aureus (MRSA) infection can quickly develop into severe, necrotizing pneumonia, causing over 50% mortality despite antibiotic treatments. In this study, we investigated the efficacy of antibiotic therapies and the impact of S. aureus alpha-toxin in a model of lethal influenza virus and MRSA coinfection. We demonstrate that antibiotics primarily attenuate alpha-toxin-induced acute lethality, even though both alpha-toxin-dependent and -independent mechanisms significantly contribute to animal mortality after coinfection. Furthermore, we found that the protein synthesis-suppressing antibiotic linezolid has an advantageous therapeutic effect on alpha-toxin-induced lung damage, as measured by protein leak and lactate dehydrogenase (LDH) activity. Importantly, using a Panton-Valentine leucocidin (PVL)-negative MRSA isolate from patient sputum, we show that linezolid therapy significantly improves animal survival from postinfluenza MRSA pneumonia compared with vancomycin treatment. Rather than improved viral or bacterial control, this advantageous therapeutic effect is associated with a significantly attenuated proinflammatory cytokine response and acute lung damage in linezolid-treated mice. Together, our findings not only establish a critical role of alpha-toxin in the extreme mortality of secondary MRSA pneumonia after influenza but also provide support for the possibility that linezolid could be a more effective treatment than vancomycin to improve disease outcomes.

scholarly journals The ClpCP Complex Modulates Respiratory Metabolism inStaphylococcus aureusand Is Regulated in a SrrAB-Dependent Manner

2019 ◽  
Vol 201 (15) ◽  
Author(s):  
Ameya A. Mashruwala ◽  
Brian J. Eilers ◽  
Amanda L. Fuchs ◽  
Javiera Norambuena ◽  
Carly A. Earle ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Energy Metabolism ◽  
Energy Homeostasis ◽  
Protein Complexes ◽  
Present Report ◽  
Bacterial Pathogen ◽  
Dependent Manner ◽  
Content Type ◽  
Fermentative Growth ◽  
Increased Sensitivity

ABSTRACTThestaphylococcalrespiratoryregulator (SrrAB) modulates energy metabolism inStaphylococcus aureus. Studies have suggested that regulated protein catabolism facilitates energy homeostasis. Regulated proteolysis inS. aureusis achieved through protein complexes composed of a peptidase (ClpQ or ClpP) in association with an AAA+family ATPase (typically, ClpC or ClpX). In the present report, we tested the hypothesis that SrrAB regulates a Clp complex to facilitate energy homeostasis inS. aureus. Strains deficient in one or more Clp complexes were attenuated for growth in the presence of puromycin, which causes enrichment of misfolded proteins. A ΔsrrABstrain had increased sensitivity to puromycin. Epistasis experiments suggested that the puromycin sensitivity phenotype of the ΔsrrABstrain was a result of decreased ClpC activity. Consistent with this, transcriptional activity ofclpCwas decreased in the ΔsrrABmutant, and overexpression ofclpCsuppressed the puromycin sensitivity of the ΔsrrABstrain. We also found that ClpC positively influenced respiration and that it did so upon association with ClpP. In contrast, ClpC limited fermentative growth, while ClpP was required for optimal fermentative growth. Metabolomics studies demonstrated that intracellular metabolic profiles of the ΔclpCand ΔsrrABmutants were distinct from those of the wild-type strain, supporting the notion that both ClpC and SrrAB affect central metabolism. We propose a model wherein SrrAB regulates energy homeostasis, in part, via modulation of regulated proteolysis.IMPORTANCEOxygen is used as a substrate to derive energy by the bacterial pathogenStaphylococcus aureusduring infection; however,S. aureuscan also grow fermentatively in the absence of oxygen. To successfully cause infection,S. aureusmust tailor its metabolism to take advantage of respiratory activity. Different proteins are required for growth in the presence or absence of oxygen; therefore, when cells transition between these conditions, several proteins would be expected to become unnecessary. In this report, we show that regulated proteolysis is used to modulate energy metabolism inS. aureus. We report that the ClpCP protein complex is involved in specifically modulating aerobic respiratory growth but is dispensable for fermentative growth.

scholarly journals Candida albicans Impacts Staphylococcus aureus Alpha-Toxin Production via Extracellular Alkalinization

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Olivia A. Todd ◽  
Mairi C. Noverr ◽  
Brian M. Peters
Keyword(s):  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Quorum Sensing ◽  
Bacterial Pathogen ◽  
Toxin Production ◽  
Extracellular Ph ◽  
Morbidity And Mortality ◽  
Alpha Toxin ◽  
Content Type

ABSTRACT Candida albicans and Staphylococcus aureus are common causes of nosocomial infections with severe morbidity and mortality. Murine polymicrobial intra-abdominal infection (IAI) with C. albicans and S. aureus results in acute mortality dependent on the secreted cytolytic effector alpha-toxin. Here, we confirmed that alpha-toxin is elevated during polymicrobial growth compared to monomicrobial growth in vitro. Therefore, this study sought to unravel the mechanism by which C. albicans drives enhanced staphylococcal alpha-toxin production. Using a combination of functional and genetic approaches, we determined that an intact agr quorum sensing regulon is necessary for enhanced alpha-toxin production during coculture and that a secreted candidal factor likely is not implicated in elevating agr activation. As the agr system is pH sensitive, we observed that C. albicans raises the pH during polymicrobial growth and that this correlates with increased agr activity and alpha-toxin production. Modulation of the pH could predictably attenuate or activate agr activity during coculture. By using a C. albicans mutant deficient in alkalinization (stp2Δ/Δ), we confirmed that modulation of the extracellular pH by C. albicans can drive agr expression and toxin production. Additionally, the use of various Candida species (C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. krusei) demonstrated that those capable of raising the extracellular pH correlated with elevated agr activity and alpha-toxin production during coculture. Overall, we demonstrate that alkalinization of the extracellular pH by the Candida species leads to sustained activation of the staphylococcal agr system. IMPORTANCE Candida albicans and Staphylococcus aureus are commonly coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Thus, they represent a significant cause of nosocomial morbidity and mortality. Yet how these organisms behave in the context of polymicrobial growth remains poorly understood. In this work, we set out to determine the mechanism by which activation of the staphylococcal agr quorum sensing system and production of its major virulence effector alpha-toxin is enhanced during coculture with C. albicans. Surprisingly, we likely ruled out that a secreted candidal factor drives this process. Instead, we demonstrated that alkalinization of the extracellular milieu by C. albicans and other Candida species correlated with elevated agr activity. Thus, we propose a mechanism where modulation of the extracellular pH by fungal opportunists can indirectly alter virulence of a bacterial pathogen. Uncovering molecular events that drive interkingdom pathogenicity mechanisms may enhance surveillance and treatment for devastating polymicrobial infections.

scholarly journals Disruption of Glycolysis by Nutritional Immunity Activates a Two-Component System That Coordinates a Metabolic and Antihost Response by Staphylococcus aureus

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Paola K. Párraga Solórzano ◽  
Jiangwei Yao ◽  
Charles O. Rock ◽  
Thomas E. Kehl-Fie
Keyword(s):  
Staphylococcus Aureus ◽  
Metabolic Flux ◽  
Bacterial Species ◽  
Critical Role ◽  
Dependent Manner ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Nutritional Immunity ◽  
Two Component

ABSTRACT During infection, bacteria use two-component signal transduction systems to sense and adapt to the dynamic host environment. Despite critically contributing to infection, the activating signals of most of these regulators remain unknown. This also applies to the Staphylococcus aureus ArlRS two-component system, which contributes to virulence by coordinating the production of toxins, adhesins, and a metabolic response that enables the bacterium to overcome host-imposed manganese starvation. Restricting the availability of essential transition metals, a strategy known as nutritional immunity, constitutes a critical defense against infection. In this work, expression analysis revealed that manganese starvation imposed by the immune effector calprotectin or by the absence of glycolytic substrates activates ArlRS. Manganese starvation imposed by calprotectin also activated the ArlRS system even when glycolytic substrates were present. A combination of metabolomics, mutational analysis, and metabolic feeding experiments revealed that ArlRS is activated by alterations in metabolic flux occurring in the latter half of the glycolytic pathway. Moreover, calprotectin was found to induce expression of staphylococcal leukocidins in an ArlRS-dependent manner. These studies indicated that ArlRS is a metabolic sensor that allows S. aureus to integrate multiple environmental stresses that alter glycolytic flux to coordinate an antihost response and to adapt to manganese starvation. They also established that the latter half of glycolysis represents a checkpoint to monitor metabolic state in S. aureus. Altogether, these findings contribute to understanding how invading pathogens, such as S. aureus, adapt to the host during infection and suggest the existence of similar mechanisms in other bacterial species. IMPORTANCE Two-component regulatory systems enable bacteria to adapt to changes in their environment during infection by altering gene expression and coordinating antihost responses. Despite the critical role of two-component systems in bacterial survival and pathogenesis, the activating signals for most of these regulators remain unidentified. This is exemplified by ArlRS, a Staphylococcus aureus global regulator that contributes to virulence and to resisting host-mediated restriction of essential nutrients, such as manganese. In this report, we demonstrate that manganese starvation and the absence of glycolytic substrates activate ArlRS. Further investigations revealed that ArlRS is activated when the latter half of glycolysis is disrupted, suggesting that S. aureus monitors flux through the second half of this pathway. Host-imposed manganese starvation also induced the expression of pore-forming toxins in an ArlRS-dependent manner. Cumulatively, this work reveals that ArlRS acts as a sensor that links nutritional status, cellular metabolism, and virulence regulation.

scholarly journals Inhibition of Staphylococcus aureus Invasion into Bovine Mammary Epithelial Cells by Contact with Live Lactobacillus casei

2012 ◽  
Vol 79 (3) ◽  
pp. 877-885 ◽  
Author(s):  
Damien S. Bouchard ◽  
Lucie Rault ◽  
Nadia Berkova ◽  
Yves Le Loir ◽  
Sergine Even
Keyword(s):  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Lactobacillus Casei ◽  
Mammary Epithelial Cells ◽  
Control Strategies ◽  
Mammary Epithelial ◽  
Dairy Herds ◽  
Dependent Manner ◽  
Content Type ◽  
Bovine Mammary Epithelial Cells

ABSTRACTStaphylococcus aureusis a major pathogen that is responsible for mastitis in dairy herds.S. aureusmastitis is difficult to treat and prone to recurrence despite antibiotic treatment. The ability ofS. aureusto invade bovine mammary epithelial cells (bMEC) is evoked to explain this chronicity. One sustainable alternative to treat or prevent mastitis is the use of lactic acid bacteria (LAB) as mammary probiotics. In this study, we tested the ability ofLactobacillus caseistrains to prevent invasion of bMEC by twoS. aureusbovine strains, RF122 and Newbould305, which reproducibly induce acute and moderate mastitis, respectively.L. caseistrains affected adhesion and/or internalization ofS. aureusin a strain-dependent manner. Interestingly,L. caseiCIRM-BIA 667 reducedS. aureusNewbould305 and RF122 internalization by 60 to 80%, and this inhibition was confirmed for two otherL. caseistrains, including one isolated from bovine teat canal. The protective effect occurred without affecting bMEC morphology and viability. Once internalized, the fate ofS. aureuswas not affected byL. casei. It should be noted thatL. caseiwas internalized at a low rate but survived in bMEC cells with a better efficiency than that ofS. aureusRF122. Inhibition ofS. aureusadhesion was maintained with heat-killedL. casei, whereas contact between liveL. caseiandS. aureusor bMEC was required to preventS. aureusinternalization. This first study of the antagonism of LAB towardS. aureusin a mammary context opens avenues for the development of novel control strategies against this major pathogen.

scholarly journals Rapid Emergence of Resistance to Linezolid and Mutator Phenotypes in Staphylococcus aureus Isolates from an Adult Cystic Fibrosis Patient

2013 ◽  
Vol 57 (10) ◽  
pp. 5186-5188 ◽  
Author(s):  
Asmaa Tazi ◽  
Jeanne Chapron ◽  
Gerald Touak ◽  
Magalie Longo ◽  
Dominique Hubert ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Cystic Fibrosis Patient ◽  
Therapeutic Option ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Mutator Phenotype ◽  
Content Type ◽  
Emergence Of Resistance ◽  
Rapid Emergence

ABSTRACTLinezolid has emerged as an important therapeutic option for the treatment ofStaphylococcus aureusin patients with cystic fibrosis. We report the rapid emergence, upon treatment with linezolid, of linezolid-resistantS. aureusclinical isolates through the accumulation of resistance-associated 23S rRNA mutations, together with acquisition of an altered mutator phenotype.

scholarly journals The Ancestral N-Terminal Domain of Big Defensins Drives Bacterially Triggered Assembly into Antimicrobial Nanonets

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Karine Loth ◽  
Agnès Vergnes ◽  
Cairé Barreto ◽  
Sébastien N. Voisin ◽  
Hervé Meudal ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Bactericidal Activity ◽  
Multidrug Resistant ◽  
High Salt ◽  
Clinical Isolates ◽  
Host Defense Peptides ◽  
Hydrophobic Domain ◽  
Content Type ◽  
Terminal Domain

ABSTRACT Big defensins, ancestors of β-defensins, are composed of a β-defensin-like C-terminal domain and a globular hydrophobic ancestral N-terminal domain. This unique structure is found in a limited number of phylogenetically distant species, including mollusks, ancestral chelicerates, and early-branching cephalochordates, mostly living in marine environments. One puzzling evolutionary issue concerns the advantage for these species of having maintained a hydrophobic domain lost during evolution toward β-defensins. Using native ligation chemistry, we produced the oyster Crassostrea gigas BigDef1 (Cg-BigDef1) and its separate domains. Cg-BigDef1 showed salt-stable and broad-range bactericidal activity, including against multidrug-resistant human clinical isolates of Staphylococcus aureus. We found that the ancestral N-terminal domain confers salt-stable antimicrobial activity to the β-defensin-like domain, which is otherwise inactive. Moreover, upon contact with bacteria, the N-terminal domain drives Cg-BigDef1 assembly into nanonets that entrap and kill bacteria. We speculate that the hydrophobic N-terminal domain of big defensins has been retained in marine phyla to confer salt-stable interactions with bacterial membranes in environments where electrostatic interactions are impaired. Those remarkable properties open the way to future drug developments when physiological salt concentrations inhibit the antimicrobial activity of vertebrate β-defensins. IMPORTANCE β-Defensins are host defense peptides controlling infections in species ranging from humans to invertebrates. However, the antimicrobial activity of most human β-defensins is impaired at physiological salt concentrations. We explored the properties of big defensins, the β-defensin ancestors, which have been conserved in a number of marine organisms, mainly mollusks. By focusing on a big defensin from oyster (Cg-BigDef1), we showed that the N-terminal domain lost during evolution toward β-defensins confers bactericidal activity to Cg-BigDef1, even at high salt concentrations. Cg-BigDef1 killed multidrug-resistant human clinical isolates of Staphylococcus aureus. Moreover, the ancestral N-terminal domain drove the assembly of the big defensin into nanonets in which bacteria are entrapped and killed. This discovery may explain why the ancestral N-terminal domain has been maintained in diverse marine phyla and creates a new path of discovery to design β-defensin derivatives active at physiological and high salt concentrations.

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