scholarly journals Bovine Staphylococcus aureus Secretes the Leukocidin LukMF′ To Kill Migrating Neutrophils through CCR1

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
M. Vrieling ◽  
K. J. Koymans ◽  
D. A. C. Heesterbeek ◽  
P. C. Aerts ◽  
V. P. M. G. Rutten ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Host Specificity ◽  
Ex Vivo ◽  
Field Isolate ◽  
Human Neutrophils ◽  
Fibrin Gel ◽  
Content Type ◽  
New Targets ◽  
Bovine Neutrophils

ABSTRACTAlthoughStaphylococcus aureusis best known for infecting humans, bovine-specific strains are a major cause of mastitis in dairy cattle. The bicomponent leukocidin LukMF′, exclusively harbored byS. aureusof ruminant origin, is a virulence factor associated with bovine infections. In this study, the molecular basis of the host specificity of LukMF′ is elucidated by identification of chemokine receptor CCR1 as its target. Bovine neutrophils, the major effector cells in the defense against staphylococci, express significant cell surface levels of CCR1, whereas human neutrophils do not. This causes the particular susceptibility of bovine neutrophils to pore formation induced by LukMF′. BovineS. aureusstrains produce high levels of LukMF′in vitro. In culture supernatant of the mastitis field isolate S1444, LukMF′ was the most important cytotoxic agent for bovine neutrophils. In a fibrin gel matrix, the effects of thein situsecreted toxins on neutrophils migrating towardS. aureuswere visualized. Under these physiologicalex vivoconditions, bovineS. aureusS1444 efficiently killed approaching neutrophils at a distance through secretion of LukMF′. Altogether, our findings illustrate the coevolution of pathogen and host, provide new targets for therapeutic and vaccine approaches to treat staphylococcal diseases in the cow, and emphasize the importance of staphylococcal toxins in general.IMPORTANCEThis study explains the mechanism of action of LukMF′, a bicomponent toxin found in bovine lineages ofS. aureusthat is associated with mastitis in cattle. At a molecular level, we describe how LukMF′ can specifically kill bovine neutrophils. Here, we demonstrate the contribution of toxins in the determination of host specificity and contribute to the understanding of mechanisms of coevolution of pathogen and host. Our study provides new targets that can be used in therapeutic and vaccine approaches to treat staphylococcal diseases in the cow. We also demonstrate the importance of toxins in specific elimination of immune cells, which has broader implications, especially in human infections.

scholarly journals Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Marloes I. Hofstee ◽  
Martijn Riool ◽  
Igors Terjajevs ◽  
Keith Thompson ◽  
Martin J. Stoddart ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Cells ◽  
Early Stage ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Maximum Size ◽  
Human Neutrophils ◽  
Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

scholarly journals Children with Invasive Staphylococcus aureus Disease Exhibit a Potently Neutralizing Antibody Response to the Cytotoxin LukAB

2013 ◽  
Vol 82 (3) ◽  
pp. 1234-1242 ◽  
Author(s):  
Isaac P. Thomsen ◽  
Ashley L. DuMont ◽  
David B. A James ◽  
Pauline Yoong ◽  
Benjamin R. Saville ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibody Response ◽  
High Titer ◽  
Humoral Response ◽  
Neutralizing Antibody ◽  
Human Neutrophils ◽  
Serum Samples ◽  
Marked Rise ◽  
Content Type

ABSTRACTDespite the importance ofStaphylococcus aureusas a common invasive bacterial pathogen, the humoral response to infection remains inadequately defined, particularly in children. The purpose of this study was to assess the humoral response to extracellular staphylococcal virulence factors, including the bicomponent leukotoxins, which are critical for the cytotoxicity ofS. aureustoward human neutrophils. Children with culture-provenS. aureusinfection were prospectively enrolled and stratified by disease type. Fifty-three children were enrolled in the study, of which 90% had invasive disease. Serum samples were obtained during the acute (within 48 h) and convalescent (4 to 6 weeks postinfection) phases, at which point both IgG titers againstS. aureusexotoxins were determined, and the functionality of the generated antibodies was evaluated. Molecular characterization of clinical isolates was also performed. We observed a marked rise in antibody titer from acute-phase to convalescent-phase sera for LukAB, the most recently describedS. aureusbicomponent leukotoxin. LukAB production by the isolates was strongly correlated with cytotoxicityin vitro, and sera containing anti-LukAB antibodies potently neutralized cytotoxicity. Antibodies toS. aureusantigens were detectable in healthy pediatric controls but at much lower titers than in sera from infected subjects. The discovery of a high-titer, neutralizing antibody response to LukAB during invasive infections suggests that this toxin is producedin vivoand that it elicits a functional humoral response.

scholarly journals Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Fatemeh Askarian ◽  
Satoshi Uchiyama ◽  
J. Andrés Valderrama ◽  
Clement Ajayi ◽  
Johanna U. E. Sollid ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Innate Immune ◽  
Human Neutrophils ◽  
Infection Model ◽  
Bacterial Survival ◽  
Bacterial Killing ◽  
Content Type ◽  
Repeat Protein

ABSTRACT Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood.

scholarly journals Bacterial Membrane-Derived Vesicles Attenuate Vancomycin Activity against Methicillin-Resistant Staphylococcus aureus

2021 ◽  
Vol 9 (10) ◽  
pp. 2055
Author(s):  
Monika Kumaraswamy ◽  
Kamilla Wiull ◽  
Bishnu Joshi ◽  
George Sakoulas ◽  
Armin Kousha ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Whole Blood ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Ex Vivo ◽  
Membrane Vesicles ◽  
World Health ◽  
Human Neutrophils ◽  
Methicillin Resistant

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved numerous antimicrobial resistance mechanisms and is identified as a serious public health threat by the World Health Organization and U.S. Centers for Disease Control and Prevention. The glycopeptide vancomycin (VAN) remains a cornerstone of therapy for severe MRSA infections despite increasing reports of therapeutic failure in hospitalized patients with bacteremia or pneumonia. Recently, the role of released bacterial-derived membrane vesicles (MVs) in antibiotic resistance has garnered attention. Here we examined the effect of exogenous MRSA-derived MVs on VAN activity against MRSA in vitro, using minimum inhibitory concentration and checkerboard assays, and ex vivo, incorporating components of host innate immunity such as neutrophils and serum complement present in blood. Additionally, the proteome of MVs from VAN-exposed MRSA was characterized to determine if protein expression was altered. The presence of MVs increased the VAN MIC against MRSA to values where clinical failure is commonly observed. Furthermore, the presence of MVs increased survival of MRSA pre-treated with sub-MIC concentrations of VAN in whole blood and upon exposure to human neutrophils but not human serum. Unbiased proteomic analysis also showed an elevated expression of MV proteins associated with antibiotic resistance (e.g., marR) or proteins that are functionally linked to cell membrane/wall metabolism. Together, our findings indicate MRSA-derived MVs are capable of lowering susceptibility of the pathogen to VAN, whole-blood- and neutrophil-mediated killing, a new pharmacodynamic consideration for a drug increasingly linked to clinical treatment failures.

scholarly journals Characterization of the Retrocyclin Analogue RC-101 as a Preventative of Staphylococcus aureus Nasal Colonization

2011 ◽  
Vol 55 (11) ◽  
pp. 5338-5346 ◽  
Author(s):  
Ryan P. Lamers ◽  
Colleen R. Eade ◽  
Alan J. Waring ◽  
Amy L. Cole ◽  
Alexander M. Cole
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Treatment Options ◽  
Nasal Colonization ◽  
Proinflammatory Response ◽  
Content Type ◽  
Bactericidal Effects ◽  
Prevention Of Nosocomial Infection

ABSTRACTNasal colonization ofStaphylococcus aureusis a risk factor for pathogenic autoinfection, particularly in postoperative patients and the immunocompromised. As such, standardized preoperative nasal decolonization ofS. aureushas become a major consideration for the prevention of nosocomial infection. However, only a few treatment options for nasal decolonization are currently available, with resistance to these approaches already a concern. Here we have identified the macrocyclic θ-defensin analogue RC-101 as a promising anti-S. aureusagent for nasal decolonization. RC-101 exhibits bactericidal effects againstS. aureuswith the use ofin vitroepithelium-free systems, while also preventing the pathogen's proliferation and attachment in anex vivohuman nasal epithelial cell adhesion model and an organotypic model of human airway epithelia. Peptide concentrations as low as 2.5 μM elicited significant reductions inS. aureusgrowth in epithelium-free systems, with 10 μM concentrations being completely bactericidal for all strains tested, including USA300. Inex vivonasal colonization models, RC-101 significantly reduced adherence, survival, and proliferation ofS. aureuson human nasal epithelia. Reductions inS. aureusviability were evident in these assays, with as little as 1 μg of peptide per tissue, while 10 μg of RC-101 completely prevented adhesion of all strains tested. Furthermore, RC-101 did not exhibit cellular toxicity to human nasal epithelia at concentrations up to 200 μM, nor did it induce a proinflammatory response in these cells. Collectively, the findings of this study identify RC-101 as a potential preventative ofS. aureusnasal colonization.

scholarly journals Povidone Iodine: Properties, Mechanisms of Action, and Role in Infection Control and Staphylococcus aureus Decolonization

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Didier Lepelletier ◽  
Jean Yves Maillard ◽  
Bruno Pozzetto ◽  
Anne Simon
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Resistance ◽  
Povidone Iodine ◽  
Ex Vivo ◽  
Cross Resistance ◽  
Content Type ◽  
Intranasal Mupirocin ◽  
Resistant Strains ◽  
Emergence Of Resistance

ABSTRACT Nasal decolonization is an integral part of the strategies used to control and prevent the spread of methicillin-resistant Staphylococcus aureus (MRSA) infections. The two most commonly used agents for decolonization are intranasal mupirocin 2% ointment and chlorhexidine wash, but the increasing emergence of resistance and treatment failure has underscored the need for alternative therapies. This article discusses povidone iodine (PVP-I) as an alternative decolonization agent and is based on literature reviewed during an expert’s workshop on resistance and MRSA decolonization. Compared to chlorhexidine and mupirocin, respectively, PVP-I 10 and 7.5% solutions demonstrated rapid and superior bactericidal activity against MRSA in in vitro and ex vivo studies. Notably, PVP-I 10 and 5% solutions were also active against both chlorhexidine-resistant and mupirocin-resistant strains, respectively. Unlike chlorhexidine and mupirocin, available reports have not observed a link between PVP-I and the induction of bacterial resistance or cross-resistance to antiseptics and antibiotics. These preclinical findings also translate into clinical decolonization, where intranasal PVP-I significantly improved the efficacy of chlorhexidine wash and was as effective as mupirocin in reducing surgical site infection in orthopedic surgery. Overall, these qualities of PVP-I make it a useful alternative decolonizing agent for the prevention of S. aureus infections, but additional experimental and clinical data are required to further evaluate the use of PVP-I in this setting.

scholarly journals Direct Microscopic Observation of Human Neutrophil-Staphylococcus aureus Interaction In Vitro Suggests a Potential Mechanism for Initiation of Biofilm Infection on an Implanted Medical Device

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Niranjan Ghimire ◽  
Brian A. Pettygrove ◽  
Kyler B. Pallister ◽  
James Stangeland ◽  
Shelby Stanhope ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Head Start ◽  
Bacterial Growth ◽  
Bacterial Biomass ◽  
Bacterial Biofilm ◽  
Confocal Scanning Laser Microscopy ◽  
Human Neutrophils ◽  
Content Type ◽  
Biofilm Infection

ABSTRACT The ability of human neutrophils to clear newly attached Staphylococcus aureus bacteria from a serum-coated glass surface was examined in vitro using time-lapse confocal scanning laser microscopy. Quantitative image analysis was used to measure the temporal change in bacterial biomass, neutrophil motility, and fraction of the surface area policed by neutrophils. In control experiments in which the surface was inoculated with bacteria but no neutrophils were added, prolific bacterial growth was observed. Neutrophils were able to control bacterial growth but only consistently when the neutrophil/bacterium number ratio exceeded approximately 1. When preattached bacteria were given a head start and allowed to grow for 3 h prior to neutrophil addition, neutrophils were unable to maintain control of the nascent biofilm. In these head-start experiments, aggregates of bacterial biofilm with areas of 50 μm2 or larger formed, and the growth of such aggregates continued even when multiple neutrophils attacked a cluster. These results suggest a model for the initiation of a biofilm infection in which a delay in neutrophil recruitment to an abiotic surface allows surface-attached bacteria time to grow and form aggregates that become protected from neutrophil clearance. Results from a computational model of the neutrophil-biofilm surface contest supported this conceptual model and highlighted the stochastic nature of the interaction. Additionally, we observed that both neutrophil motility and clearance of bacteria were impaired when oxygen tension was reduced to 0% or 2% O2.

scholarly journals The msaABCR Operon Regulates the Response to Oxidative Stress in Staphylococcus aureus

2019 ◽  
Vol 201 (21) ◽  
Author(s):  
Shanti Pandey ◽  
Gyan S. Sahukhal ◽  
Mohamed O. Elasri
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Defense Mechanism ◽  
Ex Vivo ◽  
Carotenoid Pigment ◽  
Content Type ◽  
Staphylococcal Infections ◽  
Organic Hydroperoxides

ABSTRACT Staphylococcus aureus has evolved a complex regulatory network that controls a multitude of defense mechanisms against the deleterious effects of oxidative stress stimuli, subsequently leading to the pathogen’s survival and persistence in the hosts. Previously, we characterized the msaABCR operon as a regulator of virulence, antibiotic resistance, and the formation of persister cells in S. aureus. Deletion of the msaABCR operon resulted in the downregulation of several genes involved in resistance against oxidative stress. Notably, those included carotenoid biosynthetic genes and the ohr gene, which is involved in resistance against organic hydroperoxides. These findings led us to hypothesize that the msaABCR operon is involved in resisting oxidative stress generated in the presence of both H2O2 and organic hydroperoxides. Here, we report that a protein product of the msaABCR operon (MsaB) transcriptionally regulates the expression of the crtOPQMN operon and the ohr gene to resist in vitro oxidative stresses. In addition to its direct regulation of the crtOPQMN operon and ohr gene, we also show that MsaB is the transcriptional repressor of sarZ (repressor of ohr). Taken together, these results suggest that the msaABCR operon regulates an oxidative stress defense mechanism, which is required to facilitate persistent and recurrent staphylococcal infections. Moving forward, we plan to investigate the role of msaABCR in the persistence of S. aureus under in vivo conditions. IMPORTANCE This study shows the involvement of the msaABCR operon in resisting oxidative stress by Staphylococcus aureus generated under in vitro and ex vivo conditions. We show that MsaB regulates the expression and production of a carotenoid pigment, staphyloxanthin, which is a potent antioxidant in S. aureus. We also demonstrate that MsaB regulates the ohr gene, which is involved in defending against oxidative stress generated by organic hydroperoxides. This study highlights the importance of msaABCR in the survival of S. aureus in the presence of various environmental stimuli that mainly exert oxidative stress. The findings from this study indicate the possibility that msaABCR is involved in the persistence of staphylococcal infections and therefore could be a potential antimicrobial target to overcome recalcitrant staphylococcal infections.

scholarly journals Staphylococcus aureus Elaborates Leukocidin AB To Mediate Escape from within Human Neutrophils

2013 ◽  
Vol 81 (5) ◽  
pp. 1830-1841 ◽  
Author(s):  
Ashley L. DuMont ◽  
Pauline Yoong ◽  
Bas G. J. Surewaard ◽  
Meredith A. Benson ◽  
Reindert Nijland ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Polymorphonuclear Leukocytes ◽  
The United States ◽  
Growth Conditions ◽  
Human Neutrophils ◽  
Cytotoxic Potential ◽  
Content Type ◽  
Human Pmns ◽  
Mrsa Strains

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) strains of the pulsed-field type USA300 are primarily responsible for the current community-associated epidemic of MRSA infections in the United States. The success of USA300 is partly attributed to the ability of the pathogen to avoid destruction by human neutrophils (polymorphonuclear leukocytes [PMNs]), which are crucial to the host immune response toS. aureusinfection. In this work, we investigated the contribution of bicomponent pore-forming toxins to the ability of USA300 to withstand attack from primary human PMNs. We demonstrate thatin vitrogrowth conditions influence the expression, production, and availability of leukotoxins by USA300, which in turn impact the cytotoxic potential of this clone toward PMNs. Interestingly, we also found that upon exposure to PMNs, USA300 preferentially activates the promoter of thelukABoperon, which encodes the recently identified leukocidin AB (LukAB). LukAB elaborated by extracellularS. aureusforms pores in the plasma membrane of PMNs, leading to PMN lysis, highlighting a contribution of LukAB to USA300 virulence. We now show that LukAB also facilitates the escape of bacteria engulfed within PMNs, in turn enabling the replication and outgrowth ofS. aureus. Together, these results suggest that upon encountering PMNsS. aureusinduces the production of LukAB, which serves as an extra- and intracellular weapon to protect the bacterium from destruction by human PMNs.

scholarly journals Ability of Bicarbonate Supplementation To Sensitize Selected Methicillin-Resistant Staphylococcus aureus Strains to β-Lactam Antibiotics in an Ex Vivo Simulated Endocardial Vegetation Model

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Warren E. Rose ◽  
Ana M. Bienvenida ◽  
Yan Q. Xiong ◽  
Henry F. Chambers ◽  
Arnold S. Bayer ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bactericidal Activity ◽  
Ex Vivo ◽  
Rabbit Model ◽  
Luria Bertani ◽  
Growth Media ◽  
Methicillin Resistant ◽  
Content Type ◽  
Mrsa Strains

ABSTRACT Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth [CAMHB]) with bicarbonate (NaHCO3) increases β-lactam susceptibility of selected methicillin-resistant Staphylococcus aureus (MRSA) strains (“NaHCO3 responsive”). This “sensitization” phenomenon translated to enhanced β-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO3-mediated β-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. Four previously described MRSA strains were used: two each exhibiting in vitro NaHCO3-responsive or NaHCO3-nonresponsive phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were evaluated in CAMHB with or without NaHCO3. Intra-SEV MRSA killing was determined over 72-h exposures. In both “responsive” strains, supplementation with 25 mM or 44 mM NaHCO3 significantly reduced β-lactam MICs to below the OXA susceptibility breakpoint (≤4 mg/liter) and resulted in bactericidal activity (≥3-log killing) in the model for both OXA and CFZ. In contrast, neither in vitro-defined nonresponsive MRSA strain showed significant sensitization in the SEV model to either β-lactam. At both NaHCO3 concentrations, the fractional time above MIC was >50% for both CFZ and OXA in the responsive MRSA strains. Also, in media containing RPMI plus 10% Luria-Bertani broth (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO3), both CFZ and OXA exhibited enhanced bactericidal activity against NaHCO3-responsive strains in the SEV model. Neither CFZ nor OXA exposures selected for emergence of high-level β-lactam-resistant mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO3 supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate similar enhanced susceptibility in NaHCO3-supplemented media in vitro.

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