Studying Interactions of &lt;em&gt;Staphylococcus aureus&lt;/em&gt; with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Surewaard, Bas G. J.; Strijp, Jos A. G. van; Nijland, Reindert

doi:10.3791/50788

Cited by 22 publications

(21 citation statements)

References 13 publications

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“…Neutrophils were isolated by using Percoll (1.09176 g/l) centrifugation14. Human blood was collected in heparin tubes from healthy volunteers and neutrophils were isolated by Ficoll/Histopaque centrifugation43. Blood was collected from healthy horses during the slaughter process (and immediately upon death) in tubes containing 3 mM EDTA anticoagulant.…”

Section: Methodsmentioning

confidence: 99%

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus

Koop

Vrieling

Storisteanu

et al. 2017

Sci Rep

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Bicomponent pore-forming leukocidins are a family of potent toxins secreted by Staphylococcus aureus, which target white blood cells preferentially and consist of an S- and an F-component. The S-component recognizes a receptor on the host cell, enabling high-affinity binding to the cell surface, after which the toxins form a pore that penetrates the cell lipid bilayer. Until now, six different leukocidins have been described, some of which are host and cell specific. Here, we identify and characterise a novel S. aureus leukocidin; LukPQ. LukPQ is encoded on a 45 kb prophage (ΦSaeq1) found in six different clonal lineages, almost exclusively in strains cultured from equids. We show that LukPQ is a potent and specific killer of equine neutrophils and identify equine-CXCRA and CXCR2 as its target receptors. Although the S-component (LukP) is highly similar to the S-component of LukED, the species specificity of LukPQ and LukED differs. By forming non-canonical toxin pairs, we identify that the F-component contributes to the observed host tropism of LukPQ, thereby challenging the current paradigm that leukocidin specificity is driven solely by the S-component.

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Section: Methodsmentioning

confidence: 99%

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus

Koop

Vrieling

Storisteanu

et al. 2017

Sci Rep

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“…Neutrophils were isolated from bovine blood by Percoll (1.09176 g/l) centrifugation21. Human heparin blood was collected from healthy volunteers and the isolation of neutrophils was performed by Ficoll/Histopaque centrifugation42. Purity was ≥95% and viability ≥95%, for both human and bovine neutrophils.…”

Section: Methodsmentioning

confidence: 99%

LukMF′ is the major secreted leukocidin of bovine Staphylococcus aureus and is produced in vivo during bovine mastitis

Vrieling

Boerhout

Wigcheren

et al. 2016

Sci Rep

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Staphylococcus aureus is a major human and animal pathogen and a common cause of mastitis in cattle. S. aureus secretes several leukocidins that target bovine neutrophils, crucial effector cells in the defence against bacterial pathogens. In this study, we investigated the role of staphylococcal leukocidins in the pathogenesis of bovine S. aureus disease. We show that LukAB, in contrast to the γ-hemolysins, LukED, and LukMF′, was unable to kill bovine neutrophils, and identified CXCR2 as a bovine receptor for HlgAB and LukED. Furthermore, we assessed functional leukocidin secretion by bovine mastitis isolates and observed that, although leukocidin production was strain dependent, LukMF′ was most abundantly secreted and the major toxin killing bovine neutrophils. To determine the role of LukMF′ in bovine mastitis, cattle were challenged with high (S1444) or intermediate (S1449, S1463) LukMF′-producing isolates. Only animals infected with S1444 developed severe clinical symptoms. Importantly, LukM was produced in vivo during the course of infection and levels in milk were associated with the severity of mastitis. Altogether, these findings underline the importance of LukMF′ as a virulence factor and support the development of therapeutic approaches targeting LukMF′ to control S. aureus mastitis in cattle.

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“…Wells of an eight-well Lab-Tek II chambered cover glass (Thermo Scientific, Rochester, USA) were loaded with 200 l of RPMI-HSA medium containing the mixed S. pneumoniae strains. Subsequently, 5 ϫ 10 5 freshly isolated neutrophils (36) were added (MOI of 10) to the well, and imaging was started immediately while the neutrophils were settling at the cover glass bottom of the well. Microscopic image acquisition was performed using a Leica TSC SP5 inverted microscope equipped with an HCX PL APO 40ϫ 0.85 objective (Leica Microsystems, The Netherlands).…”

Section: Methodsmentioning

confidence: 99%

Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

et al. 2015

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c Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis. Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, and pneumococcal infections (e.g., pneumonia, septicemia, and meningitis) kill more than 1 million people every year (1). Pneumococci are also quiescent colonizers of the upper respiratory tract, particularly in children, but little is known about the mechanisms underlying the transition from commensal to pathogen. It is therefore of crucial importance to understand the entire pneumococcal pathogenesis cycle in detail.The polysaccharide capsule covering the cell surface is the most central virulence factor of S. pneumoniae. The involvement of the capsule in pneumococcal pathogenesis has been appreciated since Griffith in 1928 (2) published his famous transformation experiment with rough and smooth strains of S. pneumoniae. Today, it is known that the bulky capsule, which is either negatively charged or neutral, contributes to pathogenesis by protecting pneumococci against the human immune system. For example, the capsule hinders phagocytosis and inhibits complement activity (3-6). Over 90 different pneumococcal serotypes have been identified to date (7), and the different serotypes vary in how well they protect the bacteria against phagocytosis (6). Furthermore, the amount of capsule differs between bacteria, and it has been s...

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Studying Interactions of <em>Staphylococcus aureus</em> with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Cited by 22 publications

References 13 publications

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus

LukMF′ is the major secreted leukocidin of bovine Staphylococcus aureus and is produced in vivo during bovine mastitis

Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

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