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

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

University Medical Center Utrecht

Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered.

Inactivation of staphylococcal phenol soluble modulins by serum lipoprotein particles.

Virulence de Staphylococcus aureus a &eacute;t&eacute; associ&eacute;e &agrave; la production de ph&eacute;nol modulins soluble (PSM). PSM sont connus pour activer, d&#39;attirer et de lyse des neutrophiles. Toutefois, la caract&eacute;risation fonctionnelle &eacute;tait g&eacute;n&eacute;ralement effectu&eacute;e en l&#39;absence de s&eacute;rum humain. Ici, nous d&eacute;montrons que le s&eacute;rum humain peut inhiber toutes les activit&eacute;s d&eacute;crites pr&eacute;c&eacute;demment du PSM. Nous avons observ&eacute; que s&eacute;rum peut bloquer compl&egrave;tement la lyse des cellules et le FPR2 activation des neutrophiles. Nous montrons une interaction directe entre les lipoprot&eacute;ines PSM et de s&eacute;rum dans le s&eacute;rum humain et le sang total. Une analyse ult&eacute;rieure &agrave; l&#39;aide de lipoprot&eacute;ines purifi&eacute; la haute, basse et tr&egrave;s basse densit&eacute; (VLDL, LDL et HDL) a r&eacute;v&eacute;l&eacute; qu&#39;ils neutralisent en effet PSM. Les lipoprot&eacute;ines HDL ont montr&eacute; une liaison plus forte et capacit&eacute; efficament pour PSM. De plus, nous montrons potentielle production intracellulaire du PSM par S. aureus &agrave; la phagocytose par les neutrophiles, qui ouvre un nouvel espace pour l&#39;exploration de la capacit&eacute; lytique intracellulaire du PSM. Collectivement, nos r&eacute;sultats montrent que dans un environnement de s&eacute;rum la fonction du PSM comme les toxines extracellulaires importants devrait &ecirc;tre reconsid&eacute;r&eacute;e.

Inactivation des staphylocoques ph&eacute;nol modulins soluble par les particules de lipoprot&eacute;ines s&eacute;riques.

Virulenza di Staphylococcus aureus &egrave; stato associato con la produzione di fenolo modulins solubile (PSM). PSM sono conosciuti per attivare, attrarre e lisi dei neutrofili. Tuttavia, le caratterizzazioni funzionali in genere sono state eseguite in assenza di siero umano. Qui, ci dimostrano che siero umano in grado di inibire tutte le attivit&agrave; descritte in precedenza di PSM. Abbiamo osservato che siero pu&ograve; bloccare completamente la lisi delle cellule e la FPR2 attivazione dei neutrofili. Vi mostriamo un&#39;interazione diretta tra lipoproteine PSM e siero in siero e sangue intero. Analisi successiva usando purificato alta, bassa e bassissima densit&agrave; di lipoproteine (HDL, LDL e VLDL) hanno rivelato che essi infatti neutralizzare PSM. Le lipoproteine HDL ha mostrato capacit&agrave; antagonizzando per PSM e associazione pi&ugrave; alto. Inoltre, mostriamo potenziale produzione intracellulare di PSM da S. aureus sulla fagocitosi di neutrofili, che apre una nuova area per l&#39;esplorazione della capacit&agrave; litici intracellulari di PSM. Collettivamente, i nostri dati mostrano che in un ambiente di siero la funzione di PSM come tossine extracellulari importante dovrebbe essere riconsiderata.

Inattivazione del fenolo stafilococciche solubile modulins di particelle di lipoproteina del siero.

Methicillin-resistant Staphylococcus 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 to S. aureus infection. 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 that in vitro growth 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 the lukAB operon, which encodes the recently identified leukocidin AB (LukAB). LukAB elaborated by extracellular S. aureus forms 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 of S. aureus. Together, these results suggest that upon encountering PMNs S. aureus induces the production of LukAB, which serves as an extra- and intracellular weapon to protect the bacterium from destruction by human PMNs.

Staphylococcus aureus elaborates leukocidin AB to mediate escape from within human neutrophils.

Souches d&#39;aureus (SARM) methicillin-resistant Staphylococcus du champ puls&eacute; type USA300 sont principalement responsables de l&#39;&eacute;pid&eacute;mie actuelle de communaut&eacute; associ&eacute;e &agrave; des infections &agrave; SARM aux &Eacute;tats-Unis. Le succ&egrave;s de USA300 est attribu&eacute; en partie &agrave; la capacit&eacute; de l&#39;agent pathog&egrave;ne afin d&#39;&eacute;viter la destruction par des neutrophiles humains (leucocytes polynucl&eacute;aires [PMN]), qui sont cruciales &agrave; l&#39;h&ocirc;te immuno-r&eacute;action &agrave; l&#39;infestation par S. aureus. Dans ce travail, nous avons &eacute;tudi&eacute; la contribution de filtrePediatrie formant des pores des toxines &agrave; la capacit&eacute; de USA300 pour supporter l&#39;attaque de PMN humaine primaire. Nous d&eacute;montrons que les conditions de croissance in vitro influencent l&#39;expression, la production et la disponibilit&eacute; de leukotoxins par USA300, qui tour impact le potentiel cytotoxique de ce clone vers PMN. fait int&eacute;ressant, nous avons aussi constat&eacute; qu&#39;au contact de l&#39;APF, USA300 active pr&eacute;f&eacute;rentiellement le promoteur de l&#39;op&eacute;ron lukAB, qui encode la leucocidine identifi&eacute; r&eacute;cemment AB (LukAB). LukAB &eacute;labor&eacute; par extracellulaires pores de formes S. aureus dans la membrane plasmique de l&#39;APF, conduisant &agrave; la lyse PMN, mettant en &eacute;vidence une contribution de LukAB &agrave; USA300 virulence. Nous montrons maintenant que LukAB facilite &eacute;galement l&#39;&eacute;vasion de bact&eacute;ries englouti au sein de l&#39;APF, &agrave; son tour, ce qui permet la r&eacute;plication et la croissance de S. aureus. Ensemble, ces r&eacute;sultats sugg&egrave;rent que, lorsqu&#39;il rencontre des PN S. aureus induit la production de LukAB, qui sert d&#39;extra - et intracellulaire arme pour prot&eacute;ger la bact&eacute;rie de la destruction par l&#39;APF humaine.

Staphylococcus aureus &eacute;labore leucocidine AB &agrave; la m&eacute;diation pour &eacute;chapper au sein de neutrophiles humains.

Meticillino-resistenti Staphylococcus aureus (MRSA) ceppi del tipo pulsare-campo USA300 sono principalmente responsabili per l&#39;attuale comunit&agrave; associate epidemia di infezioni da MRSA negli Stati Uniti. Il successo di USA300 &egrave; in parte attribuito alla capacit&agrave; del patogeno di evitare la distruzione di neutrofili umani (leucociti polimorfonucleari [PMN]), che sono cruciali per la risposta immunitaria all&#39;infezione da S. aureus. In questo lavoro, abbiamo studiato il contributo di bicomponente tossine formando dei pori per la capacit&agrave; di USA300 di sopportare attacco da PMN umani primari. Dimostriamo che le condizioni di crescita in vitro influenzano l&#39;espressione, la produzione e la disponibilit&agrave; di leukotoxins da USA300, che nell&#39;impatto svolta il potenziale citotossico di questo clone verso PMN. interessante, abbiamo anche trovato che all&#39;esposizione di PMN, USA300 preferenzialmente attiva il promotore dell&#39;operone lukAB, che codifica per la leucocidina recentemente identificato AB (LukAB). LukAB elaborato dai extracellulari S. aureus forme pori nella membrana del plasma di PMN, che portano alla lisi PMN, evidenziando un contributo di LukAB USA300 virulenza. Ora mostriamo che LukAB facilita anche la fuga di batteri travolto all&#39;interno di PMN, consentendo a sua volta la replica e l&#39;escrescenza di S. aureus. Insieme, questi risultati suggeriscono che su incontrando PMN S. aureus induce la produzione di LukAB, che serve come un extra - ed intracellulare arma per proteggere il batterio dalla distruzione di PMN umani.

Staphylococcus aureus elabora leucocidina AB per mediare sfuggire all&#39;interno dei neutrofili umani.

Neutrophil recruitment is essential in clearing pneumococcal infections. The first step in neutrophil extravasation involves the interaction between P-selectin on activated endothelium and P-Selectin Glycoprotein 1 (PSGL-1) on neutrophils. Here, we identify pneumococcal Zinc metalloproteinase C as a potent inhibitor of PSGL-1. ZmpC degrades the N-terminal domain of PSGL-1, thereby disrupting the initial rolling of neutrophils on activated human umbilical vein endothelial cells. Furthermore, mice infected with wild-type strain in the model of pneumococcal pneumonia showed lower lungs neutrophil infiltration compare to animals infected with ZmpC mutant. In addition, we confirmed the association of zmpC with serotype 8 and 11A and found it to be associated with serotype 33F as well. In conclusion, wereport PSGL-1 as a novel target for ZmpC and show that ZmpC inhibits neutrophil extravasation during pneumococcal pneumonia.

Pneumococcal immune evasion: ZmpC inhibits neutrophil influx.

Recrutement neutrophile est essentiel dans la gu&eacute;rison d&#39;infections &agrave; pneumocoques. La premi&egrave;re &eacute;tape dans le neutrophile extravasation implique l&#39;interaction entre la P-s&eacute;lectine sur l&#39;endoth&eacute;lium activ&eacute; et la P-s&eacute;lectine glycoprot&eacute;ine 1 (se-1) sur les neutrophiles. Ici, nous identifions des pneumocoque Zinc m&eacute;talloprot&eacute;inase C comme un puissant inhibiteur de la se-1. ZmpC d&eacute;grade le domaine N-terminal de se-1, perturbant ainsi le roulement initial des neutrophiles sur les cellules endoth&eacute;liales de veine ombilicale humaine activ&eacute;e. En outre, les souris infect&eacute;es par la souche sauvage dans le mod&egrave;le de la pneumonie &agrave; pneumocoque a montr&eacute; plus faible infiltration de neutrophiles de poumons comparent &agrave; animaux infect&eacute;s par le mutant ZmpC. En outre, nous avons confirm&eacute; l&#39;association entre zmpC et le s&eacute;rotype 8 et 11 a et trouve qu&#39;il est associ&eacute; &agrave; s&eacute;rotype 33F ainsi. En conclusion, wereport se-1 comme un roman cibler pour ZmpC et montrer que ZmpC inhibe l&#39;extravasation neutrophile au cours de la pneumonie &agrave; pneumocoque.

&Eacute;vasion immunitaire contre le pneumocoque : ZmpC inhibe l&#39;afflux de neutrophil.

Reclutamento dei neutrofili &egrave; essenziale per eliminare le infezioni da pneumococco. Il primo passo di neutrofili stravaso coinvolge l&#39;interazione tra P-Selectina sull&#39;endotelio attivato e P-Selectina glicoproteina 1 (PSGL-1) sui neutrofili. Qui, ci identifichiamo pneumococco zinco metalloproteinase C come un potente inibitore di PSGL-1. ZmpC degrada il dominio N-terminale di PSGL-1, interrompendo cos&igrave; il rotolamento iniziale dei neutrofili sulle cellule endoteliali di vena ombelicale umana attivato. Inoltre, topi infettati con selvaggio-tipo ceppo nel modello della polmonite pneumococcica ha mostrato bassa infiltrazione di neutrofili polmoni confrontare con gli animali infettati con ZmpC mutante. Inoltre, abbiamo confermato l&#39;associazione di zmpC con il sierotipo 8 e 11A e trovato per essere associato con il sierotipo 33F pure. In conclusione, wereport PSGL-1 come un romanzo di destinazione per ZmpC e mostrare che ZmpC inibisce neutrofilo stravaso durante la polmonite pneumococcica.

Evasione immune da pneumococco: ZmpC inibisce l&#39;afflusso dei neutrofili.

The pathogen Staphylococcus aureus is well adapted to its human host. Neutrophil-mediated killing is a crucial defense system against S. aureus; however, the pathogen has evolved many strategies to resist killing. We first describe the discrete steps of neutrophil activation and migration to the site of infection and the killing of microbes by neutrophils in general. We then highlight the different approaches utilized by S. aureus to resist the different steps of neutrophil attack. Various molecules are discussed in their evolutionary context. Most of the molecules secreted by S. aureus to combat neutrophil attacks at the site of infection show clear human specificity. Many elements of human neutrophil defenses appear redundant, and so the evasion strategies of staphylococci display redundant functions as well. All efforts by S. aureus to resist neutrophil-mediated killing stress the importance of these mechanisms in the pathophysiology of staphylococcal diseases. However, the highly human-specific nature of most host-pathogen interactions hinders the in vivo establishment of their contribution to staphylococcal pathophysiology. Expected final online publication date for the Annual Review of Microbiology Volume 67 is September 08, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Neutrophils Versus Staphylococcus aureus: A Biological Tug of War.

L&#39;agent pathog&egrave;ne de Staphylococcus aureus est bien adapt&eacute; &agrave; son h&ocirc;te humain. Meurtre m&eacute;di&eacute;e par les neutrophiles est un syst&egrave;me de d&eacute;fense essentiel contre Staphylococcus aureus ; Toutefois, l&#39;agent pathog&egrave;ne a &eacute;volu&eacute; de nombreuses strat&eacute;gies pour r&eacute;sister &agrave; la mise &agrave; mort. Nous d&eacute;crivons d&#39;abord les &eacute;tapes discr&egrave;tes de neutrophile d&#39;activation et de la migration sur le site de l&#39;infection et le meurtre de microbes par les neutrophiles en g&eacute;n&eacute;ral. Ensuite, nous mettons en &eacute;vidence les diff&eacute;rentes approches utilis&eacute;es par S. aureus de r&eacute;sister aux diff&eacute;rentes &eacute;tapes de l&#39;attaque de neutrophil. Diverses mol&eacute;cules sont examin&eacute;es dans leur contexte &eacute;volutif. La plupart des mol&eacute;cules s&eacute;cr&eacute;t&eacute;es par S. aureus &agrave; lutter contre les attaques de neutrophiles au site d&#39;infection montrent clairement humaine sp&eacute;cificit&eacute;. Beaucoup d&#39;&eacute;l&eacute;ments de d&eacute;fenses neutrophiles humains appara&icirc;tre redondantes, et donc, les strat&eacute;gies d&#39;&eacute;vasion des staphylocoques affichent aussi bien des fonctions redondantes. Tous les efforts de S. aureus de r&eacute;sister &agrave; la mise &agrave; mort m&eacute;di&eacute;e par les neutrophiles soulignent l&#39;importance de ces m&eacute;canismes dans la physiopathologie des maladies staphylococciques. Toutefois, la nature hautement sp&eacute;cifiques &agrave; l&#39;homme de la plupart des interactions h&ocirc;te-pathog&egrave;ne emp&ecirc;che la mise en place in vivo de leur contribution &agrave; la physiopathologie staphylococcique. Date de publication en ligne finale pr&eacute;vue pour l&#39;examen annuel de microbiologie Volume 67 est 8 septembre 2013. Veuillez consulter http://www.annualreviews.org/catalog/pubdates.aspx pour les pr&eacute;visions r&eacute;vis&eacute;es.

Bas G.J. Surewaard

Medical Microbiology

University Medical Center Utrecht

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Bas G.J. Surewaard

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Medical Microbiology

University Medical Center Utrecht

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Medical Microbiology