Anti-staphylococcal humoral immune response in persistent nasal carriers and noncarriers of Staphylococcus aureus

BACKGROUND. Persistent carriers have a higher risk of Staphylococcus aureus infections than noncarriers but a lower risk of bacteremia-related death. Here, the role played by anti-staphylococcal antibodies was studied. METHODS. Serum samples from 15 persistent carriers and 19 noncarriers were analyzed for immunoglobulin (Ig) G, IgA, and IgM binding to 19 S. aureus antigens, by means of Luminex technology. Nasal secretions and serum samples obtained after 6 months were also analyzed. RESULTS. Median serum IgG levels were significantly higher in persistent carriers than in noncarriers for toxic shock syndrome toxin (TSST)-1 (median fluorescence intensity [MFI] value, 11,554 vs. 4291; P < .001) and staphylococcal enterotoxin (SE) A (742 vs. 218; P < .05); median IgA levels were higher for TSST-1 (P < .01), SEA, and clumping factor (Clf) A and B (P < .05). The in vitro neutralizing capacity of anti-TSST-1 antibodies was correlated with the MFI value (R(2) = 0.93) and was higher in persistent carriers (90.6% vs. 70.6%; P < .05). Antibody levels were stable over time and correlated with levels in nasal secretions (for IgG, R(2) = 0.87; for IgA, R(2) = 0.77). CONCLUSIONS. Antibodies to TSST-1 ha

Funktionelle Charakterisierung neuer Virulenzfaktoren von Staphylococcus aureus

Staphylococcus (S.) aureus is the most common cause of nosocomial infections and the species is becoming increasingly resistant to antibiotics. In contrast, about 35% of the healthy population are colonized with S. aureus in the anterior nares. The genetic make-up of this species is highly diverse. Mobile genetic elements comprise about 15% of the S. aureus genome. They encode many virulence factors like the 21 different known staphylococcal superantigens (SAgs), highly potent activators of T lymphocytes. Besides their well known causative role in food poisoning and toxic shock syndrome, information about SAg involvement in pathogenesis is limited. On the other hand, the human host and its immune response are also highly diverse. This study focuses on SAgs, because they are potent virulence factors that are highly diverse and therefore mirror of the variability of the species S. aureus. The goals of this work were (i) to identify virulence determinants by comparing the prevalence of SAg genes and phages among colonizing and invasive S. aureus isolates and to correlate it with the clonal background, (ii) to determine the prevalence and the development of anti-SAg antibodies in healthy S. aureus carriers and noncarriers as well as in bacteremia patients, and (iii) to elucidate the reasons for the selective lack of neutralizing serum antibodies specific for a subgroup of SAgs, the egc SAgs. In search for a molecular-epidemiological associations between SAgs and different diseases caused by S. aureus we investigated the distribution of SAg genes and/ or bacteriophages and correlated this with the clonal background, determined by spa genotyping. The analysis of more than 700 S. aureus isolates from nasal colonization, bacteremia or furunculosis revealed that SAg-encoding mobile genetic elements and bacteriophages were strongly associated with the clonal background. As a consequence, each clonal lineage was characterized by a typical SAg gene and phage repertoire. Therefore, we suggest that the simultaneous assessment of virulence gene profiles and the genetic background strongly increases the discriminatory power of genetic investigations into the mechanisms of S. aureus pathogenesis. However, we found no association of SAg genes with bacteremia or furunculosis. While functional neutralization assays closely mimic the protective action of anti-SAg antibodies in vivo, they are labor-intensive and time-consuming. A fast and easy method for the simultaneous quantification of antibody binding to multiple staphylococcal antigens is the Luminex® technology. Using serum samples from persistent carriers and noncarriers we showed a strong correlation between antibody binding and neutralizing capacity against the SAg TSST-1. This assay confirmed the astonishing lack of antibodies against egc SAgs in healthy carriers and noncarriers, which was previously described by Holtfreter and coworkers. Since colonization is probably not sufficient to induce a robust antibody response as revealed by experimental colonization with S. aureus, we propose that (minor) infections are required to induce the high titers of non-egc SAg-neutralizing antibodies in healthy adults. To test this, we investigated whether SAgs elicit a neutralizing antibody response during S. aureus bacteremia. At the acute phase of the disease most patients already had neutralizing antibodies against non-egc SAgs, and antibody titers frequently increased during infection. Notably, egc SAgs did not elicit a boost or de novo generation of specific antibodies. The “egc gap” in the antibody response, which has now been shown in healthy adults, as well as following systemic infection with S. aureus, is astonishing. After all, egc SAgs are by far the most prevalent SAgs. In search for an explanation, the intrinsic properties of three recombinant egc (SEI, SElM, SElO) and non-egc SAgs (SEB, SElQ, TSST-1) were compared in depth. Egc and non-egc SAgs were very similar with regard to induced T cell proliferation, cytokine profiles, and gene expression of human immune cells. However, there was a striking difference in the regulation of the two groups of SAgs by S. aureus in bacterial culture. We conclude that the differential regulation of egc and non-egc SAg has an impact on the immune response. But how are SAgs regulated by S. aureus during its interaction with the host? Up until now most research on regulation of virulence factors has been performed in vitro. The immune response can help to shed light on this problem, because it is an exquisitely specific sensor for the exposure to different antigens. The high prevalence of neutralizing serum antibodies against non-egc SAgs indicates that most healthy adults have been exposed to these toxins during their encounters with S. aureus. For egc SAgs this remains an open question. However, initial data indicate that the egc SAg genes are transcribed during nasal colonization.Staphylococcus aureus ist die häufigste Ursache für nosokomiale Infektionen. Gleichzeitig besiedelt S. aureus die Nasenschleimhaut von etwa 35% der gesunden Bevölkerung. Das Genom der Spezies S. aureus ist hoch variabel - etwa 15% besteht aus mobilen genetischen Elementen. Hier werden hauptsächlich Virulenzfaktoren wie die 21 bekannten Superantigene (SAg) kodiert. SAg sind sehr potente T-Zell-Mitogene und können das Toxische-Schocksyndrom auslösen. Sie werden allerdings nicht immer wirksam, neutralisierende Antikörper können vor ihrer toxischen Wirkung schützen. SAg stehen im Mittelpunkt dieser Arbeit, da diese potenten Virulenzfaktoren mit ihrer Diversität die außergewöhnliche Variablität der Spezies S. aureus widerspiegeln. Die Arbeit hatte folgende Ziele: (i) Die Suche nach einem molekular-epidemiologischen Zusammenhang zwischen SAg und verschiedenen durch S. aureus verursachten Krankheiten, (ii) die Bestimmung der Prävalenz und Entwicklung von Antikörpern gegen SAg bei gesunden S. aureus Carriern und Nichtcarriern sowie bei Bakteriämie-Patienten und (iii) die Aufklärung der Ursache für das selektive Fehlen von neutralisierenden Antikörpern gegen eine Untergruppe von SAg, die egc-SAg. Auf der Suche nach einem molekular-epidemiologischen Zusammenhang zwischen SAg und verschiedenen durch S. aureus verursachten Krankheiten wurde die Verteilung von SAg-Genen und/ oder Bakteriophagen bei nasalen, Bakteriämie- und Furunkulose-Isolaten analysiert und mit dem genetischen Hintergrund (spa-Typisierung) der Stämme korreliert. Die SAg-kodierenden mobilen genetischen Elemente und Bakteriophagen waren nicht zufällig verteilt, sondern stark an den genetisch en Hintergrund der Isolate geknüpft. Das bedeutet, dass die parallele Bestimmung der Virulenzgen- und Bakteriophagen-Profile und des genetischen Hintergrunds die Trennschärfe von genetischen Untersuchungen zu S. aureus-Virulenzmechanismen stark erhöht. Diese Analyse zeigte keine Assoziation von SAg-Genen mit Bakteriämie oder Furunkulose.SAg Die klinisch relevante protektive Wirkung von anti-SAg-Antikörpern kann mit funktionellen Neutralisationsassays gemessen werden, allerdings sind diese Assays zeit-und arbeitsintensiv. Eine elegante Alternative ist die Luminex®-Technologie, bei der zeitgleich die Antikörperbindung an verschiedene S. aureus-Antigene quantifiziert wird. Die Analyse von Serumproben von Carriern und Nichtcarriern zeigte eine starke Korrelation der im Luminex-System gemessenen Antikörperbindung mit der neutralisierenden Kapazität gegen das SAg TSST-1. Außerdem bestätigte sich, dass Antikörper gegen egc-SAg bei gesunden Carriern und Nichtcarriern selektiv fehlen, wie bereits von Holtfreter et al. gezeigt wurde. Wir gingen davon aus, dass für die Bildung der hohen Antikörpertiter gegen nicht-egc-SAg, wie sie in gesunden Erwachsenen regelmäßig gefunden werden, Infektionen nötig sind, da experimentelle Besiedlung mit S. aureus nicht ausreichte, um eine robuste Antikörperantwort zu generieren. Um dies zu überprüfen, haben wir die Antikörperentwicklung gegen SAg während S. aureus-Bakteriämie untersucht. Bereits in der akuten Krankheitsphase hatte ein Großteil der Patienten neutralisierende Antikörper gegen nicht-egc-SAg, die häufig im Verlauf der Infektion weiter anstiegen. Interessanterweise induzierte auch eine systemische Infektion keine neutralisierenden Antikörper gegen egc-SAg. Die „egc-Lücke“ in der Antikörperantwort, die jetzt sowohl bei gesunden Erwachsenen als auch nach systemischer S. aureus-Infektion gezeigt wurde, war unerwartet, da egc-SAg bei klinischen S. aureus-Isolaten die häufigsten SAg sind. Auf der Suche nach der Ursache haben wir die intrinsischen Eigenschaften von drei rekombinanten egc- (SEI, SElM und SElO) und drei nicht-egc-SAg (SEB, SElQ und TSST-1) verglichen. Egc- und nicht-egc-SAg verhielten sich in allen untersuchten Aspekten der T-Zellaktivierung sehr ähnlich: Induktion der T-Zell-Proliferation, Zytokinsekretion und Genexpression in humanen Immunzellen. Im Gegensatz dazu wurden egc- und nicht-egc-SAg unter in vitro-Bedienungen unterschiedlich reguliert. Wir folgern daraus, dass die unterschiedliche Regulation die adaptive Immunantwort beeinflusst. Wie werden SAg bei der Wechselwirkung von S. aureus mit seinem Wirt reguliert? Die Forschung der Regulation von S. aureus-Virulenzfaktoren beschränkte sich bisher vor allem auf in vitro-Bedingungen. Die hochspezifische adaptive Immunantwort bietet einen Ausweg. Die hohe Prävalenz von neutralisierenden Antikörpern gegen nicht-egc-SAg ist ein Indiz dafür, dass die meisten gesunden Erwachsenen während ihrer Begegnungen mit S. aureus mit diesen Toxinen in Kontakt gekommen sind. Diese Antikörperantwort wird bei invasiven Episoden verstärkt. Dies zeigt, dass zumindest die nicht-egc-SAg bei einer Bakteriämie gebildet werden. Für egc-SAg lässt sich die Frage bisher nicht beantworten, doch weisen erste Daten darauf hin, dass egc-SAg während der nasalen Besiedlung transkribiert werden

Staphylococcus aureus toxins – Their functions and genetics

The outcome of encounters between Staphylococcus (S.) aureus and its human host ranges from life-threatening infection through allergic reactions to symptom-free colonization. The pan-genome of this bacterial species encodes numerous toxins, known or strongly suspected to cause specific diseases or symptoms. Three toxin families are in the focus of this review, namely (i) pore-forming toxins, (ii) exfoliative toxins and (iii) superantigens. The majority of toxin-encoding genes are located on mobile genetic elements (MGEs), resulting in a pronounced heterogeneity in the endowment with toxin genes of individual S. aureus strains. Recent population genomic analysis have provided a framework for an improved understanding of the temporal and spatial scales of the motility of MGEs and their associated toxin genes. The distribution of toxin genes among clonal lineages within the species S. aureus is not random, and phylogenetic (sub-)lineages within clonal complexes feature characteristic toxin signatures. When studying pathogenesis, this lineage association, which is caused by the clonal nature of S. aureus makes it difficult to discriminate effects of specific toxins from contributions of the genetic background and/or other associated genetic factors

Characterization of Infecting Strains and Superantigen-Neutralizing Antibodies in Staphylococcus aureus Bacteremia▿

Staphylococcus aureus superantigens (SAgs) are highly potent T cell mitogens. Antibodies against non-enterotoxin gene cluster (non-egc) SAgs are common in healthy adults, whereas neutralizing antibodies against egc SAgs are rare. We investigated the infecting S. aureus strains and the anti-SAg antibody response during S. aureus bacteremia (SAB). This prospective clinical study (www.clinicaltrials.gov, NCT00548002) included 43 injection drug users (IDUs) and 44 group-matched nonaddicts with SAB. spa genotypes and SAg gene patterns (multiplex PCR) of the S. aureus isolates were determined. The neutralizing capacities of sera obtained at the acute phase and the convalescent phase of SAB were tested against the SAg cocktail of the respective infecting strain and a panel of recombinant SAgs. The lineages CC59 and CC30 were more prevalent among bacteremia strains from IDUs than among strains from nonaddicts. SAg gene patterns in isolates from IDUs and nonaddicts were similar. At the acute phase of bacteremia, IDUs had more neutralizing antibodies against non-egc SAgs than did nonaddicts. Antibody titers frequently increased during infection. In contrast, there were no neutralizing antibodies against egc SAgs at disease onset and such antibodies were not induced by SAB. SAB triggers an antibody response only against non-egc SAgs. Preimmunization in IDU patients is probably due to previous exposure to the infecting strain

Immune cell activation by enterotoxin gene cluster (egc)-encoded and non-egc superantigens from Staphylococcus aureus

The species Staphylococcus aureus harbors 19 superantigen gene loci, six of which are located in the enterotoxin gene cluster (egc). Although these egc superantigens are far more prevalent in clinical S. aureus isolates than non-egc superantigens, they are not a prominent cause of toxic shock. Moreover, neutralizing Abs against egc superantigens are very rare, even among carriers of egc-positive S. aureus strains. In search of an explanation, we have tested two non-exclusive hypotheses: 1) egc and non-egc superantigens have unique intrinsic properties and drive the immune system into different directions and 2) egc and non-egc superantigens are released by S. aureus under different conditions, which shape the immune response. A comparison of three egc (SEI, SElM, and SElO) and three non-egc superantigens (SEB, SElQ, and toxic shock syndrome toxin-1) revealed that both induced proliferation of human PBMC with comparable potency and elicited similar Th1/Th2-cytokine signatures. This was supported by gene expression analysis of PBMC stimulated with one representative superantigen from each group (SEI and SEB). They induced very similar transcriptional changes, especially of inflammation-associated gene networks, corresponding to a very strong Th1- and Th17-dominated immune response. In contrast, the regulation of superantigen release differed markedly between both superantigen groups. Egc-encoded proteins were secreted by S. aureus during exponential growth, while non-egc superantigens were released in the stationary phase. We conclude that the distinct biological behavior of egc and non-egc superantigens is not due to their intrinsic properties, which are very similar, but caused by their differential release by S. aureus

Characterization of a Mouse-Adapted <i>Staphylococcus aureus</i> Strain

<div><p>More effective antibiotics and a protective vaccine are desperately needed to combat the ‘superbug’ <i>Staphylococcus aureus.</i> While in vivo pathogenicity studies routinely involve infection of mice with human <i>S. aureus</i> isolates, recent genetic studies have demonstrated that <i>S. aureus</i> lineages are largely host-specific. The use of such animal-adapted <i>S. aureus</i> strains may therefore be a promising approach for developing more clinically relevant animal infection models. We have isolated a mouse-adapted <i>S. aureus</i> strain (JSNZ) which caused a severe outbreak of preputial gland abscesses among male C57BL/6J mice. We aimed to extensively characterize this strain on a genomic level and determine its virulence potential in murine colonization and infection models. JSNZ belongs to the MLST type ST88, rare among human isolates, and lacks an <i>hlb</i>-converting phage encoding human-specific immune evasion factors. Naive mice were found to be more susceptible to nasal and gastrointestinal colonization with JSNZ than with the human-derived Newman strain. Furthermore, naïve mice required antibiotic pre-treatment to become colonized with Newman. In contrast, JSNZ was able to colonize mice in the absence of antibiotic treatment suggesting that this strain can compete with the natural flora for space and nutrients. In a renal abscess model, JSNZ caused more severe disease than Newman with greater weight loss and bacterial burden. In contrast to most other clinical isolates, JSNZ can also be readily genetically modified by phage transduction and electroporation. In conclusion, the mouse-adapted strain JSNZ may represent a valuable tool for studying aspects of mucosal colonization and for screening novel vaccines and therapies directed at preventing colonization.</p></div

Laboratory Mice Are Frequently Colonized with Staphylococcus aureus and Mount a Systemic Immune Response—Note of Caution for In vivo Infection Experiments

Whether mice are an appropriate model for S. aureus infection and vaccination studies is a matter of debate, because they are not considered as natural hosts of S. aureus. We previously identified a mouse-adapted S. aureus strain, which caused infections in laboratory mice. This raised the question whether laboratory mice are commonly colonized with S. aureus and whether this might impact on infection experiments. Publicly available health reports from commercial vendors revealed that S. aureus colonization is rather frequent, with rates as high as 21% among specific-pathogen-free mice. In animal facilities, S. aureus was readily transmitted from parents to offspring, which became persistently colonized. Among 99 murine S. aureus isolates from Charles River Laboratories half belonged to the lineage CC88 (54.5%), followed by CC15, CC5, CC188, and CC8. A comparison of human and murine S. aureus isolates revealed features of host adaptation. In detail, murine strains lacked hlb-converting phages and superantigen-encoding mobile genetic elements, and were frequently ampicillin-sensitive. Moreover, murine CC88 isolates coagulated mouse plasma faster than human CC88 isolates. Importantly, S. aureus colonization clearly primed the murine immune system, inducing a systemic IgG response specific for numerous S. aureus proteins, including several vaccine candidates. Phospholipase C emerged as a promising test antigen for monitoring S. aureus colonization in laboratory mice. In conclusion, laboratory mice are natural hosts of S. aureus and therefore, could provide better infection models than previously assumed. Pre-exposure to the bacteria is a possible confounder in S. aureus infection and vaccination studies and should be monitored

DNA microarray analysis of JSNZ and human CC88 isolates.

<p>DNA microarray analysis of JSNZ and human CC88 isolates.</p