Neutralization of the Staphylococcus aureus Panton-Valentine leukocidin by African and Caucasian sera.

BACKGROUND: The prevalence of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin (PVL) gene is higher in Africa (≈50%) compared to Europe (< 5%). The study aimed to measure anti-PVL-antibodies in Africans and Germans in a multi-center study and to test whether detected antibodies can neutralize the cytotoxic effect of PVL on polymorphonuclear leukocytes (PMNs). METHODS: Sera from asymptomatic Africans (n = 22, Nigeria, Gabon) and Caucasians (n = 22, Germany) were used to quantify antibody titers against PVL and α-hemolysin (in arbitrary units [AU]) by ELISA. PMNs from one African and German donor were exposed to 5 nM recombinant PVL to measure the neutralizing effect of serial dilutions of pooled sera from African and Caucasian participants, or donor sera at 0.625 and 2.5% (v/v). RESULTS: Anti-PVL-antibodies were significantly higher in Africans than in Germans (1.9 vs. 0.7 AU, p < 0.0001). The pooled sera from the study participants neutralized the cytotoxic effect of PVL on African and German PMNs in a dose dependent manner. Also, neutralization of PVL on PMNs from the African and German donors had a stronger effect with African sera (half-maximal inhibitory concentration (IC50) = 0.27 and 0.47%, respectively) compared to Caucasian sera (IC50 = 3.51 and 3.59% respectively). CONCLUSION: Africans have higher levels of neutralizing anti-PVL-antibodies. It remains unclear if or at what level these antibodies protect against PVL-related diseases.

Importance of superoxide dismutases A and M for protection of Staphylococcus aureus in the oxidative stressful environment of cystic fibrosis airways.

Staphylococcus aureus is one of the earliest pathogens that persists the airways of cystic fibrosis (CF) patients and contributes to increased inflammation and decreased lung function. In contrast to other staphylococci, S. aureus possesses two superoxide dismutases (SODs), SodA and SodM, with SodM being unique to S. aureus. Both SODs arm S. aureus for its fight against oxidative stress, a by-product of inflammatory reactions. Despite complex investigations, it is still unclear if both enzymes are crucial for the special pathogenicity of S. aureus. To investigate the role of both SODs during staphylococcal persistence in CF airways, we analysed survival and gene expression of S. aureus CF isolates and laboratory strains in different CF-related in vitro and ex vivo settings. Bacteria located in inflammatory and oxidised CF sputum transcribed high levels of sodA and sodM. Especially expression values of sodM were remarkably higher in CF sputum than in bacterial in vitro cultures. Interestingly, also S. aureus located in airway epithelial cells expressed elevated transcript numbers of both SODs, indicating that S. aureus is exposed to oxidative stress at various sites within CF airways. Both enzymes promoted survival of S. aureus during polymorphonuclear leukocyte killing and seem to act compensatory, thereby giving evidence that the interwoven interaction of SodA and SodM contributes to S. aureus virulence and facilitates S. aureus persistence within CF airways.

Conjugated Pt(II) Complexes as Luminescence-Switch-On Reporters Addressing the Microenvironment of Bacterial Biofilms.

In this work, the synthesis, structural and photophysical characterization of six phosphorescent H2O-soluble Pt(II) complexes are reported while addressing their emission maxima, photoluminescence quantum yields (ΦL), lifetimes (τ), aggregation tendency, and microenvironment sensitivity as a function of the substitution pattern on the main tridentate luminophore. Different ancillary ligands, namely, a trisulfonated phosphane and maltohexaose-conjugated pyridines (with or without amide bridges), were introduced and evaluated for the realization of switch-on-photoluminescent labels reporting on the microenvironment sensed in biofilms of Gram+ and Gram- models, namely, Staphylococcus aureus and Escherichia coli. With the aid of confocal luminescence micro(spectro)scopy, we observed that selected complexes specifically interact with the biofilms while leaving planktonic cells unlabeled. By using photoluminescence lifetime imaging microscopy, excited-state lifetimes within S. aureus biofilms were measured. The photoluminescence intensities were drastically boosted, and the excited state lifetimes were significantly prolonged upon binding to the viscous biofilm matrix, mainly due to the suppression of radiationless deactivation pathways upon shielding from physical quenching processes, such as interactions with solvent molecules and 3O2. The best performances were attained for non-aggregating complexes with maltohexaose targeting units and without amide bridges. Notably, in the absence of the maltodextrin, a hydrophobic adamantyl moiety suffices to attain a sizeable labeling capacity. Moreover, photoluminescence studies showed that selected complexes can also effectively interact with E. coli biofilms, where the bacterial cells are able to partially uptake the maltodextrin-based agents. In summary, the herein introduced concepts enable the development of specific biofilm reporters providing spatial resolution as well as lifetime- and spectrum-based readouts. Considering that most theragnostic agents reported so far mainly address metabolically active bacteria at the surface of biofilms but without reaching cells deeply immersed in the matrix, a new platform with a clear structure-property correlation is provided for the early detection of such bacterial arrays.

CD163 expression defines specific, IRF8-dependent, immune-modulatory macrophages in the bone marrow.

BACKGROUND: Scavenger receptor CD163 is exclusively expressed on monocytes/macrophages and is widely used as a marker for alternatively activated macrophages. However, the role of CD163 is not yet clear. OBJECTIVES: We sought to examine the function of CD163 in steady-state as well as in sterile and infectious inflammation. METHODS: Expression of CD163 was analyzed under normal and inflammatory conditions in mice. Functional relevance of CD163 was investigated in models of inflammation in wild-type and CD163-/- mice. RESULTS: We describe a subpopulation of bone marrow-resident macrophages (BMRMs) characterized by a high expression of CD163 and functionally distinct from classical bone marrow-derived macrophages. Development of CD163+ BMRMs is strictly dependent on IFN regulatory factor-8. CD163+ BMRMs show a specific transcriptome and cytokine secretion pattern demonstrating a specific immunomodulatory profile of these cells. Accordingly, CD163-/- mice show a stronger inflammation in allergic contact dermatitis, indicating a regulatory role of CD163. However, CD163-/- mice are highly susceptible to S aureus infections, demonstrating the relevance of CD163 for antimicrobial defense as well. CONCLUSIONS: Our data indicate that anti-inflammatory and immunosuppressive mechanisms are not necessarily associated with a decreased antimicrobial activity. In contrast, our data define a novel macrophage population that controls overwhelming inflammation on one hand but is also necessary for an effective control of infections on the other hand.

Tropical pyomyositis: an update.

Tropical pyomyositis (TP) is a life-threatening bacterial infection of the skeletal muscle that occurs particularly among children, young adults and those with immunocompromised conditions. The appropriate diagnosis and treatment are often delayed due to its non-specific signs, leading to fatal consequences. Staphylococcus aureus, especially methicillin-susceptible S. aureus, is responsible for most TP cases. However, other bacteria (i.e. streptococci, Pseudomonas aeruginosa, Escherichia coli, Klebsiella spp., Candida spp., Mycobacterium spp.) have been reported. This narrative review provides an update on the epidemiology and clinical course of TP. A special focus is laid on the role of toxins (i.e. Panton-Valentine Leucocidin and α-toxin) in the pathogenesis of TP and their implication for the clinical management of infection.

La pyomyosite tropicale (TP) est une infection bactérienne potentiellement mortelle du muscle squelettique qui survient particulièrement chez les enfants, les jeunes adultes et les personnes immunodéprimées. Le diagnostic et le traitement appropriés sont souvent retardés en raison de ses signes non spécifiques, entraînant des conséquences fatales. Staphylococcus aureus, en particulier S. aureus sensible à la méthicilline, est responsable de la plupart des cas de TP. Cependant, d'autres bactéries (ex: streptocoques, Pseudomonas aeruginosa, Escherichia coli, Klebsiella spp., Candida spp., Mycobacterium spp.) ont été rapportées. Cette revue narrative fournit une mise à jour sur l'épidémiologie et l'évolution clinique du TP. Un accent particulier est mis sur le rôle des toxines (la Leukocidine de Panton-Valentine et l'α-toxine) dans la pathogenèse du TP et leur implication pour la prise en charge clinique de l'infection.

Staphylococcus aureus triggers a shift from influenza virus-induced apoptosis to necrotic cell death.

Superinfections with Staphylococcus aureus are a major complication of influenza disease, causing excessive inflammation and tissue damage. This enhanced cell-damaging effect is also observed in superinfected tissue cultures, leading to a strong decrease in overall cell viability. In our analysis of the underlying molecular mechanisms, we observed that, despite enhanced cell damage in superinfection, S. aureus did not increase but rather inhibited influenza virus (IV)-induced apoptosis in cells on the level of procaspase-8 activation. This apparent contradiction was solved when we observed that S. aureus mediated a switch from apoptosis to necrotic cell death of IV-infected cells, a mechanism that was dependent on the bacterial accessory gene regulator ( agr) locus that promotes bacterial survival and spread. This so far unknown action may be a bacterial strategy to enhance dissemination of intracellular S. aureus and may thereby contribute to increased tissue damage and severity of disease.-Van Krüchten, A., Wilden, J. J., Niemann, S., Peters, G., Löffler, B., Ludwig, S., Ehrhardt, C. Staphylococcus aureus triggers a shift from influenza virus-induced apoptosis to necrotic cell death.

A Novel Mouse Model of Staphylococcus aureus Vascular Graft Infection: Noninvasive Imaging of Biofilm Development in Vivo.

Staphylococcus aureus causes very serious infections of vascular grafts. Knowledge of the molecular mechanisms of this disease is largely lacking because of the absence of representable models. Therefore, the aim of this study was to set up a mouse model of vascular graft infections that closely mimics the human situation. A catheter was inserted into the right carotid artery of mice, which acted as a vascular graft. Mice were infected i.v. using 8 different S. aureus strains, and development of the infection was followed up. Although all strains had varying abilities to form biofilm in vitro and different levels of virulence in mice, they all caused biofilm formation on the grafts. This graft infection was monitored using magnetic resonance imaging (MRI) and 18F-fluordeoxyglucose positron emission tomography (FDG-PET). MRI allowed the quantification of blood flow through the arteries, which was decreased in the catheter after infection. FDG-PET revealed high inflammation levels at the site of the catheter after infection. This model closely resembles the situation in patients, which is characterized by a tight interplay between pathogen and host, and can therefore be used for the testing of novel treatment, diagnosis, and prevention strategies. In addition, combining MRI and PET with microscopic techniques provides an appropriate way to characterize the course of these infections and to precisely analyze biofilm development.

Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections.

Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.

Labeling and Selective Inactivation of Gram-Positive Bacteria Employing Bimodal Photoprobes with Dual Readouts.

Carbohydrate-conjugated silicon(IV) phthalocyanines with bimodal photoactivity were developed as probes with both fluorescent labeling and photosensitizing capabilities, and the concomitant fluorescent labeling and photoinduced inactivation of Gram-positive and Gram-negative models was explored. The maltohexaose-conjugated photoprobe provides a dual readout to distinguish between both groups of pathogens, as only the Gram-positive species was inactivated, even though both appeared labeled with near-infrared luminescence. Antibiotic resistance did not hinder the phototoxic effect, as even the methicillin-resistant pathogen Staphylococcus aureus (MRSA) was completely photoinactivated. Time-resolved confocal fluorescence microscopy analysis suggests that the photoprobe sticks onto the outer rim of the microorganisms, explaining the resistance of Gram-negative species on the basis of their membrane constitution. The mannose-conjugated photoprobe yields a different readout because it is able to label and to inactivate only the Gram-positive strain.

Super-infection with Staphylococcus aureus inhibits influenza virus-induced type I IFN signalling through impaired STAT1-STAT2 dimerization.

Bacterial super-infections are a major complication in influenza virus-infected patients. In response to infection with influenza viruses and bacteria, a complex interplay of cellular signalling mechanisms is initiated, regulating the anti-pathogen response but also pathogen-supportive functions. Here, we show that influenza viruses replicate to a higher efficiency in cells co-infected with Staphylococcus aureus (S. aureus). While cells initially respond with increased induction of interferon beta upon super-infection, subsequent interferon signalling and interferon-stimulated gene expression are rather impaired due to a block of STAT1-STAT2 dimerization. Thus, S. aureus interrupts the first line of defence against influenza viruses, resulting in a boost of viral replication, which may lead to enhanced viral pathogenicity.

Staphylococcus aureus persistence in non-professional phagocytes.

S. aureus is a frequent cause of chronic and therapy-refractory infections. The ability of S. aureus to invade different types of non-professional phagocytes, to escape from the host lysosomal degradation machinery and to persist within the intracellular location for long time periods are most likely essential steps in pathogenesis. During the course from acute to chronic infection the bacteria need to dynamically react to the environmental changes and to adapt to the intracellular environment. In this context the bacteria change to SCV-like phenotypes that exhibit some characteristics of stable SCV-mutants, like upregulation of adhesins and downregulation of toxins. The exact formation mechanism and further typical features of these dynamically forming SCVs are largely unknown. In this review, recent data on the essential steps to establish chronic infections will be summarized and the clinical consequences of the dynamic bacterial adaptation mechanisms will be discussed.

Staphylococcus aureus isolates from chronic osteomyelitis are characterized by high host cell invasion and intracellular adaptation, but still induce inflammation.

Osteomyelitis is a severe inflammatory disease of the bone that is mainly caused by Staphylococcus aureus. Particularly, bone infections are difficult to treat and can develop into a chronic course with a high relapsing rate despite of antimicrobial treatments. The complex interaction of staphylococci with osseous tissue and the bacterial ability to invade host cells are thought to determine the severity of infection. Yet, defined bacterial virulence factors responsible for the pathogenesis of osteomyelitis have not been clearly identified. The aim of this study was to detect S. aureus virulence factors that are associated with osteomyelitis and contribute to a chronic course of infection. To this purpose, we collected 41 S. aureus isolates, each 11 from acute osteomyelitis (infection period less than 2 months), 10 from chronic osteomyelitis (infection period more than 12 months), 10 from sepsis and 10 from nasal colonization. All isolates were analyzed for gene expression and in functional in-vitro systems. Adhesion assays to bone matrix revealed that all isolates equally bound to matrix structures, but invasion assays in human osteoblasts showed a high invasive capacity of chronic osteomyelitis isolates. The high invasion rate could not be explained by defined adhesins, as all infecting strains expressed a multitude of adhesins that act together and determine the level of adhesion. Following host cell invasion isolates from chronic osteomyelitis induced less cytotoxicity than all other isolates and a higher percentage of Small-colony-variant (SCV)-formation, which represents an adaptation mechanism during long-term persistence. Isolates from acute and chronic osteomyelitis strongly produced biofilm and highly expressed agr and sarA that regulate secreted virulence factors and induced an inflammatory response in osteoblasts. In conclusion, chronic osteomyelitis isolates were characterized by a high host cell invasion rate, low cytotoxicity and the ability to persist and adapt within osteoblasts. Furthermore, isolates from both acute and chronic osteomyelitis strongly produced biofilm and induced high levels of host cell inflammation, which may explain tissue destruction and bone deformation observed as typical complications of long-lasting bone infections.

A novel radiolabelled salmochelin derivative for bacteria-specific PET imaging: synthesis, radiolabelling and evaluation.

For specific imaging of bacterial infections we aimed at targeting the exclusive bacterial iron transport system via siderophore-based radiotracers. De novo synthesis and radiolabeling yielded the salmochelin-based PET radiotracer [68Ga]Ga-RMA693, which showed a favourable biodistribution and a bacteria-specific uptake in an animal model of Escherichia coli infection.

The Influence of Different Sera on the Anti-Infective Properties of Silver Nitrate in Biopolymer Coatings.

The widespread prevalence of periprosthetic joint infections (PJIs) poses significant challenges in orthopedic surgeries, with pathogens such as Staphylococcus epidermidis being particularly problematic due to their capability to form biofilms on implants. This study investigates the efficacy of an innovative silver nitrate-embedded poly-L-lactide biopolymer coating designed to prevent such infections. The methods involved applying varying concentrations of silver nitrate to in vitro setups and recording the resultant bacterial growth inhibition across different serum environments, including human serum and various animal sera. Results highlighted a consistent and significant inhibition of S. epidermidis growth at all tested concentrations in each type of serum without adverse interactions with serum proteins, which commonly compromise antimicrobial efficacy. This study concludes that the silver nitrate-embedded biopolymer coating exhibits potent antibacterial properties and has potential for use in clinical settings to reduce the incidence of PJIs. Furthermore, the findings underscore the importance of considering serum interactions in the design and testing of antimicrobial implants to ensure their effectiveness in actual use scenarios. These promising results pave the way for further research to validate and refine this technology for clinical application, focusing on optimizing silver ion release and assessing biocompatibility in vivo.

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases.

OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase and endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Combined action of influenza virus and Staphylococcus aureus panton-valentine leukocidin provokes severe lung epithelium damage.

Staphylococcus aureus necrotizing pneumonia is a life-threatening disease that is frequently preceded by influenza infection. The S. aureus toxin Panton-Valentine leukocidin (PVL) is most likely causative for necrotizing diseases, but the precise pathogenic mechanisms of PVL and a possible contribution of influenza virus remain to be elucidated. In this study, we present a model that explains how influenza virus and PVL act together to cause necrotizing pneumonia: an influenza infection activates the lung epithelium to produce chemoattractants for neutrophils. Upon superinfection with PVL-expressing S. aureus, the recruited neutrophils are rapidly killed by PVL, resulting in uncontrolled release of neutrophil proteases that damage the airway epithelium. The host counteracts this pathogen strategy by generating PVL-neutralizing antibodies and by neutralizing the released proteases via protease inhibitors present in the serum. These findings explain why necrotizing infections mainly develop in serum-free spaces (eg, pulmonary alveoli) and open options for new therapeutic approaches.

On-Demand Release of Anti-Infective Silver from a Novel Implant Coating Using High-Energy Focused Shock Waves.

Implant-related infections are a significant concern in orthopedic surgery. A novel anti-infective implant coating made of bioresorbable polymer with silver nitrate was developed. A controlled release of silver ions into the vicinity of the prosthesis can be triggered on-demand by extracorporeal shock waves to effectively combat all clinically relevant microorganisms. Microscopy techniques were used to examine the effects of shock wave application on coated titanium discs. Cytotoxicity was measured using a fibroblast proliferation assay. The anti-infective effect was assessed by monitoring the growth curves of three bacterial strains and by conventional culture. Microscopic analysis confirmed surface disruption of the coatings, with a complete release of silver in the focus area after shock wave application. Spectrometry detected an increase in silver concentration in the surrounding of the discs that surpassed the minimum inhibitory concentration (MIC) for both S. epidermidis RP62A and E. coli ATCC 25922. The released silver demonstrated an anti-infective effect, significantly inhibiting bacterial growth, especially at 6% and 8% silver concentrations. Cytotoxicity testing showed decreasing fibroblast viability with increasing silver concentration in the coating, with 6% silver maintaining viability above 25%. Compared to a commonly used electroplated silver coating on the market, the new coating demonstrated superior antimicrobial efficacy and lower cytotoxicity.

Shock Wave-Activated Silver-Loaded Biopolymer Implant Coating Eliminates Staphylococcus epidermidis on the Surface and in the Surrounding of Implants.

Bacterial biofilms on foreign surfaces are considered a primary cause of implant-related infections, which are challenging to treat. A new implant coating was developed, containing anti-infective silver within a biocompatible polymer carrier substance. In addition to its passive effect on the implant surface, highly concentrated anti-infective silver can be released as needed via the application of high-energy shock waves. This intervention could be applied transcutaneously in a clinical setting without the need for additional surgery. We investigated the inhibition of biofilm formation and the effectiveness of eradication after activation of the coating via shock waves in an in vitro biofilm model using Staphylococcus epidermidis RP62A. This was performed via scanning electron microscopy and quantitative microbiology. Additionally, we examined the cytotoxicity of the new coating on normal human fibroblasts and Saos-2 osteoblast-like cells, depending on the silver concentration. All studies were compared to uncoated titanium surfaces Ti6Al4V and a conventional electroplated silver coating. Cytotoxicity toward normal human fibroblasts and Saos-2 osteoblast-like cells increased with higher silver content but remained tolerable at 6%. Compared to uncoated Ti6Al4V and the electroplated silver coating, the new coating with a silver content of 4% and 6% exhibited a significant reduction in adherent bacteria by a factor of approximately 1000. This was also evident via microscopic examination of the surface morphology of the biofilms. Furthermore, following shock wave activation, no bacteria were detectable on either the implant or in the surrounding fluid after a 24 h period.

Bacterial Lipoproteins Shift Cellular Metabolism to Glycolysis in Macrophages Causing Bone Erosion.

Belonging to a group of membrane proteins, bacterial lipoproteins (LPPs) are defined by a unique lipid structure at their N-terminus providing the anchor in the bacterial cell membrane. In Gram-positive bacteria, LPPs play a key role in host immune activation triggered through a Toll-like receptor 2 (TLR2)-mediated action resulting in macrophage stimulation and subsequent tissue damage demonstrated in in vivo experimental models. Yet the physiologic links between LPP activation, cytokine release, and any underlying switches in cellular metabolism remain unclear. In this study, we demonstrate that Staphylococcus aureus Lpl1 not only triggers cytokine production but also confers a shift toward fermentative metabolism in bone marrow-derived macrophages (BMDMs). Lpl1 consists of di- and tri-acylated LPP variants; hence, the synthetic P2C and P3C, mimicking di-and tri-acylated LPPs, were employed to reveal their effect on BMDMs. Compared to P3C, P2C was found to shift the metabolism of BMDMs and the human mature monocytic MonoMac 6 (MM6) cells more profoundly toward the fermentative pathway, as indicated by lactate accumulation, glucose consumption, pH reduction, and oxygen consumption. In vivo, P2C caused more severe joint inflammation, bone erosion, and lactate and malate accumulation than P3C. These observed P2C effects were completely abrogated in monocyte/macrophage-depleted mice. Taken together, these findings now solidly confirm the hypothesized link between LPP exposure, a macrophage metabolic shift toward fermentation, and ensuing bone destruction. IMPORTANCE Osteomyelitis caused by S. aureus is a severe infection of the bone, typically associated with severe bone function impairment, therapeutic failure, high morbidity, invalidity, and occasionally even death. The hallmark of staphylococcal osteomyelitis is the destruction of the cortical bone structures, yet the mechanisms contributing to this pathology are hitherto poorly understood. One bacterial membrane constituent found in all bacteria is bacterial lipoproteins (LPPs). Previously, we have shown that injection of purified S. aureus LPPs into wild-type mouse knee joints caused a TLR2-dependent chronic destructive arthritis but failed to elicit such effect in monocyte/macrophage-depleted mice. This observation stirred our interest in investigating the interaction of LPPs and macrophages and analyzing the underlying physiological mechanisms. This ascertainment of LPP-induced changes in the physiology of macrophages provides an important clue in the understanding of the mechanisms of bone disintegration, opening novel avenues to manage the course of S. aureus disease.

Protein Disulfide Isomerase and Extracellular Adherence Protein Cooperatively Potentiate Staphylococcal Invasion into Endothelial Cells.

Invasion of host cells is an important feature of Staphylococcus aureus. The main internalization pathway involves binding of the bacteria to host cells, e.g., endothelial cells, via a fibronectin (Fn) bridge between S. aureus Fn binding proteins and α5ß1-integrin, followed by phagocytosis. The secreted extracellular adherence protein (Eap) has been shown to promote this cellular uptake pathway of not only S. aureus, but also of bacteria otherwise poorly taken up by host cells, such as Staphylococcus carnosus. The exact mechanisms are still unknown. Previously, we demonstrated that Eap induces platelet activation by stimulation of the protein disulfide isomerase (PDI), a catalyst of thiol-disulfide exchange reactions. Here, we show that Eap promotes PDI activity on the surface of endothelial cells, and that this contributes critically to Eap-driven staphylococcal invasion. PDI-stimulated ß1-integrin activation followed by increased Fn binding to host cells likely accounts for the Eap-enhanced uptake of S. aureus into non-professional phagocytes. Additionally, Eap supports the binding of S. carnosus to Fn-α5ß1 integrin, thereby allowing its uptake into endothelial cells. To our knowledge, this is the first demonstration that PDI is crucial for the uptake of bacteria into host cells. We describe a hitherto unknown function of Eap-the promotion of an enzymatic activity with subsequent enhancement of bacterial uptake-and thus broaden mechanistic insights into its importance as a driver of bacterial pathogenicity. IMPORTANCE Staphylococcus aureus can invade and persist in non-professional phagocytes, thereby escaping host defense mechanisms and antibiotic treatment. The intracellular lifestyle of S. aureus contributes to the development of infection, e.g., in infective endocarditis or chronic osteomyelitis. The extracellular adherence protein secreted by S. aureus promotes its own internalization as well as that of bacteria that are otherwise poorly taken up by host cells, such as Staphylococcus carnosus. In our study, we demonstrate that staphylococcal uptake by endothelial cells requires catalytic disulfide exchange activity by the cell-surface protein disulfide isomerase, and that this critical enzymatic function is enhanced by Eap. The therapeutic application of PDI inhibitors has previously been investigated in the context of thrombosis and hypercoagulability. Our results add another intriguing possibility: therapeutically targeting PDI, i.e., as a candidate approach to modulate the initiation and/or course of S. aureus infectious diseases.