Minh-Thu Nguyen

Evaluation of Staphylococcus aureus Lipoproteins: Role in Nutritional Acquisition and Pathogenicity

Bacterial lipoproteins (Lpp) represent a major class of membrane proteins. They are distinguished by a lipid moiety at the N-terminus by which they are anchored either in the outer leaflet of the cytoplasmic membrane or, in Gram-negative bacteria, also in the inner leaflet of the outer membrane. In Gram-positive bacteria Lpp significantly contribute to nutrient transport, Toll-like receptor 2 activation and pathogenicity. Here we examine the Lpp of Staphylococcus aureus USA300, as a prototype for a multiple antibiotic resistant and community-acquired pathogen that is rapidly spreading worldwide. The compiled Lpp were grouped according to the postulated function and dissemination of homologs in the genus Staphylococcus and beyond. Based on this evaluation we also point out Lpp as promising vaccine candidates.

SadA-Expressing Staphylococci in the Human Gut Show Increased Cell Adherence and Internalization

A subgroup of biogenic amines, the so-called trace amines (TAs), are produced by mammals and bacteria and can act as neuromodulators. In the genus Staphylococcus, certain species are capable of producing TAs through the activity of staphylococcal aromatic amino acid decarboxylase (SadA). SadA decarboxylates aromatic amino acids to produce TAs, as well as dihydroxy phenylalanine and 5-hydroxytryptophan to thus produce the neurotransmitters dopamine and serotonin. SadA-expressing staphylococci were prevalent in the gut of most probands, where they are part of the human intestinal microflora. Furthermore, sadA-expressing staphylococci showed increased adherence to HT-29 cells and 2- to 3-fold increased internalization. Internalization and adherence was also increased in a sadA mutant in the presence of tryptamine. The α2-adrenergic receptor is required for enhanced adherence and internalization. Thus, staphylococci in the gut might contribute to gut activity and intestinal colonization.

Antigen delivery to dendritic cells shapes human CD4+ and CD8+ T cell memory responses to Staphylococcus aureus

Intracellular persistence of Staphylococcus aureus favors bacterial spread and chronic infections. Here, we provide evidence for the existence of human CD4+ and CD8+ T cell memory against staphylococcal antigens. Notably, the latter could provide a missing link in our understanding of immune control of intracellular S. aureus. The analyses showed that pulsing of monocyte-derived dendritic cells (MoDC) with native staphylococcal protein antigens induced release of Th2-associated cytokines and mediators linked to T regulatory cell development (G-CSF, IL-2 and IL-10) from both CD4+ and CD8+ T cells, thus revealing a state of tolerance predominantly arising from preformed memory T cells. Furthermore, G-CSF was identified as a suppressor of CD8+ T cell-derived IFNγ secretion, thus confirming a tolerogenic role of this cytokine in the regulation of T cell responses to S. aureus. Nevertheless, delivery of in vitro transcribed mRNA-encoded staphylococcal antigens triggered Th1-biased responses, e.g. IFNγ and TNF release from both naïve and memory T cells. Collectively, our data highlight the potential of mRNA-adjuvanted antigen presentation to enable inflammatory responses, thus overriding the existing Th2/Treg-biased memory T cell response to native S. aureus antigens.

Staphylococcal (phospho)lipases promote biofilm formation and host cell invasion

Most Staphylococcus aureus strains secrete two lipases SAL1 and SAL2 encoded by gehA and gehB. These two lipases differ with respect to their substrate specificity. Staphylococcus hyicus secretes another lipase, SHL, which is in contrast to S. aureus lipases Ca2+-dependent and has a broad-spectrum lipase and phospholipase activity. The aim of this study was to investigate the role of staphylococcal (phospho) lipases in virulence. For this we constructed a gehA-gehB double deletion mutant in S. aureus USA300 and expressed SHL in agr-positive (accessory gene regulator) and agr-negative S. aureus strains. The lipases themselves have no hemolytic or cytotoxic activity. However, in agr-negative strains SHL-expression caused an upregulation of hemolytic activity. We further show that SHL-expression significantly enhanced biofilm formation probably due to an increase of extracellular DNA release. SHL-expression also increased host cell invasion 4-6-fold. Trioleate, a main triacylglycerol component of mammalian skin, induced lipase production. Finally, in the mouse sepsis and skin colonization models the lipase producing and mutant strain showed no significant difference compared to the WT strain. In conclusion, we show that staphylococcal lipases promote biofilm formation and host cell invasion and thereby contribute to S. aureus virulence.

Lipopeptide-Induced Suicidal Erythrocyte Death Correlates with the Degree of Acylation

Background/Aims: Consequences of bacterial infection include anemia, which could result from stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Bacterial components known to stimulate eryptosis include lipopeptides. Signaling mediating the triggering of eryptosis include increased cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and cellular accumulation of ceramide. The present study aimed to define the molecular requirements for lipopeptide-induced cell membrane scrambling. Methods: Human erythrocytes were incubated for 48 hours in the absence and presence of 1 or 5 µg/ml of the synthetic lipopeptides Pam1 (lipopeptide with one fatty acid), Pam2 (lipopeptide with two fatty acids), or Pam3 (lipopeptide with three fatty acids). In the following phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fuorescence, and ceramide abundance utilizing specific antibodies. Results: Pam1 (5 µg/ml), Pam2 (5 µg/ml) and Pam3 (1 and 5 µg/ml) significantly increased the percentage of annexin-V-binding to erythrocytes in a dose dependent manner, which was largely independent of Ca2+. Pam1-3 increased the percentage of both, swollen and shrunken erythrocytes without significantly modifying the average forward scatter. They also increased reactive oxygen species (ROS) and ceramide abundance. In all assays the effect on eryptosis increased with increasing number of fatty acids, with Pam3 showing always the strongest effect. In contrast, a comparison of the effect of Pam1-3 on TLR2 dependent immune stimulation showed that not Pam3 but Pam2 displayed the strongest activity, and that immune stimulation was triggered at much lower concentrations than eryptosis. Conclusions: Lipopeptides are not only important activators of the immune system; at higher concentrations they also drive host cells into apoptosis thus aggravating a bacterial infection.

Lipid moieties on lipoproteins of commensal and non-commensal staphylococci induce differential immune responses

Lipoproteins (Lpp) of Gram-positive bacteria are major players in alerting our immune system. Here, we show that the TLR2 response induced by commensal species Staphylococcus aureus and Staphylococcus epidermidis is almost ten times lower than that induced by noncommensal Staphylococcus carnosus, and this is at least partially due to their different modifications of the Lpp lipid moieties. The N terminus of the lipid moiety is acylated with a long-chain fatty acid (C17) in S. aureus and S. epidermidis, while it is acylated with a short-chain fatty acid (C2) in S. carnosus. The long-chain N-acylated Lpp, recognized by TLR2–TLR1 receptors, silences innate and adaptive immune responses, while the short-chain N-acetylated Lpp, recognized by TLR2–TLR6 receptors, boosts it.The Lpp lipoproteins of staphylococci trigger a TLR2-dependent immune response. Here, the authors show that commensal species (S. aureus, S. epidermidis) induce a less-intense TLR2 response than non-commensal species (S. carnosus) due to differential modification of the Lpp lipid moieties.

Aspartate tightens the anchoring of staphylococcal lipoproteins to the cytoplasmic membrane

In gram‐negative bacteria, the ABC transporter LolCDE complex translocates outer membrane‐specific lipoproteins (Lpp) from the inner membrane to the outer membrane. Lpp possessing aspartate (Asp) at position +2 are not translocated because it functions as a LolCDE avoidance signal. In gram‐positive bacteria, lacking an outer membrane and the Lol system, Lpp are only anchored at the outer leaflet of the cytoplasmic membrane. However, the release of Lpp particularly in pathogenic or commensal species is crucial for immune modulation. Here, we provide evidence that in Staphylococcus aureus Asp at position +2 plays a role in withholding Lpp to the cytoplasmic membrane. Screening of published exoproteomic data of S. aureus revealed that Lpp mainly with Gly or Ser at position +2 were found in exoproteome, but there was no Lpp with Asp+2. The occurrence of Lpp with Asp+2 is infrequent in gram‐positive bacteria. In S. aureus USA300 only seven of the 67 Lpp possess Asp+2; among them five Lpp represented Lpl lipoproteins involved in host cell invasion. Our study demonstrated that replacing the Asp+2 present in Lpl8 with a Ser enhances its release into the supernatant. However, there is no different release of Asp+2 and Ser+2 in mprF mutant that lacks the positive charge of lysyl‐phosphatidylglycerol (Lys‐PG). Moreover, substitution of Ser+2 by Asp in SitC (MntC) did not lead to a decreased release indicating that in staphylococci positions +3 and +4 might also be important for a tighter anchoring of Lpp. Here, we show that Asp in position +2 and adjacent amino acids contribute in tightening the anchoring of Lpp by interaction of the negative charged Asp with the positive charged Lys‐PG.

Staphylococcus carnosus: from starter culture to protein engineering platform

Since the 1950s, Staphylococcus carnosus is used as a starter culture for sausage fermentation where it contributes to food safety, flavor, and a controlled fermentation process. The long experience with S. carnosus has shown that it is a harmless and “food grade” species. This was confirmed by the genome sequence of S. carnosus TM300 that lacks genes involved in pathogenicity. Since the development of a cloning system in TM300, numerous genes have been cloned, expressed, and characterized and in particular, virulence genes that could be functionally validated in this non-pathogenic strain. A secretion system was developed for production and secretion of industrially important proteins and later modified to also enable display of heterologous proteins on the surface. The display system has been employed for various purposes, such as development of live bacterial delivery vehicles as well as microbial biocatalysts or bioadsorbents for potential environmental or biosensor applications. Recently, this surface display system has been utilized for display of peptide and protein libraries for profiling of protease substrates and for generation of various affinity proteins, e.g., Affibody molecules and scFv antibodies. In addition, by display of fragmented antigen-encoding genes, the surface expression system has been successfully used for epitope mapping of antibodies. Reviews on specific applications of S. carnosus have been published earlier, but here we provide a more extensive overview, covering a broad range of areas from food fermentation to sophisticated methods for protein-based drug discovery, which are all based on S. carnosus.

Toll-Like Receptor 2 and Lipoprotein-Like Lipoproteins Enhance Staphylococcus aureus Invasion in Epithelial Cells

Staphylococcus aureus contains a certain subclass of lipoproteins, the so-called lipoprotein-like lipoproteins (Lpls), that not only represent Toll-like receptor 2 (TLR2) ligands but are also involved in host cell invasion. Here we addressed the question of which factors contribute to Lpl-mediated invasion of epithelial cells and keratinocytes. ABSTRACT Staphylococcus aureus contains a certain subclass of lipoproteins, the so-called lipoprotein-like lipoproteins (Lpls), that not only represent Toll-like receptor 2 (TLR2) ligands but are also involved in host cell invasion. Here we addressed the question of which factors contribute to Lpl-mediated invasion of epithelial cells and keratinocytes. For this purpose, we compared the invasiveness of USA300 and its Δlpl mutant under different conditions. In the presence of the matrix proteins IgG, fibrinogen (Fg), and fibronectin (Fn), and of fetal bovine serum (FBS), the invasion ratio was increased in both strains, and always more in USA300 than in its Δlpl mutant. Interestingly, when we compared the invasion of HEK-0 and HEK-TLR2 cells, the cells expressing TLR2 showed a 9-times-higher invasion frequency. When HEK-TLR2 cells were additionally stimulated with a synthetic lipopeptide, Pam3CSK4 (P3C), the invasion frequency was further increased. A potential reason for the positive effect of TLR2 on invasion could be that TLR2 activation by P3C also activates F-actin formation. Here we show that S. aureus invasion depends on a number of factors, on the host side as well as on the bacterial side.