RNA aggregates harness the danger response for potent cancer immunotherapy.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

Physiological Axial Tibial Rotation of the Knee During a Weightbearing Flexion.

Axial tibial rotation is a characteristic motion of the knee, but how it occurs with knee flexion is controversial. We investigated the mechanisms of tibial rotations by analyzing in vivo tibiofemoral articulations. Twenty knees of 20 living human subjects were investigated during a weightbearing flexion from full extension to maximal flexion using a dual fluoroscopic imaging system. Tibiofemoral articular contact motions on medial and lateral femoral condyles and tibial surfaces were measured at flexion intervals of 15 deg from 0 deg to 120 deg. Axial tibial rotations due to the femoral and tibial articular motions were compared. Articular contact distances were longer on femoral condyles than on tibial surfaces at all flexion intervals (p < 0.05). The articular distance on medial femoral condyle is longer than on lateral side during flexion up to 60 deg. The internal tibial rotation was 6.8 ± 4.5 deg (Mean ± SD) at the flexion interval of 0-15 deg, where 6.1 ± 2.6 deg was due to articulations on femoral condyles and 0.7 ± 5.1 deg due to articulations on tibial surfaces (p < 0.05). The axial tibial rotations due to articulations on femoral condyles are significantly larger than those on tibial surfaces until 60 deg of flexion (p < 0.05). Minimal additional axial tibial rotations were observed beyond 60 deg of flexion. The axial tibial rotations were mainly attributed to uneven articulations on medial and lateral femoral condyles. These data can provide new insights into the understanding of mechanisms of axial tibial rotations and serve as baseline knowledge for improvement of knee surgeries.

Staphylococcus lugdunensis: a Skin Commensal with Invasive Pathogenic Potential.

Staphylococcus lugdunensis is a species of coagulase-negative staphylococcus (CoNS) that causes serious infections in humans akin to those of S. aureus It was often misidentified as S. aureus, but this has been rectified by recent routine use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in diagnostic laboratories. It encodes a diverse array of virulence factors for adhesion, cytotoxicity, and innate immune evasion, but these are less diverse than those encoded by S. aureus It expresses an iron-regulated surface determinant (Isd) system combined with a novel energy-coupling factor (ECF) mechanism for extracting heme from hemoproteins. Small cytolytic S. lugdunensis synergistic hemolysins (SLUSH), peptides related to phenol-soluble modulins of S. aureus, act synergistically with ß-toxin to lyse erythrocytes. S. lugdunensis expresses a novel peptide antibiotic, lugdunin, that can influence the nasal and skin microbiota. Endovascular infections are initiated by bacterial adherence to fibrinogen promoted by a homologue of Staphylococcus aureus clumping factor A and to von Willebrand factor on damaged endothelium by an uncharacterized mechanism. S. lugdunensis survives within mature phagolysosomes of macrophages without growing and is released only following apoptosis. This differs fundamentally from S. aureus, which actively grows and expresses bicomponent leukotoxins that cause membrane damage and could contribute to survival in the infected host. S. lugdunensis is being investigated as a probiotic to eradicate S. aureus from the nares of carriers. However, this is contraindicated by its innate virulence. Studies to obtain a deeper understanding of S. lugdunensis colonization, virulence, and microbiome interactions are therefore warranted.

Farm size limits agriculture's poverty reduction potential in Eastern India even with irrigation-led intensification.

CONTEXT: Millions of people living in the Eastern Gangetic Plains (EGP) of India engage in agriculture to support their livelihoods yet are income poor, and food and climate insecure. To address these challenges, policymakers and development programs invest in irrigation-led agricultural intensification. However, the evidence for agricultural intensification to lift farmers' incomes above the poverty line remains largely anecdotal. OBJECTIVE: The main objective of this study is to use a large household survey (n = 15,572; rice: 8244, wheat: 7328; 2017/18) to assess the link between agricultural intensification and personal daily incomes from farming (FPDI) in the rice-wheat systems of the EGP - the dominant cropping system of the region. METHODS: We use the Intensification Benefit Index (IBI), a measure that relates farm size and household size to FPDI, to assess how daily incomes from rice-wheat production change with irrigation-led intensification across the EGP. RESULTS AND CONCLUSIONS: Relative to the international poverty line of 1.90 Purchasing Power Parity (PPP)$ day-1 and accounting for variations in HH size in the analysis, we found that small farm sizes limit the potential for agricultural intensification from irrigation to transform the poverty status of households in the bottom three quartiles of the IBI. The estimated median FPDI of households with intensified systems in the bottom three quartiles is only 0.51 PPP$ day-1 (a 0.15 PPP$ gain). The median FPDI increases to 2.10 PPP$ day-1 for households in the upper quartile of the IBI distribution (a 0.30 PPP$ gain). Irrigation-led agricultural intensification of rice-wheat systems in the EGP may provide substantial benefits for resilience to climatic change and food security but achieving meaningful poverty reduction will require complementary investments. SIGNIFICANCE: Transforming the poverty status of most smallholder farmers in the EGP requires diversified portfolios of rural on- and off-farm income-generating opportunities. While bolstering food- and climate security, agronomic intervention programs should consider smallholders' limited monetary incentives to invest in intensification. Irrigation-led agricultural intensification programs and policies should explicitly account for the heterogeneity in household resources, irrigation levels, and degree of dependence on agricultural income.

Investigation of femoral condyle height changes during flexion of the knee: implication to gap balance in TKA surgery.

BACKGROUND: Gap balance of the knee at 0° and 90° of flexion has been pursued in total knee arthroplasty (TKA) with the trans-epicondyle axis (TEA) as a reference. This study investigated the height changes of the tibiofemoral articulation and compared the data with the femoral condyle height changes measured using different flexion axes. MATERIALS AND METHODS: Twenty healthy knees were investigated during an in vivo weightbearing flexion using a technique combining MRI and a dual fluoroscopic imaging system (DFIS). The tibiofemoral contact points and the femoral condyle heights [measured using: TEA, geometric center axis (GCA), and iso-height axis (IHA)] were determined at each flexion angle. The height changes of the articular contact points and the femoral condyles were compared along the flexion path. RESULTS: The changes of the medial and lateral contact point heights were within 2.5 mm along the flexion path. The changes of the medial and lateral condyle heights were within 8.9 mm for TEA, within 4.2 mm for GCA and within 3.0 mm for IHA. The height changes measured by the contact points and IHA are similar (p > 0.05), and both are significantly smaller than those measured using the TEA and GCA (p < 0.05). CONCLUSIONS: The TEA and GCA measured varying femoral condyle heights, but the IHA resulted in minimal condyle height changes and could better represent the articulation characteristics of the knee. The data suggested that the IHA could be used as an alternative reference to guide surgical preparation of gap balance along the knee flexion path during TKA surgeries.

The iron-regulated surface determinant B (IsdB) protein from Staphylococcus aureus acts as a receptor for the host protein vitronectin.

Staphylococcus aureus is an important bacterial pathogen that can cause a wide spectrum of diseases in humans and other animals. S. aureus expresses a variety of virulence factors that promote infection with this pathogen. These include cell-surface proteins that mediate adherence of the bacterial cells to host extracellular matrix components, such as fibronectin and fibrinogen. Here, using immunoblotting, ELISA, and surface plasmon resonance analysis, we report that the iron-regulated surface determinant B (IsdB) protein, besides being involved in heme transport, plays a novel role as a receptor for the plasma and extracellular matrix protein vitronectin (Vn). Vn-binding activity was expressed by staphylococcal strains grown under iron starvation conditions when Isd proteins are expressed. Recombinant IsdB bound Vn dose dependently and specifically. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound Vn in a saturable manner, with KD values in the range of 16-18 nm Binding of Vn to IsdB was specifically blocked by heparin and reduced at high ionic strength. Furthermore, IsdB-expressing bacterial cells bound significantly higher amounts of Vn from human plasma than did an isdB mutant. Adherence to and invasion of epithelial and endothelial cells by IsdB-expressing S. aureus cells was promoted by Vn, and an αvß3 integrin-blocking mAb or cilengitide inhibited adherence and invasion by staphylococci, suggesting that Vn acts as a bridge between IsdB and host αvß3 integrin.

Correction: Competing for Iron: Duplication and Amplification of the isd Locus in Staphylococcus lugdunensis HKU09-01 Provides a Competitive Advantage to Overcome Nutritional Limitation.

[This corrects the article DOI: 10.1371/journal.pgen.1006246.].

There are isoheight points that measure constant femoral condyle heights along the knee flexion path.

PURPOSE: It is a challenge to evaluate the maintenance of medial and lateral soft tissue balance in total knee arthroplasty (TKA). This study aimed to determine the "isoheight" points and the "isoheight" axis (IHA) that can measure constant medial/lateral condyle heights during flexion of the knee, and compare the IHA with two major anatomical axes, the transepicondylar axis (TEA) and the geometric center axis (GCA). METHODS: Twenty-two healthy human knees were imaged using a combined MRI and dual fluoroscopic imaging system while performing a single-legged lunge (0°-120°). The isoheight points of the medial and lateral femoral condyles were defined as the locations with the least amount of changes in heights during the knee flexion; an IHA is the line connecting the medial and lateral isoheight points. The measured changes of the condyle heights using the IHA were compared with those measured using the TEA and GCA. RESULTS: Overall, the IHA was posterior and distal to the TEA, and anterior to the GCA. The isoheight points measured condyle height changes within 1.2 ± 2.3 mm at the medial and 0.7 ± 3.3 mm at the lateral sides during the knee flexion. Between 0° and 45°, the condyle height changes measured using the GCA (medial: 3.0 ± 1.8 mm, lateral: 2.3 ± 2.0 mm) were significantly larger than those of the IHA and the TEA (p < 0.05). Between 90° and 120°, the changes of the condyle heights measured using the TEA (medial: 5.3 ± 1.8 mm, lateral: 3.3 ± 1.8 mm) were significantly larger than those of the IHA and GCA (p < 0.05). CONCLUSION: There are isoheight points in the medial and lateral femoral condyles that can measure constant heights along the full range of knee flexion and could be used to formulate an "isoheight" axis (IHA) of the femur. The condyle height changes measured by the TEA and GCA were greater than the IHA measurements along the flexion path. These data could be used as a valuable reference to evaluate the condyle height changes after TKA surgeries and help achieve soft tissue balance and optimal knee kinematics along the flexion path. LEVEL OF EVIDENCE: IV.

Estimating the phenological dynamics of irrigated rice leaf area index using the combination of PROSAIL and Gaussian Process Regression.

The growth of rice is a sequence of three different phenological phases. This sequence of change in rice phenology implies that the condition of the plant during the vegetative phase relates directly to the health of leaves functioning during the reproductive and ripening phases. As such, accurate monitoring is important towards understanding rice growth dynamics. Leaf Area Index (LAI) is an important indicator of rice yields and the availability of this information during key phenological phases can support more informed farming decisions. Satellite remote sensing has been adopted as a proxy to field measurements of LAI and with the launch of freely available high resolution Satellite images such as Sentinel-2, it is imperative that accurate retrieval methods are adopted towards monitoring LAI at irrigated rice fields. Here, we evaluate the potential of a hybrid radiative transfer model (i.e., PROSAIL - Gaussian Process Regression (GPR), for estimating the phenological dynamics of irrigated rice LAI using imager derived from the Sentinel-2 multispectral instrument. LAI field measurements were obtained from an experimental rice field in Nasarawa state, Nigeria during the dry season. We used the PROSAIL radiative transfer model to create a look up table (LUT) that was subsequently used to train a GPR model. Afterwards, we evaluated the potential of the hybrid modelling approach by assessing the overall model accuracy and the extent to which LAI was able to accurately predict LAI during key rice phenological phases. We compared the predicted hybrid GPR LAI values with LAI values generated from the SNAP toolbox, based on a hybrid Artificial Neural Network (ANN) modelling approach. Our results show that the overall predictive accuracy of the hybrid GPR model (R2 = 0.82, RMSE = 1.65) was more accurate than that of the hybrid ANN model (R2 = 0.66, RMSE = 3.89) for retrieving LAI values from Sentinel-2 imagery. Both models underestimated LAI values during the reproductive and ripening phases . However, the accuracy during the phenological phases were more significant when using the hybrid GPR model (P < 0.05). During the different phenological phases, the hybrid GPR model predicted LAI more accurately during the reproductive (R2 = 0.7) and ripening (R2 = 0.59) phases compared to the hybrid ANN reproductive and ripening phases. When monitoring LAI phenological profiles of both hybrid models, the hybrid GPR and ANN models underestimated LAI during the reproductive and ripening phases. However, the ANN model underestimations were statistically significantly greater than those for the hybrid GPR model (P < 0.05). Our results highlight the potential of hybrid GPR models for estimating the phenological dynamics of irrigated rice LAI from Sentinel-2 data. They provided more accurate estimation of LAI patterns from varying nitrogen and water applications than hybrid ANN models.

Fibronectin-binding protein B (FnBPB) from Staphylococcus aureus protects against the antimicrobial activity of histones.

Staphylococcus aureus is a Gram-positive bacterium that can cause both superficial and deep-seated infections. Histones released by neutrophils kill bacteria by binding to the bacterial cell surface and causing membrane damage. We postulated that cell wall-anchored proteins protect S. aureus from the bactericidal effects of histones by binding to and sequestering histones away from the cell envelope. Here, we focused on S. aureus strain LAC and by using an array of biochemical assays, including surface plasmon resonance and ELISA, discovered that fibronectin-binding protein B (FnBPB) is the main histone receptor. FnBPB bound all types of histones, but histone H3 displayed the highest affinity and bactericidal activity and was therefore investigated further. H3 bound specifically to the A domain of recombinant FnBPB with a KD of 86 nm, ∼20-fold lower than that for fibrinogen. Binding apparently occurred by the same mechanism by which FnBPB binds to fibrinogen, because FnBPB variants defective in fibrinogen binding also did not bind H3. An FnBPB-deletion mutant of S. aureus LAC bound less H3 and was more susceptible to its bactericidal activity and to neutrophil extracellular traps, whereas an FnBPB-overexpressing mutant bound more H3 and was more resistant than the WT. FnBPB bound simultaneously to H3 and plasminogen, which after activation by tissue plasminogen activator cleaved the bound histone. We conclude that FnBPB provides a dual immune-evasion function that captures histones and prevents them from reaching the bacterial membrane and simultaneously binds plasminogen, thereby promoting its conversion to plasmin to destroy the bound histone.

Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato.

Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and ß-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to ß-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.

Exocrine Pancreas Dysfunction in Type 1 Diabetes.

OBJECTIVE: Type 1 diabetes (T1D) is characterized by autoimmune ß-cell destruction, but exocrine pancreas abnormalities may also play a role in the disease pathophysiology. Herein, we review the current evidence of exocrine damage in T1D and discuss its underlying pathophysiology, clinical evaluation, and treatment. METHOD: Extensive literature search was performed for "type 1 diabetes" and "exocrine dysfunction" on PubMed and Google Scholar databases. RESULTS: T1D pancreata are significantly smaller than controls, both in weight and volume. T cells, dendritic cells, neutrophils, and products of complement activation are seen in T1D exocrine tissues. Exocrine pancreas fibrosis, arteriosclerosis, fatty infiltration, and acinar atrophy are also observed on histology. Pancreatic exocrine insufficiency (PEI) can be assessed through direct exocrine testing, fecal elastase concentration, and measurement of serum exocrine enzymes. The prevalence of PEI in T1D varies by modality and study but is consistently greater than controls. The clinical relevance of PEI in T1D is debatable, as many patients with laboratory evidence of PEI are asymptomatic. However, in PEI-symptomatic patients reported benefits of pancreatic enzyme replacement therapy (PERT) include relief of gastrointestinal symptoms, improved quality of life, better glycemic control, and optimal nutrition. CONCLUSION: Exocrine pancreas abnormalities often occur in T1D. Whether exocrine dysfunction occurs simultaneously with ß-cell destruction, as a result of ß-cell loss, or as a combination of both remains to be definitively answered. In T1D with gastrointestinal complaints, PEI should be evaluated, usually via fecal elastase measurements. PERT is recommended for T1D patients with symptoms and laboratory evidence of PEI. ABBREVIATIONS: AAb+ = autoantibody positive; AAb- = autoantibody negative; FEC = fecal elastase concentration; PEI = pancreatic exocrine insufficiency; PERT = pancreatic enzyme replacement therapy; PP = pancreatic polypep-tide; T1D = type 1 diabetes.

Molecular interactions and inhibition of the staphylococcal biofilm-forming protein SdrC.

Staphylococcus aureus forms biofilms on indwelling medical devices using a variety of cell-surface proteins. There is growing evidence that specific homophilic interactions between these proteins represent an important mechanism of cell accumulation during biofilm formation, but the underlying molecular mechanisms are still not well-understood. Here we report the direct measurement of homophilic binding forces by the serine-aspartate repeat protein SdrC and their inhibition by a peptide. Using single-cell and single-molecule force measurements, we find that SdrC is engaged in low-affinity homophilic bonds that promote cell-cell adhesion. Low-affinity intercellular adhesion may play a role in favoring biofilm dynamics. We show that SdrC also mediates strong cellular interactions with hydrophobic surfaces, which are likely to be involved in the initial attachment to biomaterials, the first stage of biofilm formation. Furthermore, we demonstrate that a peptide derived from ß-neurexin is a powerful competitive inhibitor capable of efficiently blocking surface attachment, homophilic adhesion, and biofilm accumulation. Molecular modeling suggests that this blocking activity may originate from binding of the peptide to a sequence of SdrC involved in homophilic interactions. Our study opens up avenues for understanding the role of homophilic interactions in staphylococcal adhesion, and for the design of new molecules to prevent biofilm formation during infection.

In vivo kinematics and ligamentous function of the knee during weight-bearing flexion: an investigation on mid-range flexion of the knee.

PURPOSE: To investigate the in vivo femoral condyle motion and synergistic function of the ACL/PCL along the weight-bearing knee flexion. METHODS: Twenty-two healthy human knees were imaged using a combined MRI and dual fluoroscopic imaging technique during a single-legged lunge (0°-120°). The medial and lateral femoral condyle translation and rotation (measured using geometric center axis-GCA), and the length changes of the ACL/PCL were analyzed at: low (0°-30°), mid-range (30°-90°) and high (90°-120°) flexion of the knee. RESULTS: At low flexion (0°-30°), the strains of the ACL and the posterior-medial bundle of the PCL decreased. The medial condyle showed anterior translation and lateral condyle posterior translation, accompanied with a sharp increase in external GCA rotation (internal tibial rotation). As the knee continued flexion in mid-range (30°-90°), both ACL and PCL were slack (with negative strain values). The medial condyle moved anteriorly before 60° of flexion and then posteriorly, accompanied with a slow increase of GCA rotation. As the knee flexed in high flexion (90°-120°), only the PCL had increasingly strains. Both medial and lateral condyles moved posteriorly with a rather constant GCA rotation. CONCLUSIONS: The ACL and PCL were shown to play a reciprocal and synergistic role during knee flexion. Mid-range reciprocal anterior-posterior femoral translation or laxity corresponds to minimal constraints of the ACL and PCL, and may represent a natural motion character of normal knees. The data could be used as a valuable reference when managing the mid-range "instability" and enhancing high flexion capability of the knee after TKAs. LEVEL OF EVIDENCE: Level IV.

COVID-19 and the case for global development.

COVID-19 accentuates the case for a global, rather than an international, development paradigm. The novel disease is a prime example of a development challenge for all countries, through the failure of public health as a global public good. The COVID-19 pandemic has highlighted the falsity of any assumption that the global North has all the expertise and solutions to tackle global challenges, and has further highlighted the need for multi-directional learning and transformation in all countries towards a more sustainable and equitable world. We illustrate our argument for a global development paradigm by examining the implications of the COVID-19 pandemic across four themes or 'vignettes': global value chains, digitalisation, debt, and climate change. We conclude that development studies must adapt to a very different context from when the field emerged in the mid-20th century.

Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG.

Staphylococcus aureus surface protein SasG promotes cell-cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn(2+) strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell-cell adhesion via specific Zn(2+)-dependent homophilic bonds between ß-sheet-rich G5-E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to ∼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell-cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.

Competing for Iron: Duplication and Amplification of the isd Locus in Staphylococcus lugdunensis HKU09-01 Provides a Competitive Advantage to Overcome Nutritional Limitation.

Staphylococcus lugdunensis is a coagulase negative bacterial pathogen that is particularly associated with severe cases of infectious endocarditis. Unique amongst the coagulase-negative staphylococci, S. lugdunensis harbors an iron regulated surface determinant locus (isd). This locus facilitates the acquisition of heme as a source of nutrient iron during infection and allows iron limitation caused by "nutritional immunity" to be overcome. The isd locus is duplicated in S. lugdunensis HKU09-01 and we show here that the duplication is intrinsically unstable and undergoes accordion-like amplification and segregation leading to extensive isd copy number variation. Amplification of the locus increased the level of expression of Isd proteins and improved binding of hemoglobin to the cell surface of S. lugdunensis. Furthermore, Isd overexpression provided an advantage when strains were competing for a limited amount of hemoglobin as the sole source of iron. Gene duplications and amplifications (GDA) are events of fundamental importance for bacterial evolution and are frequently associated with antibiotic resistance in many species. As such, GDAs are regarded as evolutionary adaptions to novel selective pressures in hostile environments pointing towards a special importance of isd for S. lugdunensis. For the first time we show an example of a GDA that involves a virulence factor of a Gram-positive pathogen and link the GDA directly to a competitive advantage when the bacteria were struggling with selective pressures mimicking "nutritional immunity".

Cell Wall-Anchored Surface Proteins of Staphylococcus aureus: Many Proteins, Multiple Functions.

Staphylococcus aureus persistently colonizes about 20 % of the population and is intermittently associated with the remainder. The organism can cause superficial skin infections and life-threatening invasive diseases. The surface of the bacterial cell displays a variety of proteins that are covalently anchored to peptidoglycan. They perform many functions including adhesion to host cells and tissues, invasion of non-phagocytic cells, and evasion of innate immune responses. The proteins have been categorized into distinct classes based on structural and functional analysis. Many surface proteins are multifunctional. Cell wall-anchored proteins perform essential functions supporting survival and proliferation during the commensal state and during invasive infections. The ability of cell wall-anchored proteins to bind to desquamated epithelial cells is important during colonization, and the binding to fibrinogen is of particular significance in pathogenesis.

Molecular Interactions of Human Plasminogen with Fibronectin-binding Protein B (FnBPB), a Fibrinogen/Fibronectin-binding Protein from Staphylococcus aureus.

Staphylococcus aureus is a commensal bacterium that has the ability to cause superficial and deep-seated infections. Like several other invasive pathogens, S. aureus can capture plasminogen from the human host where it can be converted to plasmin by host plasminogen activators or by endogenously expressed staphylokinase. This study demonstrates that sortase-anchored cell wall-associated proteins are responsible for capturing the bulk of bound plasminogen. Two cell wall-associated proteins, the fibrinogen- and fibronectin-binding proteins A and B, were found to bind plasminogen, and one of them, FnBPB, was studied in detail. Plasminogen captured on the surface of S. aureus- or Lactococcus lactis-expressing FnBPB could be activated to the potent serine protease plasmin by staphylokinase and tissue plasminogen activator. Plasminogen bound to recombinant FnBPB with a KD of 0.532 µm as determined by surface plasmon resonance. Plasminogen binding did not to occur by the same mechanism through which FnBPB binds to fibrinogen. Indeed, FnBPB could bind both ligands simultaneously indicating that their binding sites do not overlap. The N3 subdomain of FnBPB contains the full plasminogen-binding site, and this includes, at least in part, two conserved patches of surface-located lysine residues that were recognized by kringle 4 of the host protein.

Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis.

Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.