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.
Summary
The molecular pathogenesis of many Staphylococcus aureus infections involves growth of bacteria as biofilm. In addition to polysaccharide intercellular adhesin (PIA) and extracellular DNA, ...surface proteins appear to mediate the transition of bacteria from planktonic growth to sessile lifestyle as well as biofilm growth, and can enable these processes even in the absence of PIA expression. However, the molecular mechanisms by which surface proteins contribute to biofilm formation are incompletely understood. Here we demonstrate that self‐association of the serine‐aspartate repeat protein SdrC promotes both bacterial adherence to surfaces and biofilm formation. However, this homophilic interaction is not required for the attachment of bacteria to abiotic surfaces. We identified the subdomain that mediates SdrC dimerization and subsequent cell‐cell interactions. In addition, we determined that two adjacently located amino acid sequences within this subdomain are required for the SdrC homophilic interaction. Comparative amino acid sequence analysis indicated that these binding sites are conserved. In summary, our study identifies SdrC as a novel molecular determinant in staphylococcal biofilm formation and describes the mechanism responsible for intercellular interactions. Furthermore, these findings contribute to a growing body of evidence suggesting that homophilic interactions between surface proteins present on neighbouring bacteria induce biofilm growth.
Staphylococcus aureus is an important opportunistic pathogen which is a leading cause of biofilm-associated infections on indwelling medical devices. The cell surface-located fibronectin-binding ...protein A (FnBPA) plays an important role in the accumulation phase of biofilm formation by methicillin-resistant S. aureus (MRSA), but the underlying molecular interactions are not yet established. Here, we use single-cell and single-molecule atomic force microscopy to unravel the mechanism by which FnBPA mediates intercellular adhesion. We show that FnBPA is responsible for specific cell-cell interactions that involve the FnBPA A domain and cause microscale cell aggregation. We demonstrate that the strength of FnBPA-mediated adhesion originates from multiple low-affinity homophilic interactions between FnBPA A domains on neighboring cells. Low-affinity binding by means of FnBPA may be important for biofilm dynamics. These results provide a molecular basis for the ability of FnBPA to promote cell accumulation during S. aureus biofilm formation. We speculate that homophilic interactions may represent a generic strategy among staphylococcal cell surface proteins for guiding intercellular adhesion. As biofilm formation by MRSA strains depends on proteins rather than polysaccharides, our approach offers exciting prospects for the design of drugs or vaccines to inhibit protein-dependent intercellular interactions in MRSA biofilms.
Staphylococcus aureus is a human pathogen that forms biofilms on indwelling medical devices, such as central venous catheters and prosthetic joints. This leads to biofilm infections that are difficult to treat with antibiotics because many cells within the biofilm matrix are dormant. The fibronectin-binding proteins (FnBPs) FnBPA and FnBPB promote biofilm formation by clinically relevant methicillin-resistant S. aureus (MRSA) strains, but the molecular mechanisms involved remain poorly understood. We used atomic force microscopy techniques to demonstrate that FnBPA mediates cell-cell adhesion via multiple, low-affinity homophilic bonds between FnBPA A domains on adjacent cells. Therefore, FnBP-mediated homophilic interactions represent an interesting target to prevent MRSA biofilms. We propose that such homophilic mechanisms may be widespread among staphylococcal cell surface proteins, providing a means to guide intercellular adhesion and biofilm accumulation.
is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign ...DNA. Here, we establish the complete genomes and methylomes for seven clinically significant, genetically diverse
isolates and perform the first systematic genomic analyses of the type I RM systems within both
and
Our analyses revealed marked differences in the gene arrangement, chromosomal location, and movement of type I RM systems between the two species. Unlike
,
type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of
Using
plasmid artificial modification (PAM) to express
, we readily overcame restriction barriers in
and achieved electroporation efficiencies equivalent to those of modification-deficient mutants. With these functional experiments, we demonstrated how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be electroporation proficient. We outline an efficient approach for the genetic manipulation of
strains from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified
BPH0736, a naturally restriction-defective, clinically significant, multidrug-resistant ST2 isolate, as an ideal candidate for molecular studies.
is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how
causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in
and demonstrate that these are distinct from those described for
Using these insights, we demonstrate an efficient approach for the genetic manipulation of
to enable the study of clinical isolates for the first time.
The bacterial pathogen Staphylococcus aureus expresses a variety of cell surface adhesion proteins that bind to host extracellular matrix proteins. Among these, the collagen (Cn)-binding protein Cna ...plays important roles in bacterium-host adherence and in immune evasion. While it is well established that the A region of Cna mediates ligand binding, whether the repetitive B region has a dedicated function is not known. Here, we report the direct measurement of the mechanical strength of Cna-Cn bonds on living bacteria, and we quantify the antiadhesion activity of monoclonal antibodies (MAbs) targeting this interaction. We demonstrate that the strength of Cna-Cn bonds in vivo is very strong (~1.2 nN), consistent with the high-affinity "collagen hug" mechanism. The B region is required for strong ligand binding and has been found to function as a spring capable of sustaining high forces. This previously undescribed mechanical response of the B region is of biological significance as it provides a means to project the A region away from the bacterial surface and to maintain bacterial adhesion under conditions of high forces. We further quantified the antiadhesion activity of MAbs raised against the A region of Cna directly on living bacteria without the need for labeling or purification. Some MAbs are more efficient in blocking single-cell adhesion, suggesting that they act as competitive inhibitors that bind Cna residues directly involved in ligand binding. This report highlights the role of protein mechanics in activating the function of staphylococcal adhesion proteins and emphasizes the potential of antibodies to prevent staphylococcal adhesion and biofilm formation.
Cna is a collagen (Cn)-binding protein from Staphylococcus aureus that is involved in pathogenesis. Currently, we know little about the functional role of the repetitive B region of the protein. Here, we unravel the mechanical strength of Cna in living bacteria. We show that single Cna-Cn bonds are very strong, reflecting high-affinity binding by the collagen hug mechanism. We discovered that the B region behaves as a nanospring capable of sustaining high forces. This unanticipated mechanical response, not previously described for any staphylococcal adhesin, favors a model in which the B region has a mechanical function that is essential for strong ligand binding. Finally, we assess the antiadhesion activity of monoclonal antibodies against Cna, suggesting that they could be used to inhibit S. aureus adhesion.
The interplanetary shock/electric field event of 5-6 November 2001 is analyzed using ACE interplanetary data. The consequential ionospheric effects are studied using GPS receiver data from the CHAMP ...and SAC-C satellites and altimeter data from the TOPEX/ Poseidon satellite. Data from ~100 ground-based GPS receivers as well as Brazilian Digisonde and Pacific sector magnetometer data are also used. The dawn-to-dusk interplanetary electric field was initially ~33 mV/m just after the forward shock (IMF BZ = -48 nT) and later reached a peak value of ~54 mV/m 1 hour and 40 min later (BZ = -78 nT). The electric field was ~45 mV/m (BZ = -65 nT) 2 hours after the shock. This electric field generated a magnetic storm of intensity DST = -275 nT. The dayside satellite GPS receiver data plus ground-based GPS data indicate that the entire equatorial and midlatitude (up to +/-50(deg) magnetic latitude (MLAT)) dayside ionosphere was uplifted, significantly increasing the electron content (and densities) at altitudes greater than 430 km (CHAMP orbital altitude). This uplift peaked ~2 1/2 hours after the shock passage. The effect of the uplift on the ionospheric total electron content (TEC) lasted for 4 to 5 hours. Our hypothesis is that the interplanetary electric field ''promptly penetrated'' to the ionosphere, and the dayside plasma was convected (by E x B) to higher altitudes. Plasma upward transport/convergence led to a ~55-60% increase in equatorial ionospheric TEC to values above ~430 km (at 1930 LT). This transport/convergence plus photoionization of atmospheric neutrals at lower altitudes caused a 21% TEC increase in equatorial ionospheric TEC at ~1400 LT (from ground-based measurements). During the intense electric field interval, there was a sharp plasma ''shoulder'' detected at midlatitudes by the GPS receiver and altimeter satellites. This shoulder moves equatorward from -54(deg) to -37(deg) MLAT during the development of the main phase of the magnetic storm. We presume this to be an ionospheric signature of the plasmapause and its motion. The total TEC increase of this shoulder is ~80%. Part of this increase may be due to a "superfountain effect." The dayside ionospheric TEC above ~430 km decreased to values ~45% lower than quiet day values 7 to 9 hours after the beginning of the electric field event. The total equatorial ionospheric TEC decrease was ~16%. This decrease occurred both at midlatitudes and at the equator. We presume that thermospheric winds and neutral composition changes produced by the storm-time Joule heating, disturbance dynamo electric fields, and electric fields at auroral and subauroral latitudes are responsible for these decreases.
Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro ...artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations.
Plasmodium falciparum cultures were synchronized with single layer Percoll during the schizont stage of the intraerythrocytic development cycle. Cultures were left to reinvade to early ring-stage and parasitaemia was quantified using flow cytometry. Cultures were diluted to 2% haematocrit and 0.5% parasitaemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700 nM of dihydroartemisinin or 0.02% dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples.
15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of - 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date.
eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.
The availability of onboard calibration for solar reflectance channels on recently launched advanced geostationary imagers provides an opportunity to revisit the calibration of the visible channels ...on past geostationary imagers, which lacked onboard calibration systems. This study used the data from the Advanced Baseline Imager (ABI) on GOES-16 and GOES-17 to calibrate the visible channels on the GOES-IP (GOES-8, -9, -10, -11, -12, -13, and -15) sensors (1994–2021). The visible channels are dominant sources of information for many of the essential climate variables from these sensors. The technique developed uses the stability of the integrated full-disk reflectance to define a calibration target that is applied to past sensors to generate new calibration equations. These equations are found to be stable and agree well with other established techniques. Given the lack of assumptions and ease of application, this technique offers a new calibration method that can be used to complement existing techniques used by the operational space agencies with the GSICS Project. In addition, its simplicity allows for its application to data that existed prior to many of the reference data employed in current calibration methods.
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