Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.     

Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.     

On the Influence of Freight Trains on Humans: A Laboratory Investigation of the Impact of Nocturnal Low Frequency Vibration and Noise on Sleep and Heart Rate

Background

A substantial increase in transportation of goods on railway may be hindered by public fear of increased vibration and noise leading to annoyance and sleep disturbance. As the majority of freight trains run during night time, the impact upon sleep is expected to be the most serious adverse effect. The impact of nocturnal vibration on sleep is an area currently lacking in knowledge. We experimentally investigated sleep disturbance with the aim to ascertain the impact of increasing vibration amplitude.

Methodology/Principal Findings

The impacts of various amplitudes of horizontal vibrations on sleep disturbance and heart rate were investigated in a laboratory study. Cardiac accelerations were assessed using a combination of polysomnography and ECG recordings. Sleep was assessed subjectively using questionnaires. Twelve young, healthy subjects slept for six nights in the sleep laboratory, with one habituation night, one control night and four nights with a variation of vibration exposures whilst maintaining the same noise exposure. With increasing vibration amplitude, we found a decrease in latency and increase in amplitude of heart rate as well as a reduction in sleep quality and increase in sleep disturbance.

Conclusions/Significance

We concluded that nocturnal vibration has a negative impact on sleep and that the impact increases with greater vibration amplitude. Sleep disturbance has short- and long-term health consequences. Therefore, it is necessary to define levels that protect residents against sleep disruptive vibrations that may arise from night time railway freight traffic.     

In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes

DNA methylation is one of the most important epigenetic alterations involved in the control of gene expression. Bisulfite sequencing of genomic DNA is currently the only method to study DNA methylation patterns at single-nucleotide resolution. Hence, next-generation sequencing of bisulfite-converted DNA is the method of choice to investigate DNA methylation profiles at the genome-wide scale. Nevertheless, whole genome sequencing for analysis of human methylomes is expensive, and a method for targeted gene analysis would provide a good alternative in many cases where the primary interest is restricted to a set of genes.Here, we report the successful use of a custom Agilent SureSelect Target Enrichment system for the hybrid capture of bisulfite-converted DNA. We prepared bisulfite-converted next-generation sequencing libraries, which are enriched for the coding and regulatory regions of 174 ADME genes (i.e. genes involved in the metabolism and distribution of drugs). Sequencing of these libraries on Illumina’s HiSeq2000 revealed that the method allows a reliable quantification of methylation levels of CpG sites in the selected genes, and validation of the method using pyrosequencing and the Illumina 450K methylation BeadChips revealed good concordance.     

The modulation rate transfer function of a harbour porpoise (Phocoena phocoena)

During echolocation, toothed whales produce ultrasonic clicks at extremely rapid rates and listen for the returning echoes. The auditory brainstem response (ABR) duration was evaluated in terms of latency between single peaks: 5.5 ms (from peak I to VII), 3.4 ms (I–VI), and 1.4 ms (II–IV). In comparison to the killer whale and the bottlenose dolphin, the ABR of the harbour porpoise has shorter intervals between the peaks and consequently a shorter ABR duration. This indicates that the ABR duration and peak latencies are possibly related to the relative size of the auditory structures of the central nervous system and thus to the animal’s size. The ABR to a sinusoidal amplitude modulated stimulus at 125 kHz (sensitivity threshold 63 dB re 1 μPa rms) was evaluated to determine the modulation rate transfer function of a harbour porpoise. The ABR showed distinct envelope following responses up to a modulation rate of 1,900 Hz. The corresponding calculated equivalent rectangular duration of 263 μs indicates a good temporal resolution in the harbour porpoise auditory system similar to the one for the bottlenose dolphin. The results explain how the harbour porpoise can follow clicks and echoes during echolocation with very short inter click intervals.     

T cell responses against microsatellite instability-induced frameshift peptides and influence of regulatory T cells in colorectal cancer

High-level microsatellite-unstable (MSI-H) colorectal carcinomas (CRC) represent a distinct subtype of tumors commonly characterized by dense infiltration with cytotoxic T cells, most likely due to expression of MSI-H-related frameshift peptides (FSP). The contribution of FSP and classical antigens like MUC1 and CEA to the cellular immune response against MSI-H CRC had not been analyzed so far. We analyzed tumor-infiltrating and peripheral T cells from MSI-H (n = 4 and n = 14, respectively) and microsatellite-stable (MSS) tumor patients (n = 26 and n = 17) using interferon gamma ELISpot assays. Responses against 4 FSP antigens and peptides derived from MUC1 to CEA were compared with and without depletion of regulatory T cells, and the results were related to the presence of the respective antigens in tumor tissue. Preexisting FSP-specific T cell responses were detected in all (4 out of 4) tumor-infiltrating and in the majority (10 out of 14) of peripheral T cell samples from MSI-H CRC patients, but rarely observed in MSS CRC patients. Preexisting T cell responses in MSI-H CRC patients were significantly more frequently directed against FSP tested in the present study than against peptides derived from classical antigens MUC1 or CEA (p = 0.049). Depletion of regulatory T cells increased the frequency of effector T cell responses specific for MUC1/CEA-derived peptides and, to a lesser extent, T cell responses specific for FSP. Our data suggest that the analyzed FSP may represent an immunologically relevant pool of antigens capable of eliciting antitumoral effector T cell responses.     

Seasonal Variation in Human Salivary Cortisol Concentration

Measurement of cortisol concentration can contribute important information about an individual's ability to adjust to various environmental demands of both physical and psychosocial origin. However, one uncertainty that affects the possibilities of correctly interpreting and designing field studies is the lack of observations of the impact of seasonal changes on cortisol excretion. For this reason, the month‐to‐month changes in diurnal cortisol concentration, the awakening cortisol response (ACR), maximum morning concentration, and fall during the day were studied in a group of 24 healthy men and women 32 to 61 yrs of age engaged in active work. On one workday for 12 consecutive months, participants collected saliva at four time points for determination of cortisol: at awakening, +30 min, +8 h, and at 21:00 h. Data were analyzed by a repeated measures design with month (12 levels) and time‐of‐day (4 levels) as categorical predictors. Cortisol concentrations were analyzed on a log scale. The diurnal pattern of cortisol was similar across months (interaction between month and time of day: p>0.4). The main effects of month and time‐of‐day were statistically significant (p <0.001). Highest concentrations were observed in February, March, and April, and lowest concentrations were observed in July and August. There were no statistically significant effects in any of the other measures, or between men and women. In conclusion, a seasonal variation in salivary cortisol concentrations was detected in an occupationally active population. Thus, seasonal variation needs to be taken into account when designing and evaluating field studies and interventions and when making comparisons across studies.     

A brief pre-exercise nap may alleviate physical performance impairments induced by short-term sustained operations with partial sleep deprivation – A field-based study

ABSTRACT

The purpose of the study was to evaluate the recuperative efficacy of pre-exercise napping on physical capacity after military sustained operations (SUSOPS) with partial sleep deprivation. Before and after a 2-day SUSOPS, 61 cadets completed a battery of questionnaires, and performed a 2-min lunges trial and a 3,000-m running time-trial. After the completion of SUSOPS, subjects were randomized to either a control [without pre-exercise nap (CON); n = 32] or a nap [with a 30-min pre-exercise nap (NAP); n = 29] group. SUSOPS enhanced perceived sleepiness and degraded mood in both groups. Following SUSOPS, the repetitions of lunges, in the CON group, were reduced by ~ 2.3%, albeit the difference was not statistically significant (p = 0.62). In the NAP group, however, the repetitions of lunges were increased by ~ 7.1% (p = 0.01). SUSOPS impaired the 3,000-m running performance in the CON group (~ 2.3%; p = 0.02), but not in the NAP group (0.3%; p = 0.71). Present results indicate, therefore, that a relatively brief pre-exercise nap may mitigate physical performance impairments ensued by short-term SUSOPS.     

Patterning and Lifetime of Plasma Membrane-Localized Cellulose Synthase Is Dependent on Actin Organization in Arabidopsis Interphase Cells

The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis.Plant cells are surrounded by a flexible yet durable extracellular matrix that makes up the cell wall. This structure offers mechanical strength that counters osmotically driven turgor pressure, is an important factor for water movement in plants, acts as a physical barrier against pathogens (Somerville et al., 2004), and is a determining factor for plant cell morphogenesis. Hence, the cell wall plays a central role in plant biology.Two main types of cell walls can typically be distinguished: the primary and the secondary cell wall. The major load-bearing component in both of these cell walls is the β-1,4-linked glucan polymer cellulose (Somerville et al., 2004). Cellulose polymers are synthesized by plasma membrane (PM)-localized cellulose synthase (CesA) complexes (Mueller and Brown, 1980), which contain several CesA subunits with similar amino acid sequences (Mutwil et al., 2008a). The primary wall CesA complexes are believed to be assembled in the Golgi and are subsequently delivered to the PM via vesicular trafficking (Gutierrez et al., 2009), sometimes associated with Golgi pausing (Crowell et al., 2009). Furthermore, the primary wall CesA complexes are preferentially inserted into the PM at sites that coincide with cortical microtubules (MTs), which subsequently guide cellulose microfibril deposition (Gutierrez et al., 2009). Hence, the cortical MT array is a determinant for multiple aspects of primary wall cellulose production.The actin cytoskeleton plays a crucial role in organized deposition of cell wall polymers in many cell types, including cellulose-related polymers and pectins in tip-growing cells, such as pollen tubes and root hairs (Hu et al., 2003; Chen et al., 2007). Thus, actin-depolymerizing drugs and genetic manipulation of ACTIN genes impair directed expansion of tip-growing cells and long-distance transport of Golgi bodies with vesicles to growing regions (Ketelaar et al., 2003; Szymanski, 2005). In diffusely growing cells in roots and hypocotyls, loss of anisotropic growth has also been observed in response to mutations to vegetative ACTIN genes and to actin-depolymerizing and -stabilizing drugs (Baluska et al., 2001; Kandasamy et al., 2009). While actin is clearly important for cell wall assembly, it is less clear what precise roles it plays.One well-known function of actin in higher plants is to support intracellular movement of cytoplasmic organelles via actomyosin-based motility (Geisler et al., 2008; Szymanski, 2009). During primary wall synthesis in interphase cells, treatment with the actin assembly inhibitor latrunculin B (LatB) led to inhibition of Golgi motility and pronounced inhomogenities in CesA density at the PM (Crowell et al., 2009; Gutierrez et al., 2009) that coincided with the density of underlying and immobile Golgi bodies (Gutierrez et al., 2009). These results suggested that Golgi motility is important for CesA distribution (Gutierrez et al., 2009). The actin cytoskeleton also appears to be important for secondary wall cellulose microfibril deposition. For example, longitudinal actin filaments (AFs) define the movement of secondary wall CesA-containing Golgi bodies in developing xylem vessels (Wightman and Turner, 2008). In addition, it has been proposed that the AFs also can regulate the delivery of the secondary wall CesA complex to the PM via pausing of the Golgi (Wightman and Turner, 2008). It is therefore clear that actin organization is important for CesA distribution and for the pattern of cellulose microfibril deposition.Despite the above findings, very few reports have undertaken detailed studies to elucidate the role of the actin cytoskeleton in the distribution and trafficking of specific proteins in plant cells. Here, we have investigated the intracellular trafficking of CesA-containing vesicles and delivery of CesAs to the PM, in the context of the actin cytoskeleton. We quantitatively demonstrate that the organization of the actin cytoskeleton regulates CesA-containing Golgi distribution and the exocytic and endocytic rate of the CesAs. However, actin organization has no effect on the localized insertion of CesAs at sites of MTs at the PM.