Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell beta1 integrins

Although Staphylococcus aureus is primarily considered an extracellular pathogen, recent evidence suggests that this bacterium can invade a variety of nonprofessional phagocytic cells. Here we investigate the early stages of cellular invasion by S. aureus and determine the bacterial and host components that are required for this process. S. aureus expresses two cell surface-associated fibronectin (FN)-binding proteins (FnbpA and FnbpB) that mediate the interaction of the bacteria with both soluble and solid-phase FN in vitro. Using a mutant of S. aureus that lacks the expression of both Fnbps, we show that the expression of either protein is necessary for efficient uptake by the mouse fibroblast line GD25beta1A. Invasion could be inhibited by soluble recombinant proteins encompassing either the FN-binding D repeat region or the A region (and B repeats) of FnbpA, suggesting that the activities of both regions are important in this process. We demonstrate that FN is also required for invasion of this cell line. In the presence of FN-depleted fetal bovine serum, the invasion level was reduced by approximately 40% compared to in the presence of whole fetal bovine serum. Invasion could be further reduced by the addition of anti-mouse FN antibodies to the assay. Finally, we utilize a mutant mouse fibroblast line, which lacks beta1 integrin expression, to demonstrate that host cell beta1 integrins are necessary for efficient cellular invasion. The level of invasion of the mutant cell line GD25 was reduced by approximately 97% compared to the beta1-expressing complemented cell line GD25beta1A. In addition, invasion of the GD25beta1A cell line could be inhibited by an RGD-containing peptide, further implicating a role for integrins in this process. Based on these observations, we put forward a model of S. aureus invasion in which host FN forms a bridge between the bacterial Fnbps and host cell beta1 integrins, leading to bacterial uptake.     

Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+) and G(q)/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+)and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.     

Control of pH and PIP2 gating in heteromeric Kir4.1/Kir5.1 channels by H-Bonding at the helix-bundle crossing

Inhibition by intracellular H(+) (pH gating) and activation by phosphoinositides such as PIP(2) (PIP(2)-gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP(2) gating and pH gating are controlled by an intra-subunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP(2) and pH gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP2 gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP(2) affinities. This indicates that Kir channel PIP(2) affinity has little impact on either the PIP(2) or pH gating kinetics.     

LKB1 inhibition of NF‐κB in B cells prevents T follicular helper cell differentiation and germinal center formation

T-cell-dependent antigenic stimulation drives the differentiation of B cells into antibody-secreting plasma cells and memory B cells, but how B cells regulate this process is unclear. We show that LKB1 expression in B cells maintains B-cell quiescence and prevents the premature formation of germinal centers (GCs). Lkb1-deficient B cells (BKO) undergo spontaneous B-cell activation and secretion of multiple inflammatory cytokines, which leads to splenomegaly caused by an unexpected expansion of T cells. Within this cytokine response, increased IL-6 production results from heightened activation of NF-κB, which is suppressed by active LKB1. Secreted IL-6 drives T-cell activation and IL-21 production, promoting T follicular helper (T_FH) cell differentiation and expansion to support a ∽100-fold increase in steady-state GC B cells. Blockade of IL-6 secretion by BKO B cells inhibits IL-21 expression, a known inducer of T_FH-cell differentiation and expansion. Together, these data reveal cell intrinsic and surprising cell extrinsic roles for LKB1 in B cells that control T_FH-cell differentiation and GC formation, and place LKB1 as a central regulator of T-cell-dependent humoral immunity.     

Lateral flow microarrays: a novel platform for rapid nucleic acid detection based on miniaturized lateral flow chromatography

Widely used nucleic acid assays are poorly suited for field deployment where access to laboratory instrumentation is limited or unavailable. The need for field deployable nucleic acid detection demands inexpensive, facile systems without sacrificing information capacity or sensitivity. Here we describe a novel microarray platform capable of rapid, sensitive nucleic acid detection without specialized instrumentation. The approach is based on a miniaturized lateral flow device that makes use of hybridization-mediated target capture. The miniaturization of lateral flow nucleic acid detection provides multiple advantages over traditional lateral flow devices. Ten-microliter sample volumes reduce reagent consumption and yield analyte detection times, excluding sample preparation and amplification, of <120s while providing sub-femtomole sensitivity. Moreover, the use of microarray technology increases the potential information capacity of lateral flow. Coupled with a hybridization-based detection scheme, the lateral flow microarray (LFM) enables sequence-specific detection, opening the door to highly multiplexed implementations for broad-range assays well suited for point-of-care and other field applications. The LFM system is demonstrated using an isothermal amplification strategy for detection of Bacillus anthracis, the etiologic agent of anthrax. RNA from as few as two B. anthracis cells was detected without thermocycling hardware or fluorescence detection systems.     

Extinctions in competitive communities forced by coloured environmental variation

Understanding the relationships between environmental fluctuations, population dynamics and species interactions in natural communities is of vital theoretical and practical importance. This knowledge is essential in assessing extinction risks in communities that are, for example, pressed by changing environmental conditions and increasing exploitation. We developed a model of density dependent population renewal, in a Lotka–Volterra competitive community context, to explore the significance of interspecific interactions, demographic stochasticity, population growth rate and species abundance on extinction risk in populations under various autocorrelation (colour) regimes of environmental forcing. These factors were evaluated in two cases, where either a single species or the whole community was affected by the external forcing. Species' susceptibility to environmental noise with different autocorrelation structure depended markedly on population dynamics, species' position in the abundance hierarchy and how similarly community members responded to external forcing. We also found interactions between demographic stochasticity and environmental noise leading to a reversal in extinction probabilities from under- to overcompensatory dynamics. We compare our results with studies of single species populations and contrast possible mechanisms leading to extinctions. Our findings indicate that abundance rank, the form of population dynamics, and the colour of environmental variation interact in affecting species extinction risk. These interactions are further modified by interspecific interactions within competitive communities as the interactions filter and modulate the environmental noise.     

Semen-derived amyloid fibrils drastically enhance HIV infection

Sexual intercourse is the major route of HIV transmission. To identify endogenous factors that affect the efficiency of sexual viral transmission, we screened a complex peptide/protein library derived from human semen. We show that naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase (PAP) form amyloid fibrils. These fibrils, termed Semen-derived Enhancer of Virus Infection (SEVI), capture HIV virions and promote their attachment to target cells, thereby enhancing the infectious virus titer by several orders of magnitude. Physiological concentrations of SEVI amplified HIV infection of T cells, macrophages, ex vivo human tonsillar tissues, and transgenic rats in vivo, as well as trans-HIV infection of T cells by dendritic or epithelial cells. Amyloidogenic PAP fragments are abundant in seminal fluid and boost semen-mediated enhancement of HIV infection. Thus, they may play an important role in sexual transmission of HIV and could represent new targets for its prevention.