A computational model of gene expression reveals early transcriptional events at the subtelomeric regions of the malaria parasite, Plasmodium falciparum

A mathematical model of the intraerythrocytic developmental cycle identifies a delay between subtelomeric and central chromosomal gene activities in the malaria parasite, Plasmodium falciparum

Bacteriocyte dynamics during development of a holometabolous insect, the carpenter ant Camponotus floridanus

<p>Abstract</p> <p>Background</p> <p>The carpenter ant <it>Camponotus floridanus </it>harbors obligate intracellular mutualistic bacteria (<it>Blochmannia floridanus</it>) in specialized cells, the bacteriocytes, intercalated in their midgut tissue. The diffuse distribution of bacteriocytes over the midgut tissue is in contrast to many other insects carrying endosymbionts in specialized tissues which are often connected to the midgut but form a distinct organ, the bacteriome. <it>C. floridanus </it>is a holometabolous insect which undergoes a complete metamorphosis. During pupal stages a complete restructuring of the inner organs including the digestive tract takes place. So far, nothing was known about maintenance of endosymbionts during this life stage of a holometabolous insect. It was shown previously that the number of <it>Blochmannia </it>increases strongly during metamorphosis. This implicates an important function of <it>Blochmannia </it>in this developmental phase during which the animals are metabolically very active but do not have access to external food resources. Previous experiments have shown a nutritional contribution of the bacteria to host metabolism by production of essential amino acids and urease-mediated nitrogen recycling. In adult hosts the symbiosis appears to degenerate with increasing age of the animals.</p> <p>Results</p> <p>We investigated the distribution and dynamics of endosymbiotic bacteria and bacteriocytes at different stages during development of the animals from larva to imago by confocal laser scanning microscopy. The number of bacteriocytes in relation to symbiont-free midgut cells varied strongly over different developmental stages. Especially during metamorphosis the relative number of bacteria-filled bacteriocytes increased strongly when the larval midgut epithelium is shed. During this developmental stage the midgut itself became a huge symbiotic organ consisting almost exclusively of cells harboring bacteria. In fact, during this phase some bacteria were also found in midgut cells other than bacteriocytes indicating a cell-invasive capacity of <it>Blochmannia</it>. In adult animals the number of bacteriocytes generally decreased.</p> <p>Conclusions</p> <p>During the life cycle of the animals the distribution of bacteriocytes and of <it>Blochmannia </it>endosymbionts is remarkably dynamic. Our data show how the endosymbiont is retained within the midgut tissue during metamorphosis thereby ensuring the maintenance of the intracellular endosymbiosis despite a massive reorganization of the midgut tissue. The transformation of the entire midgut into a symbiotic organ during pupal stages underscores the important role of <it>Blochmannia </it>for its host in particular during metamorphosis.</p

A three-phase in-vitro system for studying Pseudomonas aeruginosa adhesion and biofilm formation upon hydrogel contact lenses

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas aeruginosa </it>is commonly associated with contact lens (CL) -related eye infections, for which bacterial adhesion and biofilm formation upon hydrogel CLs is a specific risk factor. Whilst <it>P. aeruginosa </it>has been widely used as a model organism for initial biofilm formation on CLs, <it>in-vitro </it>models that closely reproduce <it>in-vivo </it>conditions have rarely been presented.</p> <p>Results</p> <p>In the current investigation, a novel <it>in-vitro </it>biofilm model for studying the adherence of <it>P. aeruginosa </it>to hydrogel CLs was established. Nutritional and interfacial conditions similar to those in the eye of a CL wearer were created through the involvement of a solid:liquid and a solid:air interface, shear forces and a complex artificial tear fluid. Bioburdens varied depending on the CL material and biofilm maturation occurred after 72 h incubation. Whilst a range of biofilm morphologies were visualised including dispersed and adherent bacterial cells, aggregates and colonies embedded in extracellular polymer substances (EPS), EPS fibres, mushroom-like formations, and crystalline structures, a compact and heterogeneous biofilm morphology predominated on all CL materials.</p> <p>Conclusions</p> <p>In order to better understand the process of biofilm formation on CLs and to test the efficacy of CL care solutions, representative <it>in-vitro </it>biofilm models are required. Here, we present a three-phase biofilm model that simulates the environment in the eye of a CL wearer and thus generates biofilms which resemble those commonly observed <it>in-situ</it>.</p

Allosteric Regulation of Fibronectin/α5β1 Interaction by Fibronectin-Binding MSCRAMMs

Citation: Liang, X. W., Garcia, B. L., Visai, L., Prabhakaran, S., Meenan, N. A. G., Potts, J. R., . . . Hook, M. (2016). Allosteric Regulation of Fibronectin/alpha(5)beta(1) Interaction by Fibronectin-Binding MSCRAMMs. Plos One, 11(7), 17. doi:10.1371/journal.pone.0159118Adherence ofmicrobes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with alpha(5)beta(1) integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/alpha(5)beta(1) integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/alpha(5)beta(1) integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/alpha(5)beta(1) on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic alpha(5)beta(1) interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/alpha(5)beta(1) affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs

Staphylococcus aureus α-Toxin Induces Inflammatory Cytokines via Lysosomal Acid Sphingomyelinase and Ceramides

Background/Aims: Staphylococcus aureus (S. aureus) infections are a major clinical problem and range from mild skin and soft-tissue infections to severe and even lethal infections such as pneumonia, endocarditis, sepsis, osteomyelitis, and toxic shock syndrome. Toxins that are released from S. aureus mediate many of these effects. Here, we aimed to identify molecular mechanisms how α-toxin, a major S. aureus toxin, induces inflammation. Methods: Macrophages were isolated from the bone marrow of wildtype and acid sphingomyelinase-deficient mice, stimulated with S. aureus α-toxin and activation of the acid sphingomyelinase was quantified. The subcellular formation of ceramides was determined by confocal microscopy. Release of cathepsins from lysosomes, activation of inflammasome proteins and formation of Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) were analyzed by western blotting, confocal microscopy and ELISA. Results: We demonstrate that S. aureus α-toxin activates the acid sphingomyelinase in ex vivo macrophages and triggers a release of ceramides. Ceramides induced by S. aureus α-toxin localize to lysosomes and mediate a release of cathepsin B and D from lysosomes into the cytoplasm. Cytosolic cathepsin B forms a complex with Nlrc4. Treatment of macrophages with α-toxin induces the formation of IL-1β and TNF-α. These events are reduced or abrogated, respectively, in cells lacking the acid sphingomyelinase and upon treatment of macrophages with amitriptyline, a functional inhibitor of acid sphingomyelinase. Pharmacological inhibition of cathepsin B prevented activation of the inflammasome measured as release of IL-1β, while the formation of TNF-α was independent of cathepsin B. Conclusion: We demonstrate a novel mechanism how bacterial toxins activate the inflammasome and mediate the formation and release of cytokines: S. aureus α-toxin triggers an activation of the acid sphingomyelinase and a release of ceramides resulting in the release of lysosomal cathepsin B and formation of pro-inflammatory cytokines

Intracellular Staphylococcus aureus Perturbs the Host Cell Ca2+^{2+} Homeostasis To Promote Cell Death

The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca$^{2+}$ increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca$^{2+}$ concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca$^{2+}$ rise led to an increase in mitochondrial Ca$^{2+}$ concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca$^{2+}$ homeostasis and induces cytoplasmic Ca$^{2+}$ overload, which results in both apoptotic and necrotic cell death in parallel or succession. IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca$^{2+}$ overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca$^{2+}$ homeostasis.