Peter Kjäll

Role of the Lipopolysaccharide-CD14 Complex for the Activity of Hemolysin from Uropathogenic Escherichia coli

ABSTRACT Bacterial pathogens produce a variety of exotoxins, which often become associated with the bacterial outer membrane component lipopolysaccharide (LPS) during their secretion. LPS is a potent proinflammatory mediator; however, it is not known whether LPS contributes to cell signaling induced by those microbial components to which it is attached. This is partly due to the common view that LPS present in bacterial component preparations is an experimental artifact. The Escherichia coli exotoxin hemolysin (Hly) is a known inducer of proinflammatory signaling in epithelial cells, and the signal transduction pathway involves fluctuation of the intracellular-Ca2+ concentration. Since LPS is known to interact with Hly, we investigated whether it is required as a cofactor for the activity of Hly. We found that the LPS/Hly complex exploits the CD14/LPS-binding protein recognition system to bring Hly to the cell membrane, where intracellular-Ca2+ signaling is initiated via specific activation of the small GTPase RhoA. Hly-induced Ca2+ signaling was found to occur independently of the LPS receptor TLR4, suggesting that the role of LPS/CD14 is to deliver Hly to the cell membrane. In contrast, the cytolytic effect triggered by exposure of cells to high Hly concentrations occurs independently of LPS/CD14. Collectively, our data reveal a novel molecular mechanism for toxin delivery in bacterial pathogenesis, where LPS-associated microbial compounds are targeted to the host cell membrane as a consequence of their association with LPS.

Biomimetic Interfaces Reveal Activation Dynamics of C-Reactive Protein in Local Microenvironments

CRP is an acute-phase reactant secreted by hepatocytes in the liver upon stimulation with endogenous proinfl ammatory cytokines and reaches a level, 100–1000-fold compared to normal conditions (0.8 mg L −1 ) in blood. [ 1 ] CRP levels are clinically measured to monitor the severity of infection, infl ammatory conditions, and cardiovascular diseases. Circulating CRP specifi cally binds to phospholipids as well as immunoglobulins in damaged tissue, [ 2 ] which enhances innate immune responses by recruiting complement components. Generally, CRP forms a pentraxin (115 kDa), a non-covalent symmetrical arrangement of fi ve identical subunits (23 kDa). [ 3 ] Each subunit possesses a PC-binding site coordinated by two calcium ions. The binding of CRP to PCs as a molecular interaction mechanism for activating CRP at the site of infection is intriguing. Classically, two Ca 2+ are believed to have a common equilibrium dissociation constant ( K D = 0.06 × 10 −3 M ) to the PC-binding pocket in CRP. [ 4 ] Recent advances revealed an intricate profi le of the CRP–Ca 2+ affi nities in which each Ca 2+ is taken up in a stepwise manner ( K D,high = 0.03 × 10 −3 M , K D,low = 5.45 × 10 −3 M ). [ 5 ]

Bacterial Nanoscale Cultures for Phenotypic Multiplexed Antibiotic Susceptibility Testing

ABSTRACT An optimal antimicrobial drug regimen is the key to successful clinical outcomes of bacterial infections. To direct the choice of antibiotic, access to fast and precise antibiotic susceptibility profiling of the infecting bacteria is critical. We have developed a high-throughput nanowell antibiotic susceptibility testing (AST) device for direct, multiplexed analysis. By processing in real time the optical recordings of nanoscale cultures of reference and clinical uropathogenic Escherichia coli strains with a mathematical algorithm, the time point when growth shifts from lag phase to early logarithmic phase (T lag) was identified for each of the several hundreds of cultures tested. Based on T lag, the MIC could be defined within 4 h. Heatmap presentation of data from this high-throughput analysis allowed multiple resistance patterns to be differentiated at a glance. With a possibility to enhance multiplexing capacity, this device serves as a high-throughput diagnostic tool that rapidly aids clinicians in prescribing the optimal antibiotic therapy.

Special issue on “Nanotechnologies: Emerging applications in biomedicine”

Nano, a word originating from the Greek word for dwarf, is a prefix that has attracted major attention over the last decade. At the engineering faculties, scientists have for long been engaged in research related to nanotechnology. This area is defined by the ability to create and control objects on the 1–100 nm scale, with the goal to develop novel materials with specific properties and functions. Compared to the size of eukaryotic cells, nanometer-sized objects are several orders of magnitudes smaller. This has generated great expectations, fromwithin the research community as well as from the society, in the novel area termed nanomedicine. This area is defined as the discipline where nanotechnological advances becomes integrated into medical applications. Whereas nanomedicine has the potential to shape, direct, and change future medical treatments in a revolutionary manner, the complexity of the human as an organism certainlymakes nanomedicine a challenging area to engage in. Yet, this area is already showing great impact on biomedical research, primarily because novel experimental platforms are created for in vitro studies. As these platforms allow novel designs of the bio-experiments, questions that previously were impossible to solve due to lack of technologies can be addressed. The scope of this special issue on “Nanotechnologies: Emerging Applications in Biomedicine” is to make biomedical researchers aware of the plethora of technologies available, which may allow each researcher to address a problem they previously believed was impossible to approach. By putting the spotlight on a number of new technologies and demonstrating how they are used to solve biomedical problems (primarily in vitro), we hope that this comprehensive issue, containing reviews that reflect the current state-of-the-art in nanomedical research, can act as a source of inspiration. We start out by demonstrating novel advancements in the field of functionalized surfaces and scaffolds that are used for cell signaling studies. The first five contributions focus on means to modulate cellular behavior, i.e. the use of novel materials and methods to steer cell growth, cell-surface interactions, and cell communication. Robert Langer and Ali Khademhosseini, pioneers in the field, begin with a comprehensive review on microfabrication technologies used to generate patterned cell co-cultures. These are used to make in vitro bio-mimics of tissue and to achieve precise control of homotypic and heterotypic cell-to-cell contact. A possibility to generate 2D and 3D scaffolds for cell cultivation is demonstrated by Masaru Tanaka et al. They show how honeycomb patterned surfaces, created by selforganization, are used to modulate cell behavior. Cell mediated assembly is a pioneering technology developed by Jeffery Brinkers group. They take advantage of the intrinsic ability of cells to organize lipid membrane nanostructures. These form coherent interface with silica mesophases, thus resulting in single, immobilized cells that act as 3D stand-alone sensors. Interesting perspectives are given as this technology opens for a possibility to pattern huge cellular arrays

Organic bioelectronics — Novel applications in biomedicine

The novel field of organic bioelectronics has grown tremendously over the last few years and it is very exciting today to see how the advantages and characteristics of this technology have paved the way for novel applications in medicine. In this special issue of BBA we discover the wide palette of biomedical applications of this technology, ranging from biosensing to biotransistor integration for diagnostics, frommolecular and electrical cell signaling in neurobiology to scaffolds and surface interfaces for regenerativemedicine to namea few. The particular characteristics and advantages of organic bioelectronics with respect to traditional bioelectronics will be showcased in the articles of this special issue. In contrast to previous special issues dedicated to this field, this issue has been guest edited by representatives of the two main fields represented by this interdisciplinary topic; biology and materials science. In this way we hope to promote this topic within the community of life scientists, by demonstrating its truly innovative nature and by illustrating with examples, the added benefit of this technology over other potential technologies for certain key biological applications. We kick off this special issue with a perspective from one of the founders of the field of organic bioelectronics, George Malliaras [1]. Malliaras introduces the history of the field and describes some of the unique characteristics of organic electronic materials and devices and their suitability for life science applications. Organic electronic materials have been shown to be unique in their ability to interface with biology. However, one particular need is the development of organic electronic materials that can be easily biofunctionalized. In this issue Martin and co-workers [2] describe the synthesis of readily biofunctionalizable conducting polymers. This opens up the way to many biomedical applications where direct incorporation of the biological component on the conducting polymer is required. The Polythiophene PEDOT has emerged as a material of choice in organic bioelectronics, both due to its relatively high conductivity and stability compared to other materials of its class. PEDOT is typically doped with either PSS or TOS. PEDOT:PSS is commercially available as a solution and thus lends itself easily to photolithography and inkjet printing (as discussed below). PEDOT:TOS however, although not solution processable, is easier to manipulate, and biological molecules can be added as dopant via electropolymerization or in a composite. This is an important consideration, as, to be truly useful for future applications in medicine, biocompatibility should be clearly demonstrated. In this issue, Karagiozaki and co-workers [3] prepared films of PEDOT: PSS and PEDOT:TOS and tested their biocompatibility with fibroblasts to address these concerns. When considering biocompatibility issues and future use of conducting polymers in biomedical applications such as tissue engineering, a clear picture of the interface formed between the biological component and the conducting polymer is required. In many of the organic electronic devices described in this special issue, the conducting polymers are in direct contact with the biological moiety. Mammalian cells

Imaging Techniques for the Study of Escherichia coli and Salmonella Infections

Infectious diseases are among the leading causes of mortality worldwide, and numerous bacterial species are included in the vast array of causative agents. This review describes microscopy-based techniques that can be used to study interactions between bacteria and infected host cells, bacterial gene expression in the infected animal, and bacteria-induced cell signaling in eukaryotic cells. As infectious model systems, urinary tract infections caused by uropathogenic Escherichia coli (UPEC) and a mouse model of typhoid fever caused by Salmonella enterica serovar Typhimurium are used. To study the interaction mechanism between bacteria and eukaryotic cells, one commonly uses cell lines, primary cells, and animal models. Within the host, bacteria can be located in various organs where they are exposed to different cell types, ranging from epithelial cells at the mucosal linings to phagocytic white blood cells. In each site, bacteria are exposed to specific sets of innate immune defense mechanisms, and to survive these threats, bacteria must rapidly adapt their gene expression profile to maximize their chance of survival in any situation. The rapid development of fluorescent reporter proteins and advances in microscopy-based techniques have provided new and promising approaches not only to locate bacteria in tissues, but also to analyze expression of virulence factors in individual bacteria and host cells during the progression of disease. These techniques enable, for the first time, studies of the complex microenvironments within infected organs and will reveal the alterations of bacterial physiology that occur during bacterial growth within a host.