Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development.

By combining X-ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.

Gradient acoustic focusing of sub-micron particles for separation of bacteria from blood lysate.

Handling of submicron-sized objects is important in many biochemical and biomedical applications, but few methods today can precisely manipulate this range of particles. We present gradient acoustic focusing that enables flow-through particle separation of submicron particles and cells and we apply it for separation of bacteria from blood lysate to facilitate their detection in whole blood for improved diagnostics. To control suspended objects below the classical 2µm size limit for acoustic focusing, we introduce a co-flowing acoustic impedance gradient to generate a stabilizing acoustic volume force that supresses acoustic streaming. The method is validated theoretically and experimentally using polystyrene particles, Staphylococcus aureus, Streptococcus pneumoniae and Escherichia coli. The applicability of the method is demonstrated by the separation of bacteria from selectively chemically lysed blood. Combined with downstream operations, this new approach opens up for novel methods for sepsis diagnostics.

Insights into the antibacterial mechanism of PEGylated nano-bacitracin A against Streptococcus pneumonia: both penicillin-sensitive and penicillin-resistant strains.

BACKGROUND: Multidrug-resistant (MDR) Streptococcus pneumonia constitute a major worldwide public health concern. MATERIALS AND METHODS: In our preliminary study, PEGylated nano-self-assemblies of bacitracin A (PEGylated Nano-BA12K) showed strong antibacterial potency against reference S. pneumonia strain (ATCC 49619). In this study, the possibility of applying PEGylated Nano-BA12K against penicillin-resistant S. pneumonia was further investigated. In addition, the underlying antibacterial mechanism of PEGylated Nano-BA12K against both sensitive and resistant S. pneumonia was also clarified systematically, since S. pneumonia was naturally resistant to its unassembled counterpart bacitracin A (BA). RESULTS: PEGylated Nano-BA12K showed strong antibacterial potency against 13 clinical isolates of S. pneumonia, including five penicillin-resistant strains. Structural changes, partial collapse, and even lysis of both penicillin-sensitive and penicillin-resistant bacteria were observed after incubation with PEGylated Nano-BA12K via transmission electron microscopy and atomic force microscopy. Thus, the cell wall or/and cell membrane might be the main target of PEGylated Nano-BA12K against S. pneumonia. PEGylated Nano-BA12K exhibited limited effect on the permeabilization and peptidoglycan content of cell wall. Surface pressure measurement suggested that PEGylated Nano-BA12K was much more tensioactive than BA, which was usually translated into a good membranolytic effect, and is helpful to permeabilize the cell membrane and damage membrane integrity, as evidenced by depolarization of the membrane potential, permeabilization of membrane and leakage of calcein from liposomes. CONCLUSION: Collectively, great cell membrane permeability and formidable membrane disruption may work together for the strong antibacterial activity of PEGylated Nano-BA12K against S. pneumonia. Taken together, PEGylated Nano-BA12K has excellent potential against both penicillin-sensitive and penicillin-resistant S. pneumonia and might be suitable for the treatment of S. pneumonia infectious diseases.

Cell division in cocci: localization and properties of the Streptococcus pneumoniae FtsA protein.

We studied the cytological and biochemical properties of the FtsA protein of Streptococcus pneumoniae. FtsA is a widespread bacterial cell division protein that belongs to the actin superfamily. In Escherichia coli and Bacillus subtilis, FtsA localizes to the septal ring after FtsZ, but its exact role in septation is not known. In S. pneumoniae, we found that, during exponential growth, the protein localizes to the nascent septa, at the equatorial zones of the dividing cells, where an average of 2200 FtsA molecules per cell are present. Likewise, FtsZ was found to localize with the same pattern and to be present at an average of 3000 molecules per cell. Consistent with the colocalization, FtsA was found to interact with FtsZ and with itself. Purified FtsA is able to bind several nucleotides, the affinity being highest for adenosine triphosphate (ATP), and lower for other triphosphates and diphosphates. The protein polymerizes in vitro, in a nucleotide-dependent manner, forming long corkscrew-like helixes, composed of 2 + 2 paired protofilaments. No nucleotide hydrolytic activity was detected. Consistent with the absence of an ATPase activity, the polymers are highly stable and not dynamic. These results suggest that the FtsA protein could also polymerize in vivo and the polymers participate in septation.

Critical-point drying: rapid method for the determination of bacterial extracellular polymer and surface structures.

The relative amount of extracellular polymer which remains about Azotobacter vinelandii, Zoogloea ramigera, Klebsiella pneumoniae, and Diplococcus pneumoniae after critical-point drying was studied by electron microscopy. The results obtained with this technique are compared to those obtained with methods that illustrate extracellular polymer, such as freeze-etching and ruthenium red staining. Comparative results indicate critical-point drying to be a rapid, reliable method for the determination of capsule-like polymer surrounding bacterial cells. In addition, critical-point drying can be used to observe morphogenetic changes, such as vesicle production.