Novel rat model of methicillin-resistant Staphylococcus aureus-infected silicone breast implants: a study of biofilm pathogenesis.

BACKGROUND: Clinical infection of breast implants occurs in 7 to 24 percent of breast reconstructions. It may persist over time in the form of biofilm without overt manifestation and is extremely difficult to eradicate. The authors' aim was to establish a novel model for biofilm infection of silicone breast implants in rats. METHODS: Fifty-six rats underwent implantation of miniature silicone breast implants and/or methicillin-resistant Staphylococcus aureus (MRSA) bacteria. Group A received implants covered with MRSA biofilm. Group B received implants and free planktonic MRSA. Group C received free planktonic MRSA without implants. A control group received sterile implants without MRSA. Each group was divided to receive either saline or vancomycin injections between days 4 and 11. Clinical evaluation, bacterial counts, and scanning electron microscopy were performed. RESULTS: The mortality rate in group B (implants infected with free planktonic MRSA) was significantly higher than that in all other groups [37 percent versus groups A and D (0 percent) and group C (7 percent)]. Treatment with vancomycin lowered temperature in groups B and C (p < 0.05) and improved wound healing in group B (p < 0.01). Vancomycin treatment reduced wound bacterial counts in free planktonic MRSA groups B and C but had no significant effect on biofilm MRSA-infected group A. CONCLUSIONS: The model successfully induced persistent breast implant infection. Free planktonic MRSA produced in situ biofilm on silicone implants. Biofilm infection has milder manifestations than free planktonic MRSA infection, which has higher rates of systemic infections and death when compared with either isolated biofilm infection or free planktonic MRSA infection without implant. Vancomycin has limited effect against mature biofilm.

Structural analysis of a metazoan nuclear pore complex reveals a fused concentric ring architecture.

The sole gateway for molecular exchange between the cytoplasm and the nucleus is the nuclear pore complex (NPC). This large supramolecular assembly mediates transport of cargo into and out of the nucleus and fuse the inner and outer nuclear membranes to form an aqueous translocation channel. The NPC is composed of eight proteinaceous asymmetric units forming a pseudo-8-fold symmetric passage. Due to its shear size, complexity, and plastic nature, dissecting the high-resolution three-dimensional structure of the NPC in its hydrated state is a formidable challenge. Toward this goal, we applied cryo-electron tomography to spread nuclear envelopes from Xenopus oocytes. To compensate for perturbations of the 8-fold symmetry of individual NPCs, we performed symmetry-independent asymmetric unit averaging of three-dimensional tomographic NPC volumes to eventually yield a refined model at 6.4 nm resolution. This approach revealed novel structural features, particularly in the spoke-ring complex and luminal domains. Fused concentric ring architecture of the spoke-ring complex was found along the translocation channel. Additionally, a comparison of the refined Xenopus model to that of its Dictyostelium homologue yielded similar pore diameters at the level of the three canonical rings, although the Xenopus NPC was found to be 30% taller than the Dictyostelium pore. This discrepancy is attributed primarily to the relatively low homology and different organization of some nucleoporins in the Dictyostelium genome as compared to that of vertebrates. Nevertheless, the experimental conditions impose a preferred axial orientation of the NPCs within spread Xenopus oocyte nuclear envelopes. This may at least in part explain the increased height of the reconstructed vertebrate NPCs compared to those obtained from tomographic reconstruction of intact Dictyostelium nuclei.

Structural analysis of the nuclear pore complex by integrated approaches.

In eukaryotic cells, the nucleus is surrounded by a double membrane system, the nuclear envelope (NE), in which the outer membrane is continuous with the endoplasmic reticulum (ER). Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes to form aqueous translocation channels that allow the free diffusion of small molecules and ions, as well as receptor-mediated transport of large macromolecules. Being the sole gateways for import and export to and from the nucleus, NPCs regulate the nucleocytoplasmic transport of macromolecules in a highly selective manner to maintain cellular functions. The large size and complexity of these multimolecular assemblies, which are composed of approximately 30 different proteins (termed nucleoporins), present a major challenge for structural biologists. Here, we discuss the latest structural findings related to the functional organization of the NPC.

The supramolecular organization of the C. elegans nuclear lamin filament.

Nuclear lamins are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. They are nuclear intermediate filament (IF) proteins forming a distinct meshwork-like layer adhering to the inner nuclear membrane, called the nuclear lamina. Here, we present for the first time, the three-dimensional supramolecular organization of lamin 10 nm filaments and paracrystalline fibres. We show that Caenorhabditis elegans nuclear lamin forms 10 nm IF-like filaments, which are distinct from their cytoplasmic counterparts. The IF-like lamin filaments are composed of three and four tetrameric protofilaments, each of which contains two partially staggered anti-parallel head-to-tail polymers. The beaded appearance of the lamin filaments stems from paired globular tail domains, which are spaced regularly, alternating between 21 nm and 27 nm. A mutation in an evolutionarily conserved residue that causes Hutchison-Gilford progeria syndrome in humans alters the supramolecular structure of the lamin filaments. On the basis of our structural analysis, we propose an assembly pathway that yields the observed 10 nm IF-like lamin filaments and paracrystalline fibres. These results serve also as a platform for understanding the effect of laminopathic mutations on lamin supramolecular organization.

Crystal structure of the Agrobacterium virulence complex VirE1-VirE2 reveals a flexible protein that can accommodate different partners.

Agrobacterium tumefaciens infects its plant hosts by a mechanism of horizontal gene transfer. This capability has led to its widespread use in artificial genetic transformation. In addition to DNA, the bacterium delivers an abundant ssDNA binding protein, VirE2, whose roles in the host include protection from cytoplasmic nucleases and adaptation for nuclear import. In Agrobacterium, VirE2 is bound to its acidic chaperone VirE1. When expressed in vitro in the absence of VirE1, VirE2 is prone to oligomerization and forms disordered filamentous aggregates. These filaments adopt an ordered solenoidal form in the presence of ssDNA, which was characterized previously by electron microscopy and three-dimensional image processing. VirE2 coexpressed in vitro with VirE1 forms a soluble heterodimer. VirE1 thus prevents VirE2 oligomerization and competes with its binding to ssDNA. We present here a crystal structure of VirE2 in complex with VirE1, showing that VirE2 is composed of two independent domains presenting a novel fold, joined by a flexible linker. Electrostatic interactions with VirE1 cement the two domains of VirE2 into a locked form. Comparison with the electron microscopy structure indicates that the VirE2 domains adopt different relative orientations. We suggest that the flexible linker between the domains enables VirE2 to accommodate its different binding partners.

Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1.

Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.

Nucleoid organization and the maintenance of DNA integrity in E. coli, B. subtilis and D. radiodurans.

For enzymatic activities to be effectively carried out, basic prerequisites must be met. Many enzymatic tasks require continuous consumption and dissipation of energy, sometimes in massive amounts. Some activities, such as DNA replication, transcription, and repair through homologous recombination rely upon templates that provide the information required for these transactions. Yet, circumstances where intracellular energy pools are severely depleted, or where intact templates are not available, frequently occur. Moreover, the fact that in order to reach their targets, enzymes must cope with an extremely crowded and viscous cellular milieu that drastically slows down their diffusion is often neglected. These impediments are particularly evident under stress conditions such as prolonged starvation or continuous exposure to DNA-damaging agents. Here we survey recent studies, which imply that when enzymatically-mediated DNA repair pathways are hindered, alternative strategies are deployed, whose common denominator is the reorganization of bacterial nucleoids into morphologies that promote DNA repair and protection.

Structure of the DNA-SspC complex: implications for DNA packaging, protection, and repair in bacterial spores.

Bacterial spores have long been recognized as the sturdiest known life forms on earth, revealing extraordinary resistance to a broad range of environmental assaults. A family of highly conserved spore-specific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP), plays a major role in mediating spore resistance. The mechanism by which these proteins exert their protective activity remains poorly understood, in part due to the lack of structural data on the DNA-SASP complex. By using cryoelectron microscopy, we have determined the structure of the helical complex formed between DNA and SspC, a characteristic member of the alpha/beta-type SASP family. The protein is found to fully coat the DNA, forming distinct protruding domains, and to modify DNA structure such that it adopts a 3.2-nm pitch. The protruding SspC motifs allow for interdigitation of adjacent DNA-SspC filaments into a tightly packed assembly of nucleoprotein helices. By effectively sequestering DNA molecules, this dense assembly of filaments is proposed to enhance and complement DNA protection obtained by DNA saturation with the alpha/beta-type SASP.

Three-dimensional reconstruction of Agrobacterium VirE2 protein with single-stranded DNA.

Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.

Nucleoid restructuring in stationary-state bacteria.

The textbook view of the bacterial cytoplasm as an unstructured environment has been overturned recently by studies that highlighted the extent to which non-random organization and coherent motion of intracellular components are central for bacterial life-sustaining activities. Because such a dynamic order critically depends on continuous consumption of energy, it cannot be perpetuated in starved, and hence energy-depleted, stationary-state bacteria. Here, we show that, at the onset of the stationary state, bacterial chromatin undergoes a massive reorganization into ordered toroidal structures through a process that is dictated by the intrinsic properties of DNA and by the ubiquitous starvation-induced DNA-binding protein Dps. As starvation proceeds, the toroidal morphology acts as a structural template that promotes the formation of DNA-Dps crystalline assemblies through epitaxial growth. Within the resulting condensed assemblies, DNA is effectively protected by means of structural sequestration. We thus conclude that the transition from bacterial active growth to stationary phase entails a co-ordinated process, in which the energy-dependent dynamic order of the chromatin is sequentially substituted with an equilibrium crystalline order.

Stress, order and survival.

Much of the sophisticated chemistry of life is accomplished by multicomponent complexes, which act as molecular machines. Intrinsic to their accuracy and efficiency is the energy that is supplied by hydrolysis of nucleoside triphosphates. Conditions that deplete energy sources should therefore cause decay and death. But studies on organisms that are exposed to prolonged stress indicate that this fate could be circumvented through the formation of highly ordered intracellular assemblies. In these thermodynamically stable structures, vital components are protected by a physical sequestration that is independent of energy consumption.