Fatty-acid binding protein 5 modulates the SAR1 GTPase cycle and enhances budding of large COPII cargoes.

COPII-coated vesicles are the primary mediators of ER-to-Golgi trafficking. Sar1, one of the five core COPII components, is a highly conserved small GTPase, which, upon GTP binding, recruits the other COPII proteins to the ER membrane. It has been hypothesized that the changes in the kinetics of SAR1 GTPase may allow for the secretion of large cargoes. Here we developed a cell-free assay to recapitulate COPII-dependent budding of large lipoprotein cargoes from the ER. We identified fatty-acid binding protein 5 (FABP5) as an enhancer of this budding process. We found that FABP5 promotes the budding of particles ∼150 nm in diameter and modulates the kinetics of the SAR1 GTPase cycle. We further found that FABP5 enhances the trafficking of lipoproteins and of other cargoes, including collagen. These data identify a novel regulator of SAR1 GTPase activity and highlight the importance of this activity for trafficking of large cargoes.

COPII-coated membranes function as transport carriers of intracellular procollagen I.

The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.

Regulation of the CUL3 Ubiquitin Ligase by a Calcium-Dependent Co-adaptor.

The ubiquitin ligase CUL3 is an essential regulator of neural crest specification whose aberrant activation has been linked to autism, schizophrenia, and hypertension. CUL3 exerts its roles by pairing with ∼90 distinct substrate adaptors, yet how the different CUL3-complexes are activated is poorly understood. Here, we show that CUL3 and its adaptor KLHL12 require two calcium-binding proteins, PEF1 and ALG2, for recognition of their substrate SEC31. PEF1 and ALG2 form a target-specific co-adaptor that translates a transient rise in cytosolic calcium levels into more persistent SEC31 ubiquitylation, which in turn triggers formation of large COPII coats and promotes collagen secretion. As calcium also instructs chondrocyte differentiation and collagen synthesis, calcium-dependent control of CUL3KLHL12 integrates collagen secretion into broader programs of craniofacial bone formation. Our work, therefore, identifies both calcium and CUL3 co-adaptors as important regulators of ubiquitylation events that control human development.

The lethal cargo of Myxococcus xanthus outer membrane vesicles.

Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs) are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including the protein encoded by MXAN_3564 (mepA), an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is part of the long-predicted (yet to date undetermined) extracellular protease suite of M. xanthus.

Death and transfiguration in static Staphylococcus epidermidis cultures.

The overwhelming majority of bacteria live in slime embedded microbial communities termed biofilms, which are typically adherent to a surface. However, when several Staphylococcus epidermidis strains were cultivated in static liquid cultures, macroscopic aggregates were seen floating within the broth and also sedimented at the test tube bottom. Light- and electron microscopy revealed that early-stage aggregates consisted of bacteria and extracellular matrix, organized in sheet-like structures. Perpendicular under the sheets hung a network of periodically arranged, bacteria-associated strands. During the extended cultivation, the strands of a subpopulation of aggregates developed into cross-connected wall-like structures, in which aligned bacteria formed the walls. The resulting architecture had a compartmentalized appearance. In late-stage cultures, the wall-associated bacteria disintegrated so that, henceforth, the walls were made of the coalescing remnants of lysed bacteria, while the compartment-like organization remained intact. At the same time, the majority of strand-containing aggregates with associated culturable bacteria continued to exist. These observations indicate that some strains of Staphylococcus epidermidis are able to build highly sophisticated structures, in which a subpopulation undergoes cell lysis, presumably to provide continued access to nutrients in a nutrient-limited environment, whilst maintaining structural integrity.

Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains.

The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 µm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities.

Electrically conductive bacterial nanowires in bisphosphonate-related osteonecrosis of the jaw biofilms.

OBJECTIVE: Bacterial biofilms play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The purpose of this preliminary study was to test the hypothesis that the extracellular filaments observed in biofilms associated with BRONJ contain electrically conductive nanowires. STUDY DESIGN: Bone samples of patients affected by BRONJ were evaluated for conductive nanowires by scanning electron microscopy (SEM) and conductive probe atomic force microscopy (CP-AFM). We created nanofabricated electrodes to measure electrical transport along putative nanowires. RESULTS: SEM revealed large-scale multispecies biofilms containing numerous filamentous structures throughout necrotic bone. CP-AFM analysis revealed that these structures were electrically conductive nanowires with resistivities on the order of 20 Ω·cm. Nanofabricated electrodes spaced along the nanowires confirmed their ability to transfer electrons over micron-scale lengths. CONCLUSIONS: Electrically conductive bacterial nanowires to date have been described only in environmental isolates. This study shows for the first time that these nanowires can also be found in clinically relevant biofilm-mediated diseases, such as BRONJ, and may represent an important target for therapy.

Quantification by energy dispersive x-ray spectroscopy of alendronate in the diseased jaw bone of patients with bisphosphonate-related jaw osteonecrosis.

OBJECTIVE: Recently, specific in vitro bisphosphonate concentrations have been established for reaching a toxic threshold that could result in the induction of bisphosphonate-related osteonecrosis of the jaw (BRONJ), but these data have not been validated in vivo. The purpose of this study was to quantify the concentration of bisphosphonates (BPs) in the diseased jaw bone of patients experiencing BRONJ. STUDY DESIGN: We hypothesized that if the average natural nitrogen content of mammalian bone is known, the excess of nitrogen in the jaw bone of BRONJ patients is likely to reflect the concentration of amino-BP. To test our hypothesis, jaw bone specimens from patients with BRONJ were acquired after sequestrectomy and analyzed by energy-dispersive X-ray spectroscopy (EDS). RESULTS: The EDS analysis of the bone demonstrated a highly linear correlation between increasing concentrations of BP and the increasing percentage of nitrogen measured at the bone surfaces (R(2) = .9851, P = .0149). CONCLUSIONS: SEM/EDS can be a valuable tool for assessing BP concentration in jaw bone and provides important insight into BP pharmacokinetics and BRONJ.

Ubiquitin-dependent regulation of COPII coat size and function.

Packaging of proteins from the endoplasmic reticulum into COPII vesicles is essential for secretion. In cells, most COPII vesicles are approximately 60-80 nm in diameter, yet some must increase their size to accommodate 300-400 nm procollagen fibres or chylomicrons. Impaired COPII function results in collagen deposition defects, cranio-lenticulo-sutural dysplasia, or chylomicron retention disease, but mechanisms to enlarge COPII coats have remained elusive. Here, we identified the ubiquitin ligase CUL3-KLHL12 as a regulator of COPII coat formation. CUL3-KLHL12 catalyses the monoubiquitylation of the COPII-component SEC31 and drives the assembly of large COPII coats. As a result, ubiquitylation by CUL3-KLHL12 is essential for collagen export, yet less important for the transport of small cargo. We conclude that monoubiquitylation controls the size and function of a vesicle coat.

Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria.

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

Development of an animal model for Aggregatibacter actinomycetemcomitans biofilm-mediated oral osteolytic infection: a preliminary study.

BACKGROUND: Biofilm-induced inflammatory osteolytic oral infections, such as periodontitis and peri-implantitis, have complex etiology and pathogenesis. A significant obstacle to research has been the lack of appropriate animal models where the inflammatory response to biofilms can be investigated. The aim of this study is to develop a novel animal model to study the host response to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans)-biofilm colonizing titanium implants. METHODS: Titanium implants were inoculated in vitro with A. actinomycetemcomitans, establishing a biofilm for 1 to 3 days. Biofilm-inoculated and control implants were transmucosally placed into rat hard palate or alveolar ridge. Analysis included documentation of clinical inflammation, polymerase chain reaction, and culture detection of A. actinomycetemcomitans and microcomputed tomography quantitation of peri-implant bone volume. RESULTS: Viable A. actinomycetemcomitans biofilm was successfully established on titanium implants in vitro, detected by confocal laser scanning microscopy. An inflammatory response characterized by clinical inflammation, bleeding, ulceration, hyperplasia, and necrosis was observed around biofilm-inoculated implants. A. actinomycetemcomitans was detected by polymerase chain reaction and culture analysis on 100% of biofilm-inoculated implants for up to 3 weeks and 25% for up to 6 weeks. Microcomputed tomography analysis demonstrated significantly lower bone volume (P <0.05) around biofilm-inoculated implants (29.6% ± 7.6%) compared to non-inoculated implants (50.5% ± 9.6%) after 6 weeks. CONCLUSIONS: These results describe a novel animal model where A. actinomycetemcomitans biofilm was established in vitro on titanium implants before placement in rat oral cavity, leading to an inflammatory response, osteolysis, and tissue destruction. This model may have potential use for investigation of host responses to biofilm pathogens and antibiofilm therapy.

Demonstration of bacterial biofilms in culture-negative silicone stent and jones tube.

PURPOSE: To demonstrate the presence of bacterial biofilms on a dacryocystorhinostomy silicone stent and a Jones tube. METHODS: One dacryocystorhinostomy silicone stent and one Jones tube were removed from 2 patients who presented with an infection of their respective nasolacrimal system. Cultures were obtained, and the implants were processed for scanning electron microscopy and confocal laser scanning microscopy, advanced microscopic methods that are applicable for detection of uncultivable biofilm organisms. RESULTS: Routine bacterial cultures revealed no growth, but bacterial biofilms on outer and inner surfaces of both implants were confirmed by advanced microscopic techniques. CONCLUSIONS: To the authors' knowledge, this is the first article that documents the presence of biofilms on a Crawford stent or a Jones tube on patients who presented with infections involving the nasolacrimal system. Although initial cultures revealed absence of any bacterial growth, confocal laser scanning microscopy and scanning electron microscopy documented bacterial colonization. Clinicians should consider the role of biofilms and the limitation of our standard culturing techniques while treating patients with device- or implant-related infections.

The role of microbial biofilms in osteonecrosis of the jaw associated with bisphosphonate therapy.

Microbial biofilms have been observed and described in bone specimens of patients with bisphosphonate (BP)-associated osteonecrosis of the jaw (BONJ) and investigators are more recently suggesting that this condition essentially represents an osteomyelitis of the jaw clinically, with greater susceptibility in some patients on BP therapy. This article explains the role of microbial biofilms in BONJ and also discusses associated factors in the disease pathogenesis, which include BP effects on bone remodeling, anti-angiogenesis, matrix necrosis, microcracks, soft tissue toxicity, and inflammation and wound healing. Recent findings suggest a key role for microbial biofilms in the pathogenesis of BONJ; this has important therapeutic implications because biofilm organisms represent a clinical target for prevention and treatment efforts aimed at reducing the significant morbidity and costs associated with this condition.

Microbial biofilms in osteomyelitis of the jaw and osteonecrosis of the jaw secondary to bisphosphonate therapy.

BACKGROUND: The authors report their observations with respect to microbial biofilms in osteomyelitis of the jaw (OMJ), compare these findings with those for osteonecrosis of the jaw (ONJ) secondary to bisphosphonate therapy and discuss recent findings that the pathogenesis of ONJ may represent a biofilm-mediated infectious disease in the context of bisphosphonate therapy. METHODS: In 2004, a program was established at the University of Southern California, Los Angeles, to evaluate, treat and monitor patients who have OMJ and ONJ. Twenty people from this cohort of study patients who were scheduled to undergo surgical debridement or sequestrectomy and who met the authors' inclusion criteria gave informed consent for the study. The authors examined bone samples histopathologically and via scanning electron microscopy, a technique applicable to biofilm characterization. RESULTS: Specimens from all patients with OMJ and ONJ exhibited large surface areas of bone occluded with well-developed biofilms comprising microbial organisms embedded in an extracellular polymeric substance. Actinomyces predominated in OMJ cases, whereas ONJ cases represented more diverse bacterial organisms in addition to fungal organisms not seen in OMJ. The authors observed resorption pits, septic clots, putative nanowires and host inflammatory cells in all specimens. CONCLUSIONS: The findings of this study support a role for microbial biofilms in both disease processes. CLINICAL IMPLICATIONS: Microbial biofilms are a potential target for therapy that includes antibiofilm modalities in the treatment and prevention of OMJ and ONJ.

Ultrastructural examination of failed molar retreatment with secondary apical periodontitis: an examination of endodontic biofilms in an endodontic retreatment failure.

A light and electron microscope examination of the resected root tip of a failing endodontically re-treated lower molar was examined. The tooth had been initially treated 10 years ago and then re-treated 2 years ago. The resected root tip was sectioned axially, and thin sections were examined through the entire length of the specimen. Thin sections were examined with a transmission electron microscope. The thin sections were randomly chosen along the isthmus areas between the mesiobuccal and mesiolingual canals. Our findings suggest that a complex, variable, multispecies biofilm was present the entire length of the specimen.

Periodontitis: an archetypical biofilm disease.

BACKGROUND: Periodontitis is a classic example of biofilm-mediated diseases. METHODS: The authors reviewed selected publications in English-language peer-reviewed journals with respect to microbial biofilms, focusing on representative works that provided a historical to a contemporary perspective on periodontal oral biofilms in the larger context of biofilm microbiology. RESULTS: Developments in advanced microscopy and molecular microbiology have allowed scientists to examine and characterize microbial biofilm-mediated diseases, such as periodontitis, more accurately than in the past. CONCLUSIONS: Periodontitis, like other biofilm infections, is refractory to antibiotic agents and host defenses because the causative microbes live in complex communities that persist despite challenges that range from targeted antibiotic agents to phagocytosis. CLINICAL IMPLICATIONS: The regular delivery of nontargeted antibiofilm agents may be an effective strategy for treating biofilms, especially if these agents include oxidative agents that dissolve the biofilm matrix.

Biofilm removal with a dental water jet.

OBJECTIVE: The objective of this study was to evaluate the effect of a dental water jet on plaque biofilm removal using scanning electron microscopy (SEM). METHODOLOGY: Eight teeth with advanced aggressive periodontal disease were extracted. Ten thin slices were cut from four teeth. Two slices were used as the control. Eight were inoculated with saliva and incubated for 4 days. Four slices were treated using a standard jet tip, and four slices were treated using an orthodontic jet tip. The remaining four teeth were treated with the orthodontic jet tip but were not inoculated with saliva to grow new plaque biofilm. All experimental teeth were treated using a dental water jet for 3 seconds on medium pressure. RESULTS: The standard jet tip removed 99.99% of the salivary (ex vivo) biofilm, and the orthodontic jet tip removed 99.84% of the salivary biofilm. Observation of the remaining four teeth by the naked eye indicated that the orthodontic jet tip removed significant amounts of calcified (in vivo) plaque biofilm. This was confirmed by SEM evaluations. CONCLUSION: The Waterpik dental water jet (Water Pik, Inc, Fort Collins, CO) can remove both ex vivo and in vivo plaque biofilm significantly.

Identification of microbial biofilms in osteonecrosis of the jaws secondary to bisphosphonate therapy.

PURPOSE: Biofilm theory has emerged to explain the etiology of the chronic infections that have come to constitute between 65% to 80% of the microbial diseases treated by physicians in the developed world. The purpose of this article is to report for the first time the observation of multispecies microbial biofilms on affected bone in patients with osteonecrosis of the jaws (ONJ) secondary to bisphosphonate therapy. PATIENTS AND METHODS: A program has been established at the University of Southern California to monitor and evaluate patients with ONJ as a multidisciplinary collaboration between the School of Dentistry, Center for Biofilms, Center for Craniofacial Molecular Biology and the Keck School of Medicine. From this cohort, 4 patients with active ONJ who were scheduled for necessary treatment in the form of sequestrectomy gave informed consent for this study. Bone samples were evaluated using conventional histopathologic techniques and scanning electron microscopy, a technique applicable to biofilm characterization. RESULTS: Bone specimens from affected sites in all patients showed large areas occluded with biofilms comprising mainly bacteria, and occasionally yeast, embedded in extracellular polymeric substance. The number of bacterial morphotypes in the biofilms ranged from 2 to 15, and they included species from the genus Fusobacterium, bacillus, actinomyces, staphylococcus, streptococcus, Selenomonas, and 3 different types of treponemes. The yeast identified was consistent with Candida species. Co-aggregation was observed between different species within the biofilms. CONCLUSION: These findings have important clinical and therapeutic implications and may suggest a role for microbial biofilms in the disease process of ONJ.

Toxic epidermal necrolysis with a rare long-term oral complication requiring surgical intervention.

BACKGROUND: Toxic epidermal necrolysis (TEN) is a rare and potentially fatal adverse dermatologic reaction, often secondary to drug intake. METHODS AND RESULTS: We report a case of an adult male who developed TEN that was complicated by a sepsis-induced coma lasting a period of 3 months. Although the patient eventually recovered, severe oral mucosal involvement during the course of disease resulted in frenum-like fibrotic bands that connected movable oral mucosa (tongue and lips) to attached oral mucosa (gingiva). This complication was painful and also affected his ability to speak and maintain adequate oral hygiene, requiring surgical intervention to restore structure and function. CONCLUSIONS: This oral manifestation in relation to TEN is rarely reported in the literature and represents a preventable complication.