Decoupling Internalization,Acidification and Phagosomal-Endosomal/lysosomal Fusion during Phagocytosis of InlA Coated Beads in Epithelial Cells

Background

Phagocytosis has been extensively examined in ‘professional’ phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in ‘non-professional’ phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells.

Methodology/Principal Findings

Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification.

Conclusions/Significance

Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23–32 min, 3–4 min and 74–120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply require fluorophore conjugation to a particle of interest, such as a pathogen or mimetic, in combination with common cell labeling dyes. As such, these methods hold promise for future measurements of receptor-mediated internalization in other cell systems, e.g. pathogen-host systems.     

Complete genome sequence of Desulfomicrobium baculatum type strain (X)

Desulfomicrobium baculatum is the type species of the genus Desulfomicrobium, which is the type genus of the family Desulfomicrobiaceae. It is of phylogenetic interest because of the isolated location of the family Desulfomicrobiaceae within the order Desulfovibrionales. D. baculatum strain X(T) is a Gram-negative, motile, sulfate-reducing bacterium isolated from water-saturated manganese carbonate ore. It is strictly anaerobic and does not require NaCl for growth, although NaCl concentrations up to 6% (w/v) are tolerated. The metabolism is respiratory or fermentative. In the presence of sulfate, pyruvate and lactate are incompletely oxidized to acetate and CO(2). Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a member of the deltaproteobacterial family Desulfomicrobiaceae, and this 3,942,657 bp long single replicon genome with its 3494 protein-coding and 72 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Acidimicrobium ferrooxidans type strain (ICP)

Acidimicrobium ferrooxidans (Clark and Norris 1996) is the sole and type species of the genus, which until recently was the only genus within the actinobacterial family Acidimicrobiaceae and in the order Acidomicrobiales. Rapid oxidation of iron pyrite during autotrophic growth in the absence of an enhanced CO(2) concentration is characteristic for A. ferrooxidans. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the order Acidomicrobiales, and the 2,158,157 bp long single replicon genome with its 2038 protein coding and 54 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Beutenbergia cavernae type strain (HKI 0122)

Beutenbergia cavernae (Groth et al. 1999) is the type species of the genus and is of phylogenetic interest because of its isolated location in the actinobacterial suborder Micrococcineae. B. cavernae HKI 0122(T) is a Gram-positive, non-motile, non-spore-forming bacterium isolated from a cave in Guangxi (China). B. cavernae grows best under aerobic conditions and shows a rod-coccus growth cycle. Its cell wall peptidoglycan contains the diagnostic L-lysine ← L-glutamate interpeptide bridge. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence from the poorly populated micrococcineal family Beutenbergiaceae, and this 4,669,183 bp long single replicon genome with its 4225 protein-coding and 53 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Leptotrichia buccalis type strain (C-1013-b)

Leptotrichia buccalis (Robin 1853) Trevisan 1879 is the type species of the genus, and is of phylogenetic interest because of its isolated location in the sparsely populated and neither taxonomically nor genomically adequately accessed family 'Leptotrichiaceae' within the phylum 'Fusobacteria'. Species of Leptotrichia are large, fusiform, non-motile, non-sporulating rods, which often populate the human oral flora. L. buccalis is anaerobic to aerotolerant, and saccharolytic. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of the order 'Fusobacteriales' and no more than the second sequence from the phylum 'Fusobacteria'. The 2,465,610 bp long single replicon genome with its 2306 protein-coding and 61 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Desulfotomaculum acetoxidans type strain (5575)

Desulfotomaculum acetoxidans Widdel and Pfennig 1977 was one of the first sulfate-reducing bacteria known to grow with acetate as sole energy and carbon source. It is able to oxidize substrates completely to carbon dioxide with sulfate as the electron acceptor, which is reduced to hydrogen sulfide. All available data about this species are based on strain 5575(T), isolated from piggery waste in Germany. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a Desulfotomaculum species with validly published name. The 4,545,624 bp long single replicon genome with its 4370 protein-coding and 100 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Rhodothermus marinus type strain (R-10)

Rhodothermus marinus Alfredsson et al. 1995 is the type species of the genus and is of phylogenetic interest because the Rhodothermaceae represent the deepest lineage in the phylum Bacteroidetes. R. marinus R-10(T) is a Gram-negative, non-motile, non-spore-forming bacterium isolated from marine hot springs off the coast of Iceland. Strain R-10(T) is strictly aerobic and requires slightly halophilic conditions for growth. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the genus Rhodothermus, and only the second sequence from members of the family Rhodothermaceae. The 3,386,737 bp genome (including a 125 kb plasmid) with its 2914 protein-coding and 48 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

The Influence of Soil Inoculum and Nitrogen Availability on Restoration of High-Elevation Steppe Communities Invaded by Bromus tectorum

Cheatgrass ( Bromus tectorum L.) is an exotic annual grass that has invaded approximately 40,000,000 ha of rangelands in the United States, including montane ecosystems that are important habitats for wildlife and livestock. In addition to well-understood mechanisms by which Cheatgrass gains competitive advantage, recent studies have shown that Cheatgrass may also change the associated soil microbial community to impact native perennial plants and promote the persistence of Cheatgrass. Furthermore, reducing plant-available N represents a tool for initiating conditions that accelerate successional change from annual- to perennial-dominated communities. At a montane, mixed shrub–grassland Cheatgrass-dominated site in Colorado, we applied sucrose to reduce available N, and we added soil from a native plant community in order to reestablish the microbial community. This approach tested the idea that intact native soil microbial communities may enhance the beneficial effect of reducing soil N availability in a restoration setting. By the end of the experiment, reduced N availability decreased Cheatgrass by 9.8%, non-native annual/biennial plant cover by 15.0%, and increased relative perennial plant cover by 13.4%; soil inoculation reduced Cheatgrass by 7.6% and increased perennial abundance by 11.3%. Soil inoculum additions and reduced N availability both contributed toward restoring a perennial-dominated community and demonstrates that addition of native soil inoculum may be a useful tool for restoration efforts.     

Mechanisms for reaching the differentiated state: Insights from neural crest-derived melanocytes

Black pigment cells, or melanocytes, are the major contributing cells to pigmentation in vertebrate organisms. Although the function of these cells is distinct depending on the organism, the events involved in their development are remarkably similar. Here, we review the mechanisms involved in the early development of melanocytes from neural crest, many of which are conserved in organisms as diverse as zebrafish, birds and humans. We also discuss recent studies that provide further insight into how melanocyte differentiation is achieved and maintained.