The moving junction protein RON8 facilitates firm attachment and host cell invasion in Toxoplasma gondii

The apicomplexan moving junction (MJ) is a highly conserved structure formed during host cell entry that anchors the invading parasite to the host cell and serves as a molecular sieve of host membrane proteins that protects the parasitophorous vacuole from host lysosomal destruction. While recent work in Toxoplasma and Plasmodium has reinforced the composition of the MJ as an important association of rhoptry neck proteins (RONs) with micronemal AMA1, little is known of the precise role of RONs in the junction or how they are targeted to the neck subcompartment. We report the first functional analysis of a MJ/RON protein by disrupting RON8 in T. gondii. Parasites lacking RON8 are severely impaired in both attachment and invasion, indicating that RON8 enables the parasite to establish a firm clasp on the host cell and commit to invasion. The remaining junction components frequently drag in trails behind invading knockout parasites and illustrate a malformed complex without RON8. Complementation of Δron8 parasites restores invasion and reveals a processing event at the RON8 C-terminus. Replacement of an N-terminal region of RON8 with a mCherry reporter separates regions within RON8 that are necessary for rhoptry targeting and complex formation from those required for function during invasion. Finally, the invasion defects in Δron8 parasites seen in vitro translate to radically impaired virulence in infected mice, promoting a model in which RON8 has a crucial and unprecedented task in committing Toxoplasma to host cell entry.     

Wastewater Treatment Effluent Reduces the Abundance and Diversity of Benthic Bacterial Communities in Urban and Suburban Rivers

In highly urbanized areas, wastewater treatment plant (WWTP) effluent can represent a significant component of freshwater ecosystems. As it is impossible for the composition of WWTP effluent to match the composition of the receiving system, the potential exists for effluent to significantly impact the chemical and biological characteristics of the receiving ecosystem. We assessed the impacts of WWTP effluent on the size, activity, and composition of benthic microbial communities by comparing two distinct field sites in the Chicago metropolitan region: a highly urbanized river receiving effluent from a large WWTP and a suburban river receiving effluent from a much smaller WWTP. At sites upstream of effluent input, the urban and suburban rivers differed significantly in chemical characteristics and in the composition of their sediment bacterial communities. Although effluent resulted in significant increases in inorganic nutrients in both rivers, surprisingly, it also resulted in significant decreases in the population size and diversity of sediment bacterial communities. Tag pyrosequencing of bacterial 16S rRNA genes revealed significant effects of effluent on sediment bacterial community composition in both rivers, including decreases in abundances of Deltaproteobacteria, Desulfococcus, Dechloromonas, and Chloroflexi sequences and increases in abundances of Nitrospirae and Sphingobacteriales sequences. The overall effect of the WWTP inputs was that the two rivers, which were distinct in chemical and biological properties upstream of the WWTPs, were almost indistinguishable downstream. These results suggest that WWTP effluent has the potential to reduce the natural variability that exists among river ecosystems and indicate that WWTP effluent may contribute to biotic homogenization.     

Structural Homology between the C-Terminal Domain of the PapC Usher and Its Plug

P pili are extracellular appendages responsible for the targeting of uropathogenic Escherichia coli to the kidney. They are assembled by the chaperone-usher (CU) pathway of pilus biogenesis involving two proteins, the periplasmic chaperone PapD and the outer membrane assembly platform, PapC. Many aspects of the structural biology of the Pap CU pathway have been elucidated, except for the C-terminal domain of the PapC usher, the structure of which is unknown. In this report, we identify a stable and folded fragment of the C-terminal region of the PapC usher and determine its structure using both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. These structures reveal a β-sandwich fold very similar to that of the plug domain, a domain of PapC obstructing its translocation domain. This structural similarity suggests similar functions in usher-mediated pilus biogenesis, playing out at different stages of the process. This structure paves the way for further functional analysis targeting surfaces common to both the plug and the C-terminal domain of PapC.Adhesive surface organelles termed pili mediate the adhesion of bacteria to host cells. Pili assembled by the chaperone-usher (CU) pathway form one of five major classes of nonflagellar surface appendages in Gram-negative bacteria, with the P pilus system from uropathogenic Escherichia coli being one of the two best-characterized CU systems. These pili are multisubunit structures consisting of two distinct subassemblies, a rigid rod with a diameter of 6.8 nm and a distal flexible tip fibrillum with a diameter of 2 nm (18, 21). In P pili the helical rod is comprised of more than 1,000 copies of the PapA subunits arranged in a right-handed helical cylinder with 3.3 subunits per turn (3, 8, 14), and the tip fibrillum is comprised of 5 to 10 copies of the PapE subunits (21). Two “adaptor” subunits, PapK and PapF, connect the PapE tip fibrillum to the PapA rod and the PapE tip fibrillum to the distal PapG adhesin (16, 21). The proximal end of the pilus is terminated by the PapH subunit (2, 50). The PapG adhesin mediates the bacterial colonization of the kidney (25, 40) by binding to the globoseries of glycolipids present in the human kidney (25, 40) (Fig. ​(Fig.1A),1A), an event that is critical in pyelonephritis.Open in a separate windowFIG. 1.(A) Schematic diagram of a P pilus assembled in the usher translocation platform. Subunits are represented by oval shapes, and N-terminal extensions are represented by short rectangular shapes. The usher homodimer is represented in the outer membrane (OM). In the usher protomer through which the nascent pilus passes, two positions of the plug are indicated by P where the plug is positioned to the side of the transmembrane barrel''s lumen and P′ where the plug has swung into the periplasmic space. (B) Domain organization of the PapC usher based on amino acid sequence. The C-terminal domain sequences are indicated in marine blue. The constructs used in this study are schematically represented underneath; all converge to a fragment containing residues 722 to 809, termed the “PapC CTD.” Ntd, N-terminal domain. (C) Identification of a discrete folding unit at the C terminus of PapC. Shown is an SDS-PAGE gel stained with Coomassie blue of the eluted PapC C-terminal fragments obtained with a construct comprising residues 641 to 809 after the first purification step. PS, prestained protein standards; Inj, loaded sample; FT, flowthrough.The assembly of pili is a coordinated process requiring two proteins: a chaperone and an outer membrane assembly platform, the usher. Pilus subunits are translocated into the periplasm via the general secretory machinery (38, 47). The binding of the PapD chaperone to the nascently translocated subunits facilitates their folding on the chaperone template. The chaperone remains bound to the folded subunits capping their interactive surfaces, thus preventing nonproductive interactions in the periplasm (7). Chaperone-subunit complexes are then targeted to the usher (PapC), where subunits polymerize in an ordered fashion and translocate across the outer membrane through the usher pore (47, 52). Subunit folding and stabilization occur when the chaperone and subunit form a complex through a mechanism termed donor strand complementation (DSC) (9, 41). In this mechanism the C-terminally truncated Ig-like fold of the pilus subunits, which contains only six of the seven β-strands that constitute the canonical Ig fold, is complemented by the donation of a β-strand from the chaperone (9, 41). Chaperone-subunit complexes are then targeted to the outer membrane usher, where the chaperone is released and subunits are noncovalently joined to preceding subunits in the nascent pilus fiber. This polymerization process is made possible by the presence of a disordered N-terminal extension sequence (NTES) in each subunit (except the adhesin) (41), which during pilus assembly displaces the strand donated by the chaperone, thereby substituting for the missing secondary structure in the previously assembled subunit. This mechanism is called donor-strand exchange (DSE) (9, 41, 42, 55). It is believed that this structural reorganization provides the driving force for pilus biogenesis, since no ATP hydrolysis or other type of external energy source is required (17, 56).DSE occurs at the outer membrane usher, which acts as a catalyst for polymerization (34). Biophysical and cryo-electron microscopy (EM) studies of the FimD usher (a close homolog of PapC) have shown that the usher is a twinned pore in both detergent and lipid bilayers (23, 46). Only one pore is used for secretion, but two pores are required for subunit recruitment (39). For PapC, both monomers and dimers have been described (15, 39). The usher has four functional domains (Fig. ​(Fig.1B):1B): a translocation domain forming a β-barrel with 24 transmembrane β-strands (15, 39), a plug domain in the middle of the translocation domain, and two periplasmic domains, one at each of the N- and C-terminal ends of the usher polypeptide (35, 48). The plug domain has a β-sandwich fold and completely occludes the pore in the inactive usher. Its function, besides gating the channel, seems to be further associated with pilus biogenesis since the deletion of the plug domain abolishes pilus formation in vitro and in vivo (15, 26, 54). The N-terminal domain selectively binds chaperone-subunit complexes (12, 33). The structure of the N-terminal domain of FimD bound to chaperone-subunit complexes indicated that the first 24 residues of FimD are involved in the recognition of chaperone-subunit complexes; the deletion of this region was shown previously to abolish pilus biogenesis (12, 32, 33).The role of the usher C-terminal domain (CTD) is not well understood. The binding of the chaperone-adhesin complex to the usher C terminus was previously demonstrated in vitro (46), while protease susceptibility in FimD shows that, following targeting to the usher N terminus, the chaperone-adhesin complex forms stable interactions with the FimD C terminus, inducing a conformational change in FimD that may be fundamental in the activation step of pilus biogenesis (29, 30, 43). The structure of the C-terminal domain is unknown and is the only part of the CU pilus biogenesis pathway not yet represented in structural terms. Here we provide evidence for the presence of a discrete folding unit in the PapC CTD and report its structure determined by nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography.     

Variation of toxigenic Vibrio cholerae O1 in the aquatic environment of Bangladesh and its correlation with the clinical strains

The diversity of toxigenic V. cholerae O1 in the aquatic environment of Bangladesh is not known. A total of 18 environmental and 18 clinical strains of toxigenic V. cholerae O1 were isolated simultaneously from four different geographical areas and tested for variation by the pulsed-field gel electrophoresis method. Environmental strains showed diversified profiles and one of the profiles was common to some environmental strains and most clinical strains. It appears that one clone has an advantage over others to cause disease. These findings suggest that the study of the molecular ecology of V. cholerae O1 in relation to its environmental reservoir is important in identifying virulent strains that cause disease.     

Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803

Primary ion pumps and antiporters exist as multigene families in the Synechocystis sp. PCC 6803 genome and show very strong homologies to those found in higher plants. The gene knock-outs of five putative Na+/H+ antiporters (slr1727, sll0273, sll0689, slr1595 and slr0415) and seven cation ATPases (sll1614, sll1920, slr0671-72, slr0822, slr1507-08-09, slr1728- 29 and slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html) were performed in this study relying on homologous recombination with mutagenenic fragments constructed using a fusion polymerase chain reaction (PCR) approach. The impacts of these gene knock-outs were evaluated in terms of Na+ and pH, and light-induced acidification and alkalization that are asso-ciated with inorganic carbon uptake. Two of the five putative antiporter mutants exhibit a characteristic interplay between the pH and Na+ dependence of growth, but only one of the antiporters appears to be necessary for high NaCl tolerance. On the other hand, the mutation of one of the two copper-trafficking ATPases produces a cell line that shows acute NaCl sensitivity. Additionally, disruptions of a putative Ca2+-ATPase and a gene cluster encoding a putative Na+-ATPase subunit also cause high NaCl sensitivity. The findings and possible mechanisms are discussed in relation to the potential roles of these transporters in Synechocystis sp. PCC 6803.     

Single-cell electroporation

Electroporation is a widely used method for the introduction of polar and charged agents such as dyes, drugs, DNA, RNA, proteins, peptides, and amino acids into cells. Traditionally, electroporation is performed with large electrodes in a batch mode for treatment of a large number of cells in suspension. Recently, microelectrodes that can produce extremely localized electric fields, such as solid carbon fiber microelectrodes, electrolyte-filled capillaries and micropipettes as well as chip-based microfabricated electrode arrays, have proven useful to electroporate single cells and subcellular structures. Single-cell electroporation opens up a new window of opportunities in manipulating the genetic, metabolic, and synthetic contents of single targeted cells in tissue slices, cell cultures, in microfluidic channels or at specific loci on a chip-based device.     

Molecular, Serological, and Virulence Characteristics of Vibrio parahaemolyticus Isolated from Environmental, Food, and Clinical Sources in North America and Asia

Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic Vibrio parahaemolyticus isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia. The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (tdh) gene. The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York. Multiplex PCR was used to confirm the species identification and the presence of tdh and to test for the tdh-related hemolysin trh. Most of the environmental, food, and clinical isolates from the United States were positive for tdh, trh, and urease production. Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only tdh. A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups. All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia. The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates. The combination of serotyping and ribotyping showed that the Pacific Coast V. parahaemolyticus population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast. The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease.     

Diurnal variation of lymphocyte subsets identified by monoclonal antibodies.

Monoclonal antibodies specific for lymphocyte subsets were used to examine circulating lymphocytes obtained at frequent intervals from healthy subjects. A diurnal rhythm was found in the total numbers of lymphocytes, T cells, inducer/helper cells, suppressor/cytotoxic cells, Ia positive cells, and B cells. The lowest levels of all subsets were seen at 0900 hours and the highest levels at 2100. In some subjects the ratio of helper to suppressor cells varied considerably during the sample period, though the ratio was relatively constant for the group as a whole.     

Assessing concordance of fossil calibration points in molecular clock studies: an example using turtles

Although still controversial, estimation of divergence times using molecular data has emerged as a powerful tool to examine the tempo and mode of evolutionary change. Two primary obstacles in improving the accuracy of molecular dating are heterogeneity in DNA substitution rates and accuracy of the fossil record as calibration points. Recent methodological advances have provided powerful methods that estimate relative divergence times in the face of heterogeneity of nucleotide substitution rates among lineages. However, relatively little attention has focused on the accuracy of fossil calibration points that allow one to translate relative divergence times into absolute time. We present a new cross-validation method that identifies inconsistent fossils when multiple fossil calibrations are available for a clade and apply our method to a molecular phylogeny of living turtles with fossil calibration times for 17 of the 22 internal nodes in the tree. Our cross-validation procedure identified seven inconsistent fossils. Using the consistent fossils as calibration points, we found that despite their overall antiquity as a lineage, the most species-rich clades of turtles diversified well within the Cenozoic. Many of the truly ancient lineages of turtles are currently represented by a few, often endangered species that deserve high priority as conservation targets.