Syphilis is a chronic disease caused by the bacterium Treponema pallidum subsp. pallidum. Treponema pallidum disseminates widely throughout the host and extravasates from the vasculature, a process that is at least partially dependent upon the ability of T. pallidum to interact with host extracellular matrix (ECM) components. Defining the molecular basis for the interaction between T. pallidum and the host is complicated by the intractability of T. pallidum to in vitro culturing and genetic manipulation. Correspondingly, few T. pallidum proteins have been identified that interact directly with host components. Of these, Tp0751 (also known as pallilysin) displays a propensity to interact with the ECM, although the underlying mechanism of these interactions remains unknown. Towards establishing the molecular mechanism of Tp0751-host ECM attachment, we first determined the crystal structure of Tp0751 to a resolution of 2.15 Å using selenomethionine phasing. Structural analysis revealed an eight-stranded beta-barrel with a profile of short conserved regions consistent with a non-canonical lipocalin fold. Using a library of native and scrambled peptides representing the full Tp0751 sequence, we next identified a subset of peptides that showed statistically significant and dose-dependent interactions with the ECM components fibrinogen, fibronectin, collagen I, and collagen IV. Intriguingly, each ECM-interacting peptide mapped to the lipocalin domain. To assess the potential of these ECM-coordinating peptides to inhibit adhesion of bacteria to host cells, we engineered an adherence-deficient strain of the spirochete Borrelia burgdorferi to heterologously express Tp0751. This engineered strain displayed Tp0751 on its surface and exhibited a Tp0751-dependent gain-of-function in adhering to human umbilical vein endothelial cells that was inhibited in the presence of one of the ECM-interacting peptides (p10). Overall, these data provide the first structural insight into the mechanisms of Tp0751-host interactions, which are dependent on the protein’s lipocalin fold.

In Toxoplasma gondii, an intracellular parasite of humans and other animals, host mitochondrial association (HMA) is driven by a gene family that encodes multiple mitochondrial association factor 1 (MAF1) proteins. However, the importance of MAF1 gene duplication in the evolution of HMA is not understood, nor is the impact of HMA on parasite biology. Here we used within- and between-species comparative analysis to determine that the MAF1 locus is duplicated in T. gondii and its nearest extant relative Hammondia hammondi, but not another close relative, Neospora caninum. Using cross-species complementation, we determined that the MAF1 locus harbors multiple distinct paralogs that differ in their ability to mediate HMA, and that only T. gondii and H. hammondi harbor HMA+ paralogs. Additionally, we found that exogenous expression of an HMA+ paralog in T. gondii strains that do not normally exhibit HMA provides a competitive advantage over their wild-type counterparts during a mouse infection. These data indicate that HMA likely evolved by neofunctionalization of a duplicate MAF1 copy in the common ancestor of T. gondii and H. hammondi, and that the neofunctionalized gene duplicate is selectively advantageous.

Apicomplexan parasites such as rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA-light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775-818) using a combination of hydrogen-deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a of 12 nm). Using the full-length MyoA motor (residues 1-831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction.

Respiratory tract infections caused by influenza A and B viruses often present nonspecifically, and a rapid, high-throughput laboratory technique that can identify influenza viruses is

Epidemiological studies suggest that the use of NSAIDs and/or a high intake of fruit and vegetables reduce the risk of oesophageal adenocarcinoma. Since COX-2 is up-regulated in Barrett's oesophageal carcinogenesis, the protective effect of NSAIDs and natural food components might reflect COX-2 inhibition. We explored the effects of quercetin, a natural flavonoid with a potent COX-2 inhibitory activity, and two commercially available selective COX-2 inhibitors (NS-398 and nimesulide) on cell proliferation, apoptosis, PGE2 production and COX-2 mRNA expression in a human oesophageal adenocarcinoma cell line (OE33). Changes in the relative numbers of adherent and floating cells were quantified and apoptotic cells were identified using ethidium bromide and acridine orange staining under fluorescence microscopy. Flow cytometric analysis of adherent and floating cells was used to quantify apoptosis and to examine the effects of the agents on the cell cycle. After 48 h exposure at concentrations of > or =1 microM both COX-2 inhibitors and quercetin suppressed cell proliferation (P < 0.01) and increased the fraction of floating apoptotic cells. At higher concentrations (50 microM) and longer exposure (48 h) the effects of quercetin were significantly greater than those of the selective COX-2 inhibitors (P < 0.01). Cell cycle analyses showed that quercetin blocked cells in S phase, while the selective COX-2 inhibitors blocked cells in G1/S interphase. COX-2 mRNA expression was suppressed by quercetin and the synthetic COX-2 inhibitors in a time- and dose-dependent manner. Quercetin and the synthetic COX-2 inhibitors (10 microM) suppressed PGE2 production by approximately 70% after 24 h exposure (P < 0.001). We conclude that OE33 is a useful model for the study of COX-2 expression and associated phenomena in human adenocarcinoma cells. Synthetic COX-2 inhibitors and the food-borne flavonoid quercetin suppress proliferation, induce apoptosis and cell cycle block in human oesophageal adenocarcinoma cells in vitro, and future studies should assess their effects in vivo.