The rfbN gene of Salmonella Typhimurium mediates phage adsorption by modulating biosynthesis of lipopolysaccharide.

The study of the interaction mechanism between bacteriophage and host is helpful in promoting development of bacteriophage applications. The mechanism of the interaction with the phage was studied by constructing the rfbN gene deletion and complemented with strains of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium, S. Typhimurium) D6. The rfbN gene deletion strain could not be lysed by phage S55 and led to a disorder of lipopolysaccharide (LPS) biosynthesis, which changed from the smooth type to rough type. Also, the RfbN protein lacking any of the three-segment amino acid (aa) sequences (90-120 aa, 121-158 aa, and 159-194 aa) produces the same result. Transmission electron microscopy and confocal microscopy assays demonstrated that phage S55 dramatically reduced adsorption to the rfbN deletion strain as compared to the wild strain D6. After co-incubation of the S55 with the purified smooth LPS, D6 could not be lysed, indicating that the smooth LPS binds to the S55 in vitro and then inhibits the cleavage activity of the S55. To sum up, the rfbN gene affects phage adsorption by regulating LPS synthesis. Furthermore, the functioning of the RfbN protein requires the involvement of multiple structures. To the best of our knowledge, this study is the first report of the involvement of the bacterial rfbN gene involved in the phage-adsorption process.

The prevention of an anomalous chromatographic behavior and the resulting successful removal of viruses from monoclonal antibody with an asymmetric charge distribution by using a membrane adsorber in highly efficient, anion-exchange chromatography in flow-through mode.

Anion exchange (AEX) chromatography in the flow-through mode is a widely employed purification process for removal of process/product-related impurities and exogenous/endogenous viruses from monoclonal antibodies (mAbs). The pH of the mobile phase for AEX chromatography is typically set at half a unit below the isoelectric point (pI) of each mAb (i.e., pI - 0.5) or lower and, in combination with a low ionic strength, these conditions are usually satisfactory for both the recovery of the mAb and removal of impurities. However, we have recently encountered a tight binding of mAb1 to AEX resins under these standard chromatographic conditions. This anomalous adsorption behavior appears to be an effect of the asymmetric charge distribution on the surface of the mAb1. We found that mAb1 did not bind to the AEX resins if the mobile phase has a much lower pH and higher ionic strength, but those conditions would not allow adequate virus removal. We predicted that the use of membrane adsorbers might provide effective mAb1 purification, since the supporting matrix has a network structure that would be less susceptible to interactions with the asymmetric charge distribution on the protein surface. We tested the Natriflo HD-Q AEX membrane adsorber under standard chromatographic conditions and found that mAb1 flowed through the membrane adsorber, resulting in successful separation from murine leukemia virus. This AEX membrane adsorber is expected to be useful for process development because mAbs can be purified under similar standard chromatographic conditions regardless of their charge distributions.

The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta.

Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta, enamel that is defective and easily damaged. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer-oligomer binding energies. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization.

Two-dimensional Ising model for microarray hybridization: cooperative interactions between bound target molecules.

The Langmuir adsorption model is widely used for description and quantification of microarray oligo-target hybridization. According to the model, the binding centers for adsorption of target molecules from solution are represented by oligo-probes. However, the Langmuir model does not consider the interactions between the targets adsorbed at the neighboring binding centers, which are possible due to high-density of array-bound probes. We have shown that the two-dimensional Ising model, which takes into account the nearest neighboring target molecules interactions, better describes the experimental data of oligo-target hybridization in comparison with the Langmuir model. Thus, we found an evidence for existence of positive cooperative interactions between adsorbed target molecules: so, binding of the first target molecules facilitates the binding of subsequent ones to the neighboring probes. Communicated by Ramaswamy H. Sarma.

Improved adsorption of an Enterococcus faecalis bacteriophage ΦEF24C with a spontaneous point mutation.

Some bacterial strains of the multidrug-resistant Gram-positive bacteria Enterococcus faecalis can significantly reduce the efficacy of conventional antimicrobial chemotherapy. Thus, the introduction of bacteriophage (phage) therapy is expected, where a phage is used as a bioagent to destroy bacteria. E. faecalis phage ΦEF24C is known to be a good candidate for a therapeutic phage against E. faecalis. However, this therapeutic phage still produces nonuniform antimicrobial effects with different bacterial strains of the same species and this might prove detrimental to its therapeutic effects. One solution to this problem is the preparation of mutant phages with higher activity, based on a scientific rationale. This study isolated and analyzed a spontaneous mutant phage, ΦEF24C-P2, which exhibited higher infectivity against various bacterial strains when compared with phage ΦEF24C. First, the improved bactericidal effects of phage ΦEF24C-P2 were attributable to its increased adsorption rate. Moreover, genomic sequence scanning revealed that phage ΦEF24C-P2 had a point mutation in orf31. Proteomic analysis showed that ORF31 (mw, 203 kDa) was present in structural components, and immunological analysis using rabbit-derived antibodies showed that it was a component of a long, flexible fine tail fiber extending from the tail end. Finally, phage ΦEF24C-P2 also showed higher bactericidal activity in human blood compared with phage ΦEF24C using the in vitro assay system. In conclusion, the therapeutic effects of phage ΦEF24C-P2 were improved by a point mutation in gene orf31, which encoded a tail fiber component.

Endurance swimming stimulates transepithelial calcium transport and alters the expression of genes related to calcium absorption in the intestine of rats.

Endurance impact exercise, e.g., running, is known to enhance the intestinal calcium absorption. However, nonimpact exercise, e.g., swimming, is more appropriate for osteoporotic patients with cardiovascular diseases or disorders of bone and joint, but the effect of swimming on the intestinal calcium transport was unknown. This study, therefore, aimed to investigate the transepithelial calcium transport and the expression of related genes in the intestine of rats trained to swim nonstop 1 h/day, 5 days/wk for 2 wk. We found that endurance swimming stimulated calcium transport in the duodenum, proximal jejunum, and cecum, while decreasing that in the proximal colon. Swimming affected neither the transepithelial potential difference nor resistance. As demonstrated by real-time PCR, the small intestine, especially the duodenum, responded to swimming by upregulating a number of genes related to the transcellular calcium transport, i.e., TRPV5, TRPV6, calbindin-D9k, PMCA1b, and NCX1, and the paracellular calcium transport, i.e., ZO-1, ZO-2, ZO-3, cingulin, occludin, and claudins, as well as nuclear receptor of 1,25(OH)2D3. In contrast, swimming downregulated those genes in the colon. Microarray analysis showed that swimming also altered the expression of duodenal genes related to the transport of several ions and nutrients, e.g., Na+, K+, Cl-, glucose, and amino acids. In conclusion, endurance swimming enhanced intestinal calcium absorption, in part, by upregulating the calcium transporter genes. The present microarray study also provided relevant information for further investigations into the intestinal nutrient and electrolyte transport during nonimpact exercise.