Latency-associated protein Acr1 impairs dendritic cell maturation and functionality: a possible mechanism of immune evasion by Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tuberculosis) in latently infected individuals survives and thwarts the attempts of eradication by the immune system. During latency, Acr1 is predominantly expressed by the bacterium. However, whether M. tuberculosis exploits its Acr1 in impairing the host immunity remains widely unexplored. Hence, currently we have investigated the role of Acr1 in influencing the differentiation and function of dendritic cells (DCs), which play a cardinal role in innate and adaptive immunity. Therefore, for the first time, we have revealed a novel mechanism of mycobacterial Acr1 in inhibiting the maturation and differentiation of DCs by inducing tolerogenic phenotype by modulating the expression of PD-L1; Tim-3; indoleamine 2, 3-dioxygenase (IDO); and interleukin 10. Furthermore, Acr1 interferes in the differentiation of DCs by targeting STAT-6 and STAT-3 pathways. Continuous activation of STAT-3 inhibited the translocation of NF-κB in Acr1-treated DCs. Furthermore, Acr1 also augmented the induction of regulatory T cells. These DCs displayed decline in their antigen uptake capacity and reduced ability to help T cells. Interestingly, M. tuberculosis exhibited better survival in Acr1-treated DCs. Thus, this study provides a crucial insight into a strategy adopted by M. tuberculosis to survive in the host by impairing the function of DCs.

Efficient ligation and cloning of DNA fragments with 2-bp overhangs.

Various methods of ligation are currently available and routinely used by molecular biologists, such as blunt end ligation, cohesive end (two and four overhangs), and ligation of Taq polymerase-derived products. However, there is no efficient method for the cloning of DNA fragments with 2-bp overhangs. We present a simple method for the efficient ligation of DNA fragments with 2-bp overhanging ends, ranging in size from 0.7 to 2.5 kbp. Our method involves the initial heating and flash freezing of the vector-insert DNA mix, and a subsequent unique ligation reaction. This method provides a new molecular biology tool for researchers.

Lactobacillus acidophilus Antimicrobial Peptide Is Antagonistic to Aeromonas hydrophila.

We identified an antimicrobial peptide (AMP) from Lactobacillus acidophilus that was antagonistic to Aeromonas hydrophila. In vitro studies such as well-diffusion and field trials revealed that the AMP was active against A. hydrophila. The field trials of AMP using A. hydrophila-infected Channa striatus with a mannone oligosaccharide (MOS) prebiotic, A. hydrophila antigens, A. hydrophila-infected fish serum, L. acidophilus, and Lactobacillus cell free-supernatant (LABS-CFS) on an indicator organism further revealed that the antimicrobial agent could protect C. striatus. Other than the AMP, none of the above were able to eliminate the infectious agent A. hydrophila, and were only able to delay the death rate for 3-4 days. Thus, we conclude that the AMP is antagonistic to A. hydrophila and may be used for treatment of A. hydrophila infections. Subsequent L. acidophilus whole-genome sequence analyses enabled an understanding of the (probable) gene arrangement and its location on the chromosome. This information may be useful in the generation of recombinant peptides to produce larger quantities for treatment.

Single-step overlap-primer-walk polymerase chain reaction for multiple mutagenesis without overlap extension.

We present a simple, single-step, single-tube, and rapid method for introducing a series of mutations into cloned DNA. Polymerase chain reaction (PCR)-based mutagenesis methods have become very prevalent due to their simplicity and efficiency for introducing mutations. Our method, overlap-primer-walk PCR, has several advantages over other published methods. It uses two common oligodeoxyribonucleotides and a series of overlapping primers specific for various mutations. Once common flanking primers are selected, two to three mutations require only one additional primer. Therefore, this method is very useful for introduction of multiple mutations in various sites of the target DNA. We illustrate the usefulness of the method by introducing several mutations into the human TNF-alpha encoding gene.

CTAB-mediated, single-step preparation of competent Escherichia coli, Bifidobacterium sp. and Kluyveromyces lactis cells.

An efficient and reproducible method for transformation depends on the competency of the organism. We have developed a simple method for the preparation of competent Escherichia coli, Kluyveromyces lactis, and Bifidobacterium sp. by using a buffer containing cetyl trimethyl ammonium bromide (CTAB) and permits efficient uptake of plasmid DNA and ligation-reaction products. Cells are harvested, washed, mixed with 1-10 µg/ml CTAB, incubated, and followed by a buffer wash. For long-term storage of competent cells, bacteria may be frozen in 10% glycerol without the addition of other components. The transformation process is very simple; plasmid DNA and the cells are mixed and incubated for 5-60 min at 4 °C; no heat pulse is required, and the duration of incubation at 4 °C is not crucial.

Draft Genome Sequence of Enterococcus raffinosus Strain CFTRI 2200, Isolated from Infant Fecal Material.

The complete genome sequence of Enterococcus raffinosus strain CFTRI 2200, isolated from the fecal material of a 7-month-old infant, is reported. The complete genome consists of 4.237 Mbp with a G+C content of 39.47% and 4,242 protein-coding genes, 54 tRNAs, and 46 rRNAs.

Expression of synthetic human tumor necrosis factor is toxic to Escherichia coli.

The overlap forward-primer-walk polymerase chain reaction method was used to synthesize the human tumor necrosis factor α (hTNF) gene in Escherichia coli cells. Growth curves for hTNF and pET23d vector cultures exhibited slower doubling rates than cultures containing the pET23d vector alone. Cell cultures transformed with hTNF reached peak densities (0.4-0.6 OD(600)) 3 to 4 h post-induction, then decreased prior to growth recovery. This inhibition occurred in the BL21DE3 strain of E. coli, whereas no inhibition of growth and no expression of hTNF were observed in the JM109 strain of E. coli containing hTNF. Induced hTNF cultures hyperexpressed the hTNF-histidine fusion protein for the first 3 to 4h of induction; subsequently, growth retardation was observed. Hyperexpression and continuous growth were observed in the extracellular expression system. Electron microscopy revealed that accumulation of hTNF inclusion bodies was apparent only in the intracellular expression system - no accumulation was observed with regard to the secretory system. The hTNF-pET23d vector was purified from cells expressing the fusion protein and from cells with recovered growth curves. Sequencing of the vector demonstrated the complete hTNF gene and T7 promoter in cells expressing the fusion protein and mutations of the T7 promoter site from recovered cells.

Identification of sulfoquinovosyl diacylglycerol as a major polar lipid in Marinococcus halophilus and Salinicoccus hispanicus and substitution with phosphatidylglycerol.

The sulfonolipid sulfoquinovosyl diacylglycerol normally associated with photosynthetic membranes was identified as a major lipid in Marinococcus halophilus, Salinicoccus hispanicus ("Marinococcus hispanicus"), and Marinococcus sp. H8 (Planococcus sp. H8). Phosphatidylglycerol and 0%-10% cardiolipin accounted for the remaining polar lipids in these moderately halophilic, Gram-positive bacteria. Negative-ion fast atom bombardment mass spectrometry was used to quantify these three polar lipids from cells grown in media containing 0.03 to 4 mol NaCl/L. All strains revealed dramatic shifts in the ratio of sulfonolipid to phospholipid dependent on the salinity of the growth media, when grown in media with low phosphate content. Highest sulfonolipid content occurred during best growth in 0.5-2 mol NaCl/L, approaching 80%-90% of the total polar lipids. It was demonstrated that growth of M. halophilus in the presence of elevated phosphate and low sulfate blocked the shift to decreased phospholipids most notably during growth in 0.5-2 mol NaCl/L, without significant influence on growth. The data suggest that in low-phosphate media the influence of NaCl concentration on growth rate (and resulting demand for phosphate by competing pathways) is the primary factor responsible for exchange between phospholipid and sulfonolipid. We conclude that sulfoquinovosyl diacylglycerol, by substitution with phospholipids, contributes to the ability of these Gram-positive cocci to adapt to changing ionic environments. A comparison of 16S rRNA established a close similarity between Planococcus sp. H8 and M. halophilus.

Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover.

To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (alpha, beta, and gamma), and also their two-domain combinations, viz. alphabeta and betagamma in Escherichia coli. After purification, alpha and beta showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs alphabeta and betagamma showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in co-factor activity between the two- and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two- and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK.HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SK-plasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK.HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels.