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Bacteriophage T4 of Escherichia coli is one of the most studied phages. Research into it has led to numerous contributions to phage biology and biochemistry. Coding about 300 gene products, this double-stranded DNA virus is the best-understood model in phage study and modern genomics and proteomics. Ranging from viral RNA polymerase, commonly found in phages, to thymidylate synthase, whose mRNA requires eukaryotic-like self-splicing, its gene products provide a pool of fine examples for phage research. However, there are still up to 130 gene products that remain poorly characterized despite being one of the most-studied model phages. With the recent advancement of cryo-electron microscopy, we have a glimpse of the virion and the structural proteins that present in the final assembly. Unfortunately, proteins participating in other stages of phage development are absent. Here, we report our systemic analysis on 22 of these structurally uncharacterized proteins, of which none has a known homologous structure due to the low sequence homology to published structures and does not belong to the category of viral structural protein. Using NMR spectroscopy and cryo-EM, we provided a set of preliminary structural information for some of these proteins including NMR backbone assignment for Cef. Our findings pave the way for structural determination for the phage proteins, whose sequences are mainly conserved among phages. While this work provides the foundation for structural determinations of proteins like Gp57B, Cef, Y04L, and Mrh, other in vitro studies would also benefit from the high yield expression of these proteins.

Bacteriophage T4 of Escherichia coli is one of the most studied phages. Research into it has led to numerous contributions to phage biology and biochemistry. Coding about 300 gene products, this double-stranded DNA virus is the best-understood model in phage study and modern genomics and proteomics. Ranging from viral RNA polymerase, commonly found in phages, to thymidylate synthase, whose mRNA requires eukaryotic-like self-splicing, its gene products provide a pool of fine examples for phage research. However, there are still up to 130 gene products that remain poorly characterized despite being one of the most-studied model phages. With the recent advancement of cryo-electron microscopy, we have a glimpse of the virion and the structural proteins that present in the final assembly. Unfortunately, proteins participating in other stages of phage development are absent. Here, we report our systemic analysis on 22 of these structurally uncharacterized proteins, of which none has a known homologous structure due to the low sequence homology to published structures and does not belong to the category of viral structural protein. Using NMR spectroscopy and cryo-EM, we provided a set of preliminary structural information for some of these proteins including NMR backbone assignment for Cef. Our findings pave the way for structural determination for the phage proteins, whose sequences are mainly conserved among phages. While this work provides the foundation for structural determinations of proteins like Gp57B, Cef, Y04L, and Mrh, other in vitro studies would also benefit from the high yield expression of these proteins. [ABSTRACT FROM AUTHOR]

A growing body of data shows that bacteriophages can interact with different kinds of immune cells. The objective of this study was to investigate whether T4 bacteriophage and T4-generated Escherichia coli lysate affect functions of monocytes, the key population of immune cells involved in antibacterial immunity. To that end we evaluated how T4 and E. coli lysate influence the expression of main costimulatory molecules including CD40, CD80 and CD86, TLR2, TLR4 on monocytes, as well as the production of IL-6 and IL-12 in cultures of peripheral blood mononuclear cells (PBMCs). Separate experiments were performed on unactivated and LPS-activated PBMCs cultures. Both studied preparations significantly increased the percentage of CD14+CD16-CD40+ and CD14+CD16-CD80+ monocytes in unactivated PBMCs cultures, as well as the concentration of IL-6 and IL-12 in culture supernates. However, neither purified T4 nor E. coli lysate had any significant effect on monocytes in LPS-activated PBMCs cultures. We conclude that LPS-activated monocytes are unresponsive to phages and products of phage-induced lysis of bacteria. This study is highly relevant to phage therapy because it suggests that in patients with infections caused by Gram-negative bacteria the administration of phage preparations to patients and lysis of bacteria by phages are not likely to overly stimulate monocytes.

Bacteriophages (phages) are viruses of bacteria. Here we evaluated the effects of T4 and A3R bacteriophages, as well as phage-generated bacterial lysates, on differentiation of human myeloid dendritic cells (DCs) from monocytes. Neither of the phages significantly reduced the expression of markers associated with differentiation of DCs and their role in the activation of T cells (CD40, CD80, CD83, CD86, CD1c, CD11c, MHC II, PD-L1, PD-L2, TLR2, TLR4, and CCR7) and phagocytosis receptors (CD64 and DEC-205). By contrast, bacterial lysate of T4 phage significantly decreased the percentages of DEC-205- and CD1c-positive cells. The percentage of DEC-205-positive cells was also significantly reduced in DCs differentiated in the presence of lysate of A3R phage. Thus while bacteriophages do not substantially affect differentiation of DCs, some products of phage-induced lysis of bacterial cells may influence the differentiation and potentially also some functions of DCs. Our results have important implications for phage therapy of bacterial infections because during infections monocytes recruited to the site of inflammation are an important source of inflammatory DCs.

Bacteriophages (phages) are viruses of bacteria. Here we evaluated the effects of T4 and A3/R bacteriophages, as well as phage-generated bacterial lysates, on differentiation of human myeloid dendritic cells (DCs) from monocytes. Neither of the phages significantly reduced the expression of markers associated with differentiation of DCs and their role in the activation of T cells (CD40, CD80, CD83, CD86, CD1c, CD11c, MHC II, PD-L1, PD-L2, TLR2, TLR4, and CCR7) and phagocytosis receptors (CD64 and DEC-205). By contrast, bacterial lysate of T4 phage significantly decreased the percentages of DEC-205- and CD1c-positive cells. The percentage of DEC-205-positive cells was also significantly reduced in DCs differentiated in the presence of lysate of A3/R phage. Thus while bacteriophages do not substantially affect differentiation of DCs, some products of phage-induced lysis of bacterial cells may influence the differentiation and potentially also some functions of DCs. Our results have important implications for phage therapy of bacterial infections because during infections monocytes recruited to the site of inflammation are an important source of inflammatory DCs. [ABSTRACT FROM AUTHOR]

A growing body of data shows that bacteriophages can interact with different kinds of immune cells. The objective of this study was to investigate whether T4 bacteriophage and T4-generated Escherichia coli lysate affect functions of monocytes, the key population of immune cells involved in antibacterial immunity. To that end, we evaluated how T4 and E. coli lysate influence the expression of main costimulatory molecules including CD40, CD80 and CD86, TLR2, TLR4 on monocytes, as well as the production of IL-6 and IL-12 in cultures of peripheral blood mononuclear cells (PBMCs). Separate experiments were performed on unactivated and LPS-activated PBMCs cultures. Both studied preparations significantly increased the percentage of CD14(+)CD16(-)CD40(+) and CD14(+)CD16(-)CD80(+) monocytes in unactivated PBMCs cultures, as well as the concentration of IL-6 and IL-12 in culture supernates. However, neither purified T4 nor E. coli lysate had any significant effect on monocytes in LPS-activated PBMCs cultures. We conclude that LPS-activated monocytes are unresponsive to phages and products of phage-induced lysis of bacteria. This study is highly relevant to phage therapy because it suggests that in patients with infections caused by Gram-negative bacteria the administration of phage preparations to patients and lysis of bacteria by phages are not likely to overly stimulate monocytes.