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The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to nucleotide-metabolizing functions. Two such genes, 48 and 50, encoding thymidylate synthase and ribonucleotide reductase (RNR), respectively, were overexpressed in Escherichia coli and the recombinant proteins were biochemically characterized. It was established that Gp50 was a class II RNR having properties similar to that of the corresponding enzyme from Lactobacillus leichmanni, whereas Gp48 was a flavin-dependent thymidylate synthase (ThyX) that resembled the Paramecium bursaria chlorella virus-1 ThyX enzyme in its properties. That both these proteins play a role in phage development was evident from the observation that they were detectable soon after the lytic phase of growth commenced. Gp48 and 50 were also found to coimmunoprecipitate, which indicates the possible existence of an L5 thymidylate synthase complex. Thymidylate synthase assays revealed that during the intracellular stage of phage growth, a significant decrease in the host thymidylate synthase (ThyA) activity occurred. It appears that synthesis of the viral enzyme (ThyX) is necessary to compensate for this loss in activity. In general, the results suggest that phage-encoded nucleotide metabolism-related functions play an important role in the lytic propagation of L5 and related mycobacteriophages.

Drug-resistant bacteria represent a significant global threat. Given the dearth of new antibiotics, host-directed therapies (HDTs) are especially desirable. As IFN-gamma (IFNγ) plays a central role in host resistance to intracellular bacteria, including Mycobacterium tuberculosis, we searched for small molecules to augment the IFNγ response in macrophages. Using an interferon-inducible nuclear protein Ipr1 as a biomarker of macrophage activation, we performed a high-throughput screen and identified molecules that synergized with low concentration of IFNγ. Several active compounds belonged to the flavagline (rocaglate) family. In primary macrophages a subset of rocaglates 1) synergized with low concentrations of IFNγ in stimulating expression of a subset of IFN-inducible genes, including a key regulator of the IFNγ network, Irf1; 2) suppressed the expression of inducible nitric oxide synthase and type I IFN and 3) induced autophagy. These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage. These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to DNA synthesis. One such gene is 65, which encodes a protein belonging to the RecA/DnaB helicase superfamily. In this study a recombinant version of the mycobacteriophage D29 gp65 was functionally characterized. The results indicated that it is not a helicase as predicted but an exonuclease that removes 3 arms from forked structures in an ATP-dependent manner. The gp65 exonuclease acts progressively from the 3 end, until the fork junction is reached. As it goes past, its progress is stalled over a stretch of seven to eight nucleotides immediately downstream of the junction. It efficiently acts on forked structures with single stranded arms. It also acts upon 5 and 3 flaps, though with somewhat relaxed specificity, but not on double-stranded forks. Sequence comparison revealed the presence of a KNRXG motif in the C-terminal half of the protein. This is a conserved element found in the RadA/Sms family of DNA repair proteins. A mutation (R203G) in this motif led to complete loss of nuclease activity. This indicated that KNRXG plays an important role in the nuclease function of not only gp65, but possibly other RadA/Sms family proteins as well. This is the first characterization of a bacteriophagederived RadA/Sms class protein. Given its mode of action, it is very likely that gp65 is involved in processing branched replication intermediates formed during the replication of phage DNA.