A small mycobacteriophage-derived peptide and its improved isomer restrict mycobacterial infection via dual mycobactericidal-immunoregulatory activities.

Mycobacteriophages express various peptides/proteins to infect Mycobacterium tuberculosis (M. tb). Particular attention has been paid to mycobacteriophage-derived endolysin proteins. We herein characterized a small mycobacteriophage-derived peptide designated AK15 with potent anti-M. tb activity. AK15 adopted cationic amphiphilic α-helical structure, and on the basis of this structure, we designed six isomers with increased hydrophobic moment by rearranging amino acid residues of the helix. We found that one of these isomers, AK15-6, exhibits enhanced anti-mycobacterial efficiency. Both AK15 and AK15-6 directly inhibited M. tb by trehalose 6,6'-dimycolate (TDM) binding and membrane disruption. They both exhibited bactericidal activity, cell selectivity, and synergistic effects with rifampicin, and neither induced drug resistance to M. tb They efficiently attenuated mycobacterial load in the lungs of M. tb-infected mice. We observed that lysine, arginine, tryptophan, and an α-helix are key structural requirements for their direct anti-mycobacterial action. Of note, they also exhibited immunomodulatory effects, including inhibition of proinflammatory response in TDM-stimulated or M. tb-infected murine bone marrow-derived macrophages (BMDMs) and M.tb-infected mice and induction of only a modest level of cytokine (tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6)) production in murine BMDMs and a T-cell cytokine (interferin-γ (IFN-γ) and TNF-α) response in murine lung and spleen. In summary, characterization of a small mycobacteriophage-derived peptide and its improved isomer revealed that both efficiently restrain M. tb infection via dual mycobactericidal-immunoregulatory activities. Our work provides clues for identifying small mycobacteriophage-derived anti-mycobacterial peptides and improving those that have cationic amphiphilic α-helices.

Dissecting the structure-function relationship in lysozyme domain of mycobacteriophage D29-encoded peptidoglycan hydrolase.

Most bacteriophages rapidly infect and kill bacteria and, therefore, qualify as the next generation therapeutics for rapidly emerging drug-resistant bacteria such as Mycobacterium tuberculosis. We have previously characterized the mycobacteriophage D29-generated endolysin, Lysin A, for its activity against mycobacteria. Here, we present a detailed characterization of the lysozyme domain (LD) of D29 Lysin A that hydrolyzes peptidoglycan of both gram-positive and gram-negative bacteria with high potency. By characterizing an exhaustive LD protein variant library, we have identified critical residues important for LD activity and stability. We further complement our in vitro experiments with detailed in silico investigations. We present LD as a potent candidate for developing phage-based broad-spectrum therapeutics.

Mycobacteriophage D29 holin C-terminal region functionally assists in holin aggregation and bacterial cell death.

Holins are phage-encoded small transmembrane proteins that perforate the bacterial cytoplasmic membrane. In most cases, this process allows the phage-encoded peptidoglycan hydrolases to act on the cell wall, resulting in host cell lysis and phage release. We report a detailed functional characterization of Mycobacterium phage D29 gp11 coding for a putative holin that, upon expression, rapidly kills both Escherichia coli and Mycobacterium smegmatis. We dissected Gp11 by making several deletions and expressing them in E. coli. The shortening of Gp11 from its C-terminus results in diminished cytotoxicity and smaller holes. Evidently, the two transmembrane domains (TMDs) present at the N-terminus of Gp11 are incapable of integrating into the cytoplasmic membrane and do not show toxicity. Interestingly, the fusion of two TMDs and a small C-terminal region that bears the coiled-coil motif resulted in restoration of the cell killing ability of the protein. We further show that the second TMD is dispensable in protein toxicity because its deletion does not abolish Gp11-mediated cell death. We conclude that Gp11 C-terminal region is necessary but not sufficient for toxicity. These results shed light on a yet undiscovered role of Gp11 C-terminal region that will help clarify the mechanism of holin-mediated membrane perforation. Finally, we abolish the toxicity of Gp11 using a specific Gly to Asp substitution in the putative loop region of the protein; the mutant protein may help to clarify how holin functions in mycobacteriophage D29.

Genomics and proteomics of mycobacteriophage patience, an accidental tourist in the Mycobacterium neighborhood.

UNLABELLED: Newly emerging human viruses such as Ebola virus, severe acute respiratory syndrome (SARS) virus, and HIV likely originate within an extant population of viruses in nonhuman hosts and acquire the ability to infect and cause disease in humans. Although several mechanisms preventing viral infection of particular hosts have been described, the mechanisms and constraints on viral host expansion are ill defined. We describe here mycobacteriophage Patience, a newly isolated phage recovered using Mycobacterium smegmatis mc(2)155 as a host. Patience has genomic features distinct from its M. smegmatis host, including a much lower GC content (50.3% versus 67.4%) and an abundance of codons that are rarely used in M. smegmatis. Nonetheless, it propagates well in M. smegmatis, and we demonstrate the use of mass spectrometry to show expression of over 75% of the predicted proteins, to identify new genes, to refine the genome annotation, and to estimate protein abundance. We propose that Patience evolved primarily among lower-GC hosts and that the disparities between its genomic profile and that of M. smegmatis presented only a minimal barrier to host expansion. Rapid adaptions to its new host include recent acquisition of higher-GC genes, expression of out-of-frame proteins within predicted genes, and codon selection among highly expressed genes toward the translational apparatus of its new host. IMPORTANCE: The mycobacteriophage Patience genome has a notably lower GC content (50.3%) than its Mycobacterium smegmatis host (67.4%) and has markedly different codon usage biases. The viral genome has an abundance of codons that are rare in the host and are decoded by wobble tRNA pairing, although the phage grows well and expression of most of the genes is detected by mass spectrometry. Patience thus has the genomic profile of a virus that evolved primarily in one type of host genetic landscape (moderate-GC bacteria) but has found its way into a distinctly different high-GC environment. Although Patience genes are ill matched to the host expression apparatus, this is of little functional consequence and has not evidently imposed a barrier to migration across the microbial landscape. Interestingly, comparison of expression levels and codon usage profiles reveals evidence of codon selection as the genome evolves and adapts to its new environment.

Data-independent acquisition (MSE) with ion mobility provides a systematic method for analysis of a bacteriophage structural proteome.

In this work, a method was developed to study the structural proteome of mycobacteriophage Marvin, a recent isolate from soil with 107 predicted coding sequences. This prototype method was applied for semi-quantitative analysis of the composition of this mycobacteriophage virion using ion mobility spectrometry and data-independent acquisition (MS(E)-IMS). MS(E)-IMS was compared to a more conventional proteomics technique employing mass spectrometry with a data-dependent acquisition strategy. MS(E)-IMS provided broad coverage of the virion proteome and high sequence coverage for individual proteins. This shotgun method does not depend on the limited sensitivity of visualization of protein bands by staining reagents inherent in gel-based methods. The method is comprehensive, provides high sequence coverage and is proposed as a particularly efficient method for the study of bacteriophage proteomes.

Pro-Gly mediated conformational switch of mycobacteriophage D29 holin transmembrane domain I is lipid concentration driven.

Biophysical and spectroscopic analysis of synthetic transmembrane domain I (1) of mycobacteriophage D29 holin shows a lipid concentration dependent conformational switch from an α-helix to a ß-sheet structure. The reversibility of this switch, upon change in the lipid-to-peptide ratio, requires a central Pro-Gly segment, and is abolished upon mutation to Ala-Ala or (D)Pro-Gly.

The secret lives of mycobacteriophages.

The study of mycobacteriophages provides insights into viral diversity and evolution, as well as the genetics and physiology of their pathogenic hosts. Genomic characterization of 80 mycobacteriophages reveals a high degree of genetic diversity and an especially rich reservoir of interesting genes. These include a vast number of genes of unknown function that do not match known database entries and many genes whose functions can be predicted but which are not typically found as components of phage genomes. Thus many mysteries surround these genomes, such as why the genes are there, what do they do, how are they expressed and regulated, how do they influence the physiology of the host bacterium, and what forces of evolution directed them to their genomic homes? Although the genetic diversity and novelty of these phages is full of intrigue, it is a godsend for the mycobacterial geneticist, presenting an abundantly rich toolbox that can be exploited to devise new and effective ways for understanding the genetics and physiology of human tuberculosis. As the number of sequenced genomes continues to grow, their mysteries continue to thicken, and the time has come to learn more about the secret lives of mycobacteriophages.

Insights into the function of the WhiB-like protein of mycobacteriophage TM4--a transcriptional inhibitor of WhiB2.

WhiB-like proteins of actinomycetes are known to co-ordinate iron-sulfur (Fe-S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB-like genes in these viruses. Here we focussed on the WhiB-like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co-ordinating a Fe-S cluster. The UV-visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe-2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA-levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apo-WhiB2 of Mycobacterium tuberculosis and apo-WhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe-S containing transcriptional regulators with DNA-binding capability.

Recombineering in Mycobacterium tuberculosis.

Genetic dissection of M. tuberculosis is complicated by its slow growth and its high rate of illegitimate recombination relative to homologous DNA exchange. We report here the development of a facile allelic exchange system by identification and expression of mycobacteriophage-encoded recombination proteins, adapting a strategy developed previously for recombineering in Escherichia coli. Identifiable recombination proteins are rare in mycobacteriophages, and only 1 of 30 genomically characterized mycobacteriophages (Che9c) encodes homologs of both RecE and RecT. Expression and biochemical characterization show that Che9c gp60 and gp61 encode exonuclease and DNA-binding activities, respectively, and expression of these proteins substantially elevates recombination facilitating allelic exchange in both M. smegmatis and M. tuberculosis. Mycobacterial recombineering thus provides a simple approach for the construction of gene replacement mutants in both slow- and fast-growing mycobacteria.

MSTF: a domain involved in bacterial metallopeptidases and surface proteins, mycobacteriophage tape-measure proteins and fungal proteins.

Here we report a novel domain, MSTF (domain involved in bacterial metallopeptidases, surface proteins and other proteins, also present in mycobacteriophage tape-measure proteins and fungal proteins), which is present in bacteria, phages and fungi. MSTF is about 67-94 amino acids in length with one HxDHxH motif and some highly conserved residues including His, Gly, Ala and Asp. Secondary structure prediction indicated that this domain contains two alpha-helices and one beta-sheet. Identification of MSTF will provide an opportunity to develop new strategies to combat pathogenic microorganisms, especially Mycobacterium tuberculosis.

Mycobacteriophage TM4: genome structure and gene expression.

Mycobacteriophage TM4 is a dsDNA-tailed phage that infects both fast-growing and slow-growing strains of mycobacteria. While TM4 has been used extensively for the construction of mycobacterial shuttle phasmids and for the delivery of reporter genes and transposons into mycobacterial cells, little is known about its genetics or molecular biology. We describe here the complete 52,797 bp genome sequence of TM4 and a map of its genome organization. While not a close relative of other mycobacteriophages, TM4 encodes several proteins with sequence similarity to those of other bacteriophages--including L5 and D29--indicating that they have common ancestry. In addition, TM4 encodes proteins with similarity to haloperoxidases, glutaredoxins and the WhiB family of transcriptional regulators. Following infection, TM4 genes are expressed in a defined temporal pattern, with the virion structural proteins expressed late in the phage growth cycle. Understanding the genetics of TM4 will greatly facilitate its use as a tool for the genetic manipulation of the mycobacteria.

Structural proteins of mycobacteriophage I3: cloning, expression and sequence analysis of a gene encoding a 70-kDa structural protein.

The structural proteins of mycobacteriophage I3 have been analysed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), radioiodination and immunoblotting. Based on their abundance the 34- and 70-kDa bands appeared to represent the major structural proteins. Successful cloning and expression of the 70-kDa protein-encoding gene of phage I3 in Escherichia coli and its complete nucleotide sequence determination have been accomplished. A second (partial) open reading frame following the stop codon for the 70-kDa protein was also identified within the cloned fragment. The deduced amino-acid sequence of the 70-kDa protein and the codon usage patterns indicated the preponderance of codons, as predicted from the high G+C content of the genomic DNA of phage I3.