Bacteriophage-encoded Proteins Research Articles

Bacteriophage-encoded protein utilization in bacterial ghost production: a mini-review.

Bacterial ghosts (BGs) are described as bacterial cell envelopes that retain their structure but lack cytoplasmic contents. The study of BGs spans multiple disciplinary domains, and the development of BG production techniques to obtain ample and stable BG samples holds significant implications for probing the biological characteristics of BGs, devising novel disease treatment strategies, and leveraging their industrial applications. Numerous products encoded within bacteriophage (phage) genomes possess the capability to lyse bacteria, thereby inducing BG formation primarily via disruption of bacterial cell wall integrity. This review comprehensively surveys the utilization of phage-encoded proteins in BG production techniques, encompassing methodologies such as phage E protein-mediated lysis, perforin protein-induced lysis, and strategies combining E protein with holin-endolysin systems. Additionally, discussions and summaries are provided on the current applications, challenges, and modification strategies associated with different techniques. Through a focused exploration of BG production techniques, with an emphasis on precise manipulation of BG formation using phage-encoded protein technologies, this study aims to furnish robust tools and methodologies for delving into the mechanisms underlying BG formation, as well as for the development of novel therapeutic strategies and applications based on BGs.

The Cytotoxic Mycobacteriophage Protein Phaedrus gp82 Interacts with and Modulates the Activity of the Host ATPase, MoxR

Approximately 70% of bacteriophage-encoded proteins are of unknown function. Elucidating these protein functions represents opportunities to discover new phage-host interactions and mechanisms by which the phages modulate host activities. Here, we describe a pipeline for prioritizing phage-encoded proteins for structural analysis and characterize the gp82 protein encoded by mycobacteriophage Phaedrus. Structural and solution studies of gp82 show it is a trimeric protein containing two domains. Co-precipitation studies with the host Mycobacterium smegmatis identified the ATPase MoxR as an interacting partner protein. Phaedrus gp82-MoxR interaction requires the presence of a loop sequence within gp82 that is highly exposed and disordered in the crystallographic structure. We show that Phaedrus gp82 overexpression in M. smegmatis retards the growth of M. smegmatis on solid medium, resulting in a small colony phenotype. Overexpression of gp82 containing a mutant disordered loop or the overexpression of MoxR both rescue this phenotype. Lastly, we show that recombinant gp82 reduces levels of MoxR-mediated ATPase activity in vitro that is required for its chaperone function, and that the disordered loop plays an important role in this phenotype. We conclude that Phaedrus gp82 binds to and reduces mycobacterial MoxR activity, leading to reduced function of host proteins that require MoxR chaperone activity for their normal activity.

Possible Mistranslation of Shiga Toxin from Pathogenic Escherichia coli as Measured by MALDI-TOF and Orbitrap Mass Spectrometry

Rationale: Shiga toxin-producing Escherichia coli (STEC) are often subjected to DNA damaging antibiotics during culturing in order to elicit the bacterial SOS response and up-regulation of bacteriophage-encoded proteins including Shiga toxin (Stx). However, such antibiotic exposure and stress may also have effects on protein expression.Methods: Escherichia coli O157:H7 strain EDL933 was grown on Luria-Bertani agar (LBA) supplemented with a sub-inhibitory concentration of ciprofloxacin. Bacterial cells were harvested, suspended in water, gently vortexed and centrifuged. Supernatants were analyzed by MALDI-TOF and nano-LC-ESI-Orbitrap mass spectrometry. A gene knockout was constructed to delete the B-subunit gene from the stx2a operon in the EDL933 strain. Results: We detected the B-subunits of Stx1a and Stx2a and also peaks in close proximity to these B-subunits. The mass difference between these variants and the Stx1a B-subunit are: -43 Da, +16 Da and +54 Da. For Stx2a B-subunit, the mass differences are: -111 Da, -91 Da, -72 Da, -59 Da, -44 Da, -29 Da, -15/-17 Da, +16 Da, +32 Da, +53/54 Da, +106 Da. When the stx2a gene knockout strain was cultured, it revealed the complete absence of the Stx2a B-subunit as well as its associated mass variants suggesting that the variants may be due to amino acid substitutions caused by translational errors. Conclusions: Our results suggest that ciprofloxacin (a fluoroquinolone antibiotic) may cause translational errors in expression of Stx. Incorporation of mistranslated B-subunit sequences into the Stx AB5 holotoxin has the potential to subtly alter its quaternary structure and its binding affinity to surface receptors of eukaryotic cells.

Real-Time Assessment of Staphylococcus aureus Biofilm Disruption by Phage-Derived Proteins.

A current focus of research is the development of new tools for removing bacterial biofilms in industrial settings. Bacteriophage-encoded proteins, such as endolysins, virion-associated peptidoglycan hydrolases, and exopolysaccharide depolymerases, have been shown to be efficient against these structures. However, the current screening techniques for the identification of antibiofilm properties of phage-derived proteins have important shortcomings. The aim of this work was to use the rapid, reproducible and accurate technology “real-time cell analyzer” for screening and comparing the antibiofilm ability of four phage-derived compounds, three lytic proteins (LysH5, CHAP-SH3b, and HydH5-SH3b) and one exopolysaccharide depolymerase (Dpo7) against Staphylococcus aureus biofilms, which have been associated with recurrent contamination of food products. The data generated after biofilm treatment allowed for the calculation of different antibiofilm parameters: (1) the minimum biofilm eradicating concentration that removes 50% of the biofilm (ranging from 3.5 ± 1.1 to 6.6 ± 0.5 μM), (2) the lowest concentration needed to observe an antibiofilm effect (∼1.5 μM for all the proteins), and (3) the specific antibiofilm activity and the percentage of biofilm removal that revealed LysH5 as the best antibiofilm compound. Overall, this technology might be used to quickly assess and compare by standardized parameters the disaggregating activity of phage antibiofilm proteins.

Bacteriophage cell lysis of Shiga toxin-producing Escherichia coli for top-down proteomic identification of Shiga toxins 1 & 2 using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.

Analysis of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) often relies upon sample preparation methods that result in cell lysis, e.g. bead-beating. However, Shiga toxin-producing Escherichia coli (STEC) can undergo bacteriophage-induced cell lysis triggered by antibiotic exposure that may allow greater selectivity of the proteins extracted. We have developed a sample preparation method for selective extraction of bacteriophage-encoded proteins and specifically Shiga toxins 1 and 2 (Stx1 & 2) expressed from STEC strains induced by DNA-damaging antibiotics. STEC strains were cultured overnight on agar supplemented with ciprofloxacin, mitomycin-C or an iron chelator to induce the bacteriophage lytic cycle with concomitant expression and release of Stx1 and/or Stx2. Sample preparation relied exclusively on bacteriophage lysis for release Stx into the extraction solution. Three clinical STEC strains were analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics analysis: E. coli O157:H7 strain EDL933, E. coli O91:H21 strain B2F1 and E. coli O26:H11 strain ECRC #05.2217. The B-subunit of Stx1a of EDL933 was detected and identified even though it was ~100-fold less abundant than the B-subunit of Stx2a that had been identified previously for this strain. Two bacteriophage-encoded proteins were also identified: L0117 and L0136. The B-subunits of Stx2d of strain B2F1 and Stx1a of strain ECRC #05.2217 were also detected and identified. Bacteriophage lysis appeared to enhance the detection sensitivity of Stx for these STEC strains compared to previous work using mechanical lysis. Detection/identification of other bacteriophage-encoded proteins (beyond Stx) tends to support the hypothesis of Stx release by bacteriophage cell lysis.

Phage as a source of antibacterial genes

Bacteriophage-encoded proteins which inhibit or modify cellular components may contribute to antibacterial drug discovery by allowing the identification of novel targets. Given their abundance and diversity, phages may have various strategies in host inhibition and therefore may possess a variety of such proteins. Using Rhodococcus equi and phage YF1, we show that a single phage possesses numerous genes that inhibit the host when introduced into the host on a plasmid. These genes mostly encode proteins of unknown function, confirming the potential that this approach may have in providing new antibacterial targets.

Streptococcus mitis Phage-Encoded Adhesins Mediate Attachment to α2-8-Linked Sialic Acid Residues on Platelet Membrane Gangliosides

The direct binding of bacteria to human platelets contributes to the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis strain SF100 is mediated in part by two bacteriophage-encoded proteins, PblA and PblB. However, the platelet membrane receptor for these adhesins has been unknown. In this study, we demonstrate that these proteins mediate attachment of bacterial cells to sialylated gangliosides on the platelet cell surface. Desialylation of human platelet monolayers reduced adherence of SF100, whereas treatment of the platelets with N- or O-glycanases did not affect platelet binding. Treatment of platelets with sialidases having different linkage specificities showed that removal of alpha2-8-linked sialic acids resulted in a marked reduction in bacterial binding. Preincubation of SF100 with ganglioside GD3, a glycolipid containing alpha2-8-linked sialic acids that is present on platelet membranes, blocked subsequent binding of this strain to these cells. In contrast, GD3 had no effect on the residual binding of platelets by strain PS344, an isogenic DeltapblA DeltapblB mutant. Preincubating platelets with specific monoclonal antibodies to ganglioside GD3 also inhibited binding of SF100 to platelets, but again, they had no effect on binding by PS344. When the direct binding of S. mitis strains SF100 and PS344 to immobilized gangliosides was tested, binding of PS344 to GD3 was reduced by 70% compared to the parent strain. These results indicated that platelet binding by SF100 is mediated by the interaction of PblA and PblB with alpha2-8-linked sialic acids on ganglioside GD3.

Phylogenetic analysis of Shiga toxin 1 and Shiga toxin 2 genes associated with disease outbreaks

BackgroundShiga toxins 1 and 2 (Stx1 and Stx2) are bacteriophage-encoded proteins that have been associated with hemorrhagic colitis, hemolytic uremic syndrome and other severe disease conditions. Stx1 and Stx2 are genetically and immunologically distinct but share the same compound toxin structure, method of entry and enzymatic function.ResultsPhylogenetic analysis was performed using Stx1 and Stx2 amino acid and nucleotide sequences from 41 strains of Escherichia coli, along with known stx sequences available from GenBank. The analysis confirmed the Stx1 and Stx2 divergence, and showed that there is generally more sequence variation among stx2 genes than stx1. The phylograms showed generally flat topologies among our strains' stx1 and stx2 genes. In the stx2 gene, 39.5% of the amino acid sites display very low nonsynonymous to synonymous substitution ratios.ConclusionThe stx1 and stx2 genes used in this phylogenetic study show sequence conservation with no significant divergence with respect to place or time. These data could indicate that Shiga toxins are experiencing purifying selection.

The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129.

Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium's nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.

The molecular basis of co-operative DNA binding between lambda integrase and excisionase.

Higher-order nucleoprotein complexes often stabilize catalytic proteins in appropriate conformations for optimal activity and contribute to regulation during reactions requiring association of proteins and DNA. Formation of such complexes, known as intasomes, is required for site-specific recombination catalysed by bacteriophage Lambda Integrase protein (Int). Int-catalysed recombination is regulated by a second bacteriophage-encoded protein, Excisionase (Xis), which both stimulates excision and inhibits integration. To exert its effect, Xis binds co-operatively with Int, thereby inducing and stabilizing a DNA bend that alters the intasome structures formed during recombination. A rare int mutant, int 2268 ts, was reported (Enquist, L.W. and Weisberg, R.A. (1984) Mol Gen Genet 195: 62-69) to be more defective for excision than integration. Here, we have determined that this mutant Int protein contains an E47K substitution, and that the resultant excision-specific defect is due, at least in part, to destabilized interactions between Int and Xis. Analysis of several engineered substitutions at Int position 47 showed that a negatively charged residue is required for co-operative DNA binding between Int and Xis, and suggest that the Int-E47 residue may contact Xis directly. Substitutions at Int position 47 also affect co-operative binding among Int proteins at arm-type DNA sites, and thereby reduce the efficiency of both integration and excision. Collectively, these results suggest that a single surface of the Int amino-terminal domain mediates two alternate types of co-operative binding interactions.

Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase.

A functional immune system depends on the production of a wide range of immunoglobulin molecules. Immunoglobulin variable region (IgV) genes are diversified after gene rearrangement by hypermutation. In the DNA deamination model, we have proposed that deamination of dC residues to dU by activation-induced deaminase (AID) triggers this diversification. In hypermutating chicken DT40 B cells, most IgV mutations are dC --> dG/dA or dG --> dC/dT transversions, which are proposed to result from replication over sites of base loss produced by the excision activity of uracil-DNA glycosylase. Blocking the activity of uracil-DNA glycosylase should instead lead to replication over the dU lesion, resulting in dC --> dT (and dG --> dA) transitions. Here we show that expression in DT40 cells of a bacteriophage-encoded protein that inhibits uracil-DNA glycosylase shifts the pattern of IgV gene mutations from transversion dominance to transition dominance. This is good evidence that antibody diversification involves dC --> dU deamination within the immunoglobulin locus itself.

Holins kill without warning.

Holins comprise the most diverse functional group of proteins known. They are small bacteriophage-encoded proteins that accumulate during the period of late-protein synthesis after infection and cause lysis of the host cell at a precise genetically programmed time. It is unknown how holins achieve temporal precision, but a conserved feature of their function is that energy poisons subvert the normal scheduling mechanism and instantly trigger membrane disruption. On this basis, timing has been proposed to involve a progressive decrease in the energized state of the membrane until a critical triggering level is reached. Here, we report that membrane integrity is not compromised after the induction of holin synthesis until seconds before lysis. The proton motive force was monitored by the rotation of individual cells tethered by a single flagellum. The results suggest an alternative explanation for the lysis "clock," in which holin concentrations build to a critical level that leads to formation of an oligomeric complex that disrupts the membrane.

Folding of coliphage T4 short tail fiber in vitro. Analysing the role of a bacteriophage-encoded chaperone.

The morphogenesis of the Escherichia coli bacteriophage T4 depends on the presence of helper proteins which are not components of the mature virion. Two bacteriophage-encoded proteins, p57 and p38, are required for the assembly of the bacteriophage T4 tail fibers. In the absence of p57, two polypeptides of the long fiber (p34 and p37) and that of the short tail fiber (p12) fail to trimerize. Instead they form water-insoluble aggregates. Co-expression of the genes 12 and 57 in vivo caused the formation of only trimeric, water-soluble p12. The function of g57 cannot be replaced by overexpression of the host proteins GroEL/ES or parvulin. The mechanism of action of this helper protein has remained unknown, mainly because it has not been possible to determine its activity in vitro. Purified p12, denatured in 7 M urea, trimerized spontaneously in a slow reaction (half-time approximately 6 h) and with low yield. Upon renaturation, p12 forms native SDS-resistant trimers as indicated by spectroscopic and hydrodynamic measurements. Addition of p57 increased the rate of folding threefold and nearly doubled the yield. These experiments demonstrate that p57 acts as a molecular chaperone during folding of T4 tail fibers.

Homologous pairing proteins encoded by the Escherichia coii recE and recT genes

Early genetic analysis of alternate recombination pathways in Escherichia coli identified the RecE recombination pathway and the required exonuclease VIII encoded by the recE gene. Observations that not all recombination events promoted by the RecE pathway require recA suggest the existence of an additional homologous pairing protein besides RecA in E. coli. Genetic and biochemical analysis of the recE gene region indicates there are two partially overlapping genes, recE and recT, encoding at least two proteins: exoVIII and the RecT protein. Biochemical analysis has shown that the RecT protein, in combination with exoVIII, promotes homologous pairing and strand exchange in reactions containing linear duplex DNA and homologous, circular, singlestranded DNA as substrates. This reaction occurs in the absence of any high-energy cofactor. These two proteins, RecT and exoVIII, appear to be members of a second class of homologous pairing proteins that are required in genetic recombination and differ from the class of homologous pairing proteins that includes RecA. Members of this second class of proteins appear to include both bacteriophage-encoded proteins and proteins from eukaryotes and their viruses.

Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins.

Based on previous in vivo genetic analysis of bacteriophage lambda growth, we have developed two in vitro lambda DNA replication systems composed entirely of purified proteins. One is termed 'grpE-independent' and consists of supercoiled lambda dv plasmid DNA, the lambda O and lambda P proteins, as well as the Escherichia coli dnaK, dnaJ, dnaB, dnaG, ssb, DNA gyrase and DNA polymerase III holoenzyme proteins. The second system includes the E.coli grpE protein and is termed 'grpE-dependent'. Both systems are specific for plasmid molecules carrying the ori lambda DNA initiation site. The major difference in the two systems is that the 'grpE-independent' system requires at least a 10-fold higher level of dnaK protein compared with the grpE-dependent one. The lambda DNA replication process may be divided into several discernible steps, some of which are defined by the isolation of stable intermediates. The first is the formation of a stable ori lambda-lambda O structure. The second is the assembly of a stable ori lambda-lambda O-lambda P-dnaB complex. The addition of dnaJ to this complex also results in an isolatable intermediate. The dnaK, dnaJ and grpE proteins destabilize the lambda P-dnaB interaction, thus liberating dnaB's helicase activity, resulting in unwinding of the DNA template. At this stage, a stable DNA replication intermediate can be isolated, provided that the grpE protein has acted and/or is present. Following this, the dnaG primase enzyme recognizes the single-stranded DNA-dnaB complex and synthesizes RNA primers. Subsequently, the RNA primers are extended into DNA by DNA polymerase III holoenzyme. The proposed model of the molecular series of events taking place at ori lambda is substantiated by the many demonstrable protein-protein interactions among the various participants.