Structure, substrate binding and activity of a unique AAA+ protein: the BrxL phage restriction factor.

Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication.

Identification and characterization of the WYL BrxR protein and its gene as separable regulatory elements of a BREX phage restriction system.

Bacteriophage exclusion ('BREX') phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ('BREX Regulator'), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer.

Genomic Diversity of Bacteriophages Infecting the Genus Acinetobacter.

The number of sequenced Acinetobacter phage genomes in the International Nucleotide Sequence Database Collaboration has increased significantly in recent years, from 37 in 2017 to a total of 139 as of January 2021 with genome sizes ranging from 31 to 378 kb. Here, we explored the genetic diversity of the Acinetobacter phages using comparative genomics approaches that included assessment of nucleotide similarity, shared gene content, single gene phylogeny, and the network-based classification tool vConTACT2. Phages infecting Acinetobacter sp. are genetically diverse and can be grouped into 8 clusters (subfamilies) and 46 sub-clusters (genera), of which 8 represent genomic singletons (additional genera). We propose the creation of five new subfamilies and suggest a reorganisation of the genus Obolenskvirus. These results provide an updated view of the viruses infecting Acinetobacter species, providing insights into their diversity.

RNA and Sugars, Unique Properties of Bacteriophages Infecting Multidrug Resistant Acinetobacter radioresistens Strain LH6.

Bacteriophages (phages) are predicted to be the most ubiquitous biological entity on earth, and yet, there are still vast knowledge gaps in our understanding of phage diversity and phage-host interactions. Approximately one hundred Acinetobacter-infecting DNA viruses have been identified, and in this report, we describe eight more. We isolated two typical dsDNA lytic podoviruses (CAP1-2), five unique dsRNA lytic cystoviruses (CAP3-7), and one dsDNA lysogenic siphovirus (SLAP1), all capable of infecting the multidrug resistant isolate Acinetobacter radioresistens LH6. Using transmission electron microscopy, bacterial mutagenesis, phage infectivity assays, carbohydrate staining, mass-spectrometry, genomic sequencing, and comparative studies, we further characterized these phages. Mutation of the LH6 initiating glycosyltransferase homolog, PglC, necessary for both O-linked glycoprotein and capsular polysaccharide (CPS) biosynthesis, prevented infection by the lytic podovirus CAP1, while mutation of the pilin protein, PilA, prevented infection by CAP3, representing the lytic cystoviruses. Genome sequencing of the three dsRNA segments of the isolated cystoviruses revealed low levels of homology, but conserved synteny with the only other reported cystoviruses that infect Pseudomonas species. In Pseudomonas, the cystoviruses are known to be enveloped phages surrounding their capsids with the inner membrane from the infected host. To characterize any membrane-associated glycoconjugates in the CAP3 cystovirus, carbohydrate staining was used to identify a low molecular weight lipid-linked glycoconjugate subsequently identified by mutagenesis and mass-spectrometry as bacterial lipooligosaccharide. Together, this study demonstrates the isolation of new Acinetobacter-infecting phages and the determination of their cell receptors. Further, we describe the genomes of a new genus of Cystoviruses and perform an initial characterization of membrane-associated glycoconjugates.

The characteristics and genome analysis of vB_ApiP_XC38, a novel phage infecting Acinetobacter pittii.

Acinetobacter pittii is an important pathogen causing nosocomial infection worldwide. In this study, a multidrug-resistant A. pittii ABC38 was used as host bacterium to isolate the lytic phage vB_ApiP_XC38. The biological characteristics of vB_ApiP_XC38 were studied and the genome was sequenced and analyzed. vB_ApiP_XC38 belonged to Podoviridae family. The phage had double-stranded genome, which comprised 79,328 bp with 39.58% G+C content displaying very low similarity (< 1% identity) with published genomes of other phages and bacteria. A total of 97 open reading frames (ORFs) were predicted and contained nucleotide metabolism and replication module, structural components module, and lysis module. The ANI, AAI, and phylogenetic analysis indicated that all phages were found distant from vB_ApiP_XC38. Altogether, morphological, genomics, and phylogenetic analysis suggest that vB_ApiP_XC38 is more likely a novel phage of A. pittii.

Bacteriophage of the Skin Microbiome in Patients with Psoriasis and Healthy Family Controls.

The bacteriophage (phage) component of the skin microbiome in patients with psoriasis has not been systematically explored. The purpose of this study is to investigate phage and bacterial components of the skin microbiome in patients with psoriasis and in healthy family controls. Lesional skin swabs of four different locations (elbow, forearm, knee, and scalp) were taken from patients with psoriasis. Healthy skin swabs of matched locations were taken from contralateral non-lesional skin and healthy family controls. Skin microbiomes were investigated using next-generation shotgun metagenomics sequencing. 81 skin microbiome samples (27 lesional skin samples and 54 healthy skin samples from contralateral non-lesional skin and family controls) obtained from 16 subjects with psoriasis and 16 matched family controls were sequenced and analyzed. Among phage species with abundant host bacteria, two significantly differential abundant phage species, Acinetobacter phage Presley and Pseudomonas phage O4 (adjusted P < 0.05), between psoriasis lesional skin and healthy skin were identified. Samples with high levels of these phage species had their host bacteria abundance suppressed (P = 0.03 and P < 0.001). Differential phage composition between lesional skin in patients with psoriasis and healthy skin from contralateral non-lesional sites and family controls, as well as the suppression of bacteria host of the respective phage, suggest possible avenues for probiotic phage therapeutics.

Virus-like particle-display of the enterotoxigenic Escherichia coli heat-stable toxoid STh-A14T elicits neutralizing antibodies in mice.

Enterotoxigenic Escherichia coli (ETEC) causes diarrhoea by secreting enterotoxins into the small intestine. Human ETEC strains may secrete any combination of three enterotoxins: the heat-labile toxin (LT) and the heat-stable toxins (ST), of which there are two variants, called human ST (STh) and porcine ST (STp). Strains expressing STh, either alone or in combination with LT and/or STp, are among the four most important diarrhoea-causing pathogens affecting children in low- and middle-income countries. ST is therefore an attractive target for ETEC vaccine development. To produce a safe ST-based vaccine, several challenges must be solved. ST must be rendered immunogenic and non-toxic, and antibodies elicited by an ST vaccine should neutralize ST but not cross-react with the endogenous ligands uroguanylin and guanylin. Virus-like particles (VLPs) tend to be highly immunogenic and are increasingly being used as carriers for presenting heterologous antigens in new vaccines. In this study, we have coupled native STh and the STh-A14T toxoid to the coat protein of Acinetobacter phage AP205 by using the SpyCatcher system and immunized mice with these VLPs without the use of adjuvants. We found that both STs were efficiently coupled to the VLP, that both the STh and STh-A14T VLPs were immunogenic in mice, and that the resulting serum antibodies could completely neutralize the toxic activities of native STh. The serum antibodies showed a high degree of immunological cross-reaction to STp, while there was little or no unwanted cross-reaction to uroguanylin and guanylin. Moreover, compared to native STh, the STh-A14T mutation did not seem to negatively impact the immunogenicity of the construct or the neutralizing ability of the resulting sera. Taken together, these findings demonstrate that VLPs are suitable carriers for making STs immunogenic, and that the STh-A14T-coupled AP205 VLP represents a promising ETEC vaccine candidate.

Characterization of vB_ApiM_fHyAci03, a novel lytic bacteriophage that infects clinical Acinetobacter strains.

We present here the isolation and characterization of Acinetobacter pittii phage vB_ApiM_fHyAci03 (fHyAci03), which belongs to the family Myoviridae. The fHyAci03 genome was found to be 165,975 bp in length and predicted to contain 255 genes. While the whole genome was 92.4% identical to Acinetobacter baumannii phage KARL-1, phylogenetic analysis based on phage long distal tail fiber amino acid sequences assigned fHyAci03 and KARL-1 to different subclusters, reflecting their different host species. Together with phylogenetic analysis, genome comparisons indicated that fHyAci03 is a novel member of the subfamily Tevenvirinae. Host range experiments revealed that fHyAci03 could infect two clinical strains of Acinetobacter nosocomialis and six clinical strains of A. pittii. Thus, fHyAci03 is a novel lytic phage that infects clinical Acinetobacter strains and represents a potential new candidate to be used in phage therapy.

Expression and replication of virus-like circular DNA in human cells.

The consumption of bovine milk and meat is considered a risk factor for colon- and breast cancer formation, and milk consumption has also been implicated in an increased risk for developing Multiple Sclerosis (MS). A number of highly related virus-like DNAs have been recently isolated from bovine milk and sera and from a brain sample of a MS patient. As a genetic activity of these Acinetobacter-related bovine milk and meat factors (BMMFs) is unknown in eukaryotes, we analyzed their expression and replication potential in human HEK293TT cells. While all analyzed BMMFs show transcriptional activity, the MS brain isolate MSBI1.176, sharing homology with a transmissible spongiform encephalopathy-associated DNA molecule, is transcribed at highest levels. We show expression of a replication-associated protein (Rep), which is highly conserved among all BMMFs, and serological tests indicate a human anti-Rep immune response. While the cow milk isolate CMI1.252 is replication-competent in HEK293TT cells, replication of MSBI1.176 is complemented by CMI1.252, pointing at an interplay during the establishment of persistence in human cells. Transcriptome profiling upon BMMF expression identified host cellular gene expression changes related to cell cycle progression and cell viability control, indicating potential pathways for a pathogenic involvement of BMMFs.

A prokaryotic viral sequence is expressed and conserved in mammalian brain.

A natural and permanent transfer of prokaryotic viral sequences to mammals has not been reported by others. Circular "SPHINX" DNAs <5 kb were previously isolated from nuclease-protected cytoplasmic particles in rodent neuronal cell lines and brain. Two of these DNAs were sequenced after Φ29 polymerase amplification, and they revealed significant but imperfect homology to segments of commensal Acinetobacter phage viruses. These findings were surprising because the brain is isolated from environmental microorganisms. The 1.76-kb DNA sequence (SPHINX 1.8), with an iteron before its ORF, was evaluated here for its expression in neural cells and brain. A rabbit affinity purified antibody generated against a peptide without homology to mammalian sequences labeled a nonglycosylated ∼41-kDa protein (spx1) on Western blots, and the signal was efficiently blocked by the competing peptide. Spx1 was resistant to limited proteinase K digestion, but was unrelated to the expression of host prion protein or its pathologic amyloid form. Remarkably, spx1 concentrated in selected brain synapses, such as those on anterior motor horn neurons that integrate many complex neural inputs. SPHINX 1.8 appears to be involved in tissue-specific differentiation, including essential functions that preserve its propagation during mammalian evolution, possibly via maternal inheritance. The data here indicate that mammals can share and exchange a larger world of prokaryotic viruses than previously envisioned.

Isolation and characterization of a virulent bacteriophage infecting Acinetobacter johnsonii from activated sludge.

A double-stranded DNA phage named AJO1, infecting Acinetobacter johnsonii, which plays an important role in wastewater treatment, was isolated from activated sludge in a full-scale municipal wastewater treatment plant. Based on morphological taxonomy, AJO1, with an icosahedral head 55 ± 2 nm in diameter and a non-contractile tail 8 ± 2 nm in length, was classified as a member of the Podoviridae family. Bacterial infection characteristics were as follows: no polyvalent infectivity, optimal multiplicity of infection of 10-2; eclipse and burst size of 30 min and 51.2 PFU-infected cells-1, respectively. It showed considerable infectivity under a neutral pH condition (pH 6.0-9.0) and relatively high temperature (55 °C). Whole-genome sequencing of AJO1 revealed a linearly permuted DNA (41 437 bp) carrying 54 putative open reading frames and 4 repeats. This is the first report of isolation of an A. johnsonii phage, whose bacteriophage distribution and population dynamics are not well known. The results of this study could contribute to subsequent research on the interaction between bacteriophages and their hosts during wastewater treatment. In addition, AJO1 may become a candidate for potential therapy against A. johnsonii infection in clinical applications, since this species is an opportunistic pathogen.

Comparative Analysis of 37 Acinetobacter Bacteriophages.

Members of the genus Acinetobacter are ubiquitous in the environment and the multiple-drug resistant species A. baumannii is of significant clinical concern. This clinical relevance is currently driving research on bacterial viruses infecting A. baumannii, in an effort to implement phage therapy and phage-derived antimicrobials. Initially, a total of 42 Acinetobacter phage genome sequences were available in the international nucleotide sequence databases, corresponding to a total of 2.87 Mbp of sequence information and representing all three families of the order Caudovirales and a single member of the Leviviridae. A comparative bioinformatics analysis of 37 Acinetobacter phages revealed that they form six discrete clusters and two singletons based on genomic organisation and nucleotide sequence identity. The assignment of these phages to clusters was further supported by proteomic relationships established using OrthoMCL. The 4067 proteins encoded by the 37 phage genomes formed 737 groups and 974 orphans. Notably, over half of the proteins encoded by the Acinetobacter phages are of unknown function. The comparative analysis and clustering presented enables an updated taxonomic framing of these clades.

Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses.

BACKGROUND: Malaria, caused by Plasmodium falciparum, continues to have a devastating impact on global health, emphasizing the great need for a malaria vaccine. The circumsporozoite protein (CSP) is an attractive target for a malaria vaccine, and forms a major component of RTS,S, the most clinically advanced malaria vaccine. The clinical efficacy of RTS,S has been moderate, yet has demonstrated the viability of a CSP-based malaria vaccine. In this study, a vaccine comprised of the full-length CSP antigen presented on a virus-like particle (VLP) is produced using a split-intein conjugation system (SpyTag/SpyCatcher) and the immunogenicity is tested in mice. METHODS: Full-length 3d7 CSP protein was genetically fused at the C-terminus to SpyCatcher. The CSP-SpyCatcher antigen was then covalently attached (via the SpyTag/SpyCatcher interaction) to Acinetobacter phage AP205 VLPs which were modified to display one SpyTag per VLP subunit. To evaluate the VLP-display effect, the immunogenicity of the VLP vaccine was tested in mice and compared to a control vaccine containing AP205 VLPs plus unconjugated CSP. RESULTS: Full-length CSP was conjugated at high density (an average of 112 CSP molecules per VLP) to AP205 SpyTag-VLPs. Vaccination of mice with the CSP Spy-VLP vaccine resulted in significantly increased antibody titres over a course of 7 months as compared to the control group (2.6-fold higher at 7 months after immunization). Furthermore, the CSP Spy-VLP vaccine appears to stimulate production of IgG2a antibodies, which has been linked with a more efficient clearing of intracellular parasite infection. CONCLUSION: This study demonstrates that the high-density display of CSP on SpyTag-VLPs, significantly increases the level and quality of the vaccine-induced humoral response, compared to a control vaccine consisting of soluble CSP plus AP205 VLPs. The SpyTag-VLP platform utilized in this study constitutes a versatile and rapid method to develop highly immunogenic vaccines. It might serve as a generic tool for the cost-effective development of effective VLP-vaccines, e.g., against malaria.

NMR Spectroscopic Assignment of Backbone and Side-Chain Protons in Fully Protonated Proteins: Microcrystals, Sedimented Assemblies, and Amyloid Fibrils.

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100-111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 µg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.

Phage T4-induced DNA breaks activate a tRNA repair-defying anticodon nuclease.

The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4-excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction-modification (RM) protein and turned on by a phage-encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double-stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi RloC expressed in Escherichia coli is activated by wild-type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction.

Genome organisation of the Acinetobacter lytic phage ZZ1 and comparison with other T4-like Acinetobacter phages.

BACKGROUND: Phage ZZ1, which efficiently infects pathogenic Acinetobacter baumannii strains, is the fifth completely sequenced T4-like Acinetobacter phage to date. To gain a better understanding of the genetic characteristics of ZZ1, bioinformatics and comparative genomic analyses of the T4 phages were performed. RESULTS: The 166,687-bp double-stranded DNA genome of ZZ1 has the lowest GC content (34.4%) of the sequenced T4-like Acinetobacter phages. A total of 256 protein-coding genes and 8 tRNA genes were predicted. Forty-three percent of the predicted ZZ1 proteins share up to 73% amino acid identity with T4 proteins, and the homologous genes generally retained the same order and transcriptional direction. Beyond the conserved structural and DNA replication modules, T4 and ZZ1 have diverged substantially by the acquisition and deletion of large blocks of unrelated genes, especially in the first halves of their genomes. In addition, ZZ1 and the four other T4-like Acinetobacter phage genomes (Acj9, Acj61, 133, and Ac42) share a well-organised and highly conserved core genome, particularly in the regions encoding DNA replication and virion structural proteins. Of the ZZ1 proteins, 70, 64, 61, and 56% share up to 86, 85, 81, and 83% amino acid identity with Acj9, Acj61, 133, and Ac42 proteins, respectively. ZZ1 has a different number and types of tRNAs than the other 4 Acinetobacter phages, although some of the ZZ1-encoded tRNAs share high sequence similarity with the tRNAs from these phages. Over half of ZZ1-encoded tRNAs (5 out of 8) are related to optimal codon usage for ZZ1 proteins. However, this correlation was not present in any of the other 4 Acinetobacter phages. CONCLUSIONS: The comparative genomic analysis of these phages provided some new insights into the evolution and diversity of Acinetobacter phages, which might elucidate the evolutionary origin and host-specific adaptation of these phages.

Acinetobacter phage genome is similar to Sphinx 2.36, the circular DNA copurified with TSE infected particles.

While analyzing plasmids of Acinetobacter sp. DS002 we have detected a circular DNA molecule pTS236, which upon further investigation is identified as the genome of a phage. The phage genome has shown sequence similarity to the recently discovered Sphinx 2.36 DNA sequence co-purified with the Transmissible Spongiform Encephalopathy (TSE) particles isolated from infected brain samples collected from diverse geographical regions. As in Sphinx 2.36, the phage genome also codes for three proteins. One of them codes for RepA and is shown to be involved in replication of pTS236 through rolling circle (RC) mode. The other two translationally coupled ORFs, orf106 and orf96, code for coat proteins of the phage. Although an orf96 homologue was not previously reported in Sphinx 2.36, a closer examination of DNA sequence of Sphinx 2.36 revealed its presence downstream of orf106 homologue. TEM images and infection assays revealed existence of phage AbDs1 in Acinetobacter sp. DS002.

Complete genome sequence of bacteriophage phiAC-1 infecting Acinetobacter soli strain KZ-1.

To date, a number of bacteriophages (phages) infecting Acinetobacter species have been reported and characterized. However, Acinetobacter phages which infect A. soli have not been investigated yet. Here, we report the complete genome sequence of Acinetobacter phage phiAC-1, which belongs to the Myoviridae, infecting Acinetobacter soli strain KZ-1.

Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages.

The complete nucleotide sequence of ssRNA phage AP205 propagating in Acinetobacter species is reported. The RNA has three large ORFs, which code for the following homologues of the RNA coliphage proteins: the maturation, coat and replicase proteins. Their gene order is the same as that in coliphages. RNA coliphages or Leviviridae fall into two genera: the alloleviviruses, like Q(beta), which have a coat read-through protein, and the leviviruses, like MS2, which do not have this coat protein extension. AP205 has no read-through protein and may therefore be classified as a levivirus. A major digression from the known leviviruses is the apparent absence of a lysis gene in AP205 at the usual position, overlapping the coat and replicase proteins. Instead, two small ORFs are present at the 5' terminus, preceding the maturation gene. One of these might encode a lysis protein. The other is of unknown function. Other new features concern the 3'-terminal sequence. In all ssRNA coliphages, there are always three cytosine residues at the 3' end, but in AP205, there is only a single terminal cytosine. Distantly related viruses, like AP205 and the coliphages, do not have significant sequence identity; yet, important secondary structural features of the RNA are conserved. This is shown here for the 3' UTR and the replicase-operator hairpin. Interestingly, although AP205 has the genetic map of a levivirus, its 3' UTR has the length and RNA secondary structure of an allolevivirus. Sharing features with both MS2 and Q(beta) suggests that, in an evolutionary sense, AP205 should be placed between Q(beta) and MS2. A phylogenetic tree for the ssRNA phages is presented.

Classification of Acinetobacter phages.

Eight phage species and type viruses are proposed. They belong to the Myoviridae, Siphoviridae, and Podoviridae families of tailed phages and are characterized by a combination of morphological and physicochemical properties. An unusual siphovirus species has an elongated head and transverse tail disks.