The saclayvirus Aci01-1 very long and complex fiber and its receptor at the Acinetobacter baumannii surface

The Acinetobacter baumannii bacteriophage Aci01-1, which belongs to the genus Saclayvirus of the order Caudoviricetes, has an icosahedral head and a contractile rigid tail. We report that Aci01-1 has, attached to the tail conical tip, a remarkable 146-nm-long flexible fiber with seven beads and a terminal knot. Its putative gene coding for a 241.36-kDa tail fiber protein is homologous to genes in Aci01-1-related and unrelated phages. Analysis of its 3D structure using AlphaFold provides a structural model for the fiber observed by electron microscopy. We also identified a putative receptor of the phage on the bacterial capsule that is hypothesized to interact with the Aci01-1 long fiber.


Introduction
Acinetobacter baumannii is an opportunistic pathogen that has become a major human health threat in hospital (nosocomial) and community settings, due to the proliferation of antibiotic-resistant strains [1]. This has led the World Health Organization to assign critical priority to development of antibacterial strategies to control those infections [2]. Bacterial viruses that kill bacterial cells with high specificity may become central actors for diversifying the panel of strategies for effective antibacterial treatments [3,4]. Our research aims at characterizing bacteriophages infecting A. baumannii and the interactions between these phages as part of an effort to deliver agents for controlling nosocomial infections caused by this human pathogen.
Previously, we described three virulent phages of the genus Saclayvirus, Aci01-1, Aci02-2, and Aci05, that infect A. baumannii strain Ab09 [5]. These phages have closely related genomes of 103 kbp, 104 kbp, and 103 kbp, respectively. Here, we purified and imaged by electron microscopy (EM) virions of Aci01-1 and Aci05. Their most notable feature is their very long and complex tail fiber. We also isolated bacteria resistant to Aci01-1 infection, revealing that the bacterial capsule plays a role in the sensitivity of A. baumannii to Aci01-1.
Bacteriophages Aci01-1 and Aci05 were isolated in Abidjan, Côte d'Ivoire, and their genome sequence was reported previously [5]. A. baumannii strain Ab09 was isolated from a clinical sample in France [6]. Phage amplification was performed using the double-layer agar technique. For this, 10 6 plaque-forming units (PFU) of the phage were mixed with 10 9 A. baumannii Ab09 cells in 200 µL of LB medium supplemented with 10 mM MgSO 4 , incubated at room temperature for 20 min, mixed with melted soft agar (7 g/L LB medium), and then layered onto freshly poured and solidified LB agar plates (15 g/L LB medium). After a 6-8 h incubation period at 37°C, phage particles were recovered in 4 mL of phosphate-buffered saline (PBS), reaching titers of 10 10 -10 11 PFU/mL. The suspension was treated with DNase I (50 µg/mL) and RNase A (10 µg/mL) for 1 h at 37°C, and phage particles were precipitated with 6% PEG 8000 overnight at 4°C. After centrifugation, the pellet was resuspended in phage TBT buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 10 mM MgCl 2 ). Three chloroform extractions were performed, the supernatant was passed through a 0.45-µm filter, and phage particles were pelleted by ultracentrifugation at 154,000 g for 1 h at 4°C. Phage particles were then loaded onto a preformed caesium chloride (CsCl) step gradient and centrifuged at 287,000 g for 3 h at 20 °C [7]. A band containing the phages was recovered by puncturing the tube with a needle, and the suspension was dialyzed against TBT buffer. For this, the dialysis bag containing the phage suspension was placed inside a beaker containing 1 L of TBT buffer. The buffer was changed after 4 h and 16 h. Phages were recovered after four additional hours of dialysis. To perform electron microscopy (EM) observations, three microliters of CsCl-purified phage particles were adsorbed onto a grid coated with a carbon film (EMS, Hatfield, PA, USA), washed with distilled water, and negatively stained with 2% uranyl acetate (Sigma, St. Louis, MO, USA) before imaging [8].
A. baumannii variants resisting phage infection were obtained following infection on solid medium at an input multiplicity of 0.01 Aci01-1 phages/bacterium for 48 h at 37°C. Colonies of different sizes and shapes were picked and purified by two rounds of colony isolation before testing for susceptibility to Aci01-1 infection.
For DNA purification, A. baumannii bacteria were lysed in lysis buffer (10 mM Tris-HCl, pH 7.8, 10 mM EDTA, 10 mM NaCl, and 0.5% SDS) and treated with proteinase K at 50 µg/mL -1 for 2 h at 50°C, followed by one phenol and one chloroform extraction. DNA was precipitated with ethanol and resuspended in TE buffer (10 mM Tris-HCl, pH 7.8, and 1 mM EDTA). The DNA was sequenced in an Illumina MiSeq 300-bp paired-end run with a TruSeq 900-bp insert library, producing 1.4 to 3.9 million reads. Quality control was performed using FastQC version 0.11.5, and reads were trimmed using GeneiousR11 (Biomatters Ltd., Auckland, New Zealand) with default parameters. To identify mutations in phage-resistant variants, we compared the genomes of the parental strain to those of the variants using 2-kupl as described [9].
EM observation of CsCl-purified Aci01-1 phage particles showed that they were of the myovirus morphotype, composed of an icosahedral head 80 ± 3 nm in diameter and a 128 ± 2 nm contractile tail, the sizes of which were obtained from the measurements of 10 virions (Fig. 1a). These particles were indistinguishable from Aci05 phage particles (not shown). Particles with extended and contracted tails were present in the Aci01-1 preparation ("E" and "C", respectively, in Fig. 1a,). In the contracted state, the external shaft of the tail had a reduced length, and the internal tail tube became visible. Phage tails featured a 146 ± 3 nm-long flexible fiber attached to the conical tail tip distal from the head that was no longer visible in the contracted state of the tail. Seven bead-like structural elements (arrowheads labeled 1 to 7 in Fig. 1b) were precisely positioned along the fiber, which ended with an elongated complex structure (bracket in Fig. 1b). This fiber was perfectly visible in highly purified phage particles but more difficult to detect in phage particles of crude or partially purified phage preparations. Phages lambda [16], T5 [17], and SPP1 [18] also have an axial tail fiber anchored in the conical tip of the tail tube, but the Aci01-1 fiber is much longer, and its structure is more complex.
We then searched for putative genes in the Aci01-1 genome (ID NC_048074) that could code for fiber proteins. A very long gene encoding a 2211-amino-acid (aa) protein (Aci11_031) was identified within the structural protein genes of Aci01-1. Equally long proteins are encoded by phage Aci05 (genome ID NC_048080) (2223 aa long; Aci05_030) and other Acinetobacter phages. Using different bioinformatics tools, proteins that were more distantly related exhibited a modular organization with alternating regions with and without homology to the Aci01-1 protein.
These are all annotated as putative tail fiber or tail proteins. The putative tail fiber protein of Aci11-031 has 34to 35-aa repeats, as identified using the Rapid Automatic Detection and Alignment of Repeats in protein sequences (RADAR) tool at EBI (Fig. 2a) [19]. These repeats are predicted to form coiled-coils separated by ~250 aa, as shown using REPeats and PERiodicity (REPPER) (Fig. 2b) [20]. Prediction of the 3D structure was performed using Alpha-Fold 2 and AlphaFold-Multimer [13,15]. Analysis of the full protein provided low-confidence models. As phage fibers and spikes are often organized as elongated homotrimers, we performed AlphaFold-Multimer predictions for trimers of its predicted alpha-helix and beta-sheetforming regions, as shown in Supplementary Fig. S1a and b, respectively. High-confidence trimer models of alphahelical coiled-coil regions (Supplementary Fig. S1a) were obtained for sequences containing the amino acid repeats identified using RADAR (Fig. 2a), whereas trimers of beta-sheet regions formed globular elements (Supplementary Fig. S1b). Those globular regions were interpreted to form fiber beads. Supplementary Figure S2 shows the distribution of the different domains whose structure was investigated using AlphaFold, and Supplementary Fig.  S3 shows a schematic representation of the fiber deduced from measurement of the stems and beads observed by EM (Supplementary Table S1). Next, we divided the complete Aci11-031 into three fragments with an overlapping end and performed an AlphaFold analysis of trimers. The degree of confidence for the predicted structures was high, except for the amino terminus and the region of bead 7 (Fig. 3a). Remarkably, the predicted structure revealed the presence of stems and beads organized exactly as observed by EM of the phage fiber (Figs. 1b and 3b and Supplementary Fig. S3). The N-terminal region, made of 60 hydrophobic residues that likely attach the fiber to the tail, were disorganized, whereas the C-terminal region, which presumably recognizes the bacterial receptor, showed a more complex organization. Comparison with known protein structures obtained from the Protein Data Bank (PDB) were performed using Phyre2 [21] and Dali [22]. They showed that beads 2, 4, 5, and 6 displayed similarities to sugar-binding proteins and to endoglucanases that degrade carbohydrates. Additional analysis will be necessary to better resolve the fine structure of the fiber and identify the function of each of the domains. It is also likely that an additional protein, a tail fiber assembly chaperone, binds to the C-terminal tip of the fiber in order to form the trimer, as has been observed previously in other phage fibers [23].
In three A. baumannii phages closely related to Aci01-1, vB_AbaM_phiAbaA1 (NC_031280), vB_AbaM_P1 (OL960030), and Abp53 (JF317274), the homologous proteins are 3259, 2902, and 1176 aa long, respectively. Lee et al. [24] showed that phage Abp53 protein ORF1176 (AEQ18751) has similarities to the tail protein gp21 (3433 aa) of Klebsiella oxytoca siphovirus phiKO2 (NC_005857), a phage that is otherwise very different from Abp53. Interestingly, by inspecting electron micrographs of phiKO2 virions, we could detect a fiber, with bead-like elements, attached to the conical tail tip [25]. Similar proteins with size ranging from 1676 to 3702 aa can be found in the genome of different lytic or temperate phages, some of them containing repeats predicted to form coiled-coil structures [25]. These include the 1300-aa-long protein J of the phage lambda tail fiber, which contacts the receptor LamB on the Escherichia coli cell surface [26,27].
The original features of the Aci01-1 tail fiber prompted us to attempt to identify its bacterial receptor. In order to identify A. baumannii genes involved in susceptibility to phage infection, we recovered Ab09 colonies surviving infection by Aci01-1. In three out of eight phage-resistant clones, lysis was observed in dense growth zones on LB agar plates, suggesting the presence of the Aci01-1 phage genome in those Aci01-1-resistant bacteria (carrier state). We performed whole-genome sequencing of one resistant strain without virus, of one resistant carrier strain, and of the Ab09 parental culture used to select for resistant strains. In the non-carrier strain, a single point mutation resulting in a threonine-to-methionine amino acid substitution was found in gna, one of the K capsule biosynthesis genes involved in D-GalpNAcA synthesis (equivalent to WpbO in P. aeruginosa). The capsule biosynthesis gene cluster of A. baumannii Ab09, assembled from reads mapped onto the reference strain ACICU (GenBank no. CP031380), was shown to be most closely related to that of strain NIPH KL30 (GenBank no. MN166189). Using the Bautype Capsule Prediction Tool [28], we confirmed that the capsular type of Ab09 was K30.
In the resistant carrier strain, no mutation could be detected in the bacterial genome, but we found large amounts of non-integrated viral DNA that could be assembled into a complete 103-kb Aci01-1 genome sequence. This suggested that resistance resulted from episomal maintenance of the Aci01-1 phage DNA in the bacterium, as observed previously with pseudo-lysogens of P. aeruginosa [29,30].
We report here that the myovirus Aci01-1 has a remarkably long and complex fiber fixed to the conical tip of the tail tube, distal from the head, a structure that has not been described so far. Very long fibers have been found in flagellotropic phages that curl around the flagella of the host, such as in Salmonella phage chi [31], but their structure is different from that of the saclayviruses. A. baumannii lacks flagella, but it can express very long cell extensions such as the fimbrial CsuA/BABCDE-dependent pili that play an essential role in adhesion and biofilm formation on abiotic surfaces [32,33]. It might be interesting in the future to test the susceptibility to phage Aci01-1 of A. baumannii strains deficient in the production of these pili. Sequencing of bacterial strains resisting infection by phage Aci01-1 identified a point mutation in an enzyme involved in biosynthesis of the polysaccharide capsule surrounding the A. baumannii surface [34], suggesting that this structure is the receptor for phage Aci01-1. It is likely that the long fiber helps the phage to reach its capsular receptor through the thick extracellular material in biofilms and to hydrolyze the capsule Fig. 2 Analysis of the Aci11_031 protein structure. (a) Detection by RADAR of repeats, showing the first and last residue numbers in the sequence, the alignment scores as described [44], and repeat sequences colored according to residue sequence conservation. (b) REPPER secondary structure prediction using PSIPRED [45] and PCOILS [46] polysaccharide, digging the way for the tail tip to reach the bacterial membrane. The capsule was reported previously to be the receptor of different A. baumannii phages [35,36]. The capsular type of strain Ab09 is K30, and it will be important to test whether phage Aci01-1 can infect other strains with the same or another type. It is also possible that the phage can lyse strains of other types from outside, as reported previously for phage vB_AbaM_B9 myovirus [37].
Interestingly, we found that several Ab09 phage-resistant variants did not bear any chromosomal mutations but that they were carriers of phage DNA. A similar observation was made for strains resistant to bacteriophages of P. aeruginosa [29,30], Staphylococcus aureus [38], and Bacteroides intestinalis [39]. This extra-chromosomal persistence mechanism of the genomes of lytic phages has been called "carrier state" or "pseudo-lysogeny", having been originally described for starved cells carrying lytic phage genomes [40,41]. Our findings suggest that this mechanism may be even more widely represented in phagebacteria interactions and not limited to stationary-phase bacteria. Further experiments will be necessary to assess the role of the bacterial growth phase in the arrest of the phage multiplication cycle. The above observations illustrate the complex relationship between virulent phages and bacterial populations [42,43].

Author contributions
All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by CP, MO, PT, and CE. The first draft of the manuscript was written by CP, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.   Reconstruction of the full protein 3D structure. R1 to R13 represent the repeats identified using RADAR. B1 to B7 are the beads observed by EM on the phage fiber, whereas b0 is a small bead not detected by EM. The tip of the fiber is hypothesized to bind to the phage receptor. Structures are colored according to the model confidence from blue (high confidence) to red (low confidence).

Data availability
The data presented in this study are available in Supplementary Figures S2-S3. The nucleotide sequences reported in this work have been deposited in the GenBank database under accession numbers NC_048074 and Genbank ID NC_048080, for Aci01-1 and Aci05 respectively. The raw reads archives of phages Aci01-1 (ERR2822810) and phage Aci05 (ERR2822812) have been deposited in the European Nucleotide Archive (ENA) under study PRJEB28456. The read archives of strain Ab09 (ERR10922808) and of the phageresistant variants (ERR10922809 and ERR10922810) have been deposited under study PRJEB60153.