The branched receptor binding complex ofAckermannviridaephages promotes adaptative host recognition

The branched receptor binding complex of Ackermannviridae phages promotes adaptative host recognition | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The branched receptor binding complex of Ackermannviridae phages promotes adaptative host recognition View ORCID Profile Anders Nørgaard Sørensen , View ORCID Profile Cedric Woudstra , Dorottya Kalmar , View ORCID Profile Jorien Poppeliers , View ORCID Profile Rob Lavigne , View ORCID Profile Martine Camilla Holst Sørensen , View ORCID Profile Lone Brøndsted doi: https://doi.org/10.1101/2024.03.21.586117 Anders Nørgaard Sørensen 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Stigbøjlen 4, 1870 Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anders Nørgaard Sørensen Cedric Woudstra 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Stigbøjlen 4, 1870 Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Cedric Woudstra Dorottya Kalmar 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Stigbøjlen 4, 1870 Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jorien Poppeliers 2 Laboratory of Gene Technology , KU Leuven. Kasteelpark Arenberg 21 box 2462, 3001 Heverlee, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jorien Poppeliers Rob Lavigne 2 Laboratory of Gene Technology , KU Leuven. Kasteelpark Arenberg 21 box 2462, 3001 Heverlee, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rob Lavigne Martine Camilla Holst Sørensen 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Stigbøjlen 4, 1870 Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Martine Camilla Holst Sørensen Lone Brøndsted 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Stigbøjlen 4, 1870 Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lone Brøndsted For correspondence: Lobr{at}sund.ku.dk Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Bacteriophages may express multiple receptor binding proteins, enabling the recognition of distinct and diverse bacterial receptors for infection of a broad range of strains. Ackermannviridae phages recognize diverse O-antigens or K-antigens as receptors by expressing multiple tail spike proteins (TSPs). These TSPs interact and form a branched protein complex protruding from the baseplate attached to the distal tail. Here, we aimed to mimic the evolution of the TSP complex by studying the acquisition of new TSPs without disrupting the functionality of the complex. Using kuttervirus phage S117 as a backbone, we demonstrated the acquisition of entire tsp genes from Kuttervirus and Agtrevirus phages within the Ackermannviridae family. A fifth TSP was designed to interact with the complex and provide new host recognition to expand the branched TSP complex. Interestingly, the acquisition of tsp5 resulted in new variants of the branched TSP complex due to the exchange or deletion of tsp genes. Overall, our study provides novel insight into the development of the branched TSP complex, enabling Ackermannviridae phages to adapt to new hosts. Introduction Bacteriophages (phages) express receptor binding proteins that form long or short tail fibers (TF) or tail spike proteins (TSPs) attached as distal structures to the phage tail. When infecting a bacterial host, the phage receptor binding proteins recognize the surface receptor of the bacterial host. The specificity of this initial phage-host interaction ensures the infection of proper hosts, and receptors have been suggested as the major determinant of the host range of phages 1 . On the phage side, most characterized phages typically use a single receptor binding protein to recognize their bacterial host. Yet, the rise of whole genome sequencing has identified an increasing number of phages that encode multiple receptor binding proteins. For example, gamaleyavirus phage G7C expresses two tail spike proteins (TSPs) interacting in a complex where one of the TSPs deacetylates the O-antigen on E. coli 2 . Even more complex is the network of receptor binding proteins of recently characterized phages; for instance, phage LKp24 encodes 14 TSPs, each proposed to recognize a distinct K-antigen of Klebsiella , including K2, K1, K25, K35, and KN4 3 . The multiple receptor binding proteins of these phages often form a branched complex by interacting through defined structural domains or entire proteins conserved even across phage families 4 , 5 . For example, TSPs of Klebsiella phages carrying a branched TSP complex share similarities to domains 2 and 3 found in Gp10 of the T4 phage 3 , 4 . In phage T4, Gp10 is part of the peripheral baseplate and consists of four domains that form an X shape and are crucial for assembling the tail fiber complex. Domains 2 and 3 of Gp10 adopt a conserved ß-jellyroll fold interacting with the short tail fiber and the baseplate protein Gp9, which further interacts with the long tail fiber 6 , 7 . Therefore, the ability to mediate protein-protein interactions may explain why Gp10-like domains are widespread in phages expressing multiple receptor binding proteins arranged in a branched complex. Phages of the Ackermannviridae family are another example of multiple TSPs linked together in a branched complex using Gp10-like domains. In these phages, Gp10-like domains are found as N-terminal conserved domains named XD 8 . Most Ackermannviridae phages encode up to four different TSPs (hereafter referred to as TSP1 to TSP4). Interestingly, only two of them, TSP4 and TSP2, contain the conserved XD domains necessary for linking the four TSPs into a complex. Both TSP2 and TSP4 express an XD2 domain that interact to form the branched complex. Furthermore, TSP4 and TSP2 also express an XD3 domain interacting with TSP1 and TSP3, respectively. Finally, all four TSPs contain tandem repeat domains (TD), where the TD1 domain forms the N-terminal structural domain in TSP1 and TSP3. The role of the TD1 domains is to interact with the XD3 domains of TSP2 and TSP4. To assemble the complex, TSP4 can interact with TSP1 before or after the interaction with TSP2. In contrast, TSP3 can only interact with the XD3 domain of TSP2 following the formation of the TSP4-TSP2 complex 8 , 9 . Taken together, the four TSPs of Ackermannviridae phages encode several conserved N-terminal domains essential to establish the TSP complex. The host recognition of Ackermannviridae phages is mediated by the variable C-terminal of the four TSPs. This domain recognizes polysaccharides as receptors like lipopolysaccharide (LPS), exopolysaccharides (EPS), and capsular polysaccharides (CPS), allowing the phages to infect diverse bacteria belonging to the Enterobacteriaceae 8 , 10 – 14 . We previously analyzed the TSP diversity in the Kuttervirus , Agtrevirus , Limestonevirus , and Taipeivirus genera of the Ackermannviridae family 12 , 15 . Interestingly, a large pool of genetic diversity of receptor binding domains was revealed, suggesting that phage receptor binding proteins have been diversified to match the multitude of variations of O-antigen and K-antigens expressed by Enterobacteriaceae 8 , 10 , 12 , 13 . While genetic variation of TSP receptor binding domains has been demonstrated, especially in the Kuttervirus genus, it was also observed that many of the phages express similar TSPs 12 . For instance, 53 of the 69 Kuttervirus phages analyzed express a similar TSP3 that binds to Salmonella enterica subspecies expressing either the O4 or O9 O-antigens 12 . In contrast, phages in the Agtrevirus genus express unique receptor binding proteins, meaning that only a few phages share similar TSPs 16 . However, the high degree of conservation of the XD and TD domains may serve as sites for homologous recombination and allow for acquiring new TSPs and diversifying the branched complex of Ackermannviridae phages. However, this remains to be experimentally proven. This study aims to mimic the evolution of the branched TSP complex of Ackermannviridae phages by investigating the acquisition of novel TSPs and their impact on the assembly and functionality of the complex. We used kuttervirus phage S117 as a model phage and showed that this phage can functionally acquire entire tsp genes originating from phages belonging to the Kuttervirus and Agtrevirus genera of the Ackermannviridae family. To expand the branched TSP complex, we designed a tsp5 gene containing N-terminal domains interacting with the complex and the C-terminal of kuttervirus phage Det7, recognizing a novel host. Interestingly, this led to recombination between tsp5 with tsp4 , or deletion of tsp3 and the truncation of tsp4 . Our results give insight into the branched TSP network, allowing Ackermannviridae phages to adapt to new hosts. Results Conservation of the N-terminal TSP domains allows phage S117 to acquire entire tsp genes from another kuttervirus phage The structural N-termini domains of the TSPs in Ackermannviridae phages are essential for assembly and, consequently, functionality of the TSP complex. At the same time, the conserved N-termini of the tsp genes may allow for homologous recombination between tsp genes, thus altering host recognition 8 , 9 , 12 . To investigate the exchange of tsp genes between phages within the same genus, we used kuttervirus S117 as a model phage for acquiring novel tsp genes, whereas kuttervirus CBA120 was a source of such tsp genes. Analyzing the tsp gene cluster of phages S117 and CBA120 showed that tsp1 and tsp2 are highly similar (93.25 and 99.02% identity, respectively), whereas tsp3 and tsp4 only show similarity in the N-termini sequences ( Figure 1A ). For tsp3 , the similarity corresponds to the TD1 and TD2 domains, whereas the anchor domain, the three XD domains, and the TD1 and TD2 of TSP4 are conserved between the two phages (Figure S1 and S2). Therefore, due to the conservation of these important N-termini domains, we hypothesized that phage S117 could acquire the entire tsp3 or tsp4 genes of phage CBA120 and still maintain the integrity of the TSP complex ( Figure 1B ). Since the C-terminal receptor binding domains of newly acquired tsps are different, the functionality of the TSP complex can be verified by determining the host range of the recombinant phages. For this, we used previous data showing that TSP1 and TSP3 allow S117 to infect Salmonella enterica subspecies Minnesota O21 and Typhimurium O4 O-antigens, respectively, whereas TSP2 binds to the O157 O-antigen of E. coli. The host receptor for TSP4 has yet to be identified 12 . Similarly, TSP3 and TSP4 of phage CBA120 allow infection of E. coli O77 or E. coli O78, respectively (Plattner et al. 2019). To allow phage S117 to acquire tsp3 or tsp4 gene from phage CBA120, we cloned each of the tsp3 and tsp4 genes on a plasmid flanked with 500 bp sequences homologous to phage S117 and used a CRISPR-Cas9 system as counter selection. Homologous recombination was promoted by infecting Salmonella Typhimurium (LT2c ΔStyLTI), carrying the homologous template and the CRISPR-Cas9 system with phage S117. Salmonella Typhimurium LT2c previously deleted for the StyLTI restriction-modification system was used to maintain the two plasmids in the host 17 . Subsequently, single plaques were picked and screened by PCR for the presence of the tsp3 or tsp4 genes of CBA120, respectively. Recombinant phages S117- tsp3* and S117- tsp4* were then isolated and shown to form plaques on the new hosts, E. coli O77 or E. coli O78, respectively. In addition, we confirmed that the TSP complex was still functional by showing that the recombinant phages S117- tsp3* and S117- tsp4* could still form plaques on the remaining phage S117 hosts with an efficiency of plating similar to the wildtype S117 phage ( Figure 1C and D ). Furthermore, sequencing the recombinant phages S117- tsp3* and S117- tsp4* confirmed the exchange of tsp genes. Overall, we showed that phage S117 can acquire entire tsp3 and tsp4 genes from a phage within the genus and still preserve the functionality of the TSP complex. Download figure Open in new tab Figure 1: Phage S117 acquisition of tsp3 and tsp4 from Kuttervirus phage CBA120. A) Comparison of the tsp locus of phages CBA120 and S117 demonstrate that the phages encode similar tsp1 and tsp2 genes, whereas the tsp3 and tsp4 genes only share similarity in the N-termini. The alignment of the tsp gene cluster was generated by Clinker version 0.0.10 with the percentage identity between the genes indicated by the grey scale. B) Overview of the branched TSP complex of S117 before and after the acquisition of tsp genes from Kuttervirus CBA120. C) Host range analysis of S117- wt and recombinant phages S117- tsp3 * and S117- tsp4 * by plating 10-fold dilutions of phage stock on each host as indicated and observing plaque formation. The S117- tsp3* phage infects the new TSP3 host ( E. coli O77) and the TSP1 and TSP2 hosts of S117. Phage S117- tsp4* infects the new TSP4 host ( E. coli O78) and the TSP1, TSP2, and TSP3 hosts of S117. D) Efficiency of plating (EOP) of S117- wt , the recombinant S117- tsp3 * and S117- tsp4 * and CBA120. EOPs were calculated by dividing the PFU/mL of the tested strains by the PFU/mL of the propagation strain (indicated by light green and EOP of 1). Phage S117 can acquire a new tsp2 gene from agtrevirus phage AV101 Our previous analysis of the diversity of TSPs of Ackermannviridae phages revealed that similar TSPs were associated with phage genera, suggesting that exchange only occurs between phages in the same genus 12 . However, we have recently shown that the receptor binding domain of TSP4 of agtrevirus AV101 shared similarity towards kuttervirus LPST94. As a consequence, an exchange may be possible between different genera within this family 16 . Indeed, the N-termini of tsp genes are highly similar, including the conserved XD domains of TSP2 and TSP4 and the TD1 domains of TSP1 and TSP3. We, therefore, speculated if tsp genes of agtrevirus AV101 belonging to another genus in the Ackermannviridae family could be exchanged by kuttervirus phage S117 and still form a functional TSP complex. Agtrevirus phage AV101 infects Extended Spectrum β-lactamase (ESBL) producing E. coli strains [18], and we previously showed that the four TSPs recognize E. coli O8, O82, O153, and O159 O-antigens, respectively 16 . Alignment of the tsp gene clusters of phages AV101 and S117 show sequence similarity in the N-termini conserved domains in all four TSPs ( Figure 2 ). For example, the XD2, XD3, and TD1 domains of TSP2 are conserved (Figure S3), suggesting that TSP2 of AV101 can be incorporated into the S117 TSP complex. To demonstrate the functionality of this recombinant TSP complex, we used the same approach described above. Still, instead of screening for the new tsp2 gene with PCR, we directly selected for plaque formation on the new host, E. coli O82. The recombinant S117- tsp2* phage did indeed infect the new E. coli O82 host of AV101 TSP2 as well as the hosts of TSP1 and TSP3 of S117, Salmonella Minnesota O21 and E. coli O157, respectively ( Figure 2B and C ). In conclusion, these results demonstrate that a tsp2 gene originating from Agtrevirus within the Ackermannviridae family can be exchanged across genera and still produce infectious phages. Download figure Open in new tab Figure 2: Phage S117 acquisition of tsp2 from Agtrevirus phage AV101. A) Comparison of the tsp locus of phages AV101 and S117 showed that all genes are similar in the N-termini, and only other short regions are similar between the tsp1 , tsp3, and tsp4 genes of the two phages. The alignment of the tsp gene cluster was generated by Clinker version 0.0.10 with the percentage identity between the genes indicated by the grey scale. B) Overview of the branched TSP complex of S117 before and after the acquisition of tsp2 from Agtrevirus phage AV101. C) Host range analysis of recombinant phage S117- tsp2 * by plating 10-fold dilutions of phage stock on each host as indicated and observing plaque formation. The S117- tsp2* phage infects the new TSP2 host ( E. coli O82) and the TSP1 and TSP3 hosts of S117. D) Efficiency of plating (EOP) of S117- wt , the recombinant S117- tsp2 *, and AV101. EOPs were calculated by dividing the PFU/mL of the tested strains by the PFU/mL of the propagation strain (indicated by light green and EOP of 1). An attempt to add a tsp5 gene led to recombination between tsp4 and tsp5 The XD domains in TSP2 and TSP4 are crucial for assembling the branched TSP complex since the XD2 domains of TSP2 and TSP4 interact ( Figure 3B ) 8 , 9 . We speculated if an additional TSP (TSP5) could be incorporated into the complex by carrying an XD2 domain, for example by addition this to an existing TSP2, as we reasoned that a TSP2 containing two XD2 domains could interact with both TSP4 and TSP2 ( Figure 3B ). To create such a novel tsp5 , we added the XD2 domain of tsp4 of S117 upstream of the entire tsp2 gene originating from Kuttervirus Det7 (recognizing the O3 O-antigen from Salmonella Anatum) 18 . A schematic representation of the synthetical construct is presented in Figure S4. To prevent disrupting the transcription of the remaining tsp genes, we aimed to insert the novel tsp5 downstream of the tsp gene cluster following the successful strategy described above ( Figure 3A ). To isolate recombinant phages, we utilized the same methods as before and spotted the stock containing phages with potential recombinant genomes directly on the new S. Anatum host and isolated plaques representing S117 carrying TSP5. Indeed, a recombinant phage S117- tsp5* was able to infect the new S. Anatum host and the hosts recognized by TSP1, TSP2, and TSP3 ( Figure 3C and D ). As we do not know the receptor of TSP4, and hence the host of S117, we verified by PCR that all tsp genes, including tsp5 , were present in the recombinant phage S117- tsp5 *. Our results showed that amplification of the region for the tsp5 gene insert did not correspond to the expected size (3658 bp) but was identical to the wild type S117 (1000 bp) ( Figure 3E ). In addition, the tsp4 gene of the recombinant S117- tsp5* genome could not amplify ( Figure 3E ). We hypothesized that a recombinant event between tsp4 and tsp5 may have created a phage able to infect the new host. To investigate this further, we sequence-verified the genome of phage S117- tsp5*. We found that the receptor binding domain of tsp4 was replaced by the receptor binding domain of tsp5 , indeed suggesting recombination between tsp4 and tsp5 ( Figure 3F ). Overall, we did not manage to introduce an additional tsp gene into the genome of S117 that could interact with the TSP complex. Instead, the recombination event created a phage with a tsp4 containing the novel receptor binding domain of phage Det7, allowing the phage to infect S . Anatum. Download figure Open in new tab Figure 3: Selection for phage S117- tsp5* results in recombination between S117 tsp4 and tsp5 genes. A) Location of the additional tsp5 gene in the genome of phage S117. B) Overview of the designing of the synthetic tsp5 gene composed of the XD2 domain of tsp4 of phage S117 fused to the entire tsp2 gene of phage Det7 and model for the expanded TSP complex. C) Host range analysis of recombinant phage S117- tsp5 * by plating tenfold dilutions of phage stock on each host as indicated and observing plaque formation. The S117- tsp5* phage infects the new TSP5 host ( S . Anatum) and the TSP1, TSP2, and TSP3 host of S117. D) Efficiency of plating (EOP) of S117- wt and the recombinant S117- tsp5 *. EOPs were calculated by dividing the PFU/mL of the tested strains by the PFU/mL of the propagation strain (indicated by light green and EOP of 1). E) PCR using primers as indicated in materials and methods to detect the four tsp genes and determine the location of the tsp5 genes. F) Whole genome sequencing of recombinant S117- tsp5 * demonstrates a recombination event between tsp4 and tsp5 , resulting in a chimeric TSP4 protein containing the N-terminal of Tsp4 and the receptor binding domain of the theTSP5. Acquisition of a tsp5 gene resulted in deletion of tsp3 and truncation of tsp4 In a second attempt to create S117 phages carrying an additional tsp5 gene, we used the previously isolated recombinant phage S117- tsp4* expressing TSP4 from CBA120 instead of wildtype S117, thus allowing for screening for all five TSP hosts ( Figure 3 ). We indeed isolated a recombinant phage (S117- tsp4* - tsp5* ) that infects S. Anatum ( Figure 4A ). Unexpectedly, phage S117- tsp4* - tsp5* could not infect hosts recognized by TSP2, TSP3, and TSP4 and could barely infect the TSP1 host ( Figure 4A and B ). These results suggest that the additional XD2 domain of tsp5 may disturb the assembly of the TSP complex. Sequencing of the tsp cluster demonstrated that tsp5 was correctly inserted into the genome and no reads mapped to the tsp3 gene or the C-terminal part of the tsp4 gene, while the recombinant phage genome could not be fully closed ( Figure 4C ). Yet, further analysis confirmed that the N-terminal sequences encoding the anchor domain and the XD1 and XD2 domains of the tsp4 gene were present in our sequencing data ( Figure 4D ). Combining the sequencing data and host range results suggests that S117- tsp4* - tsp5* only carries the functional receptor binding domains of TSP1 and TSP5 ( Figure 4E ). Overall, the acquisition of a tsp5 into the genome resulted in the deletion of tsp3 and truncation of tsp4 but still expressed the TSP4 domains required for assembly and functionality of the TSP complex. Download figure Open in new tab Figure 4: Acquisition of tsp5 leads to deletion of tsp3 and most of tsp4. A) Host range analysis of recombinant phage S117- tsp4 *- tsp5 * by plating tenfold dilutions of phage stock on each host as indicated and observing plaque formation. The new modified S117- tsp4* - tsp5 * phage infects the host of TSP5 ( S. Anatum. B). Efficiency of plating (EOP) of S117- wt and the recombinant S117- tsp4*-tsp5 * were calculated by dividing PFU/mL of the tested strains by the PFU/mL of the propagation strain (EOP of 1). Recombinant phage S117- tsp4* - tsp5 * infects S. Anatum with a high EOP compared to the TSP1 host S . Minnesota and showed no infection of TSP2, TSP3 and TSP4 hosts. C) Whole genome sequencing of phage S117- tsp4* - tsp5 * revealed a deletion of tsp3 and most of the tsp4 gene. D) Sequence alignment of the N-terminal domains of TSP4 of S117- wt and the TSP4 of S117- tsp4* - tsp5 * showed high similarity until the XD2 domain. Only the XD domains necessary for interactions within the TSP network were conserved in the S117- tsp4* - tsp5 * phage. E) Proposed TSP complex of recombinant phage S117- tsp4* - tsp5 *. Discussion Tail spike proteins (TSPs) allow phages to successfully infect their host by recognizing specific polysaccharides bacterial receptors like the LPS or CPS 8 , 11 , 14 , 19 . However, bacteria express a high diversity of these polysaccharides within a species 20 – 22 . For instance, E. coli isolates combined display an array of 185 different O-antigens of LPS 21 . Therefore, phages expressing TSPs may be limited in their host range compared to phages that recognize a protein receptor of a more conserved nature within a bacterial species. For instance, the receptor binding protein of gelderlandvirus phage S16 recognizes the outer membrane protein OmpC as the receptor, and due to the conserved nature of the protein, the phage can infect a broad range of Salmonella enterica serotypes 23 . To allow infection of a broader selection of hosts, phages using TSPs as receptor binding proteins have evolved different mechanisms to adapt to the large diversity of polysaccharide receptors. Here, we studied Ackermannviridae phages expressing four TSPs forming a branched complex protruding the baseplate. We aimed to mimic diverse mechanisms that allow these phages to acquire novel TSPs and investigated the impact of novel TSPs on the assembly and functionality of the complex. Bacterial receptors are the major determinant of phage susceptibility, even more than internal phage resistance mechanisms 1 . Hence, phages have evolved different strategies to overcome the diversity of phage receptors. One approach to adapting to a new host is to exchange receptor binding domains between phages through homologous recombination 24 – 27 . Indeed, in silico analysis of tsp genes in the Ackermannviridae family has suggested that tsp genes undergo homologous recombination due to the conserved N-termini of the four genes 12 . Our study showed that homologous recombination can exchange tsp genes and still produce infectious phages without disrupting the TSP complex, similar to a recent study 28 . While we showed exchange within the family, other in-silico analyses of TSPs in the Ackermannviridae family have demonstrated that the receptor binding domains are exchanged with distant related lytic phages and prophages 11 , 12 , 16 . For instance, the receptor binding domain of TSP4 of AV101 is similar to kayfunavirus ST31 and phapecoctavirus Ro121c4YLVW that both are distantly related phages 16 . While domain exchange seems to be prevalent in the family, the frequency of exchange is not known. Co-evolution studies of bacteria and phages often show that phages adapt to new hosts by point mutations in the receptor binding domain and not through domain exchange 29 , 30 . This may be due to the limited number of bacteria and phages in a given co-evolution experiment, thereby limiting the natural complexity. Thus, while we observe and show that exchanging whole genes or domains is a possible strategy for adaption to new hosts, the exchange frequency is unknown and remains to be determined. While the exchange of genes of domains of receptor binding proteins is one way of adapting to a new environment, phages have also evolved to express multiple receptor binding proteins 2 – 4 , 31 . Phages in the Stephanstirmvirinae subfamily, e.g. phage phi92, encode up to five receptor binding proteins including both tail spike proteins (TSPs) and tail fibers (TFs) each protruding the baseplate 32 , 33 . Other phages encode proteins or domains that anchor the receptor binding proteins, like the Gp10-like domains in Ackermannviridae phages, to form a complex 4 , 8 , 9 . In Ackermannviridae phages, it was previously suggested that the phages have evolved by gene duplication from a single TSP (TSP4) and that acquisition of the Gp10-like domains allowed multiple TSPs to interact in a complex with TSP4 8 , 12 . In most Limestonevirus phages, the TSP4 does not carry a receptor binding domain but only the N-terminal structural domains necessary for assembling the TSP complex, including the remaining TSPs. Furthermore, Limestonevirus phages do not encode a tsp3 gene 34 – 36 . Similarly, we observed that attempting to introduce a fifth TSP resulted in a recombinant phage without a TSP3 and a TSP4 with only the N-termini structural domains. Thus, TSP4 may have been an adaptor protein that established the ability to form a complex and gained a receptor binding domain through evolution. While we did not successfully add a fifth tsp gene in the genome of S117, two recent studies suggested that Taipeivirus phages like KpS110 and Menlow encode five TSPs 4 , 37 . Bioinformatic analysis of these phages showed that all five genes in the tsp cluster encode proteins that adopt a β-helix fold common for all TSPs. Neither of the studies investigated the N-termini structural domains of the fifth TSP nor showed if the TSP is incorporated in the TSP complex. Still, overall, it may suggest that it is, in fact, possible to add a fifth TSP into the complex. In our TSP5 design, we incorporated an XD2 domain into a tsp4 gene of Det7. The XD domains of TSP5 showed a shared sequence similarity with the XD domains present in TSP2 and TSP4 of S117. This overall sequence similarity could be the reason behind the observed different recombination events. Overall, our results also demonstrate that it is possible to engineer phages with multiple TSPs and could be used in further studies with complexes that carry Gp10-like domains. Method and materials Strains, phages, and plasmids Bacterial strains, phages, and plasmids used in this study are found in Table 1 . View this table: View inline View popup Download powerpoint Table 1: Bacterial strains, plasmids, and phages used in the study. Bioinformatic analysis The genomes of Kuttervirus phages S117 (accession number MH370370 ) and CBA120 ( NC_016570 ), and Agtrevirus phage AV101(OQ973471) were used for analysis of their tsp gene cluster and for designing primers for tsp exchange. Sequence similarities of the tsp gene clusters were visualized using EasyFig version 2.2.5 with default settings. Alignments of individual tsp genes of CBA120, AV101, and S117 were also done in CLC Workbench 22 with default settings. Phage propagation Kuttervirus phages S117, CBA120, and agtrevirus phage AV101 were propagated on the host S. Typhimurium (LT2c), E. coli (NCTC12900) and E. coli (ESBL040) strains, respectively, as described earlier 12 . Single colonies of the propagating strains were inoculated into LB medium (Lysogeny Broth, Merck, Darmstadt, Germany) and incubated at 37°C shaking at 170 rpm until reaching the exponential phase (approximately five hours). Previous stocks of the phages were diluted to 10 3 -10 5 PFU/mL, and 330 µL of the phage dilutions and 330 µL of the hosts were mixed before 12mL top agar (Lbov; LB broth with 0,6% Agar bacteriological no.1, Oxoid) were added to the mixture and poured onto three LA plates (LB with 1,2% agar). After the top agar was solidified, they were incubated overnight at 37°C. The next day, 5 mL SM buffer (0.1 M NaCl, 8 mM MgSO4·7H2O, 50 mM Tris-HCl, pH 7.5) was added to each of the three plates followed by incubation of the plates overnight at 4°C and shaken at 50 rpm. The SM buffer was collected and centrifuged at 11,000 rpm at 4°C for 15 minutes and filtered twice with 0.2 µM filters. The new phage stocks were stored at 4°C before use. The titer of the stocks was determined with a plaque assay (described below). Phage host range analysis To determine the host range of CBA120, AV101, and S117 phages, plaque assays were carried out 38 . Briefly, single colonies of strains of interest were inoculated into a 5 mL medium and incubated at 37°C shaking at 180 rpm for approximately five hours to reach the exponential phase. Subsequently, 100 µL of the strain was mixed with 4 mL of top agar and poured onto LA plates. The plates were dried, and 10-fold serial dilutions of the phage stocks were spotted onto the top agar plates. After overnight incubation, the plates were screened for single plaques, and the efficiency of plating was calculated by comparing the PFU/mL of the tested strains by the PFU/mL of the propagation strain. Phage DNA isolation Phages DNAs were isolated before engineering or DNA sequencing 12 . Rnase and Dnase (Thermo Fischer Scientific) were added to the sample with a final concentration of 10 µg/mL and 20 µg/mL, respectively, prior to the nucleic acid extraction. The sample mixture was incubated for 20 min at 37°C followed by the addition of sterile EDTA (pH 8) (Thermo Fischer Scientific) at a concentration of 20 mM. To degrade the phage capsid, 50 µg/mL of Proteinase K (Thermo Fischer Scientific) was added to the sample and incubated for two hours at 56°C. The release of the DNA was verified on a 1% agarose gel. Following, the genomic DNA Clean & ConcentratorTM-10 kit (Zymo Research) was used to isolate the phage genome using the manufacturer’s instructions. The concentration of the isolated DNA was measured with Qubit (Thermo Fisher Scientific), and the quality of the DNA was verified on a 1% agarose gel. Phage engineering Genetic engineering of phages was performed through CRISPR/Cas9, as previously described 17 . Briefly, we used a recently developed two plasmids system, pEcCas (addgene #73227) and pEcgRNA (addgene #166581), where Cas9 was encoded by the pEcCas plasmid, and the Cas9 RNA guide could be cloned into the pEcgRNA plasmid as well as the recombinant template used to modify the phage genomes. Furthermore, the pEcCas plasmid also expressed the Lambda Red system under an inducible arabinose promoter to increase recombination efficiency 39 . Guide RNA efficiency Guide RNA (gRNA) targeting the four S117 TSPs were designed using the CRISPR tool on the Benchling® website. A list of the efficient guides for each tsp gene is presented in Table 2 . The guides were introduced into the pEcgRNA vector through reverse PCR using the CloneAmp HiFi PCR Premix (Takara Bio) following the manufacturer’s instructions. Primers for cloning the guides into pEcgRNA are presented in the Appendix (Table S1). The PCR products were purified using Zymo PCR purification kit (Zymo Research) and directly transformed into Stellar™ chemical competent cells (Takara Bio) and plated on spectinomycin (50 µg/ml) plates. The pEcgRNA-guide plasmids from an overnight culture of Stellar cells expressing the plasmids grown in LB medium with spectinomycin (50 µg/ml) were extracted using GeneJET Plasmid Miniprep Kit (Thermo Scientific™). The pEcgRNA-guide plasmids were individually cloned into LT2c ΔStyLTI competent cells harboring the pEcCas plasmid and plated on both spectinomycin for selection of pEcgRNA (50 µg/ml) and kanamycin for selection of pEcgRNA-guide plates (50 µg/ml). The next day, the presence of both plasmids was screened by colony PCR using DreamTaq Green PCR Master Mix (Thermo Fischer™). Guide efficiency was evaluated by plaque assay on LT2c ΔStyLTI containing pEcgRNA-guide and pEcCas and on LT2c ΔStyLTI containing only pEcCas as a control (Figure S4). View this table: View inline View popup Download powerpoint Table 2: CRISPR guides used for tsp exchange. Construction of the recombinant templates The guides with the highest efficiency were further used for tsp gene exchange. All primers used are presented in Table S1. To exchange the tsp genes in Kuttervirus S117, we constructed a recombinant template (RT). More specifically, the tsp gene and 500 base pair upstream and downstream of the S117 tsp gene that should be exchanged (Left and Right Homology Arms (LHA and RHA)) were amplified by PCR using CloneAmp HiFi PCR Premix (Takara Bio). LHA and RHA primers carried overhangs with homology to the tsp exchanged gene. After PCR purification, the RT was constructed by Splicing by Overhang Extension (SOE) PCR. Briefly, the LHA and RHA PCR products were used as primers for amplifying the tsp exchange gene using CloneAmp HiFi PCR Premix (Takara Bio). After five PCR cycles, primers amplifying the whole new construct were added, and the PCR was continued for 30 additional cycles. To verify the correct assembly of the three fragments 5 µL of the PCR reaction was run on a 1% agarose gel. Correct amplicons were isolated by Zymo PCR purification kit (Zymo Research). The PCR products were cloned into a PCR linearized pEcgRNA-guide plasmid to make the pEcgRNA-guide-RT plasmid using In-fusion® HD-cloning kit (Takara Bio) following the manufacturer’s instructions. Exchange of tsp genes The LT2c expressing both pEcCas and the different pEcgRNA-guide-RT plasmids were grown to an OD600=0.2 before 0.1% arabinose was added to induce the expression of the Lambda Red system. The strains were further grown for two hours followed by a plaque assay to perform the recombination between the phage and the pEcgRNA-guide-RT plasmid. The next day, plaques were screened for the presence of the new tsp genes with DreamTaq Green PCR Master Mix (Thermo Fischer™). Positive plaques were then used for a new plaque assay with the new host to test if the recombinant phage could infect the host. The recombinant phages were further propagated on the new host. Construction of TSP5 engineered phage To construct a phage with an additional tsp gene ( tsp5 ) we designed a tsp2 gene from kuttervirus phage Det7 (accession number NC_027119 ) with an additional XD2 domain from S117 tsp4 gene in the start of the gene (Figure S5). Furthermore, the promoter for the tsp2 of Det7 was added to the construct. The designed gene was ordered from Twist Bioscience. To insert the tsp5 gene into the genome of S117 we utilized the same method as for exchanging the tsp genes (see Phage engineering section). Guide RNAs are presented in Table 2 and primers for cloning into pEcgRNA are presented in Table S1. Phage DNA sequencing Using a Qubit 2.0 instrument and the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific), the quality of the DNA preparations of genomes of S117 and recombinant S117 was evaluated. The NEBNext Ultra DNA Library Prep kit from New England Biolabs was used to prepare the genomes for Illumina sequencing, and the HiSeq 4000 machine (Illumina) was used to sequence the samples. The CLC Genomics Workbench 21 (Qiagen, Aarhus) with default settings was used to assemble the raw reads after trimming. CRediT authorship contribution statement Anders Nørgaard Sørensen: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Dorottya Kalmar: Validation, Formal analysis, Investigation. Cedric Woudstra: Conceptualization, Methodology, Writing – review and editing. Jorien Poppeliers : Formal analysis, writing - review & editing. Rob Lavigne : Writing - review & editing, Funding acquisition. Martine Camilla Holst Sørensen: Conceptualization, Writing – review & editing, Funding acquisition. Lone Brøndsted: Conceptualization, Project administration, Supervision, Visualization, Writing - review & editing, Funding acquisition. Acknowledgments This work was supported by the Danish Council for Independent Research (9041-00159B). Abbreviations CPS Capsular polysaccharide EPS Exopolysaccharide ESBL Extended spectrum ß-lactamase LB Luria-Bertani LHA Left homology arm LPS Lipopolysaccharide PFU Plaque formation unit RBP Receptor binding protein RHA Right homology arm RT Recombinant template TD Tandem repeat TF Tail fiber TSP Tail spike protein References 1. ↵ Gaborieau , B. , Vaysset , H. , Tesson , F. , Charachon , I. , Dib , N. , Bernier , J. , Dequidt , T. , Georjon , H. , Clermont , O. , Hersen , P. , et al. ( 2023 ). Predicting phage-bacteria interactions at the strain level from genomes . bioRxiv , 2023.11.22.567924 . doi: 10.1101/2023.11.22.567924 . OpenUrl Abstract / FREE Full Text 2. ↵ Prokhorov , N.S. , Riccio , C. , Zdorovenko , E.L. , Shneider , M.M. , Browning , C. , Knirel , Y.A. , Leiman , P.G. , and Letarov , A. V . ( 2017 ). Function of bacteriophage G7C esterase tailspike in host cell adsorption . Mol Microbiol 105 , 385 – 398 . doi: 10.1111/mmi.13710 . 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Share The branched receptor binding complex of Ackermannviridae phages promotes adaptative host recognition Anders Nørgaard Sørensen , Cedric Woudstra , Dorottya Kalmar , Jorien Poppeliers , Rob Lavigne , Martine Camilla Holst Sørensen , Lone Brøndsted bioRxiv 2024.03.21.586117; doi: https://doi.org/10.1101/2024.03.21.586117 Share This Article: Copy Citation Tools The branched receptor binding complex of Ackermannviridae phages promotes adaptative host recognition Anders Nørgaard Sørensen , Cedric Woudstra , Dorottya Kalmar , Jorien Poppeliers , Rob Lavigne , Martine Camilla Holst Sørensen , Lone Brøndsted bioRxiv 2024.03.21.586117; doi: https://doi.org/10.1101/2024.03.21.586117 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7644) Biochemistry (17728) Bioengineering (13916) Bioinformatics (42037) Biophysics (21488) Cancer Biology (18636) Cell Biology (25552) Clinical Trials (138) Developmental Biology (13401) Ecology (19940) Epidemiology (2067) Evolutionary Biology (24367) Genetics (15621) Genomics (22545) Immunology (17764) Microbiology (40475) Molecular Biology (17208) Neuroscience (88744) Paleontology (667) Pathology (2842) Pharmacology and Toxicology (4834) Physiology (7659) Plant Biology (15175) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9834) Zoology (2272)