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Ssrna Phage Penetration Triggers Detachment of the F-Pilus

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Ssrna Phage Penetration Triggers Detachment of the F-Pilus ssRNA phage penetration triggers detachment of the F-pilus Laith Harba,b, Karthik Chamakuraa,b, Pratick Kharac, Peter J. Christiec, Ry Younga,b, and Lanying Zenga,b,1 aDepartment of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843; bCenter for Phage Technology, Texas A&M University, College Station, TX 77843; and cDepartment of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Houston, TX 77030 Edited by Thomas J. Silhavy, Princeton University, Princeton, NJ, and approved August 27, 2020 (received for review June 9, 2020) Although the F-specific ssRNA phage MS2 has long had paradigm of the gRNA from the capsid and its penetration into the cell status, little is known about penetration of the genomic RNA (gRNA) (13), a reduction in cellular nucleoside triphosphates (14), and into the cell. The phage initially binds to the F-pilus using its matu- cleavage of the 44-kDa Mat into two peptides that remain as- ration protein (Mat), and then the Mat-bound gRNA is released from sociated with the cell (15). the viral capsid and somehow crosses the bacterial envelope into The role of F-pili in the transfer of gRNA from the virion into the cytoplasm. To address the mechanics of this process, we fluo- the cell is mysterious. Two prevailing models account for the rescently labeled the ssRNA phage MS2 to track F-pilus dynamics mechanism of transport by F-pili. In the “pilus conduction” during infection. We discovered that ssRNA phage infection triggers model initially proposed by Brinton in 1965 (16), the pilus- the release of F-pili from host cells, and that higher multiplicity of docked phage delivers its gRNA into the central channel of infection (MOI) correlates with detachment of longer F-pili. We also the F-pilus for conveyance to the cell interior. Although there is report that entry of gRNA into the host cytoplasm requires the little direct evidence for this model, the pilus lumen contains – F-plasmid encoded coupling protein, TraD, which is located at the basic residues that potentially interact with the negatively cytoplasmic entrance of the F-encoded type IV secretion system charged gRNA (17). In the “pilus retraction” model, the pilus- (T4SS). However, TraD is not essential for pilus detachment, indicat- docked phage gains access to the bacterial cell surface through ing that detachment is triggered by an early step of MS2 engage- retraction of the pilus, whereupon the Mat-gRNA complex ment with the F-pilus or T4SS. We propose a multistep model in passes across the cell envelope through the T4SS or another MICROBIOLOGY which the ssRNA phage binds to the F-pilus and through pilus re- route (18). Although not reported for F-pili, RNA phage parti- traction engages with the distal end of the T4SS channel at the cell cles have been shown to accumulate at the base of pili elaborated surface. Continued pilus retraction pulls the Mat-gRNA complex out by IncP and IncC plasmids during infection, suggesting that pilus of the virion into the T4SS channel, causing a torsional stress that retraction has occurred (12, 19, 20). More support for this model breaks the mature F-pilus at the cell surface. We propose that P. aeruginosa phage-induced disruptions of F-pilus dynamics provides a selective comes from the finding that cells infected with the advantage for infecting phages and thus may be prevalent among ssRNA phage PP7 had a 50% reduction in overall type IV pilus the phages specific for retractile pili. lengths and that phage particles were often seen at the bases of pili, as observed on electron microscopy (21). In the absence of ssRNA phage MS2 | F-pilus | pili retraction | pili detachment | firm experimental support for either model, how the Mat-gRNA genomic RNA entry complex passes through the bacterial envelope into the cell in- terior remains unknown. ingle-stranded (ss) RNA phages have been identified for Sdiverse bacterial hosts, including Escherichia coli (1, 2), Significance Pseudomonas aeruginosa (3, 4), Caulobacter crescentus (5), and Acinetobacter spp. (6). All known ssRNA phages infect their Many pathogenic bacteria utilize dynamic appendages called hosts by initially binding to retractile pili, such as the conjugative pili to facilitate important functions, such as gene transfer and pili associated with conjugative DNA transfer and the type IV motility. These processes contribute to the spread of antibiotic pili (which are not phylogenetically related to conjugative pili) resistance genes and support persistence in bacterial infec- mediating DNA uptake and twitching motility (7). Most of our tions, making them prime targets for therapeutic purposes. The knowledge regarding RNA phage biology stems from studies of single-stranded (ss) RNA bacteriophages all use dynamic pili to the ssRNA phages that infect enteric bacteria carrying the F sex facilitate infection; however, key mechanistic details describing factor plasmids (8). These plasmids code for type IV secretion how the pilus promotes viral entry into the host cell have remained elusive. Here we used fluorescence microscopy to systems (T4SSs) and associated F-pili, which are hollow, filamen- uncover a telling phenomenon associated with ssRNA phage tous, and dynamic appendages that extend and retract to initiate infection: initial penetration of the viral payload causes donor–recipient cell contacts during bacterial conjugation (9). breakage of host pili. This provides a selective advantage for The infection process has been best studied with the ssRNA the infecting phage, and thus this phenomenon may be phage MS2 and its derivatives. MS2 has a ∼3.5-kb genomic RNA widespread among other pilus-specific phage systems. (gRNA) that encodes four proteins: the maturation protein (Mat) used for host recognition, the coat protein (Coat) that Author contributions: L.H. and L.Z. designed research; L.H., K.C., and P.K. performed re- forms the capsid, the lysis protein (L), and the replicase (Rep), search; L.H. and L.Z. analyzed data; and L.H., K.C., P.K., P.J.C., R.Y., and L.Z. wrote the viral subunit of the RNA-dependent RNA replicase. The the paper. virions are ∼27 nm in diameter (10) and consist of 178 copies of The authors declare no competing interest. Coat encapsidating the gRNA, which is bound to a single copy of This article is a PNAS Direct Submission. Mat (11). Infection is initiated when MS2 adsorbs to the side of Published under the PNAS license. the F-pilus via the Mat protein. The binding of MS2 to piliated 1To whom correspondence may be addressed. Email: [email protected]. cells at 4 °C or free F-pili at 4 to 37 °C is reversible and does not This article contains supporting information online at https://www.pnas.org/lookup/suppl/ lead to loss of infectivity in the viral particles (12); however, at doi:10.1073/pnas.2011901117/-/DCSupplemental. 37 °C, the interaction of MS2 with piliated cells leads to release www.pnas.org/cgi/doi/10.1073/pnas.2011901117 PNAS Latest Articles | 1of8 Downloaded by guest on September 29, 2021 In this study, we used fluorescently labeled MS2 phage to are inhibited on ice (27, 28), the addition of MS2-GFP to characterize F-pilus dynamics during ssRNA phage infection. quantify pili does not promote further infection. Strikingly, we discovered that MS2 triggers the release of F-pili by Remarkably, the MS2-infected culture showed a marked in- a mechanism requiring retraction. Our findings strongly support crease in the amount of free F-pili in the media (i.e., F-pili not the “pilus retraction” model and suggest a novel mechanism for attached to a cell) compared with the buffer-only control culture. superinfection exclusion resulting from ssRNA phage infection. As shown in Fig. 2B, the frequency of detached pili increased with time and reached a plateau after ∼10 min (∼45%), which is Results when gRNA penetration is essentially complete (29). This sug- Detection of F-pili by Fluorescent Labeling of MS2 Particles. Previ- gests that F-pilus detachment is related to MS2 gRNA entry into ously, ssRNA phage particles labeled with residue-specific fluo- the cell. Notably, we did not see any increase in detached pili in rescent dyes have been used to identify F-pili by fluorescence the uninfected culture, suggesting that the binding of high microscopy (22, 23). Although this method produces labeled numbers of MS2-GFP to the pili does not lead to shearing or virions, we noted a significant titer loss of 50% after labeling, breakage during imaging. possibly due to the labeling of Mat by the dye (23). To avoid To determine whether F-pilus detachment is MS2-specific, we perturbing the binding interface of the phage particles, we tagged tested whether other F-specific phages induce detachment. Like β MS2 virions with fluorescent proteins to illuminate F-pili, as was MS2, the ssRNA phage Q (an Allolevivirus) was seen to bind to previously done for labeling of phage lambda and P1 (24, 25). the side of the F-pilus. In contrast, ssDNA phage M13 was bound Cells harboring a plasmid encoding the MS2 Coat fused to exclusively to the pilus tip. Similarly to MS2, we observed an β sfGFP (Fig. 1A) were infected with MS2 and subsequently in- increase in detached pili when cells were infected with Q , al- though ultimately only ∼30% of the total pili were detached duced for production of the Coat-sfGFP fusion protein. During β assembly, the phage incorporates fluorescent Coat proteins, (Fig. 2B). In contrast to MS2 and Q , infection by M13 yielded a resulting in mosaic particles containing both native and GFP- similar basal level of pilus detachment as the uninfected control.
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