Key Residues of S. flexneri OmpA Mediate Infection by Bacteriophage Sf6

Abstract

Many viruses, including bacteriophage, have the inherent ability to utilize several types of proteinaceous receptors as an attachment mechanism to infect cells, yet the molecular mechanisms that drive receptor binding have not been elucidated. Using bacteriophage Sf6 and its host, Shigella flexneri, we investigated how Sf6 utilizes outer membrane protein A (OmpA) for infection. Specifically, we identified that surface loops of OmpA mediate Shigella infection. We further characterized which residues in the surface loops are responsible for Sf6 binding and productive infection using a combination of in vivo and in vitro approaches including site-directed mutagenesis, phage plaque assays, circular dichroism spectroscopy, and in vitro genome ejection assays. Our data indicate that Sf6 can productively interact with other bacterial OmpAs as long as they share homology in loops 2 and 4, suggesting that these loops may determine host specificity. Our data provide a model in which Sf6 interacts with OmpA using the surface of the protein and new insights into viral attachment through binding to membrane protein receptors.

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

“…The most obvious possible effect that could lead to P22 resistance would be a low O-antigen polysaccharide amount or length, since its synthesis requires the secretion of several periplasmic proteins. In order to determine whether the yajC deletion has an effect on LPS production, we extracted LPS from the parental strain 14028s and the isogenic DyajC mutant and visualized the resulting preparations in silver-stained SDS polyacrylamide electrophoresis gels (Parent et al, 2014;Porcek and Parent, 2015). There was no effect on LPS functionality as measured by in vitro genome ejection caused by purified LPS nor was there any change in the ability of P22 to adsorb to DyajC cells in vivo using phage depletion methods (see Supporting Information Fig.…”

“…In addition to primary receptors, such as LPS, which mediate initial binding of virions to cells, many phages also must recognize a second cell envelope feature, in order to complete DNA delivery (Casjens and Molineux, 2012). Recently, studies with a close relative of P22 identified multiple outer membrane proteins (Omps) that, in addition to O-antigen, are important for mediating phage Sf6 DNA delivery into Shigella flexneri (Parent et al, 2012(Parent et al, , 2014Porcek and Parent, 2015). OmpA and OmpC were identified as critical for attachment after Sf6 virions conclude their initial interactions with the primary LPS receptor (Verma et al, 1991;Clark et al, 1991;Chua et al, 1999).…”

Bacteriophage P22 ejects all of its internal proteins before its genome

“…Hybrid P22 with either an Sf6 tail needle knob or a T4 foldon tail were induced from prophage, and were a gift from Sherwood Casjens (University of Utah). LPS was purified using a kit (Bulldog Bio), and OmpA was purified as described (Parent et al ., 2014; Porcek and Parent, 2015). Each type of virus with final concentration of 10 9 PFU/ml was incubated with LPS (0.25 mg/ml), or OmpA (0.2 mg/ml), or both LPS and OmpA, all in the presence of detergent Triton X-100 [1.06% (w/v)].…”

Kinetic Analysis of Bacteriophage Sf6 Binding to Outer Membrane Protein A Using Whole Virions

“…Sf6 infection of Shigella flexneri is a two-step process that utilizes both glycans and proteins during infection. First, Sf6 reversibly recognizes and then hydrolyzes LPS via its tailspikes [23,24]. Second, Sf6 interacts irreversibly with a protein receptor to commit to infection [25].…”

“…Our previous work showed that OmpA surface loops mediate Sf6 infection and confer host range [23]. Individual amino acid substitutions in OmpA loops result in a range of Sf6 infection efficiencies [23].…”