scholarly journals A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU

2021 ◽  
Author(s):  
Andrea N.W. Lim ◽  
Minmin Yen ◽  
Kimberley D. Seed ◽  
David W. Lazinski ◽  
Andrew Camilli
Keyword(s):  
Host Range ◽  
Vibrio Cholerae ◽  
Receptor Binding ◽  
Mutant Phenotype ◽  
Tail Fiber ◽  
Missense Mutations ◽  
Wild Type ◽  
Mutant Strains ◽  
Stool Samples ◽  
Receptor Binding Protein

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host-range mutants within infant rabbits infected with a mixture of wild type and OmpU mutant strains. ICP2 host-range mutants, that can now infect OmpU mutant strains, had missense mutations in putative tail fiber gene gp25 and putative adhesin gp23. Using site-specific mutagenesis we show that single or double mutations in gp25 are sufficient to generate the host-range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to give a host-range mutant phenotype. All ICP2 host-range mutants retained the ability to plaque on wild type V. cholerae cells. The strength of binding of host-range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host-range mutants evolve by a two-step process where, first, gp25 mutations are selected for their broad host-range, albeit accompanied by low level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near wild type efficiencies of adsorption and subsequent phage multiplication. Importance Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to a renewed interest in phage biology and their potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular co-evolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

scholarly journals A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU

2021 ◽  
Author(s):  
Andrea N.W. Lim ◽  
Minmin Yen ◽  
Kimberley D. Seed ◽  
David W. Lazinski ◽  
Andrew Camilli
Keyword(s):  
Host Range ◽  
Vibrio Cholerae ◽  
Receptor Binding ◽  
Binding Protein ◽  
Mutant Phenotype ◽  
Tail Fiber ◽  
Missense Mutations ◽  
Wild Type ◽  
Stool Samples ◽  
Receptor Binding Protein

AbstractICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host-range mutants within infant rabbits infected with a mixture of wild type and OmpU mutant strains. ICP2 host-range mutants had missense mutations in putative tail fiber gene gp25 and putative adhesin gp23. Using site-specific mutagenesis we show that single or double mutations in gp25 are sufficient to generate the host-range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to give a host-range mutant phenotype. All ICP2 host-range mutants retained the ability to plaque on wild type V. cholerae cells. The strength of binding of host-range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host-range mutants evolve by a two-step process where, first, gp25 mutations are selected for their broad host-range, albeit accompanied by low level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near wild type efficiencies of adsorption and subsequent phage multiplication.ImportanceConcern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to a renewed interest in phage biology and their potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies a ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular co-evolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

scholarly journals GENETICS OF YEAST HEXOKINASE

Genetics ◽  
1977 ◽  
Vol 86 (4) ◽  
pp. 727-744
Author(s):  
Zita Lobo ◽  
P K Maitra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
The Other ◽  
Missense Mutations ◽  
Structural Genes ◽  
Wild Type ◽  
Hexokinase Activity ◽  
Mutant Strains ◽  
Mutant Genes ◽  
Yeast Hexokinase ◽  
Respective Wild Type

ABSTRACT Two independent isolates of Saccharomyces cerevisiae lacking hexokinase activity (EC 2.7.1.1) are described. Both mutant strains grow on glucose but are unable to grow on fructose, and contain two mutant genes h×k1 and h×k2 each. The mutations are recessive and noncomplementing. Genetic analysis suggests that these two unlinked genes h×k1 and h×k2 determine, independently of each other, the synthesis of hexokinase isozymes P1 and P2, respectively. h×k1 is located on chromosome VIR distal to met10, and h×k2 is on chromosome IIIR distal to MAL2. Of four hexokinase-positive spontaneous reversions, one is very tightly linked to h×k1 and the other three to the h×k2 locus. The reverted enzymes are considerably more thermolabile than the respective wild-type enzymes, and in one case show altered immunological properties. Data are presented which suggest that the h×k1 and h×k2 mutations are missense mutations in the structural genes of hexokinase P1 and hexokinase P2, respectively. These are presumably the only enzymes that allow S. cerevisiae to grow on fructose.

scholarly journals The Virulence Regulatory Protein ToxR Mediates Enhanced Bile Resistance in Vibrio cholerae and Other PathogenicVibrio Species

2000 ◽  
Vol 68 (3) ◽  
pp. 1491-1497 ◽  
Author(s):  
Daniele Provenzano ◽  
Darren A. Schuhmacher ◽  
Justin L. Barker ◽  
Karl E. Klose
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Outer Membrane Proteins ◽  
Regulatory Protein ◽  
Wild Type ◽  
Outer Membrane Porins ◽  
Mutant Strains ◽  
Bile Resistance ◽  
El Tor

ABSTRACT The transmembrane regulatory protein ToxR is required for expression of virulence factors in the human diarrheal pathogenVibrio cholerae, including cholera toxin (CT) and the toxin coregulated pilus (TCP). ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, which is the direct transcriptional activator of CT and TCP genes. However, ToxR, independent of ToxT, directly activates and represses transcription of the outer membrane porins OmpU and OmpT, respectively. The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic elements, while the toxR, ompU, andompT genes are apparently in the ancestralVibrio chromosome. The contribution of ToxR-dependent modulation of outer membrane porins to cholera pathogenesis has remained unknown. We demonstrate that ToxR mediates enhanced bile resistance in a ToxT-independent manner. In both classical and El Tor biotypes of V. cholerae, a toxR mutant strain has a reduced minimum bactericidal concentration (MBC) of bile, the bile component deoxycholate (DC), and the anionic detergent sodium dodecyl sulfate (SDS) compared to both wild-type and toxTmutant strains. Classical and El Tor toxR mutant strains also exhibit reduced growth rates at subinhibitory concentrations of DC and SDS. Growth of either V. cholerae biotype in subinhibitory concentrations of bile or DC induces increased ToxR-dependent production of a major 38-kDa outer membrane protein, which was confirmed to be OmpU by Western blot. Measurement of transcription of a ompUp-lacZ fusion in both biotypes reveals stimulation (about two- to threefold) of ToxR-dependent ompU transcription by the presence of bile or DC, suggesting that ToxR may respond to the presence of bile. ThetoxR mutant strains of three additional human intestinal pathogenic Vibrio species, V. mimicus, V. fluvialis, and V. parahaemolyticus, display lower MBCs of bile, DC, and SDS and have altered outer membrane protein profiles compared to the parental wild-type strains. Our results demonstrate a conserved role for ToxR in the modulation of outer membrane proteins and bile resistance of pathogenic Vibriospecies and suggest that these ToxR-dependent outer membrane proteins may mediate enhanced resistance to bile. We speculate that ToxR-mediated bile resistance was an early step in the evolution ofV. cholerae as an intestinal pathogen.

scholarly journals Balance between promiscuity and specificity in phage λ host range

2020 ◽  
Author(s):  
Bryan Andrews ◽  
Stanley Fields
Keyword(s):  
Host Range ◽  
Host Resistance ◽  
Fitness Landscape ◽  
Tail Fiber ◽  
Wild Type ◽  
E Coli ◽  
General Ability ◽  
Novel Host ◽  
Selection For ◽  
Resistance To Viruses

AbstractAs hosts acquire resistance to viruses, viruses must overcome that resistance to re-establish infectivity, or go extinct. Despite the significant hurdles associated with adapting to a resistant host, viruses are evolutionarily successful and maintain stable coevolutionary relationships with their hosts. To investigate the factors underlying how pathogens adapt to their hosts, we performed a deep mutational scan of the region of the λ tail fiber tip protein that mediates contact with the λ host, E. coli. Phages harboring amino acid substitutions were subjected to selection for infectivity on wild type E. coli, revealing a highly restrictive fitness landscape, in which most substitutions completely abrogate function. By comparing this lack of mutational tolerance to evolutionary diversity, we highlight a set of mutationally intolerant and diverse positions associated with host range expansion. Imposing selection for infectivity on three λ-resistant hosts, each harboring a different missense mutation in the λ receptor, reveals hundreds of adaptive variants in λ. We distinguish λ variants that confer promiscuity, a general ability to overcome host resistance, from those that drive host-specific infectivity. Both processes may be important in driving adaptation to a novel host.

scholarly journals Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity

2015 ◽  
Vol 90 (5) ◽  
pp. 2418-2433 ◽  
Author(s):  
James Weger-Lucarelli ◽  
Matthew T. Aliota ◽  
Nathan Wlodarchak ◽  
Attapon Kamlangdee ◽  
Ryan Swanson ◽  
...  
Keyword(s):  
Host Range ◽  
Receptor Binding ◽  
Chikungunya Virus ◽  
Semliki Forest Virus ◽  
Binding Protein ◽  
Protein Domain ◽  
Cdna Clones ◽  
E2 Protein ◽  
Receptor Binding Protein

ABSTRACTAlphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicityin vitroandin vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination inAedes aegyptimosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis.IMPORTANCEChikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat, little is known about the role the receptor binding protein (E2) plays on disease outcome in an infected host. To study this, our laboratory generated chimeric CHIKV containing corresponding regions of the Semliki Forest virus (SFV) E2 (domains A, B, and C) substituted into the CHIKV genome. Our results demonstrate that each domain of E2 likely plays a critical, but dissimilar role in the viral life cycle. Our experiments show that manipulation of E2 domains can be useful for studies on viral pathogenesis and potentially the production of vaccines and/or antivirals.

scholarly journals Porcine Endogenous Retroviruses Infect Cells Lacking Cognate Receptors by an Alternative Pathway: Implications for Retrovirus Evolution and Xenotransplantation

2004 ◽  
Vol 78 (16) ◽  
pp. 8868-8877 ◽  
Author(s):  
Dimitri Lavillette ◽  
David Kabat
Keyword(s):  
Host Range ◽  
Receptor Binding ◽  
Envelope Glycoprotein ◽  
Alternative Pathway ◽  
Leukemia Virus ◽  
Endogenous Retroviruses ◽  
Wild Type ◽  
Porcine Endogenous Retroviruses ◽  
Gibbon Ape Leukemia Virus ◽  
Porcine Tissues

ABSTRACT A PHQ motif near the amino termini of gammaretroviral envelope glycoprotein surface (SU) subunits is important for infectivity but not for incorporation into virions or binding to cognate receptors. The H residue of this motif is most critical, with all substitutions we tested being inactive. Interestingly, porcine endogenous retroviruses (PERVs) of all three host-range groups, A, B, and C, lack full PHQ motifs, but most members have an H residue at position 10. H10A PERV mutants are noninfectious but were efficiently transactivated by adding to the assays a PHQ-containing SU or receptor-binding subdomain (RBD) derived from a gibbon ape leukemia virus (GALV). A requirement of this transactivation was a functional GALV receptor on the cells. In contrast to this heterologous transactivation, PERV RBDs and SUs were inactive in all tested cells, including porcine ST-IOWA cells. Surprisingly, transactivation by GALV RBD enabled wild-type or H10A mutant PERVs of all three host-range groups to efficiently infect cells from humans and rodents that lack functional PERV receptors and it substantially enhanced infectivities of wild-type PERVs, even for cells with PERV receptors. Thus, PERVs can suboptimally infect cells that contain cognate receptors or they can employ a transactivation pathway to more efficiently infect all cells. This ability to infect cells lacking cognate receptors was previously demonstrated only for nontransmissible variant gammaretroviruses with recombinant and mutant envelope glycoproteins. We conclude that some endogenously inherited mammalian retroviruses also have a receptor-independent means for overcoming host-range and interference barriers, implying a need for caution in xenotransplantation, especially of porcine tissues.

Phenotype microarray screening of carbon sources used by Vibrio cholerae identifies genes regulated by the cAMP receptor protein

2013 ◽  
Vol 59 (7) ◽  
pp. 472-478 ◽  
Author(s):  
Baoli Chen ◽  
Weili Liang ◽  
Rui Wu ◽  
Pu Liang ◽  
Biao Kan
Keyword(s):  
Carbon Source ◽  
Vibrio Cholerae ◽  
High Throughput Screening ◽  
Carbon Sources ◽  
Experimental Studies ◽  
Receptor Protein ◽  
Phenotype Microarray ◽  
Wild Type ◽  
Rt Pcr ◽  
Mutant Strains

The cyclic AMP receptor protein (CRP) regulates genes involved in carbon source metabolism, iron uptake, and virulence in bacteria. Identifying the carbon sources utilized by bacteria that are regulated by CRP will help elucidate the CRP regulation cascade and associated responses to environmental stimuli. CRP-dependent regulation of carbon source metabolism in Vibrio cholerae is not thoroughly understood. To identify the candidate carbon sources utilized by V. cholerae that are affected by CRP, we used high-throughput screening to compare the metabolic differences between wild-type and CRP mutant strains of V. cholerae O1 El Tor. Phenotype microarray was used for primary screening of the wild-type and mutant strains, followed by minimal media growth assays and quantitative RT-PCR to validate the candidate carbon sources. In total, 24 carbon sources were subject to CRP regulation, 11 of which have not been previously reported in bacteria. The genes known to be involved in the metabolism of 4 of the carbon sources identified were verified by quantitative RT-PCR. In addition, gel shift experiments showed that CRP bound directly to VCA0053 and VC0391 promoters. Overall, this comprehensive analysis of CRP-mediated catabolite control in V. cholerae has identified new candidate carbon sources for in-depth experimental studies.

scholarly journals The Presence of Two Receptor-Binding Proteins Contributes to the Wide Host Range of Staphylococcal Twort-Like Phages

2016 ◽  
Vol 82 (19) ◽  
pp. 5763-5774 ◽  
Author(s):  
Ippei Takeuchi ◽  
Keita Osada ◽  
Aa Haeruman Azam ◽  
Hiroaki Asakawa ◽  
Kazuhiko Miyanaga ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Host Range ◽  
Receptor Binding ◽  
Polyclonal Antibody ◽  
Binding Proteins ◽  
Host Recognition ◽  
Resistant Bacteria ◽  
Tail Fiber ◽  
Content Type ◽  
Wide Host Range

ABSTRACTThanks to their wide host range and virulence, staphylococcal bacteriophages (phages) belonging to the genusTwortlikevirus(staphylococcal Twort-like phages) are regarded as ideal candidates for clinical application forStaphylococcus aureusinfections due to the emergence of antibiotic-resistant bacteria of this species. To increase the usability of these phages, it is necessary to understand the mechanism underlying host recognition, especially the receptor-binding proteins (RBPs) that determine host range. In this study, we found that the staphylococcal Twort-like phage ΦSA012 possesses at least two RBPs. Genomic analysis of five mutant phages of ΦSA012 revealed point mutations inorf103, in a region unique to staphylococcal Twort-like phages. Phages harboring mutated ORF103 could not infectS. aureusstrains in which wall teichoic acids (WTAs) are glycosylated with α-N-acetylglucosamine (α-GlcNAc). A polyclonal antibody against ORF103 also inhibited infection by ΦSA012 in the presence of α-GlcNAc, suggesting that ORF103 binds to α-GlcNAc. In contrast, a polyclonal antibody against ORF105, a short tail fiber component previously shown to be an RBP, inhibited phage infection irrespective of the presence of α-GlcNAc. Immunoelectron microscopy indicated that ORF103 is a tail fiber component localized at the bottom of the baseplate. From these results, we conclude that ORF103 binds α-GlcNAc in WTAs, whereas ORF105, the primary RBP, is likely to bind the WTA backbone. These findings provide insight into the infection mechanism of staphylococcal Twort-like phages.IMPORTANCEStaphylococcusphages belonging to the genusTwortlikevirus(called staphylococcal Twort-like phages) are considered promising agents for control ofStaphylococcus aureusdue to their wide host range and highly lytic capabilities. Although staphylococcal Twort-like phages have been studied widely for therapeutic purposes, the host recognition process of staphylococcal Twort-like phages remains unclear. This work provides new findings about the mechanisms of host recognition of the staphylococcal Twort-like phage ΦSA012. The details of the host recognition mechanism of ΦSA012 will allow us to analyze the mechanisms of infection and expand the utility of staphylococcal Twort-like phages for the control ofS. aureus.

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