scholarly journals Phage Proteins Required for Tail Fiber Assembly Also Bind Specifically to the Surface of Host Bacterial Strains

2020 ◽  
Vol 203 (3) ◽  
Author(s):  
Olesia I. North ◽  
Alan R. Davidson
Keyword(s):  
Cell Surface ◽  
Host Range ◽  
Large Family ◽  
Cell Surface Receptor ◽  
Tail Fiber ◽  
Bacterial Strains ◽  
Surface Binding ◽  
Content Type ◽  
Fiber Assembly ◽  
Phage Mu

ABSTRACT To initiate their life cycle, phages must specifically bind to the surface of their bacterial hosts. Long-tailed phages often interact with the cell surface using fibers, which are elongated intertwined trimeric structures. The folding and assembly of these complex structures generally requires the activity of an intra- or intermolecular chaperone protein. Tail fiber assembly (Tfa) proteins are a very large family of proteins that serve as chaperones for fiber folding in a wide variety of phages that infect diverse species. A recent structural study showed that the Tfa protein from Escherichia coli phage Mu (TfaMu) mediates fiber folding and stays bound to the distal tip of the fiber, becoming a component of the mature phage particle. This finding revealed the potential for TfaMu to also play a role in cell surface binding. To address this issue, we have here shown that TfaMu binds to lipopolysaccharide (LPS), the cell surface receptor of phage Mu, with a similar strength as to the fiber itself. Furthermore, we have found that TfaMu and the Tfa protein from E. coli phage P2 bind LPS with distinct specificities that mirror the host specificity of these two phages. By comparing the sequences of these two proteins, which are 93% identical, we identified a single residue that is responsible for their distinct LPS-binding behaviors. Although we have not yet found conditions under which Tfa proteins influence host range, the potential for such a role is now evident, as we have demonstrated their ability to bind LPS in a strain-specific manner. IMPORTANCE With the growing interest in using phages to combat antibiotic-resistant infections or manipulate the human microbiome, establishing approaches for the modification of phage host range has become an important research topic. Tfa proteins are a large family of proteins known previously to function as chaperones for the folding of phage fibers, which are crucial determinants of host range for long-tailed phages. Here, we reveal that some Tfa proteins are bi-functional, with the additional activity of binding to LPS, the surface binding receptor for many phages. This discovery opens up new potential avenues for altering phage host range through engineering of the surface binding specificity of Tfa proteins.

scholarly journals Differences in Lactococcal Cell Wall Polysaccharide Structure Are Major Determining Factors in Bacteriophage Sensitivity

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Stuart Ainsworth ◽  
Irina Sadovskaya ◽  
Evguenii Vinogradov ◽  
Pascal Courtin ◽  
Yann Guerardel ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Host Range ◽  
Variable Region ◽  
Cell Surface Receptor ◽  
Host Recognition ◽  
Cell Wall Polysaccharide ◽  
Subtype C ◽  
Content Type ◽  
Type C

ABSTRACTAnalysis of the genetic locus encompassing a cell wall polysaccharide (CWPS) biosynthesis operon of eight strains ofLactococcus lactis, identified as belonging to the same CWPS type C genotype, revealed the presence of a variable region among the strains examined. The results allowed the identification of five subgroups of the C type named subtypes C1to C5. This variable region contains genes encoding glycosyltransferases that display low or no sequence homology between the subgroups. In this study, we purified an acidic polysaccharide from the cell wall ofL. lactis3107 (subtype C2) and confirmed that it is structurally different from the previously established CWPS of subtype C1L. lactisMG1363. The CWPS ofL. lactis3107 is composed of pentasaccharide repeating units linked by phosphodiester bonds with the structure 6-α-Glc-3-β-Galf-3-β-GlcNAc-2-β-Galf-6-α-GlcNAc-1-P. Combinations of genes from the variable region of subtype C2were introduced into a mutant of subtype C1L. lactisNZ9000 deficient in CWPS biosynthesis. The resulting recombinant mutant synthesized a polysaccharide with a composition characteristic of that of subtype C2L. lactis3107 and not wild-type C1L. lactisNZ9000. By challenging the recombinant mutant with various lactococcal phages, we demonstrated that CWPS is the host cell surface receptor of tested bacteriophages of both the P335 and 936 groups and that differences between the CWPS structures play a crucial role in determining phage host range.IMPORTANCEDespite the efforts of nearly 80 years of lactococcal phage research, the precise nature of the cell surface receptors of the P335 and 936 phage group receptors has remained elusive. This work demonstrates the molecular nature of a P335 group receptor while bolstering the evidence of its role in host recognition by phages of the 936 group and at least partially explains why such phages have a very narrow host range. The information generated will be instrumental in understanding the molecular mechanisms of how phages recognize specific saccharidic receptors located on the surface of their bacterial host.

scholarly journals Sibling Rivalry in Myxococcus xanthus Is Mediated by Kin Recognition and a Polyploid Prophage

2016 ◽  
Vol 198 (6) ◽  
pp. 994-1004 ◽  
Author(s):  
Arup Dey ◽  
Christopher N. Vassallo ◽  
Austin C. Conklin ◽  
Darshankumar T. Pathak ◽  
Vera Troselj ◽  
...  
Keyword(s):  
Cell Surface ◽  
Kin Recognition ◽  
Myxococcus Xanthus ◽  
Laboratory Strain ◽  
Cell Surface Receptor ◽  
Form Complex ◽  
Content Type ◽  
Recognition Specificity ◽  
Surface Receptor ◽  
Evolutionary Event

ABSTRACTMyxobacteria form complex social communities that elicit multicellular behaviors. One such behavior is kin recognition, in which cells identify siblings via their polymorphic TraA cell surface receptor, to transiently fuse outer membranes and exchange their contents. In addition, outer membrane exchange (OME) regulates behaviors, such as inhibition of wild-typeMyxococcus xanthus(DK1622) from swarming. Here we monitored the fate of motile cells and surprisingly found they were killed by nonmotile siblings. The kill phenotype required OME (i.e., was TraA dependent). The genetic basis of killing was traced to ancestral strains used to construct DK1622. Specifically, the kill phenotype mapped to a large “polyploid prophage,” Mx alpha. Sensitive strains contained a 200-kb deletion that removed two of three Mx alpha units. To explain these results, we suggest that Mx alpha expresses a toxin-antitoxin cassette that uses the OME machinery ofM. xanthusto transfer a toxin that makes the population “addicted” to Mx alpha. Thus, siblings that lost Mx alpha units (no immunity) are killed by cells that harbor the element. To test this, an Mx alpha-harboring laboratory strain was engineered (bytraAallele swap) to recognize a closely related species,Myxococcus fulvus. As a result,M. fulvus, which lacks Mx alpha, was killed. These TraA-mediated antagonisms provide an explanation for how kin recognition specificity might have evolved in myxobacteria. That is, recognition specificity is determined by polymorphisms intraA, which we hypothesize were selected for because OME with non-kin leads to lethal outcomes.IMPORTANCEThe transition from single cell to multicellular life is considered a major evolutionary event. Myxobacteria have successfully made this transition. For example, in response to starvation, individual cells aggregate into multicellular fruiting bodies wherein cells differentiate into spores. To build fruits, cells need to recognize their siblings, and in part, this is mediated by the TraA cell surface receptor. Surprisingly, we report that TraA recognition can also involve sibling killing. We show that killing originates from a prophage-like element that has apparently hijacked the TraA system to deliver a toxin to kin. We hypothesize that this killing system has imposed selective pressures on kin recognition, which in turn has resulted in TraA polymorphisms and hence many different recognition groups.

scholarly journals Adenovirus Fiber Shaft Contains a Trimerization Element That Supports Peptide Fusion for Targeted Gene Delivery

2006 ◽  
Vol 80 (24) ◽  
pp. 12324-12331 ◽  
Author(s):  
Jiali Li ◽  
Sonya Lad ◽  
Guang Yang ◽  
Yunping Luo ◽  
Milena Iacobelli-Martinez ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Cell Surface ◽  
Chimeric Protein ◽  
Cell Surface Receptor ◽  
Cell Surface Receptors ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Surface Receptors ◽  
Fiber Assembly ◽  
Fiber Protein

ABSTRACT Adenoviral (Ad) vectors have been widely used in human gene therapy clinical trials. However, their application has frequently been restricted by the unfavorable expression of cell surface receptors critical for Ad infection. Infections by Ad2 and Ad5 are largely regulated by the elongated fiber protein that mediates its attachment to a cell surface receptor, coxsackie and adenovirus receptor (CAR). The fiber protein is a homotrimer consisting of an N-terminal tail, a long shaft, and a C-terminal knob region that is responsible for high-affinity receptor binding and Ad tropism. Consequently, the modification of the knob region, including peptide insertion and C-terminal fusion of ligands for cell surface receptors, has become a major research focus for targeting gene delivery. Such manipulation tends to disrupt fiber assembly since the knob region contains a stabilization element for fiber trimerization. We report here the identification of a novel trimerization element in the Ad fiber shaft. We demonstrate that fiber fragments containing the N-terminal tail and shaft repeats formed stable trimers that assembled onto Ad virions independently of the knob region. This fiber shaft trimerization element (FSTE) exhibited a capacity to support peptide fusion. We showed that Ad, modified with a chimeric protein by direct fusion of the FSTE with a growth factor ligand or a single-chain antibody, delivered a reporter gene selectively. Together, these results indicate that the shaft region of Ad fiber protein contains a trimerization element that allows ligand fusion, which potentially broadens the basis for Ad vector development.

scholarly journals Sinorhizobium meliloti Phage ΦM9 Defines a New Group of T4 Superfamily Phages with Unusual Genomic Features but a Common T=16 Capsid

2015 ◽  
Vol 89 (21) ◽  
pp. 10945-10958 ◽  
Author(s):  
Matthew C. Johnson ◽  
Kelsey B. Tatum ◽  
Jason S. Lynn ◽  
Tess E. Brewer ◽  
Stephen Lu ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Open Reading Frames ◽  
Host Cells ◽  
Close Relative ◽  
Tail Fiber ◽  
Nitrogen Fixing ◽  
Related Factors ◽  
Content Type ◽  
Fiber Assembly ◽  
Host Infection

ABSTRACTRelatively little is known about the phages that infect agriculturally important nitrogen-fixing rhizobial bacteria. Here we report the genome and cryo-electron microscopy structure of theSinorhizobium meliloti-infecting T4 superfamily phage ΦM9. This phage and its close relativeRhizobiumphage vB_RleM_P10VF define a new group of T4 superfamily phages. These phages are distinctly different from the recently characterized cyanophage-likeS. melilotiphages of the ΦM12 group. Structurally, ΦM9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the ΦM9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. ΦM9 also has the same T=16 capsid architecture as the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, ΦM9 andS. melilotiphage ΦM12 have a small number of open reading frames in common that appear to encode structural proteins involved in interaction with the host and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins, and glycanases that cleave host exopolysaccharide.IMPORTANCEDespite recent advances in the phylogenetic and structural characterization of bacteriophages, only a small number of phages of plant-symbiotic nitrogen-fixing soil bacteria have been studied at the molecular level. The effects of phage predation upon beneficial bacteria that promote plant growth remain poorly characterized. First steps in understanding these soil bacterium-phage dynamics are genetic, molecular, and structural characterizations of these groups of phages. The T4 superfamily phages are among the most complex phages; they have large genomes packaged within an icosahedral head and a long, contractile tail through which the DNA is delivered to host cells. This phylogenetic and structural study ofS. meliloti-infecting T4 superfamily phage ΦM9 provides new insight into the diversity of this family. The comparison of structure-related genes in both ΦM9 andS. meliloti-infecting T4 superfamily phage ΦM12, which comes from a completely different lineage of these phages, allows the identification of host infection-related factors.

scholarly journals Genetic definition of two functional elements in a bacteriophage T4 host-range "cassette".

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 471-479
Author(s):  
M Snyder ◽  
W B Wood
Keyword(s):  
Host Range ◽  
Bacteriophage T4 ◽  
Temperature Sensitive ◽  
Tail Fiber ◽  
Neighboring Gene ◽  
Distal Half ◽  
Bacterial Surface ◽  
Fiber Assembly ◽  
Proximal Half ◽  
Carboxy Terminal

Abstract Gene 37 of T4 encodes the major subunit of the distal half of the tail fiber. The distal tip of the fiber, comprised of the carboxy-terminal ends of two molecules of gene 37 product (gp37), carries the principal determinant of the phage host range. The gp37 carboxyl termini recognize the bacterial surface during infection, and, in addition, include a site required for interaction with the product of gp38 during distal half-fiber assembly. In the absence of interaction with gp38, gp37 polypeptides do not dimerize. Eleven temperature-sensitive mutants with defects located near the promoter-distal end of gene 37 were tested at nonpermissive temperatures for production of an antigen that is diagnostic of distal half-fiber assembly. Six of the mutations prevent distal half-fiber assembly. The other five allow assembly of distal half fibers, which combine with proximal half fibers and attach to phage particles, but the resulting phage do not adsorb to bacteria. These two classes of mutations define two adjacent but separate genetic regions, corresponding to two different functional domains in gp37. These two regions and the neighboring gene 38 comprise a functional unit that can be considered as a host-range "cassette," with features that are strikingly similar to corresponding functional units in other unrelated as well as related phages.

scholarly journals Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry

2016 ◽  
Vol 90 (13) ◽  
pp. 6097-6111 ◽  
Author(s):  
Bethany A. Rhein ◽  
Rachel B. Brouillette ◽  
Grace A. Schaack ◽  
John A. Chiorini ◽  
Wendy Maury
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Ebola Virus ◽  
Virus Entry ◽  
Binding Pocket ◽  
Cell Surface Receptor ◽  
Target Cells ◽  
Virus Binding ◽  
Content Type ◽  
Amino Terminal

ABSTRACTPhosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of theFiloviridaefamily of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion–TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions.IMPORTANCEWith more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses, including EBOV. TIM family member TIM-4 is expressed on macrophages and dendritic cells, which are early cellular targets during EBOV infection. Here, we performed a mutagenesis screening of the IgV domain of murine and human TIM-4 to identify residues that are critical for EBOV entry. Surprisingly, we identified more human than murine TIM-4 IgV domain residues that are required for EBOV entry. Defining the TIM IgV residues needed for EBOV entry clarifies the virus-receptor interactions and paves the way for the development of novel therapeutics targeting virus binding to this cell surface receptor.

Ephrin-B3 binds to a sulfated cell-surface receptor

2010 ◽  
Vol 433 (1) ◽  
pp. 215-223 ◽  
Author(s):  
Halvor L. Holen ◽  
Lillian Zernichow ◽  
Kristine E. Fjelland ◽  
Ida M. Evenroed ◽  
Kristian Prydz ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Site Directed Mutagenesis ◽  
Cell Surface Receptor ◽  
Binding Property ◽  
Heparin Binding ◽  
Surface Binding ◽  
Cellular Signalling

The ephrins are a family of proteins known to bind the Eph (erythropoietin-producing hepatocellular) receptor tyrosine kinase family. In the present paper, we provide data showing that ephrin-B3 binds a sulfated cell-surface protein on HEK-293T (human embryonic kidney-293 cells expressing the large T-antigen of simian virus 40) and HeLa cells, a binding that is nearly completely blocked by treatment of these cell lines with chlorate or heparinase, or by addition of the heavily sulfated glycosaminoglycan heparin. This indicates that heparan sulfate on these cells is essential for cell-surface binding of ephrin-B3. Heparin did not affect ephrin-B3 binding to EphB receptors expressed on transfected HEK-293T cells, indicating further that ephrin-B3 binds an alternative receptor which is a heparan sulfate proteoglycan. Site-directed mutagenesis analysis revealed that Arg178 and Lys179 are important for heparin binding of ephrin-B3 and also for ephrin-B3 binding to cells. These amino acids, when introduced in the non-heparin-binding ephrin-B1, conferred the heparin-binding property. Functional studies reveal that ephrin-B3 binding to cells induces cellular signalling and influences cell rounding and cell spreading. In conclusion, our data provide evidence for an unknown ephrin-B3-binding cell-surface proteoglycan involved in cellular signalling.

scholarly journals Strain-Specific Features of Extracellular Polysaccharides and Their Impact on Lactobacillus plantarum-Host Interactions

2016 ◽  
Vol 82 (13) ◽  
pp. 3959-3970 ◽  
Author(s):  
I-Chiao Lee ◽  
Graziano Caggianiello ◽  
Iris I. van Swam ◽  
Nico Taverne ◽  
Marjolein Meijerink ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lactobacillus Plantarum ◽  
Surface Properties ◽  
Gene Clusters ◽  
Host Cells ◽  
Extracellular Polysaccharides ◽  
Bacterial Strains ◽  
Strain Specificity ◽  
Content Type ◽  
Health Promoting

ABSTRACTLactobacilli are found in diverse environments and are widely applied as probiotic, health-promoting food supplements. Polysaccharides are ubiquitously present on the cell surface of lactobacilli and are considered to contribute to the species- and strain-specific probiotic effects that are typically observed. TwoLactobacillus plantarumstrains, SF2A35B and Lp90, have an obvious ropy phenotype, implying high extracellular polysaccharide (EPS) production levels. In this work, we set out to identify the genes involved in EPS production in theseL. plantarumstrains and to demonstrate their role in EPS production by gene deletion analysis. A modelL. plantarumstrain, WCFS1, and its previously constructed derivative that produced reduced levels of EPS were included as reference strains. The constructed EPS-reduced derivatives were analyzed for the abundance and sugar compositions of their EPS, revealingcps2-like gene clusters in SF2A35B and Lp90 responsible for major EPS production. Moreover, these mutant strains were tested for phenotypic characteristics that are of relevance for their capacity to interact with the host epithelium in the intestinal tract, including bacterial surface properties as well as survival under the stress conditions encountered in the gastrointestinal tract (acid and bile stress). In addition, the Toll-like receptor 2 (TLR2) signaling and immunomodulatory capacities of the EPS-negative derivatives and their respective wild-type strains were compared, revealing strain-specific impacts of EPS on the immunomodulatory properties. Taken together, these experiments illustrate the importance of EPS inL. plantarumstrains as a strain-specific determinant in host interaction.IMPORTANCEThis study evaluates the role of extracellular polysaccharides that are produced by different strains ofLactobacillus plantarumin the determination of the cell surface properties of these bacteria and their capacity to interact with their environment, including their signaling to human host cells. The results clearly show that the consequences of removal of these polysaccharides are very strain specific, illustrating the diverse and unpredictable roles of these polysaccharides in the environmental interactions of these bacterial strains. In the context of the use of lactobacilli as health-promoting probiotic organisms, this study exemplifies the importance of strain specificity.

scholarly journals Human-Specific Bacterial Pore-Forming Toxins Induce Programmed Necrosis in Erythrocytes

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Timothy J. LaRocca ◽  
Elizabeth A. Stivison ◽  
Eldad A. Hod ◽  
Steven L. Spitalnik ◽  
Peter J. Cowan ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Host Range ◽  
Acid Sphingomyelinase ◽  
Gardnerella Vaginalis ◽  
Bacterial Strains ◽  
Programmed Necrosis ◽  
Content Type ◽  
Cell Death Pathway ◽  
Human Specific

ABSTRACT A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. IMPORTANCE In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC death pathway that shares important components with necroptosis, a programmed necrosis module that occurs in nucleated cells, these findings expand our understanding of RBC biology and RBC-pathogen interactions. In addition, our work provides a link between cholesterol-dependent cytolysin (CDC) host restriction and promotion of bacterial growth in the presence of RBCs, which may provide a selective advantage to human-associated bacterial strains that elaborate such toxins and a potential explanation for the narrowing of host range observed in this toxin family.

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