Publications by authors named "Nikolai S Prokhorov"

14 Publications

  • Page 1 of 1

Quantitative description of a contractile macromolecular machine.

Sci Adv 2021 Jun 11;7(24). Epub 2021 Jun 11.

Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics (SCSB), The University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA.

Contractile injection systems (CISs) [type VI secretion system (T6SS), phage tails, and tailocins] use a contractile sheath-rigid tube machinery to breach cell walls and lipid membranes. The structures of the pre- and postcontraction states of several CISs are known, but the mechanism of contraction remains poorly understood. Combining structural information of the end states of the 12-megadalton R-type pyocin sheath-tube complex with thermodynamic and force spectroscopy analyses and an original modeling procedure, we describe the mechanism of pyocin contraction. We show that this nanomachine has an activation energy of 160 kilocalories/mole (kcal/mol), and it releases 2160 kcal/mol of heat and develops a force greater than 500 piconewtons. Our combined approach provides a quantitative and experimental description of the membrane penetration process by a CIS.
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http://dx.doi.org/10.1126/sciadv.abf9601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8195476PMC
June 2021

Reprogramming bacteriophage host range: design principles and strategies for engineering receptor binding proteins.

Curr Opin Biotechnol 2021 04 18;68:272-281. Epub 2021 Mar 18.

Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX, USA.

Bacteriophages (phages) use specialized tail machinery to deliver proteins and genetic material into a bacterial cell during infection. Attached at the distal ends of their tails are receptor binding proteins (RBPs) that recognize specific molecules exposed on host bacteria surfaces. Since the therapeutic capacity of naturally occurring phages is often limited by narrow host ranges, there is significant interest in expanding their host range via directed evolution or structure-guided engineering of their RBPs. Here, we describe the design principles of different RBP engineering platforms and draw attention to the mechanisms linking RBP binding and the correct spatial and temporal attachment of the phage to the bacterial surface. A deeper understanding of these mechanisms will directly benefit future engineering of more effective phage-based therapeutics.
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http://dx.doi.org/10.1016/j.copbio.2021.02.006DOI Listing
April 2021

Action of a minimal contractile bactericidal nanomachine.

Nature 2020 04 15;580(7805):658-662. Epub 2020 Apr 15.

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA, USA.

R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics. Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold. Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage. Here we report the atomic model of the complete R2 pyocin in its pre-contraction and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the following sequence of events during pyocin contraction: tail fibres trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; and this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.
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http://dx.doi.org/10.1038/s41586-020-2186-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513463PMC
April 2020

Structure and Function of the Branched Receptor-Binding Complex of Bacteriophage CBA120.

J Mol Biol 2019 09 17;431(19):3718-3739. Epub 2019 Jul 17.

Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-0647, USA. Electronic address:

Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120-TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1-TSP2-TSP3-TSP4 branched complex in CBA120 and its related ViI-like phages.
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http://dx.doi.org/10.1016/j.jmb.2019.07.022DOI Listing
September 2019

High-throughput LPS profiling as a tool for revealing of bacteriophage infection strategies.

Sci Rep 2019 02 27;9(1):2958. Epub 2019 Feb 27.

Winogradsky Institute of Microbiology, Research Center of Biotechnology of the Russian Academy of Sciences, prosp. 60-letiya Oktyabrya, 7 bld. 2, 117312, Moscow, Russian Federation.

O-antigens of Gram-negative bacteria modulate the interactions of bacterial cells with diverse external factors, including the components of the immune system and bacteriophages. Some phages need to acquire specific adhesins to overcome the O-antigen layer. For other phages, O-antigen is required for phage infection. In this case, interaction of phage receptor binding proteins coupled with enzymatic degradation or modification of the O-antigen is followed by phage infection. Identification of the strategies used by newly isolated phages may be of importance in their consideration for various applications. Here we describe an approach based on screening for host LPS alterations caused by selection by bacteriophages. We describe an optimized LPS profiling procedure that is simple, rapid and suitable for mass screening of mutants. We demonstrate that the phage infection strategies identified using a set of engineered E. coli 4 s mutants with impaired or altered LPS synthesis are in good agreement with the results of simpler tests based on LPS profiling of phage-resistant spontaneous mutants.
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http://dx.doi.org/10.1038/s41598-019-39590-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393563PMC
February 2019

Escherichia coli bacteriophage Gostya9, representing a new species within the genus T5virus.

Arch Virol 2019 Mar 1;164(3):879-884. Epub 2018 Dec 1.

Federal State Institution «Federal Research Centre «Fundamentals of Biotechnology» of the Russian Academy of Sciences», Winogradsky Institute of Microbiology, prosp. 60-letiya Oktyabrya, 7/2, 117312, Moscow, Russian Federation.

Escherichia coli bacteriophage Gostya9 (genus T5virus) was isolated from horse feces collected in Moscow, Russia, in 2013. This phage was associated in a single plaque with the previously reported phage 9g and was subsequently purified. Analysis of the complete genomic sequence of Gostya9 revealed that it is closely related to the T5-like bacteriophage DT57C, which had been isolated at the same location in 2007. These two viruses share 79.5% nucleotide sequence identity, which is below the 95% threshold applied currently to demarcate bacteriophage species. The most significant features distinguishing Gostya9 from DT57C include 1) the presence of one long tail fiber protein gene, 122c (ltf), instead of the two genes, ltfA and ltfB, that are present in DT57C; 2) the absence of the gene for the receptor-blocking lytic conversion lipoprotein precursor llp; and 3) the divergence of the receptor-recognition protein, pb5, which is only distantly related at the amino acid sequence level. The observed features of the Gostya9 adsorption apparatus are suggestive of a possible novel specificity for the final receptor and make this phage interesting for possible direct application in phage therapy of E. coli infections or as a source of receptor-recognition protein for engineering new phage specificities.
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http://dx.doi.org/10.1007/s00705-018-4113-2DOI Listing
March 2019

Corrigendum to "Structure of the O-polysaccharide of Escherichia coli O87" [Carbohydr. Res. 412 (2015) 15-18].

Carbohydr Res 2018 07 25;464. Epub 2018 May 25.

N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991, Moscow, Russia.

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http://dx.doi.org/10.1016/j.carres.2018.04.013DOI Listing
July 2018

Function of bacteriophage G7C esterase tailspike in host cell adsorption.

Mol Microbiol 2017 Aug 19;105(3):385-398. Epub 2017 Jun 19.

Research Center of Biotechnology, Russian Academy of Sciences, Winogradsky Institute of Microbiology, 7b2 pr. 60-letiya Oktyabrya, Moscow, 117312, Russia.

Bacteriophages recognize and bind to their hosts with the help of receptor-binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C - tailspikes gp63.1 and gp66 - are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and function of gp63.1 and show that unlike any previously described RPB, gp63.1 deacetylates surface polysaccharides of E. coli 4s leaving the backbone of the polysaccharide intact. We demonstrate that gp63.1 and gp66 form a stable complex, in which the N-terminal part of gp66 serves as an attachment site for gp63.1 and anchors the gp63.1-gp66 complex to the G7C tail. The esterase domain of gp63.1 as well as domains mediating the gp63.1-gp66 interaction is widespread among all three families of tailed bacteriophages.
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http://dx.doi.org/10.1111/mmi.13710DOI Listing
August 2017

Structure of the T4 baseplate and its function in triggering sheath contraction.

Nature 2016 05;533(7603):346-52

École Polytechnique Fédérale de Lausanne (EPFL), BSP-415, 1015 Lausanne, Switzerland.

Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.
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http://dx.doi.org/10.1038/nature17971DOI Listing
May 2016

Branched Lateral Tail Fiber Organization in T5-Like Bacteriophages DT57C and DT571/2 is Revealed by Genetic and Functional Analysis.

Viruses 2016 Jan 21;8(1). Epub 2016 Jan 21.

Winogradsky Institute of Microbiology, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, build. 2, Moscow 119071, Russia.

The T5-like siphoviruses DT57C and DT571/2, isolated from horse feces, are very closely related to each other, and most of their structural proteins are also nearly identical to T5 phage. Their LTFs (L-shaped tail fibers), however, are composed of two proteins, LtfA and LtfB, instead of the single Ltf of bacteriophage T5. In silico and mutant analysis suggests a possible branched structure of DT57C and DT571/2 LTFs, where the LtfB protein is connected to the phage tail via the LtfA protein and with both proteins carrying receptor recognition domains. Such adhesin arrangement has not been previously recognized in siphoviruses. The LtfA proteins of our phages are found to recognize different host O-antigen types: E. coli O22-like for DT57C phage and E. coli O87 for DT571/2. LtfB proteins are identical in both phages and recognize another host receptor, most probably lipopolysaccharide (LPS) of E. coli O81 type. In these two bacteriophages, LTF function is essential to penetrate the shield of the host's O-antigens. We also demonstrate that LTF-mediated adsorption becomes superfluous when the non-specific cell protection by O-antigen is missing, allowing the phages to bind directly to their common secondary receptor, the outer membrane protein BtuB. The LTF independent adsorption was also demonstrated on an O22-like host mutant missing O-antigen O-acetylation, thus showing the biological value of this O-antigen modification for cell protection against phages.
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http://dx.doi.org/10.3390/v8010026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728585PMC
January 2016

Complete genome sequences of T5-related Escherichia coli bacteriophages DT57C and DT571/2 isolated from horse feces.

Arch Virol 2015 Dec 9;160(12):3133-7. Epub 2015 Sep 9.

Winogradsky Institute of Microbiology, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave 33, build. 2, Moscow, 119071, Russia.

We report the complete genome sequencing of two Escherichia coli T5-related bacteriophages, DT57C and DT571/2, isolated from the same specimen of horse feces. These two isolates share 96% nucleotide sequence identity and can thus be considered representatives of the same novel species within the genus T5likevirus. The observed variation in the ltfA gene of these phages, resulting from a recent recombination event, may explain the observed host-range differences, suggesting that a modular mechanism makes a significant contribution to the short-term evolution (or adaptation) of T5-like phage genomes in the intestinal ecosystem. Comparison of our isolates to their closest relative, coliphage T5, revealed high overall synteny of the genomes and high conservation of the sequences of almost all structural proteins as well as of the other proteins with identified functions. At the same time, numerous alterations and non-orthologous replacements of non-structural protein genes (mostly of those with unknown functions) as well as substantial differences in tail fiber locus organization support the conclusion that DT57C and DT571/2 form a species-level group clearly distinct from bacteriophage T5.
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http://dx.doi.org/10.1007/s00705-015-2582-0DOI Listing
December 2015

Structure of the O-polysaccharide of Escherichia coli O87.

Carbohydr Res 2015 Aug 1;412:15-8. Epub 2015 May 1.

N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russia.

The following structure of the O-polysaccharide of Escherichia coli HS1/2 serving as a primary receptor for bacteriophage DT57-12 was elucidated by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy: This structure is shared by E. coli O87 type strain. Putatively assigned functions of genes in the O-antigen gene cluster of E. coli O87 are consistent with the O-polysaccharide structure established.
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http://dx.doi.org/10.1016/j.carres.2015.04.014DOI Listing
August 2015

Genomic sequencing and biological characteristics of a novel Escherichia coli bacteriophage 9g, a putative representative of a new Siphoviridae genus.

Viruses 2014 Dec 19;6(12):5077-92. Epub 2014 Dec 19.

Laboratory of microbial viruses, Winogradsky Institute of Microbiology, Russian Academy of Sciences, prosp. 60-letiya Oktyabrya, 7/2, 117312 Moscow, Russia.

Bacteriophage 9 g was isolated from horse feces using Escherichia coli C600 as a host strain. Phage 9 g has a slightly elongated capsid 62 × 76 nm in diameter and a non-contractile tail about 185 nm long. The complete genome sequence of this bacteriophage consists of 56,703 bp encoding 70 predicted open reading frames. The closest relative of phage 9 g is phage PhiJL001 infecting marine alpha-proteobacterium associated with Ircinia strobilina sponge, sharing with phage 9 g 51% of amino acid identity in the main capsid protein sequence. The DNA of 9 g is resistant to most restriction endonucleases tested, indicating the presence of hypermodified bases. The gene cluster encoding a biosynthesis pathway similar to biosynthesis of the unusual nucleoside queuosine was detected in the phage 9 g genome. The genomic map organization is somewhat similar to the typical temperate phage gene layout but no integrase gene was detected. Phage 9 g efficiently forms stable associations with its host that continues to produce the phage over multiple passages, but the phage can be easily eliminated via viricide treatment indicating that no true lysogens are formed. Since the sequence, genomic organization and biological properties of bacteriophage 9 g are clearly distinct from other known Enterobacteriaceae phages, we propose to consider it as the representative of a novel genus of the Siphoviridae family.
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http://dx.doi.org/10.3390/v6125077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276943PMC
December 2014

Variations in O-antigen biosynthesis and O-acetylation associated with altered phage sensitivity in Escherichia coli 4s.

J Bacteriol 2015 Mar 15;197(5):905-12. Epub 2014 Dec 15.

S. N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmental Escherichia coli isolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen of E. coli O22, differing only in side-chain α-D-glucosylation in the former, mediated by a gtr locus on the chromosome. Spontaneous mutations of E. coli 4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions of E. coli 4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.
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http://dx.doi.org/10.1128/JB.02398-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4325112PMC
March 2015
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