scholarly journals Maintenance of the Shigella sonnei virulence plasmid is dependent on its repertoire and amino acid sequence of toxin:antitoxin systems

2022 ◽  
Author(s):  
Jessica E. Martyn ◽  
Giulia Pilla ◽  
Sarah Hollingshead ◽  
Kristoffer S. Winther ◽  
Susan Lea ◽  
...  
Keyword(s):  
Amino Acid ◽  
Vaccine Development ◽  
Shigella Flexneri ◽  
Amino Acid Sequences ◽  
Shigella Sonnei ◽  
Single Amino Acid ◽  
Multi Drug Resistance ◽  
Virulence Plasmid ◽  
Closely Related Species ◽  
Single Amino Acid Polymorphisms

Shigella sonnei is a major cause of bacillary dysentery, and of increasing concern due to the spread of multi-drug resistance. S. sonnei harbours pINV, a ∼ 210 kb plasmid that encodes a Type III secretion system (T3SS), which is essential for virulence. During growth in the laboratory, avirulence arises spontaneously in S. sonnei at high frequency, hampering studies on and vaccine development against this important pathogen. Here we investigated the molecular basis for the emergence of avirulence in S. sonnei , and show that avirulence mainly results from pINV loss, consistent with previous findings. Ancestral deletions have led to the loss from S. sonnei pINV of two toxin:antitoxin (TA) systems involved in plasmid maintenance, CcdAB and GmvAT, which are found on pINV in Shigella flexneri . We show that introduction of these TA systems into S. sonnei pINV reduced but did not eliminate pINV loss, while single amino acid polymorphisms found in the S. sonnei VapBC TA system compared with S. flexneri VapBC also contribute to pINV loss. Avirulence also results from deletions of T3SS-associated genes on pINV through recombination between insertion sequences (ISs) on the plasmid; these events differ from those observed in S. flexneri due to the different distribution and repertoire of ISs. Our findings demonstrate that TA systems and ISs influence plasmid dynamics and loss in S. sonnei , and could be exploited for the design and evaluation of vaccines. IMPORTANCE Shigella sonnei is the major cause of shigellosis in high-income and industrialising countries, and an emerging multi-drug resistant pathogen. A significant challenge when studying this bacterium is that it spontaneously becomes avirulent during growth in the laboratory, through loss of its virulence plasmid (pINV). Here we decipher the mechanisms leading to avirulence in S. sonnei and how the limited repertoire and amino acid sequences of plasmid-encoded toxin:antitoxin (TA) systems make the maintenance of pINV in this bacterium less efficient compared with Shigella flexneri . Our findings highlight how subtle differences in plasmids in closely-related species have marked effects and could be exploited to reduce plasmid loss in S. sonnei . This should facilitate research on this bacterium and vaccine development.

scholarly journals Structures suggest an approach for converting weak self-peptide tumor antigens into superagonists for CD8 T cells in cancer

2021 ◽  
Vol 118 (23) ◽  
pp. e2100588118
Author(s):  
Pengcheng Wei ◽  
Kimberly R. Jordan ◽  
Jonathan D. Buhrman ◽  
Jun Lei ◽  
Hexiang Deng ◽  
...  
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
Amino Acid Substitution ◽  
Cd8 T Cells ◽  
Tumor Antigens ◽  
Vaccine Development ◽  
Single Amino Acid ◽  
Cell Repertoire ◽  
Subtle Change

Tumors frequently express unmutated self-tumor–associated antigens (self-TAAs). However, trial results using self-TAAs as vaccine targets against cancer are mixed, often attributed to deletion of T cells with high-affinity receptors (TCRs) for self-TAAs during T cell development. Mutating these weak self-TAAs to produce higher affinity, effective vaccines is challenging, since the mutations may not benefit all members of the broad self-TAA–specific T cell repertoire. We previously identified a common weak murine self-TAA that we converted to a highly effective antitumor vaccine by a single amino acid substitution. In this case the modified and natural self-TAAs still raised very similar sets of CD8 T cells. Our structural studies herein show that the modification of the self-TAA resulted in a subtle change in the major histocompatibility complex I–TAA structure. This amino acid substitution allowed a dramatic conformational change in the peptide during subsequent TCR engagement, creating a large increase in TCR affinity and accounting for the efficacy of the modified self-TAA as a vaccine. These results show that carefully selected, well-characterized modifications to a poorly immunogenic self-TAA can rescue the immune response of the large repertoire of weakly responding natural self-TAA–specific CD8 T cells, driving them to proliferate and differentiate into functional effectors. Subsequently, the unmodified self-TAA on the tumor cells, while unable to drive this response, is nevertheless a sufficient target for the CD8 cytotoxic effectors. Our results suggest a pathway for more efficiently identifying variants of common self-TAAs, which could be useful in vaccine development, complementing other current nonantigen-specific immunotherapies.

scholarly journals Functional Annotation and Curation of Hypothetical Proteins Present in A Newly Emerged Serotype 1c of Shigella flexneri: Emphasis on Selecting Targets for Virulence and Vaccine Design Studies

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 340
Author(s):  
Tanuka Sen ◽  
Naresh K. Verma
Keyword(s):  
Diarrheal Disease ◽  
Shigella Flexneri ◽  
Amino Acid Sequences ◽  
New Drugs ◽  
Homology Search ◽  
Multi Drug Resistance ◽  
Hypothetical Proteins ◽  
Signal Transducers ◽  
Bioinformatics Databases ◽  
Regulatory Functions

Shigella flexneri is the principal cause of bacillary dysentery, contributing significantly to the global burden of diarrheal disease. The appearance and increase in the multi-drug resistance among Shigella strains, necessitates further genetic studies and development of improved/new drugs against the pathogen. The presence of an abundance of hypothetical proteins in the genome and how little is known about them, make them interesting genetic targets. The present study aims to carry out characterization of the hypothetical proteins present in the genome of a newly emerged serotype of S. flexneri (strain Y394), toward their novel regulatory functions using various bioinformatics databases/tools. Analysis of the genome sequence rendered 4170 proteins, out of which 721 proteins were annotated as hypothetical proteins (HPs) with no known function. The amino acid sequences of these HPs were evaluated using a combination of latest bioinformatics tools based on homology search against functionally identified proteins. Functional domains were considered as the basis to infer the biological functions of HPs in this case and the annotation helped in assigning various classes to the proteins such as signal transducers, lipoproteins, enzymes, membrane proteins, transporters, virulence, and binding proteins. This study contributes to a better understanding of growth, survival, and disease mechanism at molecular level and provides potential new targets for designing drugs against Shigella infection.

scholarly journals Effect of single amino acid substitutions on the formation of the PlA and Bak alloantigenic epitopes

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 681-687 ◽  
Author(s):  
A Goldberger ◽  
M Kolodziej ◽  
M Poncz ◽  
JS Bennett ◽  
PJ Newman
Keyword(s):  
Amino Acid ◽  
Expression System ◽  
Single Amino Acid ◽  
Expression Vectors ◽  
Membrane Glycoproteins ◽  
Amino Acid Polymorphism ◽  
Specific Expression ◽  
Mammalian Expression ◽  
Single Amino Acid Polymorphisms ◽  
Necessary And Sufficient

Abstract The subunits that comprise the platelet-specific integrin alpha IIb beta 3 are polymorphic in nature, with several allelic forms present in the human gene pool. Minor changes in the secondary and tertiary structures of platelet membrane glycoproteins (GP) IIb and IIIa encoded by these alleles can result in an alloimmune reaction after transfusion or during pregnancy. To better understand the molecular structure of the PlA alloantigen system, located on GPIIIa, and the Bak alloantigen on GPIIb, we used a heterologous mammalian expression system to express these integrin subunits in their known polymorphic forms. An expression vector containing the PlA1 form of a GPIIIa cDNA, which encodes a leucine at amino acid 33 (Leu33), was modified to express the PlA2- associated form encoding a proline at amino acid 33 (Pro33). Similarly, a Baka GPIIb cDNA expressing an isoleucine at amino acid 843 (IIe843) was modified to express the Bakb form containing a serine at the same position (Ser843). Transfection of these vectors into COS cells resulted in the synthesis of GPIIb and GPIIIa molecules that were identical in size to those present in platelet lysates. Immunoprecipitation of the GPIIIa-transfected COS lysates with PlA)- specific alloantisera indicated that the Leu33 form was recognized only by anti-PIA1 sera while the Pro33 form was bound only by anti-PlA2 sera, showing that single amino acid polymorphisms are necessary and sufficient to direct the formation of the PlA1 and PlA2 alloepitopes. Similar experiments with Bak allele-specific expression vectors indicated that while the amino acid polymorphism (IIe843 in equilibrium Ser843) was necessary, posttranslational processing of pro-IIb was required for efficient exposure of both the Baka and Bakb alloepitopes.

SERPINS: ANTITHROMBIN AND OTHER INHIBITORS OF COAGULATION AND FIBRINOLYSIS. EVIDENCE FROM AMINO ACID SEQUENCES

1987 ◽  
Author(s):  
R W Carrell ◽  
P D Christey ◽  
D R Boswell
Keyword(s):  
Amino Acid ◽  
Inhibitory Activity ◽  
Inflammatory Responses ◽  
Three Dimensional ◽  
Activated Protein C ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
General Function ◽  
Topological Features ◽  
Coagulation And Fibrinolysis

A number of the key inhibitors of coagulation and fibrinolysis have recently been shown to be members of the same superfamily of serine protease inhibitors, the serpins. The archetypes of the group are alpha-l-antitrypsin and antithrombin and it includes antiplasmin, C1-inhibitor, heparin cofactor II and the newly recognised inhibitors of plasminogen activators and activated Protein C. Alignment of their structures shows that they have the same skeletal three-dimensional conformation and, by inference, the same general function mechanisms.The serpins have a reactive centre, primarily dependent on a single amino acid, exteriorly placed on a stressed peptide loop. This functions by offering the cognate protease a high-affinity substrate that resists complete cleavage to form a tight 1:1 complex of inhibitor and protease that is subsequently removed from the circulation. The loop is vulnerable to cleavage with resulting loss of inhibitory activity. This irreversible switch is utilised: pathologically by venom and invasive bacterial proteases; and physiologically by the neutrophil leucocyte to modify local inflammatory responses. These mechanisms contribute to the changes seen in DIC and the shock syndromes.Modelling of antithrombin indicates the likely topological features involved in the binding of heparin, namely a sphere of positive charge centred on the A and D helices and involving Arg 47, Lys 125, Arg 129 and probably Arg 132 and Lys 133.Because the serpins are largely dependent for their specificityon a single amino acid it is now possible to precisely tailor inhibitory activity by site specific mutation. This has been used to produce recombinant antitrypsins that function as an improved inhibitor of neutrophil proteases (valine or leucine reactive centre), or as an analogue of antithrombin (arginine reactive centre). An elegant application of this approach is the engineered mutants of antiplasmin recently described by Holmes, Collen and colleagues (Leuven).

scholarly journals Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function.

1987 ◽  
Vol 7 (6) ◽  
pp. 2231-2242 ◽  
Author(s):  
J E Rudolph ◽  
M Kimble ◽  
H D Hoyle ◽  
M A Subler ◽  
E C Raff
Keyword(s):  
Amino Acid ◽  
Sequence Divergence ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Structural Constraints ◽  
Wild Type ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Single Amino Acid Residue ◽  
Beta 2

The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.

Sign in / Sign up

Export Citation Format

Share Document