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1.
J Bacteriol ; 204(3): e0051921, 2022 03 15.
Article in English | MEDLINE | ID: mdl-34978459

ABSTRACT

Shigella sonnei is a major cause of bacillary dysentery and an increasing concern due to the spread of multidrug resistance. S. sonnei harbors pINV, an ∼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 showed that avirulence mainly results from pINV loss, which is 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 showed that the introduction of these TA systems into S. sonnei pINV reduced but did not eliminate pINV loss, while the single amino acid polymorphisms found in the S. sonnei VapBC TA system compared with S. flexneri VapBC also contributed to pINV loss. Avirulence also resulted from deletions of T3SS-associated genes in pINV through recombination between insertion sequences (ISs) on the plasmid. These events differed from those observed in S. flexneri due to the different distribution and repertoire of ISs. Our findings demonstrated that TA systems and ISs influenced 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 industrializing countries and is an emerging, multidrug-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 deciphered 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 highlighted 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.


Subject(s)
Antitoxins , Dysentery, Bacillary , Toxin-Antitoxin Systems , Amino Acid Sequence , Antitoxins/genetics , DNA Transposable Elements , Dysentery, Bacillary/microbiology , Dysentery, Bacillary/prevention & control , Humans , Plasmids/genetics , Shigella flexneri/genetics , Shigella sonnei/genetics , Toxin-Antitoxin Systems/genetics , Virulence/genetics
2.
Mol Microbiol ; 111(1): 221-236, 2019 01.
Article in English | MEDLINE | ID: mdl-30315706

ABSTRACT

Type II toxin-antitoxin (TA) modules, which are important cellular regulators in prokaryotes, usually encode two proteins, a toxin that inhibits cell growth and a nontoxic and labile inhibitor (antitoxin) that binds to and neutralizes the toxin. Here, we demonstrate that the res-xre locus from Photorhabdus luminescens and other bacterial species function as bona fide TA modules in Escherichia coli. The 2.2 Å crystal structure of the intact Pseudomonas putida RES-Xre TA complex reveals an unusual 2:4 stoichiometry in which a central RES toxin dimer binds two Xre antitoxin dimers. The antitoxin dimers each expose two helix-turn-helix DNA-binding domains of the Cro repressor type, suggesting the TA complex is capable of binding the upstream promoter sequence on DNA. The toxin core domain shows structural similarity to ADP-ribosylating enzymes such as diphtheria toxin but has an atypical NAD+ -binding pocket suggesting an alternative function. We show that activation of the toxin in vivo causes a depletion of intracellular NAD+ levels eventually leading to inhibition of cell growth in E. coli and inhibition of global macromolecular biosynthesis. Both structure and activity are unprecedented among bacterial TA systems, suggesting the functional scope of bacterial TA toxins is much wider than previously appreciated.


Subject(s)
Bacterial Toxins/metabolism , Escherichia coli/growth & development , NAD/metabolism , Photorhabdus/genetics , Toxin-Antitoxin Systems , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Protein Conformation , Protein Multimerization , Pseudomonas putida/genetics
3.
Nucleic Acids Res ; 45(5): 2875-2886, 2017 03 17.
Article in English | MEDLINE | ID: mdl-27998932

ABSTRACT

Expression of bacterial type II toxin-antitoxin (TA) systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo-palindromic sequences on operator DNA. In this context, the toxin functions as a co-repressor by stimulating DNA binding through direct interaction with the antitoxin. Here, we determine crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to its cognate DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions in which C-terminal extended structures of the antitoxin VapB1 swap positions to interlock the complex in the DNA-bound state. We further show that a pseudo-palindromic protein sequence in the antitoxin is responsible for this interaction and required for binding and inactivation of the VapC1 toxin dimer. Sequence analysis of 4127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread and unique to bacterial and archaeal VapB antitoxins suggesting a general principle governing regulation of VapBC TA systems. Finally, a structure of C-terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for toxin activation in vivo.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Caulobacter crescentus/genetics , DNA, Bacterial/chemistry , DNA-Binding Proteins/chemistry , Membrane Glycoproteins/chemistry , Operator Regions, Genetic , Amino Acid Motifs , Bacterial Proteins/metabolism , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/metabolism , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Membrane Glycoproteins/metabolism , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Domains
4.
Nat Commun ; 4: 2796, 2013.
Article in English | MEDLINE | ID: mdl-24225902

ABSTRACT

The highly persistent and often lethal human pathogen, Mycobacterium tuberculosis contains at least 88 toxin-antitoxin genes. More than half of these encode VapC PIN domain endoribonucleases that inhibit cell growth by unknown mechanisms. Here we show that VapC20 of M. tuberculosis inhibits translation by cleavage of the Sarcin-Ricin loop (SRL) of 23S ribosomal RNA at the same position where Sarcin and other eukaryotic ribotoxins cleave. Toxin-inhibited cells can be rescued by the expression of the antitoxin, thereby raising the possibility that vapC20 contributes to the extreme persistence exhibited by M. tuberculosis. VapC20 cleavage is inhibited by mutations in the SRL that flank the cleavage site but not by changes elsewhere in the loop. Disruption of the SRL stem abolishes cleavage; however, further mutations that restore the SRL stem structure restore cleavage, revealing that the structure rather than the exact sequence of the SRL is important for this activity.


Subject(s)
Bacterial Proteins/metabolism , Endoribonucleases/metabolism , Fungal Proteins/metabolism , Mycobacterium tuberculosis/enzymology , RNA, Ribosomal, 23S/metabolism , Ricin/metabolism , Virulence Factors/toxicity , Bacterial Proteins/chemistry , Bacterial Proteins/toxicity , Endoribonucleases/antagonists & inhibitors , Endoribonucleases/chemistry , Endoribonucleases/genetics , Fungal Proteins/chemistry , Mutation , RNA, Ribosomal, 23S/chemistry , Ricin/chemistry , Virulence Factors/chemistry
5.
Article in English | MEDLINE | ID: mdl-23832203

ABSTRACT

Upon release from the stable complex formed with its antitoxin VapB, the toxin VapC (MvpT) of the Gram-negative pathogen Shigella flexneri is capable of globally down-regulating translation by specifically cleaving initiator tRNA(fMet) in the anticodon region. Recombinant Shigella flexneri VapC(D7A) harbouring an active-site mutation was overexpressed in Escherichia coli, purified to homogeneity and crystallized by the vapour-diffusion technique. A preliminary X-ray crystallographic analysis shows that the crystals diffracted to at least 1.9 Å resolution at a synchrotron X-ray source and belonged to the trigonal space group in the hexagonal setting, H3, with unit-cell parameters a = b = 120.1, c = 52.5 Å, α = ß = 90, γ = 120°. The Matthews coefficient is 2.46 Å(3) Da(-1), suggesting two molecules per asymmetric unit and corresponding to a solvent content of 50.0%.


Subject(s)
RNA, Transfer, Met/metabolism , Shiga Toxin/chemistry , Shiga Toxin/isolation & purification , Shigella flexneri/enzymology , Catalytic Domain , Crystallization , Crystallography, X-Ray , Dysentery, Bacillary/genetics , Dysentery, Bacillary/metabolism , Dysentery, Bacillary/microbiology , Shiga Toxin/metabolism , Synchrotrons
6.
Nucleic Acids Res ; 40(10): 4347-57, 2012 May.
Article in English | MEDLINE | ID: mdl-22287572

ABSTRACT

Toxin-antitoxin (TA) loci encode inhibitors of translation, replication or cell wall synthesis and are common elements of prokaryotic plasmids and chromosomes. Ten TA loci of Escherichia coli K-12 encode mRNases that cumulatively contribute to persistence (multidrug tolerance) of the bacterial cells. The mechanisms underlying induction and reversion of the persistent state are not yet understood. The vapBC operon of Salmonalla enterica serovar Typhimurium LT2 encodes VapC, a tRNase that reversibly inhibits translation by site-specific cleavage of tRNA(fMet). VapB is an antitoxin that interacts with and neutralizes VapC via its C-terminal tail and regulate TA operon transcription via its N-terminal DNA binding domain that recognize operators in the vapBC promoter region. We show here that transcription of the vapBC operon of S. enterica is controlled by a recently discovered regulatory theme referred to as 'conditional cooperativity': at low T/A ratios, the TA complex binds cooperatively to the promoter region and represses TA operon transcription whereas at high T/A ratios, the excess toxin leads to destabilization of the TA-operator complex and therefore, induction of transcription. We present evidence that an excess of VapC toxin leads to operator complex destabilization by breaking of toxin dimers.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial , Membrane Glycoproteins/metabolism , Salmonella typhimurium/genetics , Transcription, Genetic , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Dimerization , Homeostasis , Mutation , Operator Regions, Genetic , Operon , Protease La/metabolism , Protein Binding , Ribonucleases/metabolism
7.
J Mol Biol ; 414(5): 713-22, 2011 Dec 16.
Article in English | MEDLINE | ID: mdl-22037005

ABSTRACT

Toxin-antitoxin (TA) loci are common in archaea and prokaryotes and allow cells to rapidly adapt to changing environmental conditions through release of active regulators of metabolism. Many toxins are endonucleases that target cellular mRNA and tRNAs, while the antitoxins tightly wrap around the toxins to inhibit them under normal circumstances. The antitoxins also bind to operators in the promoter regions of the cognate TA operon and thereby regulate transcription. For enteric vapBC TA loci, the VapC toxins specifically cleave tRNA(fMet) and thus down-regulate protein synthesis. Here, we describe the crystal structure of the intact Shigella flexneri VapBC TA complex, determined to 2.7 Å resolution. Both in solution and in the crystal structure, four molecules of each protein combine to form a large and globular hetero-octameric assembly with SpoVT/AbrB-type DNA-binding domains at each end and a total molecular mass of about 100 kDa. The structure gives new insights into the inhibition of VapC toxins by VapB and provides the molecular basis for understanding transcriptional regulation through VapB dimerization.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , DNA-Binding Proteins/chemistry , Membrane Glycoproteins/chemistry , Shigella flexneri , Bacterial Proteins/antagonists & inhibitors , Bacterial Toxins/antagonists & inhibitors , Crystallography, X-Ray , DNA-Binding Proteins/antagonists & inhibitors , Membrane Glycoproteins/antagonists & inhibitors , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary
8.
Proc Natl Acad Sci U S A ; 108(18): 7403-7, 2011 May 03.
Article in English | MEDLINE | ID: mdl-21502523

ABSTRACT

Eukaryotic PIN (PilT N-terminal) domain proteins are ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. The majority of prokaryotic PIN-domain proteins are encoded by the abundant vapBC toxin--antitoxin loci and inhibit translation by an unknown mechanism. Here we show that enteric VapCs are site-specific endonucleases that cleave tRNA(fMet) in the anticodon stem-loop between nucleotides +38 and +39 in vivo and in vitro. Consistently, VapC inhibited translation in vivo and in vitro. Translation-reactions could be reactivated by the addition of VapB and extra charged tRNA(fMet). Similarly, ectopic production of tRNA(fMet) counteracted VapC in vivo. Thus, tRNA(fMet) is the only cellular target of VapC. Depletion of tRNA(fMet) by vapC induction was bacteriostatic and stimulated ectopic translation initiation at elongator codons. Moreover, addition of chloramphenicol to cells carrying vapBC induced VapC activity. Thus, by cleavage of tRNA(fMet), VapC simultaneously may regulate global cellular translation and reprogram translation initiation.


Subject(s)
Bacterial Proteins/metabolism , Endoribonucleases/metabolism , Gene Expression Regulation, Bacterial/physiology , Membrane Glycoproteins/metabolism , RNA, Transfer, Met/metabolism , Salmonella typhimurium/enzymology , Shigella flexneri/enzymology , Blotting, Northern , Blotting, Western , Chloramphenicol , Gene Expression Regulation, Bacterial/genetics , Luciferases , Oligonucleotides/genetics , Plasmids/genetics , Ultracentrifugation
9.
Mol Microbiol ; 72(4): 918-30, 2009 May.
Article in English | MEDLINE | ID: mdl-19400780

ABSTRACT

Toxin-antitoxin loci have been identified in almost all free-living prokaryotes, often in high copy numbers. The biological function and molecular targets of the abundant vapBC loci are not yet known. Here we analyse the vapBC loci of Salmonella LT2 and Shigella plasmid pMYSH6000. Both loci encode putative PIN (PilT N-terminal) domain toxins, and antitoxins that may regulate vapBC transcription. We show that vapBC(LT2) and vapBC(pMYSH) are bona fide TA loci: (i) both VapCs inhibited cell growth very efficiently and were counteracted by the cognate VapBs; (ii) both VapCs inhibited translation; (iii) transcription of the vapBC loci was induced by amino acid starvation and chloramphenicol, consistent with the proposal that VapB is an unstable inhibitor of vapBC transcription; (iv) ectopic expression of both VapCs induced a bacteriostatic condition that could be reversed by the cognate antitoxins. Unexpectedly, induction of vapC in Escherichia coli resulted in mRNA cleavage at stop-codons. Surprisingly, these cleavages depended on the yefM yoeB locus, indicating cross-activation between different toxins, that is, VapC activated YoeB mRNA interferase. Activation of YoeB depended on Lon, indicating that Lon degrades YefM antitoxin. Based on these results we present a model that explains activation of YoeB.


Subject(s)
Bacterial Toxins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Membrane Glycoproteins/metabolism , RNA, Bacterial/metabolism , Bacterial Toxins/genetics , Codon, Terminator , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Membrane Glycoproteins/genetics , Plasmids , Protease La/metabolism , RNA, Messenger/metabolism , Salmonella/genetics , Salmonella/metabolism , Shigella/genetics , Shigella/metabolism , Transcriptional Activation
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