Structure of FitAB from Neisseria gonorrhoeae Bound to DNA Reveals a Tetramer of Toxin-Antitoxin Heterodimers Containing Pin Domains and Ribbon-Helix-Helix Motifs

Abstract

Neisseria gonorrhoeae is a sexually transmitted pathogen that initiates infections in humans by adhering to the mucosal epithelium of the urogenital tract. The bacterium then enters the apical region of the cell and traffics across the cell to exit into the subepithelial matrix. Mutations in the fast intracellular trafficking (fitAB) locus cause the bacteria to transit a polarized epithelial monolayer more quickly than the wild-type parent and to replicate within cells at an accelerated rate. Here, we describe the crystal structure of the toxin-antitoxin heterodimer, FitAB, bound to a high affinity 36-bp DNA fragment from the fitAB promoter. FitA, the antitoxin, binds DNA through its ribbonhelix-helix motif and is tethered to FitB, the toxin, to form a heterodimer by the insertion of a four turn ␣-helix into an extensive FitB hydrophobic pocket. Neisseria gonorrhoeae (GC) 3 is the agent of the sexually transmitted disease, gonorrhea. The mechanisms used by GC to initiate infection have been very well characterized. Gonococci adhere via a multistep cascade and subsequently enter cells forming the epithelial barrier of the urogenital tract, traffic across these cells and exit into the subepithelial matrix (1, 2). Although studies have identified many of the molecular mechanisms used by GC to adhere to and enter cells, our knowledge of the mechanisms that operate in the later stages of infection is limited.GC are able to survive and grow within epithelial cells (3); they also traverse the epithelial monolayer to infect the stromal tissue of the subepithelium (2). The immune response to bacteria in the subepithelium produces the inflammation and purulent discharge characteristic of gonorrhea (4, 5). On occasion, GC establish a carrier state in which an asymptomatic individual harbors culturable and transmissible bacteria. These carriers are key to the spread of gonococcal disease, as humans are the only known reservoir for GC (6). The mechanisms by which GC maintains this persistent state are unknown. One hypothesis is that the organism resides within the epithelial cells instead of crossing into the subepithelium, thus evading the host immune response. The gene product(s) that affect GC intracellular growth and transcytosis are therefore important for the maintenance of gonococci in the human population.The fitAB operon was identified in a screen for GC mutants with a fast intracellular trafficking phenotype across polarized epithelial monolayers (3). A GC mutant that lacks fitAB grows normally extracellularly, but has an accelerated rate of intracellular replication with a concomitant increase in the rate at which this mutant traverses a monolayer of polarized epithelial cells. Thus, either FitA or FitB, or their complex, is hypothesized to slow intracellular replication and intracellular trafficking of GC.FitA is an 8.4-kDa protein with a predicted N-terminal ribbon-helix-helix (RHH) DNA binding motif (7,8). FitB is a 15.3-kDa protein with a predicted PIN (PilT-N terminus) domain according to the BLAST search t...

Structure and Proposed Activity of a Member of the VapBC Family of Toxin-Antitoxin Systems

“…The closest structural homolog is FitAB from Neisseria gonorrhoeae (% identity VapC-5/FitB ϭ 21% and % identity VapB-5/FitA ϭ 14%) for which the structure of the complex was determined with and without DNA bound to the N terminus of FitA (34). Superimposing VapC-5 on Ngo FitB shows the same deviation of helix ␣-2 compared with Pae VapC (Fig.…”

“…While no nuclease activity could be detected for Ngo FitAB or Ngo FitB in vitro (34), VapBC-5 clearly shows low nuclease activity on dsRNA substrates as well as a magnesium dependence consistent with a two metal ion catalytic mechanism. Although no binding constant could be obtained, the difficulty to separate VapB-5 from VapC-5 as well as the high salt content in the buffer during purification let us hypothesize that the complex formed is tight.…”

Structure of the Escherichia coli Antitoxin MqsA (YgiT/b3021) Bound to Its Gene Promoter Reveals Extensive Domain Rearrangements and the Specificity of Transcriptional Regulation

“…These domain rearrangements are unprecedented for DNA binding by a bacterial antitoxin. To date, three antitoxin⅐DNA structures have been reported: the chromosome-encoded hipBA module from E. coli (27), the plasmid-encoded ccdAB module from E. coli (55), and the chromosome-encoded fitAB module from Neisseria gonorrhoeae (56). HipB, like MqsA, is one of the few antitoxins identified to date that recognizes DNA via an XRE-HTH DNA-binding motif (19,27,57,58).…”

“…However, it is well established across many TA systems, including MqsR⅐MqsA (35), that TA complexes bind the operon promoter with higher affinity than the antitoxin alone. Two toxin⅐antitoxin⅐DNA structures are currently available (FitB⅐FitA⅐DNA (56) and HipA⅐HipB⅐DNA (27)), and each displays a different mechanism of toxin co-regulation. In the case of the FitAB module, two FitB toxin monomers bind to two FitA antitoxin dimers to form a heterotetramer.…”

“…Previously identified residues that comprise the MqsR active site are Lys 56 , Gln 68 , Tyr 81 , and Lys 96 , all of which are accessible when bound to the MqsA-N domain (19). Unexpectedly, in the MqsR⅐MqsA⅐PmqsRA complex model, these residues are now facing inward, toward the pseudo-2-fold rotation axis of the MqsA dimer and underneath the bound DNA (Fig.…”