Isolation of
            <i>Neisseria gonorrhoeae</i>
            Mutants That Show Enhanced Trafficking across Polarized T84 Epithelial Monolayers

Hopper, Sylvia; Wilbur, J. Scott; Vasquez, Brandi; Larson, Jason; Clary, Susan; Mehr, Ian J.; Seifert, H. Steven; So, Magdalene

doi:10.1128/iai.68.2.896-905.2000

Cited by 62 publications

(64 citation statements)

References 63 publications

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“…Bicistronic operons where an RHH DNA-binding protein is juxtaposed with a PIN domain have been proposed to form one family of toxin/antitoxin systems (13). The PIN domain containing protein is thus predicted to act as a toxin; this is in agreement with the role of FitB in slowing GC replication when the bacteria are within epithelial cells (3).…”

supporting

confidence: 65%

“…Four such FitAB heterodimers associate into a novel tetrameric structure that binds to the IR36 sequence from the fitAB promoter region with high affinity. Many PIN domain-containing proteins are involved in nucleic acid metabolism and/or remodeling, with the prokaryotic FitAB and its homologues responsible for controlling rates of DNA replication and/or plasmid maintenance (3,27,28). This structure illustrates the mechanism by which antitoxins with RHH motifs are able to block the activity of PIN domain toxins in prokaryotes (46).…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Mattison

Wilbur

et al. 2006

Journal of Biological Chemistry

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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...

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supporting

confidence: 65%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Mattison

Wilbur

et al. 2006

Journal of Biological Chemistry

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119

160

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“…This contradicted findings from gentamicin protection assays (Shaw & Falkow, 1988), which are commonly used as a measure of intracellular GC. According to this assay, P+, Opa-non-expressing MS11 do not invade cells until 4-5 h post-infection (Hopper et al, 2000;Lin et al, 1997). This quandary was solved when we examined the entire series of thin sections from the same block.…”

Section: D Serial Sectioning To Probe the Intracellularity Of Gcmentioning

confidence: 99%

Influence of type IV pilus retraction on the architecture of the Neisseria gonorrhoeae-infected cell cortex

et al. 2009

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Early in infection, Neisseria gonorrhoeae can be observed to attach to the epithelial cell surface as microcolonies and induce dramatic changes to the host cell cortex. We tested the hypothesis that type IV pili (Tfp) retraction plays a role in the ultrastructure of both the host cell cortex and the bacterial microcolony. Using serial ultrathin sectioning, transmission electron microscopy and 3D reconstruction of serial 2D images, we have obtained what we believe to be the first 3D reconstructions of the N. gonorrhoeae-host cell interface, and determined the architecture of infected cell microvilli as well as the attached microcolony. Tfp connect both wild-type (wt) and Tfp retraction-deficient bacteria with each other, and with the host cell membrane. Tfp fibres and microvilli form a lattice in the wt microcolony and at its periphery. Wt microcolonies induce microvilli formation and increases of surface area, leading to an approximately ninefold increase in the surface area of the host cell membrane at the site of attachment. In contrast, Tfp retractiondeficient microcolonies do not affect these parameters. Wt microcolonies had a symmetrical, dome-shaped structure with a circular 'footprint', while Tfp retraction-deficient microcolonies were notably less symmetrical. These findings support a major role for Tfp retraction in microvilli and microcolony architecture. They are consistent with the biophysical attributes of Tfp and the effects of Tfp retraction on epithelial cell signalling. INTRODUCTIONNeisseria gonorrhoeae (the gonococcus, or GC) remains a significant public health concern. The Gram-negative diplococcus causes a million cases of gonorrhoea annually in Western Europe and North America, and over 62 million cases worldwide (World Health Organization). Humans are the only known reservoir for GC. The exquisite tropism of GC for man and the ability of GC to establish a carrier state (Turner et al., 2002) reflect a highly evolved relationship between pathogen and host.Gonococcal attachment is promoted by type IV pili (Tfp), peritrichous fibres that are 6 nm in width and up to several micrometres in length. The Tfp filament is composed of helical polymers of pilin subunits (Craig et al., 2006;Parge et al., 1995) and minor proteins (Carbonnelle et al., 2005;Winther-Larsen et al., 2005). Tfp participate in important biological processes such as DNA uptake/genetic exchange, twitching motility, attachment, and host cell signalling (Mattick, 2002;. These activities require, or are enhanced by, the retraction of Tfp fibres (Wolfgang et al., 2000).Gonococcal Tfp retraction is an activity that requires PilT, the Tfp retraction motor subunit (Wolfgang et al., 1998). Retracting Tfp generate strong 'pull' forces, ranging from 50 picoNewtons (pN) to 1 nanoNewton (nN) (Biais et al., 2008;Opitz et al., 2009). Repeated cycles of Tfp extension, substrate attachment and Tfp retraction allow GC to crawl over surfaces and aggregate into microcolonies. Tfp retraction from bacteria in a microcolony allows the community to craw...

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The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site

et al. 2012

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VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 Å resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA. The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg 21 ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg 21 ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 Å closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.

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Isolation of Neisseria gonorrhoeae Mutants That Show Enhanced Trafficking across Polarized T84 Epithelial Monolayers

Cited by 62 publications

References 63 publications

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

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

Influence of type IV pilus retraction on the architecture of the Neisseria gonorrhoeae-infected cell cortex

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site

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Isolation of Neisseria gonorrhoeae Mutants That Show Enhanced Trafficking across Polarized T84 Epithelial Monolayers

Cited by 62 publications

References 63 publications

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

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

Influence of type IV pilus retraction on the architecture of the Neisseria gonorrhoeae-infected cell cortex

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA‐binding site

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The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site