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Mobile DNA in the Pathogenic Neisseria


Mobile DNA in the Pathogenic Neisseria

Kyle P Obergfell et al. Microbiol Spectr.


The genus Neisseria contains two pathogenic species of notable public health concern: Neisseria gonorrhoeae and Neisseria meningitidis. These pathogens display a notable ability to undergo frequent programmed recombination events. The recombination mediated pathways of transformation and pilin antigenic variation in the Neisseria are well studied systems that are critical for pathogenesis. Here we will detail the conserved and unique aspects of transformation and antigenic variation in the Neisseria. Transformation will be followed from initial DNA binding through recombination into the genome with consideration to the factors necessary at each step. Additional focus is paid to the unique type IV secretion system that mediates donation of transforming DNA in the pathogenic Neisseria. The pilin antigenic variation system uses programed recombinations to alter a major surface determinant which allows immune avoidance and promotes infection. We discuss the trans- and cis- acting factors which facilitate pilin antigenic variation and present the current understanding of the mechanisms involved in the process.


Figure 1
Figure 1. Type IV Pilus and DNA Uptake
A. Type IV Pilus – The Tfp is a several micron long, 60 angstrom wide fiber anchored in the inner membrane by PilG that extends through the PilQ secretin pore. Composed mainly of the major pilin PilE (pilin), which is processed by a dedicated protease, PilD. The PilF and PilT NTPases mediate extension and retraction of the pilus through polymerization and depolymerization of the pilin subunits. B. Competence Pseudopilus – Hypothesized pseudopilus that could mediate transformation. Utilizes the type IV pilus complex including the PilQ pore but is not an extended fiber. Possible localization of ComP to the pseudopilus could mediate specific DNA binding. C. DNA Uptake Model - Retraction of the (pseudo)pilus mediated by PilT brings the initial length of DNA into the periplasm. DNA is then bound by a protein or protein complex possibly containing ComE which mediates import of the remaining length of DNA into the periplasm. The inner membrane protein ComA facilitates DNA entry into the cytoplasm.
Figure 2
Figure 2. Type IV Secretion System Model
ParA and ParB recruit the chromosomal DNA to the type IV secretion system. TraI relaxase nicks the DNA at the oriT site and the DNA is unwound possiblibyl by the Yea helicase. The resulting single stranded DNA possibly still bound by TraI is then secreted through the type IV secretion complex into the extracellular mileu in a contact-independent manner. The inner membrane complex is predicted to consist of TraG, TraD and TraC with TraB spanning both the inner and outer membranes to form a channel for the DNA. The transglycosylases AtlA and LtgX create localized breaks in the peptidoglycan to allow the system to assemble. The outer membrane complex consists of TraB, TraK and TraV.
Figure 3
Figure 3. Molecular Description of Antigenic Variation
The pilE and pilS loci have regions of sequence microhomology (grey) and variability (colored). Sequence from a nonexpressed pilS loci copy is transferred into the expression locus with the pilS sequence not changing. Recombination can occur A. in just a section of the gene resulting in a pilE-pilS hybrid, B. across the entire pilS gene resulting in an entirely new variable region of pilE, or C. multiple times with different silent copies resulting in a new pilE sequence containing information from different silent copies throughout the variable regions.
Figure 4
Figure 4. The pilE Guanine Quartet (G4)
A. Gene map showing the location of the pilE-associated G4 forming sequence and the sRNA promoter required for antigenic variation at the pilE locus. B. The sequence upstream of pilE that forms a G4. Mutation of the boxed guanine residues lead to loss of antigenic variation implicating the G4 in antigenic variation. C. The parallel G4 structure of the pilE G4 as solved by NMR analysis.
Figure 5
Figure 5. Proposed Recombination Pathways
A. Unequal Crossing Over Model – A dsDNA break occurs at the pilE locus and I. the 5′ ends are resected by RecBCD to leave 3′ overhangs. II. A single 3′ end mediated by RecA, invades the pilS locus forming a D-loop. III. The 3′ ends are extended by DNA polymerase using the pilS gene as a template. IV. Resolution of the double Holliday junctions results in a new pilE sequence without altering the donor pilS sequence. B. Successive Half Crossing Over Model – Recombination begins with a dsDNA break or single-stranded gap in pilE in a region of homology. I. A RecA and RecOR mediated half crossing over event occurs linking the pilE and a pilS locus on a sister chromosome. II. A second half crossing over event occurs in another region of microhomology downstream of the first event between the pilE:pilS hyrbid and the original pilE locus. III. This recombination event leads to a new sequence at the pilE locus and destruction of the donor chromosome. C. Hybrid Intermediate Model – Similar to the half crossing over model, recombination initiates with a double stranded break or single-stranded gap at pilE and I. a half crossing over event with a donor pilS on the same chromosome. II. This results in a pilE:pilS hybrid intermediate and the loss of the donor chromosome. III. The hybrid intermediate then undergoes two recombination events with the recipient pilE on a different chromosome. The first recombination event would occur in the extensive region of homology upstream of the genes while the second even would utilize microhomology within the variable regions of the genes. IV. Resolution of the Holliday junction intermediates leads to a new pilE sequence on the recipient chromosome.
Figure 6
Figure 6. Proposed Antigenic Variation Initiation Pathway
Transcription initiation at the sRNA upstream of pilE melts the DNA allowing the G4 structure to form. An unknown protein may bind the G4 to stabilize the structure. A single stranded nick may occur on the strand opposite the G4 due to a stalled replication fork. RecQ could unwind the G4 structure. RecJ resects the 5′ nicked end allowing RecA to mediate recombination, possibly enhanced by binding the G4 structure, with RecOR using regions of homology between pilE and the donor pilS, possibly through a recombination mechanism detailed in Figure 5. RecG and RuvABC then process and resolve the recombination intermediate.

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