RIC: Research Interest Comparator

Smith, Alexandra, Ph.D.
Affiliation: Microbiology
Email: xandra.smith@utsouthwestern.edu
Home Page: not provided
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 Abstract:
Lyme borreliosis (Lyme disease), caused by Borrelia burgdorferi, remains the most prevalent arthropod-borne infection in the United States. In addition to its clinical importance, many features of Lyme disease continue to be poorly understood, including those traits of B. burgdorferi that allow it to subsist in nature in both ticks and mammals. It is now widely held that the spirochete must undergo profound adaptive changes in order to accommodate its two very diverse niches in nature. Clearly, elucidating key aspects of such adaptive responses is critical for understanding many enigmatic features of B. burgdorferi related to vector colonization, spirochete transmission, mammalian infection, Lyme disease progression (pathogenesis), and bacterial chronicity. It is now well documented that key membrane lipoproteins of B. burgdorferi are vital for maintaining the spirochete in its enzootic transmission cycle between ticks and mammals. We were the first to report that the alternative sigma factor RpoS regulates the expression of a number of these lipoproteins, two of which are outer surface protein C (OspC) and A (OspA). However, we also have contended that RpoS first must be activated by another transcription factor, RpoN (resulting in an RpoN-RpoS regulatory network), before RpoS can promote the expression of these virulence-associated lipoproteins. In this regard, we have hypothesized that RpoN directly controls the expression of RpoS by binding to the rpoS promoter. Bases -78 to -63 upstream of the rpoS open reading frame comprise a theoretical RpoN-dependent -24/-12 promoter (5'-TGGCACAGTTTTTGCA-3'). However, to date there have been little experimental data to substantiate such predictions. 5'-RACE (Rapid amplification of cDNA 5' ends) was used to determine the transcriptional initiation site for rpoS. 5'-RACE experiments have revealed that the initiation of transcription for rpoS is located at residue -50 upstream of rpoS and 13 base pairs downstream of the putative promoter. This is the region we had predicted to be involved, inasmuch as in most cases, initiation of transcription tends to be at nucleotides 11, 12, or 13 downstream of the promoter. To further confirm the significance of the predicted RpoN binding site we determined the effect of mutations in the promoter sequence on rpoS transcription. Site-directed mutagenesis of plasmids containing the wild-type rpoS promoter was carried out to yield the following mutations: G -76 T and C -64 A. An RpoS-deficient mutant of B. burgdorferi was complemented with the mutant plasmids, and the rpoS transcripts within the resulting borrelial strains were assessed by quantitative real-time RT-PCR. Complementation of the RpoS-deficient mutant with the wild-type rpoS promoter resulted in rpoS transcript levels of 77% (0.62-0.95%) relative to normal expression (87-116%). The G -76 T mutation in the -24 region of the rpoS promoter resulted in decreased rpoS transcription of 36% (23-55%). This latter result agreed with our hypothesis, in that the -24 region appears to be essential for binding of RpoN (i.e., a mutation in this region should result in decreased RpoN binding). Although not anticipated, the C -64 A mutation in the -12 region of the rpoS promoter resulted in increased rpoS transcription of 146% (124-171%). This -12 region is involved in early melting of the DNA, thereby resulting in an open complex form, from which transcription can then proceed. Although not universal, mutations in this region have been shown in some circumstances to result in increased transcription of RpoN-dependent genes of enteric bacteria. There appears to be a stringent requirement for the -24/-12 elements to be on the same face of the DNA helix, inasmuch as deletions of one or more nucleotides in enteric bacteria abolish RpoN-dependent promoter function. Recently, we have constructed two such deletion mutations in the rpoS promoter, and complementation experiments using the RpoS-deficient mutant of B. burgdorferi are in progress. We have expended significant efforts towards preparing soluble recombinant RpoN protein. Recombinant borrelial RpoN protein is necessary for experiments to show evidence of direct binding of the RpoN protein to the rpoS promoter region. Previous results from our lab have shown that E. coli RpoN protein specifically binds to the borrelial rpoS promoter in a dose-dependent fashion (in electrophoretic mobility gel shift assays [EMSAs]). Success in the purification of borrelial RpoN, however, has been problematic, inasmuch as borrelial RpoN tends to be insoluble and also negatively affects the growth of the E. coli host harboring the borrelial rpoN gene for hyperexpression. To circumvent this obstacle, we have recently had success in constructing an MBP fusion containing the C-terminal portion of borrelial RpoN. In this truncated fusion protein, the activator-interacting domain is not present, but both the putative core-binding and presumptive DNA-binding domains remain intact. EMSAs are now being developed and performed using this fusion protein to assess the direct binding of borrelial RpoN to the borrelial rpoS promoter. Understanding the molecular details of the RpoN-RpoS regulatory pathway has far reaching implications for elucidating many aspects of B. burgdorferi gene regulation, particularly as it pertains to governing infectivity and virulence expression. Discerning the elements of this pathway, and the molecular mechanisms involved, also has the potential to uncover regulatory and virulence-associated genes that contribute to many as yet unknown aspects of B. burgdorferi's complex parasitic strategy. All of these issues relate directly to efforts towards vaccine development, inasmuch as the regulation of virulence-associated genes needs to be understood before a rational approach to vaccine development can be realized.
Keywords extracted from the abstract: [ eliminated words list ]
Count Word
19.481 -12
19.481 -24
6.000 -50
6.000 -63
12.198 -64
12.198 -76
6.000 -78
8.665 -race
6.000 -tggcacagtttttgca-3
6.000 124-171
6.000 23-55
6.000 62-0
6.000 87-116
6.000 [emsas]
3.087 abolish
3.211 accommodate
1.675 activated
6.000 activator-interacting
4.968 adaptive
1.265 addition
2.206 affects
2.732 agreed
2.025 allow
1.777 alternative
2.132 amplification
2.838 anticipated
3.338 appears
1.396 approach
4.387 arthropod-borne
4.695 aspects
Count Word
1.813 assays
1.668 assess
1.542 assessed
3.289 bacteria
1.148 bacterial
1.420 base
2.376 bases
4.065 binding
2.104 binds
2.845 borrelia
21.066 borrelial
3.503 borreliosis
12.983 burgdorferi
2.160 c-terminal
1.721 carried
0.984 cases
1.399 caused
1.805 cdna
1.053 changes
3.424 chronicity
2.559 circumstances
3.472 circumvent
1.992 clearly
0.816 clinical
2.765 coli
2.565 colonization
5.130 complementation
3.110 complemented
2.525 complex
2.845 comprise
Count Word
2.128 confirm
2.169 constructed
3.238 constructing
3.004 containing
4.448 contended
2.400 continue
1.831 contribute
1.388 controls
4.566 core-binding
1.660 critical
1.557 cycle
0.847 data
2.231 date
2.535 decreased
1.834 deletion
2.433 deletions
2.565 details
1.359 determine
1.283 determined
1.308 developed
2.033 development
3.130 direct
3.580 directly
4.203 discerning
2.168 disease
2.273 diverse
1.664 dna
1.757 dna-binding
2.114 documented
1.563 domain
Count Word
1.874 domains
2.064 dose-dependent
4.694 downstream
1.222 early
0.890 effect
4.629 efforts
2.385 electrophoretic
3.517 elements
6.458 elucidating
4.383 emsas
2.512 ends
3.669 enigmatic
5.440 enteric
3.605 enzootic
1.630 essential
3.858 expended
1.303 experimental
4.130 experiments
3.315 expression
2.163 face
1.973 factor
2.039 far
2.331 fashion
3.199 features
2.254 first
1.204 following
1.398 form
2.348 frame
1.313 further
4.396 fusion
Count Word
1.515 gel
1.825 gene
3.114 genes
2.907 governing
0.909 growth
2.995 harboring
2.499 held
2.480 helix
1.776 host
4.351 hyperexpression
1.717 hypothesis
2.355 hypothesized
1.803 implications
1.705 importance
11.660 inasmuch
1.327 including
1.897 increased
2.396 infection
2.798 infectivity
5.226 initiation
2.846 insoluble
1.794 intact
3.737 involved
1.930 issues
3.816 key
2.175 lab
1.919 latter
0.931 levels
4.823 lipoproteins
1.709 little
Count Word
1.773 located
8.996 lyme
2.393 maintaining
1.879 mammalian
4.681 mammals
2.759 mbp
1.429 mechanisms
2.949 melting
1.050 membrane
2.140 mobility
1.923 molecular
0.902 more
2.157 most
3.522 must
1.979 mutagenesis
5.124 mutant
3.856 mutation
5.101 mutations
3.442 nature
1.717 necessary
1.924 needs
2.343 negatively
2.040 network
3.886 niches
1.028 normal
4.944 now
3.799 nucleotides
3.319 obstacle
3.556 open
1.503 order
Count Word
3.694 ospa
4.019 ospc
2.006 outer
2.107 pairs
2.470 parasitic
1.752 particularly
1.848 pathogenesis
3.215 pathway
1.222 performed
4.032 pertains
3.684 plasmids
2.168 poorly
2.123 portion
1.292 potential
3.787 predicted
2.553 predictions
2.982 preparing
2.982 presumptive
2.476 prevalent
1.550 previous
2.970 problematic
3.031 proceed
2.514 profound
2.146 progress
1.766 progression
2.216 promote
9.164 promoter
3.024 protein
1.218 purification
4.124 putative
Count Word
1.718 quantitative
1.579 rapid
2.657 rational
2.503 reaching
2.044 reading
2.540 real-time
2.967 realized
3.435 recently
2.721 recombinant
2.108 regard
5.919 region
2.349 regulates
2.674 regulation
4.902 regulatory
2.659 relate
1.284 related
1.501 relative
2.078 remain
1.835 remains
0.947 report
2.301 requirement
2.088 residue
1.318 responses
4.183 resulted
4.682 resulting
0.741 results
1.318 revealed
22.536 rpon
15.234 rpon-dependent
11.899 rpon-rpos
Count Word
22.701 rpos
15.472 rpos-deficient
2.279 rt-pcr
1.283 same
0.904 sequence
2.030 shift
2.342 sigma
1.765 significance
0.990 significant
2.796 site
2.291 site-directed
1.931 soluble
1.887 specifically
10.115 spirochete
0.575 states
1.285 strains
1.997 strategy
3.152 stringent
5.131 subsist
3.437 substantiate
4.158 success
1.231 surface
6.096 tends
1.972 theoretical
2.236 thereby
5.537 ticks
4.141 towards
2.503 traits
2.328 transcript
6.060 transcription
Count Word
1.961 transcriptional
2.141 transcripts
3.270 transmission
1.881 truncated
3.549 uncover
2.245 undergo
3.638 understanding
4.592 understood
0.576 united
2.600 universal
1.878 unknown
4.575 upstream
0.946 used
1.801 using
3.520 vaccine
2.098 vector
2.097 virulence
10.959 virulence-associated
2.321 vital
2.045 widely
3.757 wild-type
2.047 yield
RIC Statistics:
Extraction Method: Keyword Count with Lexical Variants Added
Eliminated words list: MedlinePlus List
Similarity Method: Weighted keyword count
Weighting Method: Term Frequency * Inverse Document Frequency
Database: Medline Updates from current month
Publication Type: All
Score Calculation Method: Cosine Similarity Method
Sort by: Score
Submission date and time: 3-8-2006, 13:21:40
Computation time: 00:00:04

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Last updated: Wednesday, 08-Mar-2006 13:21:44 CST