Borrelia burgdorferi (Bb), the Lyme disease spirochete, undergoes dramatic adaptive changes as it cycles in nature between its diverse tick and mammalian hosts. In 2001, we discovered that the alternative sigma factor RpoS (? controls the synthesis of key outer membrane lipoproteins that confer mammalian infectivity and pathogenicity to Bb. We additionally discovered that RpoS itself is controlled by another alternative sigma factor, RpoN (? ?), that binds to the rpoS promoter. We named this novel pathway in Bb the RpoN-RpoS regulatory pathway. This pathway is the first example of an alternative sigma factor regulatory cascade controlling virulence in a bacterial pathogen. The RpoN-RpoS pathway is activated in Bb by various environmental stimuli that accompany tick feeding, and is sustained during Bb's mammalian infection phase. Over the past funding interval, we made the additional striking discovery that in addition to the enhancer- binding protein (Rrp2) needed for RpoN activation, another protein known as BosR (BB0647) functions as a second activator to promote RpoN-dependent rpoS transcription in Bb. BosR is the first example, in any bacterium, of an additional activator essential for ?-dependent gene transcription. This system represents a new paradigm of ?-mediated gene control in bacteria. We have further determined that BosR is a Zn+2- containing DNA-binding protein that binds to a core sequence (BosR box) of ATTTAANTTAAAT. Bioinformatics and in vitro electrophoretic mobility shift assays have revealed three potential BosR binding sites within rpoS, one of which is located immediately adjacent to the RpoN binding site. However, there remain many unanswered questions concerning how BosR functions, particularly when Bb is in its native environments of ticks and mammals.
In Aim 1 of this proposal, we shall assess the biological role(s) of the three BosR binding sites in rpoS. Selected rpoS promoter mutations/deletions will be examined for their influence on rpoS expression in growing Bb, and selected rpoS promoter mutants also will be assessed for their ability to escape from ticks and infect and disseminate in mice.
In Aim 2, we shall structurally character BosR, using both NMR spectroscopy and X-ray crystallography, to examine parameters explaining how BosR coordinates metal, has a predilection to form homodimers, participates in protein-protein interactions, and how it binds to DNA conformationally.
In Aim 3, we shall determine the regulons influenced by BosR, RpoN, and RpoS when Bb is within its native settings of ticks and mammals; these have not been previously compared directly. New gene targets of interest also will be assessed for their roles in the life cycle of Bb. These combined studies will (i) provide new insights into potentially blocking the transmission of Bb, (ii) clarify the novel mechanism by which BosR activates the central RpoN-RpoS pathway or other RpoN-independent genes for Bb's virulence; and (iii) further elucidate this new paradigm of ?-mediated bacterial gene control. Resultant findings could lead to new intervention strategies (vaccines, therapeutics) for Lyme disease.

Public Health Relevance

This project seeks to understand better how the Lyme disease bacterium uses a novel method of gene regulation (alternative sigma factor regulatory cascade) to strategically govern the expression of a number of its virulence traits critical to its transmission between arthropod (tick) and mammalian (human) hosts. Emphasis will be placed on clarifying the role of a newly defined activator (BosR) for this pathway, and determining how BosR functions in tick transmission, infectivity for mammals, and induction of disease. Understanding the role of BosR in transmission and pathogenesis processes could lead to new intervention strategies (vaccines, therapeutics) for Lyme disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI059062-14
Application #
9416054
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Ilias, Maliha R
Project Start
2004-05-01
Project End
2020-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
14
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Ouyang, Zhiming; Zhou, Jianli; Norgard, Michael V (2014) Synthesis of RpoS is dependent on a putative enhancer binding protein Rrp2 in Borrelia burgdorferi. PLoS One 9:e96917
Ouyang, Zhiming; Zhou, Jianli; Norgard, Michael V (2014) CsrA (BB0184) is not involved in activation of the RpoN-RpoS regulatory pathway in Borrelia burgdorferi. Infect Immun 82:1511-22
Brautigam, Chad A; Ouyang, Zhiming; Deka, Ranjit K et al. (2014) Sequence, biophysical, and structural analyses of the PstS lipoprotein (BB0215) from Borrelia burgdorferi reveal a likely binding component of an ABC-type phosphate transporter. Protein Sci 23:200-12
Ouyang, Zhiming; Narasimhan, Sukanya; Neelakanta, Girish et al. (2012) Activation of the RpoN-RpoS regulatory pathway during the enzootic life cycle of Borrelia burgdorferi. BMC Microbiol 12:44
Ouyang, Zhiming; Deka, Ranjit K; Norgard, Michael V (2011) BosR (BB0647) controls the RpoN-RpoS regulatory pathway and virulence expression in Borrelia burgdorferi by a novel DNA-binding mechanism. PLoS Pathog 7:e1001272
Ouyang, Zhiming; Haq, Shayma; Norgard, Michael V (2010) Analysis of the dbpBA upstream regulatory region controlled by RpoS in Borrelia burgdorferi. J Bacteriol 192:1965-74
Becker, Amy M; Blevins, Jon S; Tomson, Farol L et al. (2010) Invariant NKT cell development requires a full complement of functional CD3 zeta immunoreceptor tyrosine-based activation motifs. J Immunol 184:6822-32
Ouyang, Zhiming; Kumar, Manish; Kariu, Toru et al. (2009) BosR (BB0647) governs virulence expression in Borrelia burgdorferi. Mol Microbiol 74:1331-43
Ouyang, Zhiming; He, Ming; Oman, Tara et al. (2009) A manganese transporter, BB0219 (BmtA), is required for virulence by the Lyme disease spirochete, Borrelia burgdorferi. Proc Natl Acad Sci U S A 106:3449-54
He, Ming; Oman, Tara; Xu, Haijun et al. (2008) Abrogation of ospAB constitutively activates the Rrp2-RpoN-RpoS pathway (sigmaN-sigmaS cascade) in Borrelia burgdorferi. Mol Microbiol 70:1453-64

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