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Biosynthesis of mannose-containing cell wall components important in Mycobacterium tuberculosis virulence

Keiser, Tracy Lynn
http://rave.ohiolink.edu/etdc/view?acc_num=osu1397762377

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Microbiology.
Tuberculosis (TB) remains a worldwide scourge. Mycobacterium tuberculosis (M.tb) is one of the oldest and most successful pathogens in human history. It has developed a plethora of ways to subvert the host immune response and make a home out of what should be its undoing, its host cell niche, the macrophage phagosome. Mycobacterium species are compositionally unique organisms belonging to the phyla, Actinomycetes, which also includes Corynebacterium, Rhodococcus, Nocardia and Streptomyces. Their cell envelope includes structural components of both Gram negative and Gram positive bacteria in addition to molecules exclusive to mycobacterium, like mycolic acids. This unique cell envelope configuration provides M.tb with a physical barrier to environmental insults and is responsible for the variable retention of the Gram stain. M.tb coats itself with mannosylated molecules such as the abundant mannosylated lipoglycans which have mannosyl motifs that resemble those of mammalian glycoproteins. This molecular mimicry enables M.tb to take advantage of alveolar macrophages that have up-regulated surface receptors like the C-type lectin mannose receptor to gain entry into the cell and enhance its survival. Synthesis of the mannosylated lipoglycans involves several enzymes and pathways, and thus is difficult to study as a whole. The emphasis of this thesis will be on synthesis of the mannose donor molecules for these lipoglycans. The terminal mannose caps as well as the mannan structures in the core of these molecules are synthesized through a variety of specific mannosyltransferases that use the donors GDP-mannose and polyprenyl phosphate mannose (PPM) that are products of the mannose donor biosynthesis pathway. The putative genes of this pathway in M.tb are orthologs with 100% sequence identity to those in the attenuated vaccine strain M. bovis BCG and include manA (an isomerase), manB (a phosphomannomutase), manC (a GDP-mannose pyrophosphorylase), and ppm1 (polyprenyl-phosphate mannose synthase). Additionally, there are several other neighboring genes, like whiB2 (Fe-S clustering molecule and transcriptional regulator), Rv3256c, Rv3258c (hypothetical proteins), and Rv3253c (a postulated membrane flipase) whose functions are unknown, but are potentially contributing members of the mannose donor biosynthesis pathway. In this thesis we examined expression and some functional characterization of genes involved in the putative mannose donor biosynthetic pathway. Our results for the transcriptional profile in broth show that there are differences in expression of certain genes between M.tb and BCG under the same growth conditions despite their identical sequences. The expression profile of M.tb and BCG mannose donor biosynthesis genes in human macrophages is of particular importance not only because macrophages are the natural host cell niche for M.tb but also because the expression profiles are highly reproducible among different donors. It is of particular interest that the genes Rv3256c, Rv3258c and ppm1 were highly expressed in M.tb 2 hours post-infection of macrophages and then gradually decreased. Due to this up regulation at early time points post infection, characterization of the genes Rv3256c, Rv3258c and ppm1was of great interest and a focus of this work. Ppm1over-expressiom in M. smegmatis or M. tuberculosis provided no phenotype while over-expression of Rv3258c was unstable in the surrogate organism, M. smegmatis, and also showed no phenotype in M.tb. On the other hand, over-expression of Rv3256c reduced rather than increased cell wall mannosylated lipoglycans compared to the vector control. Despite the decrease in these molecules, over-expression of Rv3256c showed an increase in association with and survival in human macrophages. Thus, although the function still remains a mystery, evidence is provided for the role of Rv3256c in virulence. TB has become romanticized in our culture claiming the lives of some of our most beloved characters. Although we understand that the disease is caused solely by this unicellular organism, the interaction between host and microbe is incredibly complex. A more complete understanding of the entry and acclimation of M.tb into host cell macrophages could possibly allow us to develop new ways to prevent infection, limit dissemination following primary infection and possibly help enhance immune protection.
Larry Schlesinger, MD (Advisor)
Samantha King, PhD (Committee Member)
Robert Munson, PhD (Committee Member)
Stephanie Seveau, PhD (Committee Member)
134 p.
Genetics; Microbiology
Tuberculosis; Mycobacterium tuberculosis; pathogenesis; microbial genetics; mannosylated lipoglycans; glucosamine six phosphate synthase; polyprenol monophosphatemannose

Recommended Citations

Citations

  • Keiser, T. L. (2014). Biosynthesis of mannose-containing cell wall components important in Mycobacterium tuberculosis virulence [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397762377

    APA Style (7th edition)

  • Keiser, Tracy. Biosynthesis of mannose-containing cell wall components important in Mycobacterium tuberculosis virulence. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1397762377.

    MLA Style (8th edition)

  • Keiser, Tracy. "Biosynthesis of mannose-containing cell wall components important in Mycobacterium tuberculosis virulence." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397762377

    Chicago Manual of Style (17th edition)

osu1397762377
904
© 2014, some rights reserved.Creative Commons License Biosynthesis of mannose-containing cell wall components important in Mycobacterium tuberculosis virulence by Tracy Lynn Keiser is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.