Host control of intracellular bacterial infections

In this thesis various immune mechanisms regulating control of infections with the intracellular bacteria Listeria monocytogenes and Chlamydia pneumoniae have been studied. These bacteria infect macrophages in which they can successfully grow. However, macrophages are potent killers of intracellular bacteria.

CD44 is expressed in immune and parenchymal cells and is involved in various inflammatoryimmune processes. Here we have described a novel function of this molecule in infection with L. monocytogenes. CD44-/- bone marrow derived macrophages (BMM) showed a dramatically decreased intracellular numbers of L. monocytogenes compared to wild type (WT) BMM.

CD44 was not required for listerial uptake, escape from the phagosome into the cytosol, cell-to-cell spread or formation of actin tails, but probably facilitated listerial growth in the cytoplasm. Furthermore, both hernatopoietic and non-hematopoietic cells mediated a CD44-dependent increased susceptibility to L. monocytogenes in mice. Cell extracts from CD44-/- BMM contained molecules that inhibited listerial growth. Such extracts also inhibited growth of Francisella tularensis, an intracellular bacterium that also escapes from the phagosome into the cytosol, and Salmonella typhimurium, which resides in vacuolar compartments. Growth of F. tularensis, but not S. typhimurium, was impaired in CD44-/-BMM, indicating that the growthpromoting role of CD44 is not restricted to L. monocytogenes, and that the intracytoplasmatic bacterial localization might here be of importance.

L. monocytogenes, have flagella, involved in motility. Flagellin is encoded by flaA, and is the major subunit of flagella. CheY and CheA regulate flagella-mediated chemotaxis in various bacteria. We generated listerial mutants to study whether the gene products of flaA, cheA and cheY affected the outcome of infection with L. monocytogenes. We found that flagellin and the cheY and cheA gene products were important for adherence to and invasion of epithelial cells. However, murine infection with these mutants suggested that flagellin also contributes to expression of TNF-alpha. We suggest a dual role for flagella in the biology of L. monocytogenes infection: inducing protective immune responses, but also aiding L. monocytogenes to invade host cells. Infection of BMM with C. pneumoniae induces IFN-gamma that control bacterial growth. Such IFN-gamma expression is controlled by TLR4-MyD88-IFN-alphabeta signaling, as well as by a TLR4-independent pathway resulting in NF-kappaB activation.

We tried to unravel the signaling pathways involved in such expression. We found that IL-1R-associated kinase 4 (IRAK4), an adaptor molecule involved in TLR4-MyD88 signaling, was not necessary for induction of interferons, NF-kappaB and proinflammatory cytokines in BMM after infection with C. pneumoniae. Furthermore, IRAK4 played no role in control of infection. On the contrary, IFNalpha/beta expression was dependent on the interferon regulatory factor 3 and IFN-beta. However, IFN-alpha expression was also dependent on an IFN-beta-independent signal mediated by MyD88. Expression of IFN-gamma is dependent on IFN-alpha/betaR signaling but also on NFkappaB activation. Binding of microbial molecules to nucleotide-binding oligomerization domain (NOD) proteins lead to NF-kappaB activation. However, NF-kappaB activation or accumulation of proinflammatory cytokines in C. pneumoniae infected NOD-/- or control BMM were similar. Surprisingly, NOD I -'- BMM or mice showed higher C. pneumoniae levels than WT controls. NOD I also controlled levels of L. monocytogenes in infections of both BMM and mice.

List of scientific papers

I. Eriksson E, Dons L, Rothfuchs AG, Heldin P, Wigzell H, Rottenberg ME (2003). CD44-regulated intracellular proliferation of Listeria monocytogenes. Infect Immun. 71(7): 4102-11.
https://pubmed.ncbi.nlm.nih.gov/12819101

II. Eriksson E, Wigzell H, Rottenberg ME (2004). CD44 facilitates intracellular growth of Gram-positive and Gram-negative bacteria. [Manuscript]

III. Dons L, Eriksson E, Jin Y, Rottenberg ME, Kristensson K, Larsen CN, Bresciani J, Olsen JE (2004). Role of flagellin and the two-component CheA/CheY system of Listeria monocytogenes in host cell invasion and virulence. Infect Immun. 72(6): 3237-44.
https://pubmed.ncbi.nlm.nih.gov/15155625

IV. Trumstedt C, Eriksson E, Gigliotti Rothfuchs A, Gigliotti D, Wigzell H, Rottenberg ME (2004). Role of NOD1- and TLR-signaling in resistance against intracellular bacterial infections. [Manuscript]