Dr. M. Böhm
Research Group Böhm
Campylobacter jejuni, a spiral-shaped Gram-negative pathogen, is a highly frequent cause of gastrointestinal foodborne illness in humans worldwide. Clinical outcome of C. jejuni infections ranges from mild to severe diarrheal disease, and some other complications including reactive arthritis and Guillain-Barré syndrome. It has been proposed that crossing the intestinal epithelial barrier and host cell entry by C. jejuni are considered the primary reasons for damage to the intestinal tissue. The proposed signaling mechanisms involved in human host cell invasion and their potential role in the development of C. jejuni-mediated disease are controversially discussed in the literature, and it is not yet clear if C. jejuni enters host cells by the „zipper“ or „trigger“ mechanism of invasion. C. jejuni adherence to cells has been shown to involve a variety of different outer membrane adhesins, including JlpA, PEB1 and the fibronectin binding proteins CadF and FlpA. C. jejuni invasion of INT-407 and other intestinal cell lines has been observed to induce rearrangements of the host cytoskeleton by small Rho GTPases, which are directly linked to bacterial uptake. We have provided various lines of evidence indicated that the GTPase members Rac1 and Cdc42, but not RhoA, are activated during infection and play a significant role in the invasion process. Using knockout cell lines derived from fibronectin(-/-), integrin beta1(-/-), and focal adhesion kinase (FAK)(-/-) deficient mice and corresponding wild-type (WT) controls, we are studying C. jejuni-induced signaling cascades involved in the bacterial invasion process. High resolution scanning electron microscopy, GTPase pull-downs, G-LISA, and gentamicin protection assays were applied and experiments indicated that each of the above host cell factors is indeed required for activation of the small Rho GTPase members Rac1 and Cdc42 as well as maximal host cell invasion of this pathogen. Interestingly, membrane ruffling, filipodia formation, tight engulfment of bacteria and invasion were only seen during infection of WT control cells, but not in fibronectin(-/-), integrin beta1(-/-), and FAK(-/-) knockout cell lines. The detailed signaling pathways leading to the activation of Rac1 and Cdc42 involve CadF targeting of fibronectin and require certain receptors (integrins, EGF and PDGF receptors), kinases (FAK, Src and PI3-kinase) and guanine exchange factors (DOCK180, Vav2 and Tiam-1). We could show that C. jejuni triggers two major pathways: (i) the CadF→fibronectin →integrinb1→ FAK/Src→ EGFR/PDGFR → PI3-kinase→Vav2 and (ii) CadF→fibronectin→integrin-b1→FAK→DOCK180/Tiam-1 signalling cascades to activate Cdc42 and Rac1, respectively. How these concerted interactions stimulate actin and/or microtubule rearrangements and subsequently provide the pulling forces for bacterial uptake is still not fully clear and currently under investigation in the lab. Future work should also clarify the contradictory role of some previously identified other factors, and should identify and characterize novel virulence determinants.
1. Boehm M, Krause-Gruszczynska M, Rohde M, Tegtmeyer N, Takahashi S, Oyarzabal
OA & Backert S (2011) Role of fibronectin, integrin beta1, FAK, Tiam-1, DOCK180 in
activating Rho GTPase Rac1. Front in Cell. Infect. Microbiol. 1:17.
2. Krause-Gruszczynska* M, Boehm* M, Rohde M, Tegtmeyer N, Takahashi S, Buday
L,Oyarzabal OA & Backert S (2011) The signaling pathway of Campylobacter jejuni
induced Cdc42 activation: Role of fibronectin integrin beta1, tyrosine kinases and
guanine exchange factor Vav2. Cell Commun. and Signal. 9:32.
3. Hoy B, Geppert T, Boehm M, Reisen F, Plattner P, Gadermaier G, Sewald N, Ferreira F,
Briza P, Schneider G, Backert S & Wessler S (2012) Distinct roles of secreted HtrA
proteases from Gram-negative pathogens in cleaving the junctional protein and tumor
suppressor E-cadherin. J. Biol. Chem. 287 (13): 10115-10120.
4. Boehm M, Hoy B, Rohde M, Tegtmeyer N, Bæk KT, Oyarzabal OA, Brøndsted L,
Wessler S & Backert S (2012) Rapid paracellular transmigration of Campylobacter jejuni
across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA
cleaving E-cadherin but not fibronectin. Gut Pathogens 4:3.
5. Backert S, Boehm M, Wessler S & Tegtmeyer N (2013) Transmigration route of
Campylobacter jejuni across polarized intestinal epithelial cells: paracellular, transcellular
or both? Cell Commun. and Signal. 11:72.
6. Boehm M, Haenel I, Hoy B, Brøndsted L, Smith TG, Hoover T, Wessler S & Tegtmeyer
N (2013) Extracellular secretion of protease HtrA from Campylobacter jejuni is highly
efficient and independent of its protease activity and flagellum. Eur. J. Microbiol.
Immunol. 3:3 163-173.
7. Heimesaat MM, Alutis M, Grundmann U, Fischer A, Tegtmeyer N, Boehm M, Kühl AA,
Göbel UB, Backert S & Bereswill S (2014) The role of serine protease HtrA in acute
ulcerative enterocolitis and extra-intestinal immune responses during Campylobacter
jejuni infection of gnotobiotic IL-10 deficient mice. Front in Cell. Infect. Microbiol. 4:77.
8. Heimesaat MM, Fischer A, Alutis M, Grundmann U, Boehm M, Tegtmeyer N, Göbel
UB, Kühl AA, Bereswill S & Backert S (2014) The impact of serine protease HtrA in
apoptosis, intestinal immune responses and extra-intestinal histopathology during
Campylobacter jejuni infection of infant mice. Gut Pathogens 6.16 .
9. Boehm M, Lind J, Backert S, Tegtmeyer N. (2015) Campylobacter jejuni serine protease
HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion,
invasion, and transmigration. Eur J Microbiol Immunol (Bp). 5: 68-80.
10. Perna AM, Rodrigues T, Schmidt TP, Boehm M, Stutz K, Reker D, Pfeiffer B, Altmann
KH, Backert S, Wessler S, Schneider G. (2015) Fragment-Based De Novo Design
Reveals a Small-Molecule Inhibitor of Helicobacter Pylori HtrA. Angew Chem Int Ed.
* equal contribution (shared first authorship)