Expression of fluorescent proteins in bifidobacteria for analysis of host-microbe interactions.

Bifidobacteria are an important component of the human gastrointestinal microbiota and are frequently used as probiotics. The genetic inaccessibility and lack of molecular tools commonly used in other bacteria have hampered a detailed analysis of the genetic determinants of bifidobacteria involved in their adaptation to, colonization of, and interaction with the host. In the present study, a range of molecular tools were developed that will allow the closing of some of the gaps in functional analysis of bifidobacteria. A number of promoters were tested for transcriptional activity in Bifidobacterium bifidum S17 using pMDY23, a previously published promoter probe vector. The promoter of the gap gene (Pgap) of B. bifidum S17 yielded the highest promoter activity among the promoters tested. Thus, this promoter and the pMDY23 backbone were used to construct a range of vectors for expression of different fluorescent proteins (FPs). Successful expression of cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry could be shown for three strains representing three different Bifidobacterium spp. The red fluorescent B. bifidum S17/pVG-mCherry was further used to demonstrate application of fluorescent bifidobacteria for adhesion assays and detection in primary human macrophages cultured in vitro. Furthermore, pMGC-mCherry was cloned by combining a chloramphenicol resistance marker and expression of the FP mCherry under the control of Pgap. The chloramphenicol resistance marker of pMGC-mCherry was successfully used to determine gastrointestinal transit time of B. bifidum S17. Moreover, B. bifidum S17/pMGC-mCherry could be detected in fecal samples of mice after oral administration.

CD14-dependent monocyte isolation enhances phagocytosis of listeria monocytogenes by proinflammatory, GM-CSF-derived macrophages.

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

Tissue inhibitor of metalloproteinases-1-induced scattered liver metastasis is mediated by hypoxia-inducible factor-1α.

The "protease web", representing the network of proteases, their inhibitors, and effector molecules, arises as a pivotal determinant of tissue homeostasis. Imbalances of this network, for instance caused by elevated host levels of tissue inhibitor of metalloproteinases-1 (TIMP-1), have been shown to increase the susceptibility of target organs to scattered metastasis by inducing the hepatocyte growth factor (HGF) pathway. Increased expression of the hypoxia-inducible factor-1α-subunit (HIF-1α) is also associated with tumour progression and is also known to induce HGF-signaling via up-regulation of the HGF-receptor Met, namely under canonical stress conditions like lack of oxygen. Here, we aimed to identify a possible metastasis-promoting connection between TIMP-1, HIF-1α, and HGF-signaling. We found that HIF-1α and HIF-1-signaling were increased during liver metastasis of L-CI.5s T-lymphoma cells in TIMP-1 overexpressing syngeneic DBA/2 mice. In vitro, exposure of L-CI.5s cells to recombinant TIMP-1 revealed that TIMP-1 itself was able to induce HIF-1α and HIF-1-signaling. Knock-down of HIF-1α identified tumour cell-derived HIF-1α as mediator of this TIMP-1-induced invasiveness in vitro. In vivo, HIF-1α knock-down significantly impaired Met expression as well as Met phosphorylation and inhibited scattered liver metastasis. Furthermore, HGF-dependent TIMP-1-promoted Met phosphorylation and HGF-dependent TIMP-1-induced invasiveness in vitro was mediated by HIF-1α. We conclude that elevated levels of TIMP-1 in the microenvironment of tumour cells can promote metastasis by inducing HIF-1α-dependent HGF-signaling. This connection between a protease inhibitor (TIMP-1) and a classically stress-related factor (HIF-1α) is a so far undiscovered impact of the "protease web" on tissue homeostasis with important implications for metastasis.