Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under-the control of a strong promoter (PL, from bacteriophage A) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines.Bacteroides nodosus is the essential causative agent of ovine footrot (4, 11). This anaerobe contains numerous surface filaments, about 6 nm in diameter and ranging up to several micrometers in length (14, 46, 50), termed fimbriae (or common pili), which play a central role in both pathogenesis and immunity (for a recent review, see reference 29).Fimbriae have adherent functions and appear to be a mechanism for the colonization of epithelial tissues in eucaryotic hosts. The properties of B. nodosus fimbriae (14) suggest that they belong in the category of type 4, as proposed by Ottow (38), citing Pseudomonas aeruginosa (6) as a prototype. Fimbriae of this type have a polar location on the cell and appear to be involved in surface translocation by a phenomenon known as twitching motility (21). The same characteristics are also observed in the fimbriae found in a broad range of gram-negative species classified within the genera Acinetobacter, Alteromonas, Bacteroides, Eikenella, Moraxella, Neisseria, and Pseudomonas, among others (6,17,20,21).This grouping is supported by recent protein and DNA sequence analyses of the structural subunits of the fimbriae of B. nodosus (12, 31), Moraxella nonliquefaciens (16), Moraxella bovis (28), Neisseria gonorrhoeae (22,33,44), Neisseria meningitidis (22, 36), and P. aeruginosa (42). These subunits, which range in size from about 145 to 160 amino acids among different species and serotypes, all share the distinctive feature of an unusual modified amino acid, methylphenylalanine (MeF), as the first residue in the mature protein, as well as a striking degree of sequence conservation throughout the amino-terminal region. This region is highly hydrophobic and exhibits at least 90% homology with the following 32-amino-acid consensus sequence:MeF T L I E L M I V

Key words: xenograft; matrix metalloproteinases; MMP-13; collagenase-3; breast tumour; stroma; bone metastasis; microenvironment Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes with the ability to degrade extracellular matrix (ECM) components as well as numerous secreted and membrane-bound cell modulators and thus are considered key to the tissue rearrangements associated with malignancy. There are 24 soluble and membrane-anchored members of the MMP family in humans that demonstrate extensive sequence homology and overlapping but distinct substrate specificities. 1 Proteolytic activity of the MMPs is regulated at several levels, most notably via gene transcription, activation via proteolysis of a propeptide, cell compartmentalisation and inhibition by the endogenous tissue inhibitors of metalloproteinases (TIMPs). 2 Although endowed with pro-invasive properties, the functions of MMPs have been shown to be much more widespread than simply facilitating migration/invasion. They also are involved in tumour initiation and progression, activation of chemokines and growth factors, angiogenesis and apoptosis induction to name a few. 1,3 It is thus not surprising that many MMPs have been documented in cancer tissue. 2,4,5 Although MMPs have been implicated in several key steps of tumour progression, clinical trials using synthetic MMP inhibitors have not led to significant therapeutic benefit. This is in contrast to marked inhibition of tumour growth and metastasis in animal models. 6,7 A number of reasons have been proposed for these shortcomings, including the high tumour burden of selected patients for clinical trials, a lack of surrogate markers for dose efficacy and the revelation that some MMPs can inhibit tumour growth. 8,9 It is thus clear that our understanding of the source and specific function of each MMP in the various stages of tumour progression is not complete and that this knowledge is crucial to effectively apply MMP inhibitors in the treatment of cancer. 1,2,6,7 A tumour is defined as a local uncontrolled growth of abnormal tissue consisting of transformed cells, as well as other cell types including fibroblasts, macrophages, endothelial cells and connective tissue components known as the stroma. 10 Tumour cells are known to influence and manipulate the stroma such that the latter produces a permissive and supportive environment, which helps facilitate the growth of the carcinoma. Breast cancer (BrCa) tissue has been shown to contain most MMPs including MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-13, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, [11][12][13][14][15][16][17][18][19][20] BrCa cells frequently and selectively metastasise to the bone, resulting in osteolysis, pathologic fracture, pain and significant clinical morbidity. 21 Numerous MMPs have been implicated in bone turnover, 22,23 and more recently bone metastasis in experimental breast and prostate cancer models were found to be sensitive to MMP inhibition. 24 -26 Determining which MMPs are more influential i...

The ability to activate pro-matrix metalloproteinase (pro-MMP)-2 via membrane type-MMP is a hallmark of human breast cancer cell lines that show increased invasiveness, suggesting that MMP-2 contributes to human breast cancer progression. To investigate this, we have stably transfected pro-MMP-2 into the human breast cancer cell line MDA-MB-231, which lacks MMP-2 expression but does express its cell surface activator, membrane type 1-MMP. Multiple clones were derived and shown to produce pro-MMP-2 and to activate it in response to concanavalin A. In vitro analysis showed that the pro-MMP-2-transfected clones exhibited an increased invasive potential in Boyden chamber and Matrigel outgrowth assays, compared with the parental cells or those transfected with vector only. When inoculated into the mammary fat pad of nude mice, each of the MMP-2-tranfected clones grew faster than each of the vector controls tested. After intracardiac inoculation into nude mice, pro-MMP-2-transfected clones showed a significant increase in the incidence of metastasis to brain, liver, bone, and kidney compared with the vector control clones but not lung. Increased tumor burden was seen in the primary site and in lung metastases, and a trend toward increased burden was seen in bone, however, no change was seen in brain, liver, or kidney. This data supports a role for MMP-2 in breast cancer progression, both in the growth of primary tumors and in their spread to distant organs. MMP-2 may be a useful target for breast cancer therapy when refinement of MMP inhibitors provides for MMP-specific agents.