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The Pla surface protease of Yersinia pestis activates human plasminogen and is a central virulence factor in bubonic and pneumonic plague. Pla is a transmembrane ␤-barrel protein and member of the omptin family of outer membrane proteases which require bound lipopolysaccharide (LPS) to be proteolytically active. Plas

The Pla surface protease of Yersinia pestis, encoded by the Y. pestis-specific plasmid pPCP1, is a versatile virulence factor. In vivo studies have shown that Pla is essential in the establishment of bubonic plague, and in vitro studies have demonstrated various putative virulence functions for the Pla molecule. Pla is a surface protease of the omptin family, and its proteolytic targets include the abundant, circulating human zymogen plasminogen, which is activated by Pla to the serine protease plasmin. Plasmin is important in cell migration, and Pla also proteolytically inactivates the main circulating inhibitor of plasmin, alpha2-antiplasmin. Pla also is an adhesin with affinity for laminin, a major glycoprotein of mammalian basement membranes, which is degraded by plasmin but not by Pla. Together, these functions create uncontrolled plasmin proteolysis targeted at tissue barriers. Other proteolytic targets for Pla include complement proteins. Pla also mediates bacterial invasion into human endothelial cell lines; the adhesive and invasive charateristics of Pla can be genetically dissected from its proteolytic activity. Pla is a 10-stranded antiparallel beta-barrel with five surface-exposed short loops, where the catalytic residues are oriented inwards at the top of the beta-barrel. The sequence of Pla contains a three-dimensional motif for protein binding to lipid A of the lipopolysaccharide. Indeed, the proteolytic activity of Pla requires rough lipopolysaccharide but is sterically inhibited by the O antigen in smooth LPS, which may be the selective advantage of the loss of O antigen in Y. pestis. Members of the omptin family are highly similar in structure but differ in functions and virulence association. The catalytic residues of omptins are conserved, but the variable substrate specificities in proteolysis by Pla and other omptins are dictated by the amino acid sequences near or at the surface loops, and hence reflect differences in substrate binding. The closest orthologs of Pla are PgtE of Salmonella and Epo of Erwinia, which functionally differ from Pla. Pla gives a model of how a horizontally transferred protein fold can diverge into a powerful virulence factor through adaptive mutations.

In this study we show the expression of flavin mononucleotide (FMN)-based fluorescent protein (FbFP) BS2 as a marker for gene expression in the opportunistic human anaerobic pathogen Bacteroides fragilis. B. fragilis 638R strain carrying osu::bs2 constructs showed inducible fluorescence following addition of maltose anaerobically compared to non-fluorescent cells under glucose repressed conditions. Bacteria carrying ahpC::bs2 or dps::bs2 constructs were fluorescent following induction by oxygen compared to non-fluorescent cells from the anaerobic control cultures. In addition, when these transcriptional fusion constructs were mobilized into B. fragilis IB263, a constitutive peroxide response strain, fluorescent BS2 was detected in both anaerobic and aerobic cultures confirming the unique properties of the FbFP BS2 to yield fluorescent signal in B. fragilis in the presence and absence of oxygen. Moreover, intracellular expression of BS2 was also detected when cell culture monolayers of J774.1 macrophages were incubated with B. fragilis ahpC::bs2 or dps::bs2 strains within an anaerobic chamber. This suggests that ahpC and dps are induced following internalization by macrophages. Thus, we show that BS2 is a suitable tool for the detection of gene expression in obligate anaerobic bacteria in in vivo studies.