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Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis. doi: 10.1128/JB.00458-10

The mat (or ecp) fimbrial operon is ubiquitous and conserved in Escherichia coli, but its functions remain poorly described. In routine growth media newborn meningitis isolates of E. coil express the meningitis-associated and temperature-regulated (Mat) fimbria, also termed E. coli common pilus (ECP), at 20[degrees]C, and here we show that the six-gene (matABCDEF)-encoded Mat fimbria is needed for temperature-dependent biofilm formation on abiotic surfaces. The matBCDEF deletion mutant of meningitis E. coil IHE 3034 was defective in an early stage of biofilm development and consequently unable to establish a detectable biofilm, contrasting with IHE 3034 derivatives deleted for flagella, type 1 fimbriae or S-fimbriae, which retained the wild-type biofilm phenotype. Furthermore, induced production of Mat fimbriae from expression plasmids enabled biofilm-deficient E. coil K-1 2 cells to form biofilm at 20[degrees]C. No biofilm was detected with IHE 3034 or MG1655 strains grown at 37[degrees]C. The surface expression of Mat fimbriae and the frequency of Mat-positive cells in the IHE 3034 population from 20 [degrees]C were high and remained unaltered during the transition from planktonic to biofilm growth and within the matured biofilm community. Considering the prevalence of the highly conserved mat locus in E. coil genomes, we hypothesize that Mat fimbria-mediated biofilm formation is an ancestral characteristic of E. coil DOI 10.1099/mic.0.039610-0

The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. The primary structure of the Pla [beta]-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis, which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtus and Angola carries the single amino acid change T259I located in surface loop 5 of the [beta]-barrel. Recombinant Y. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259I was impaired in fibrinolysis and in plasminogen activation. Lack of detectable generation of the catalytic light chain of plasmin and inactivation of plasmin enzymatic activity by the Pla T259I construct indicated that Microtus Pla cleaved the plasminogen molecule more unspecifically than did common Pla. The isoform pattern of the Pla T259I molecule was different from that of the common Pla molecule. Microtus Pla was more efficient than wild-type Pla in [[alpha].sub.2]-antiplasmin inactivation. Pla of Y. pestis and PgtE of Salmonella enterica have evolved from the same omptin ancestor, and their comparison showed that PgtE was poor in plasminogen activation but exhibited efficient antiprotease inactivation. The substitution [sup.259]IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, altered proteolysis in favor of plasmin formation, whereas the reverse substitution [sup.259]TIDKN/IIDKT in Pla altered proteolysis in favor of [[alpha].sub.2]-antiplasmin inactivation. The results suggest that Microtus Pla represents an ancestral form of Pla that has evolved into a more efficient plasminogen activator in the pandemic Y. pestis lineages.

The plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase of Lactobacillus crispatus were localized on the cell surface at pH 5 but released into the medium at an alkaline pH. These proteins bound to lipoteichoic acids at a pH below their isoelectric point. The results indicate that lactobacilli rapidly modify their surface properties in response to changes in pH.

The abundant proteolytic plasminogen (Plg)/plasmin system is important in several physiological functions in mammals and also engaged by a number of pathogenic microbial species to increase tissue invasiveness or to obtain nutrients. This paper reports that a commensal bacterium, Lactobacillus crispatus, interacts with the Pig system. Strain ST1 of L. crispatus enhanced activation of human Plg by the tissue-type Pig activator (tPA), whereas enhancement of the urokinase-mediated Plg activation was lower. ST1 cells bound Pig, plasmin and tPA only poorly, and the Plg-binding and activation-enhancing capacities were associated with extracellular material released from the bacteria into buffer. The extracellular proteome of L. crispatus ST1 contained enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as major components. The enolase and the GAPDH genes of ST1 were cloned, sequenced and expressed in recombinant Escherichia coli as [His.sub.6]-fusion proteins, which bound Plg and enhanced its activation by tPA. Variable levels of secretion of enolase and GAPDH proteins as well as of the Pig activation cofactor function were detected in strains representing major taxonomic groups of the genus Lactobacillus. So far, interference with the Plg system has been addressed with pathogenic microbes. The results reported here demonstrate a novel interaction between a member of the microbiota and a major proteolytic system in humans.

Yersinia pestis is a species that emerged recently from Yersinia pseudotuberculosis and gained an exceptional pathogenicity potential. Among the major genetic differences between the plague bacillus and its ancestor is the acquisition of the pPla plasmid, which has been associated with the increased virulence of Y. pestis. In a previous study, introduction of pPla into Y. pseudotuberculosis did not lead to any modification of the virulence of the host bacterium. However, it was subsequently demonstrated that the presence of smooth lipopolysaccharide (LPS) inhibits the activity of Pla. In this study, pPla was introduced into a Y. pseudotuberculosis strain expressing smooth LPS, and into a variant in which a mutation that abrogates the formation of O-antigen (O-Ag) repeats (as in natural isolates of Y. pestis) was generated. It was found that in both strains, Pla was synthesized, exported to the bacterial membrane and processed as in Y. pestis. However, the ability of Pla to activate plasminogen was weak and observed only at 37 [degrees]C in the smooth strain, while this activity was similar to that of Y. pestis and expressed at both 28 and 37 [degrees]C in the O-Ag mutant strain. Similarly, Pla-mediated inactivation of the antiprotease [[alpha].sub.2]-antiplasmin was not detected in the smooth Y. pseudotuberculosis strain grown at 28 [degrees]C, but was expressed at both temperatures in the O-Ag mutant strain. Despite the more efficient activity of Pla, the Y. pseudotuberculosis O-Ag mutant strain exhibited a lower pathogenicity upon subcutaneous infection of mice. The results thus indicate that, although abrogation of O side chain synthesis in a Y. pseudotuberculosis strain harbouring pPla potentiates the two proteolytic activities of Pla, this is not sufficient to confer to Y. pseudotuberculosis a higher pathogenicity potential. These results also suggest that acquisition of pPla may not have been sufficient to confer an immediate higher pathogenic potential to the ancestor Y. pestis strain.

The plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria expresses a type III secretion system that is necessary for both pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Here we show that X. campestris pv. vesicatoria produces filamentous structures, the Hrp pili, at the cell surface under hrp-inducing conditions. Analysis of purified Hrp pili and immunoelectron microscopy revealed that the major component of the Hrp pilus is the HrpE protein which is encoded in the hrp gene cluster. Sequence homologues of hrpE are only found in other xanthomonads. However, hrpE is syntenic to the hrpY gene from another plant pathogen, Ralstonia solanacearum. Bioinformatic analyses suggest that all major Hrp pilus subunits from gram-negative plant pathogens may share the same structural organization, i.e., a predominant alpha-helical structure. Analysis of nonpolar mutants in hrpE demonstrated that the Hrp pilus is essential for the productive interaction of X. campestris pv. vesicatoria with pepper host plants. Furthermore, a functional Hrp pilus is required for type III-dependent protein secretion. Immunoelectron microscopy revealed that type III-secreted proteins, such as HrpF and AvrBs3, are in close contact with the Hrp pilus during and/or after their secretion. By systematic analysis of nonpolar hrp/hrc (hrp conserved) and hpa (hrp associated) mutants, we found that Hpa proteins as well as the translocon protein HrpF are dispensable for pilus assembly, while all other Hrp and Hrc proteins are required. Hence, there are no other conserved Hrp or Hrc proteins that act downstream of HrpE during type III-dependent protein translocation.

Cell adhesion analysis shows that fresh lactobacillus cells adhere to the epithelium and in a strain-dependent manner, the cells adhere to follicle-associated epithelium and the apical surfaces of mature endocytes of intestinal villi.

Research demonstrates that the S-layer of the SlpA surface protein mediattes Lactobacillus brevis ATCC 8287 adhesion to human epithelial cells and fibronectin. The receptor-binding region is confined to a region of 81 amino acids in the N-terminal part of the S-layer.

Results show that the matB fimbrial gene is expressed only in a geneticlly conserved, virulent Escherichica coli strain. Data suggest that regulation of Mat fimbria expression involves transcriptional and posttranslational events.

Research describes construction and purification of gaf gene cluster encoded GafD lectin from Escherichia coli for structure-function analysis of the fimbrial adhesisn protein. Results show that GafD is a soluble non-fusion form of the lectin and its protease-resistant subdomain exhibits receptor-binding specificity.

Research has been conducted on the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810. The cbsA gene encoding of this protein has been expressed in Lactobacillus casei ATCC 393 (super)T and the results indicate that S-protein was secreted into the medium of the recombinant bacteria.

Results show that the collagen-binding S-layer protein CbsA is a polypeptide of 410 amino acids with a stronger collagen-binding region at the N-terminus and a weaker one at the C-terminus. Data further indicate that collagen-binding requires a crystal structure with regular array of CbsA.

Research describes construction of a flagellum carrying two different recombinant flagellins of flagellin subunits in Escherichia coli. Data demonstrate that a fibronectin-binding 115-mer peptide and a 302-mer collagen-binding peptide can be inserted into a flagellum as revealed by adhesion and immunochemical tests.

The Xanthomonas capestris fimA gene encoding the bundle-forming fimbriae and cell aggregation was analyzed to characterize the role of fimbriae in Xanthomonas capestris infections. The nucleotide sequence of the fimA gene of Xanthomonas campestris was composed of two open reading frames that encode FimA and FimB proteins. Mutations in the fimA gene also impaired cell aggregation and reduced the virulence of Xanthomonas campestris cells in infected tomato leaves.

Lactobacillus acidophilus commonly inhabits the intestinal tract of both man and animal. The S-layer of Lactobacillus crispatus JCM 5810 of A2 strain acts as specific bacterial adhesin and initiates bacterial adhesion to the mammalian extracellular matrix (ECM), thereby providing direct evidence that normal intestinal bacterial flora can adhere to the subintestinal ECM. S layers commonly occur in lactic acid bacteria. JCM 5810 of the S-layer has high affinity towards I and IV collagen and exhibits functional heterogeneity.

An analysis of the outer membrane protein (OMP) and lipopolysaccharide (LPS) patterns of 54 isolates of 16 distinct pathovars of Xanthomonas campestris reveals that the strains within a single pathovar are almost identical to isolates from other pathovars. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) aids the analysis of the characteristic features of OMPs and LPSs of X. campestris. Within X. campestris pv. translucens, X. campestris pv. campestris and X campestris pv. vesicatoria, profiles of LPSs and OMPs differ from each other.

Abstract Background Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic Erwinia pyrifoliae exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of Yersinia pestis. We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells. Results Pla and Epo expressed in Escherichia coli are both functional endopeptidases and cleave human serine protease inhibitors, but Epo failed to activate plasminogen and to mediate invasion into a human endothelial-like cell line. Swapping of ten amino acid residues at two surface loops of Pla and Epo introduced plasminogen activation capacity in Epo and inactivated the function in Pla. We also compared the structure of Pla and the modeled structure of Epo to analyze the structural variations that could rationalize the different proteolytic activities. Epo-expressing bacteria managed to invade human cells only after all extramembranous residues that differ between Pla and Epo and the first transmembrane β-strand had been changed. Conclusions We describe molecular adaptation of a protease from an environmental setting towards a virulence factor detrimental for humans. Our results stress the evolvability of bacterial β-barrel surface structures and the environment as a source of progenitor virulence molecules of human pathogens.

The applicability of the capping protein FliD for display purposes and the flagellum display was developed into a multihybrid surface display system in which three different foreign peptides are expressed simultaneously within the same flagellum that is at the tip and along the filament. It showed that FliD is successfully used in bacterial surface display.

Lactobacillus crispatus is a common member of the beneficial microbiota present in the vertebrate gastrointestinal and human genitourinary tracts. Here, we report the genome sequence of L. crispatus ST1, a chicken isolate displaying strong adherence to vaginal epithelial cells. doi: 10.1128/JB.00399-10