Structural and functional studies of the metalloregulator Fur identify a promoter-binding mechanism and its role in Francisella tularensis virulence.

Francisella tularensis is a Gram-negative bacterium causing tularaemia. Classified as possible bioterrorism agent, it may be transmitted to humans via animal infection or inhalation leading to severe pneumonia. Its virulence is related to iron homeostasis involving siderophore biosynthesis directly controlled at the transcription level by the ferric uptake regulator Fur, as presented here together with the first crystal structure of the tetrameric F. tularensis Fur in the presence of its physiological cofactor, Fe2+. Through structural, biophysical, biochemical and modelling studies, we show that promoter sequences of F. tularensis containing Fur boxes enable this tetrameric protein to bind them by splitting it into two dimers. Furthermore, the critical role of F. tularensis Fur in virulence and pathogenesis is demonstrated with a fur-deleted mutant showing an attenuated virulence in macrophage-like cells and mice. Together, our study suggests that Fur is an attractive target of new antibiotics that attenuate the virulence of F. tularensis.

In vitro and in vivo evaluation of fluoroquinolone resistance associated with DNA gyrase mutations in Francisella tularensis, including in tularaemia patients with treatment failure.

Fluoroquinolones (FQs) are highly effective for treating tularaemia, a zoonosis caused by Francisella tularensis, but failures and relapses remain common in patients with treatment delay or immunocompromised status. FQ-resistant strains of F. tularensis harboring mutations in the quinolone-resistance determining region (QRDR) of gyrA and gyrB, the genes encoding subunits A and B of DNA gyrase, have been selected in vitro. Such mutants have never been isolated from humans as this microorganism is difficult to culture. In this study, the presence of FQ-resistant mutants of F. tularensis was assessed in tularaemia patients using combined culture- and PCR-based approaches. We analyzed 42 F. tularensis strains and 82 tissue samples collected from 104 tularaemia cases, including 32 (30.7%) with FQ treatment failure or relapse. Forty F. tularensis strains and 55 clinical samples were obtained before any FQ treatment, while 2 strains and 15 tissue samples were collected after treatment. FQ resistance was evaluated by the minimum inhibitory concentration (MIC) for the bacterial strains, and by newly developed PCR-based methods targeting the gyrA and gyrB QRDRs for both the bacterial strains and the clinical samples. None of the F. tularensis strains displayed an increased MIC compared with FQ-susceptible controls. Neither gyrA nor gyrB QRDR mutation was found in bacterial strains and tissue samples tested, including those from patients with FQ treatment failure or relapse. Further phenotypic and genetic resistance traits should be explored to explain the poor clinical response to FQ treatment in such tularaemia patients.

Serological microarray for a paradoxical diagnostic of Whipple's disease.

Whipple's disease is a systemic chronic infection caused by Tropheryma whipplei. Asymptomatic people may carry T. whipplei in their digestive tract and this can be determined by PCR, making serological diagnosis useful to distinguish between carriers and patients. Putative antigenic proteins were selected by computational analysis of the T. whipplei genome, immunoproteomics studies and from literature. After expression, putative T. whipplei antigens were screened by microimmunofluorescence with sera of immunized rabbit. Selected targets were screened by microarray using sera from patients and carriers. Paradoxically, with 19 tested recombinant proteins and a glycosylated native protein of T. whipplei, a higher immune response was observed with asymptomatic carriers. In contrast, quantification of human IgA exhibited a higher reaction in patients than in carriers against 10 antigens. These results were used to design a diagnostic test with a cut-off value for each antigen. A blind test assay was performed and was able to diagnose 6/8 patients and 11/12 carriers. Among people with positive T. whipplei PCR of the stool, patients differ from carriers by having positive IgA detection and a negative IgG detection. If confirmed, this serological test will distinguish between carriers and patients in people with positive PCR of the stool.

High expression of Tim-3 mRNA in urinary cells from kidney transplant recipients with acute rejection.

TIM-3 is a recently described molecule specifically expressed on Th1 differentiated T cells. We explored the usefulness of urinary mRNA profiles in the diagnosis of renal acute rejection (AR). Sixty urinary samples from renal transplant recipients simultaneously collected to allograft biopsy, (AR = 30 and No-AR =30), and 12 urinary samples from stable renal transplants were analyzed. Urinary mRNA encoding for TIM-3 and IFN-gamma were quantified using real time RT-PCR. TIM-3 mRNA was highly expressed in AR (559.19 +/- 644.41) compared to No-AR (3.78 +/- 7.20), and stable transplants (0.54 +/- 0.76), p < 0.001. To a lesser degree, IFN-gamma mRNA transcripts were also increased in AR (50.40 +/- 38.71), compared with No-AR (4.69 +/- 12.62), and stable transplants (0.38 +/- 0.44) p < 0.001. The highest expression of TIM-3 in AR makes it a promising noninvasive test for its diagnosis.

Rickettsia felis, from culture to genome sequencing.

Rickettsia felis has been recently cultured in XTC2 cells. This allows production of enough bacteria to create a genomic bank and to sequence it. The chromosome of R. felis is longer than that of previously sequenced rickettsiae and it possess 2 plasmids. Microscopically, this bacterium exhibits two forms of pili: one resembles a conjugative pilus and another forms hair-like projections that may play a role in pathogenicity. R. felis also exhibits several copies of ankyrin-repeat genes and tetratricopeptide encoding gene that are specifically linked to pathogenic host-associated bacteria. It also contains toxin-antitoxin system encoding genes that are extremely rare in intracellular bacteria and may be linked to plasmid maintenance.

Preliminary transcriptional analysis of spoT gene family and of membrane proteins in Rickettsia conorii and Rickettsia felis.

Rickettsiae survival implicates adaptation to different environmental conditions. We hypothesized that multiple copies of genes in bacteria with reduced genomes might account for such a process. Transcription of spoT and sca paralogs was thus analyzed in R. conorii and R. felis.

From genes to proteins: in vitro expression of rickettsial proteins.

The availability of the complete genome sequences of several organisms allows the comparative analysis of genomes, a branch of bioinformatics known as genomics. With this approach, much can be learned about the biology of organisms that are difficult to culture, even when few, if any, of their proteins have been isolated and studied directly. We have focused our interest on Rickettsia conorii, an obligate intracellular bacterium responsible for Mediterranean spotted fever, a disease endemic in southern Europe. While bioinformatic annotation of the complete genome of this bacteria has allowed identification of 1,374 genes, a large number of them remain functionally uncharacterized. The final goal of many experiments in molecular biology is to use biological systems to synthesize the protein encoded by the gene being studied. Because three-dimensional structures are more resilient to evolution and change than amino acid sequences, structure determination of some open reading frames should also exhibit structural similarity to previously described protein families. We have thus initiated a systematic expression and structure determination program for the proteins encoded by rickettsial genes of interest. We have cloned different genes of R. conorii by recombinational cloning (GATEWAY), Invitrogen) a method that uses in vitro site-specific recombination to accomplish a directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. The constructions in p-Dest17 yielded several clones able to express recombinant proteins with a C-terminal histidine tag. Expression of corresponding proteins was then performed using a cell-free protein expression system (Rapid Translation System, RTS, Roche Diagnostics). The recombinational cloning approach coupled to RTS provides an approach to rapid optimization of protein expression and is very useful to express rickettsial proteins. Moreover, this system is able to overcome some of the limitations encountered with rickettsial proteins highly toxic for E. coli or insect cells.

Use of rpoB gene analysis for detection and identification of Bartonella species.

Identification of Bartonella species is of increasing importance as the number of infections in which these bacteria are involved increases. To date, these gram-negative bacilli have been identified by various serological, biochemical, and genotypic methods. However, the development of alternative tools is required, principally to circumvent a major risk of contamination during sample manipulation. The aim of our study was to investigate the possible identification of various Bartonella species by comparison of RNA polymerase beta-subunit gene (rpoB) sequences. This approach has previously been shown to be useful for the identification of members of the family Enterobacteriaceae (C. M. Mollet, M. Drancourt, and D. Raoult, Mol. Microbiol. 26:1005-1011, 1997). Following PCR amplification with specific oligonucleotides, a 825-bp region of the rpoB gene was sequenced from 13 distinct Bartonella strains. Analysis of these sequences allowed selection of three restriction enzymes (ApoI, AluI, and AflIII) useful for discerning the different strains by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. To confirm the potential value of such an approach for identification of Bartonella, the rpoB PCR was then applied to 94 clinical samples, and the results obtained were identical to those obtained by our reference PCR method. Twenty-four isolates were also adequately identified by PCR-RFLP analysis. In all cases, our results were in accordance with those of the reference method. Moreover, conserved regions of DNA were chosen as suitable primer targets for PCR amplification of a 439-bp fragment which can be easily sequenced.

rpoB gene analysis as a novel strategy for identification of spirochetes from the genera Borrelia, Treponema, and Leptospira.

Spirochetes are emerging pathogens for which culture and identification are partly unresolved. In fact, 16S rRNA-based sequencing is by far the most widely used PCR methodology that is able to detect such uncultivable pathogens. However, this assay actually has some limitations linked to potential problems of contamination, which hampers diagnosis. To circumvent this, we have devised a simple PCR strategy involving targeting of the gene encoding the RNA polymerase beta subunit (rpoB), a highly conserved enzyme. The complete sequence of the Leptospira biflexa (serovar patoc) rpoB gene was determined and compared with the published sequences for Borrelia burgdorferi and Treponema pallidum. From the resulting analysis, degenerate nucleotide primers were designed and tested for their ability to amplify a portion of the rpoB gene from various spirochetes. Using two different pairs of these primers, we succeeded in obtaining specific rpoB-amplified fragments for all members of the genera Leptospira, Treponema, and Borrelia tested and no other bacteria. Our findings may have significant implications for the development of a new tool for the identification of spirochetes, especially if clinical samples are contaminated or when the infecting strain is uncultivable.

Determination of the rpoB gene sequences of Bartonella henselae and Bartonella quintana for phylogenic analysis.

Using the Genome Walker procedure, which allows PCR amplification of genomic DNA using a single gene-specific primer and direct automated sequencing methodology, we obtained the nucleotide sequence of the RNA polymerase beta subunit (rpoB) from Bartonella henselae and Bartonella quintana. A phylogenetic tree constructed from these data and other rpoB sequences available in GenBank is, in part, consistent with those previously derived from 16S rRNA gene sequences and confirms the position of Bartonella within the alpha subdivision of Proteobacteria. In fact, this analysis showed that rpoB data are similar to 16S rRNA data for the alpha, beta and gamma subdivisions of Proteobacteria. In contrast, concerning other bacteria included in our study, the topologies of phylogenetic trees were different. Based on the bootstrap values derived from rpoB phylogenic analysis, we believe that this molecule should contribute to better understanding the evolutionary process.

Systemic administration of endotoxin induces bronchopulmonary hyperreactivity dissociated from TNF-alpha formation and neutrophil sequestration into the murine lungs.

Bronchopulmonary hyperreactivity (BHR), an increased responsiveness to nonspecific bronchoconstrictor agents, is a well-known characteristic of bronchial asthma. It has been recently suggested that the severity of this disease is related to the endotoxin content of house dust. In the present report, it is shown that the i.p. administration of bacterial LPS to mice is followed by a marked early dose-dependent BHR in response to methacholine. The microscopic examination showed no ultrastructural lesions of the lungs or of the airways, but a marked neutrophil accumulation in the capillaries, as confirmed by an increase of the lung content in the neutrophil enzyme marker myeloperoxidase. In parallel, high levels of TNF-alpha were found in plasma as well as its transcripts in the lung tissues. Using immunologic (anti-TNF-alpha and anti-granulocyte Abs), and pharmacologic (dexamethasone and vinblastine) tools, it is demonstrated that BHR is apparently neither related to the presence of neutrophils in the pulmonary microvasculature nor to the synthesis of TNF-alpha.

Interaction between Entamoeba histolytica and intestinal epithelial cells involves a CD44 cross-reactive protein expressed on the parasite surface.

This study shows that Entamoeba histolytica binds hyaluronic acid. The binding molecule was identified as an 80-kDa membrane protein and was recognized by anti-CD44 monoclonal antibodies. These data indicate that a CD44 cross-reacting adherence molecule is expressed on E. histolytica.

Specific inhibition of thrombin-induced cell activation by the neutrophil proteinases elastase, cathepsin G, and proteinase 3: evidence for distinct cleavage sites within the aminoterminal domain of the thrombin receptor.

The aim of this study was to investigate the inhibitory effects of human leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR3) on the activation of endothelial cells (ECs) and platelets by thrombin and to elucidate the underlying mechanisms. Although preincubation of ECs with HLE or Cat G prevented cytosolic calcium mobilization and prostacyclin synthesis induced by thrombin, these cell responses were not affected when triggered by TRAP42-55, a synthetic peptide corresponding to the sequence of the tethered ligand (Ser42-Phe55) unmasked by thrombin on cleavage of its receptor. Using IIaR-A, a monoclonal antibody directed against the sequence encompassing this cleavage site, flow cytometry analysis showed that the surface expression of this epitope was abolished after incubation of ECs with HLE or Cat G. Further experiments conducted with platelets indicated that not only HLE and Cat G but also PR3 inhibited cell activation induced by thrombin, although they were again ineffective when TRAP42-55 was the agonist. Similar to that for ECs, the epitope for IIaR-A disappeared on treatment of platelets with either proteinase. These results suggested that the neutrophil enzymes proteolyzed the thrombin receptor downstream of the thrombin cleavage site (Arg41-Ser42) but left intact the TRAP42-55 binding site (Gln83-Ser93) within the extracellular aminoterminal domain. The capacity of these proteinases to cleave five overlapping synthetic peptides mapping the portion of the receptor from Asn35 to Pro85 was then investigated. Mass spectrometry studies showed several distinct cleavage sites, i.e., two for HLE (Val(72)-Ser73 and Ile74-Asn75), three for Cat G (Arg41-Ser42, Phe55-Trp56 and Tyr69-Arg70), and one for PR3 (Val(72)-Ser73). We conclude that this singular susceptibility of the thrombin receptor to proteolysis accounts for the ability of neutrophil proteinases to inhibit cell responses to thrombin.

Inhibition of neutrophil-endothelial cell adhension by a neutrophil product, cathepsin G.

In the present study we investigated the modulation of the polymorphonuclear neutrophil (PMN)-endothelial cell adhesion process by the two main proteinases released from activated PMN during their adhesion to endothelium. Our results showed that, in contrast with elastase, cathepsin G was a powerful inhibitor of PAIN adhesion to interleukin-1 (IL-1)-treated human umbilical vein endothelial cells. This inhibitory effect was linked to the enzymatic activity of the proteinase and was selectively directed against PMN. Because the viability and the reactivity of PMN were not modified by cathepsin G, we looked for a possible effect on adhesion molecules. L-selectin was not cleaved by cathepsin G, whereas it was by chymotrypsin, a closely related proteinase. Cathepsin G blocked PMN adhesion to activated endothelial cells, but also to serum- or fibrinogen-coated plates, three adhesion processes mediated by CD11b/CD18. However, by FACScan analysis or by immunoprecipitation, we failed to find evidence of modifications of CD11b/CD18 expression. Although the precise molecular target(s) of cathepsin G remain(s) to be defined, these data indicate that this proteinase, which is known as an inflammatory mediator, can also be considered as a potential down-regulator of adhesion reactions involved in the inflammatory process.

Proteolysis of thrombospondin during cathepsin-G-induced platelet aggregation: functional role of the 165-kDa carboxy-terminal fragment.

The serine-proteinase cathepsin G (CG) is a potent agonist of platelet aggregation inducing the release and surface expression of alpha-granule adhesive proteins such as fibrinogen (Fg) and thrombospondin-1 (TSP-1). Because Fg and TSP-1 are potential substrates for the enzymatic activity of CG, we investigated the fate of these proteins during CG-induced platelet aggregation using an immunoblot technique. Only a small proportion of secreted Fg was proteolyzed by CG and platelet aggregation was efficiently inhibited by anti-fibrinogen Fab fragments. In contrast, TSP-1 was extensively proteolyzed on aggregated platelets releasing in the milieu a fragment with Mr approximately 28 000, corresponding to the amino-terminal heparin-binding domain (HBD). Several antibodies, directed against the cell-associated carboxy-terminal TSP-1f fragment (Mr approximately 165000) impaired the formation of stable macroaggregates, indicating that this fragment may contribute to platelet aggregation in the absence of the HBD.

Protective effect of platelet activating factor antagonists on cultured endothelial cell lysis induced by elastase or activated neutrophils.

1. The mechanism(s) responsible for injury of endothelial cells induced by human leukocyte elastase (HLE) was investigated in an immortalized venous human endothelial cell line (IVEC). 2. First, the proteinase concentrations and incubation delays necessary to trigger a significant IVEC cytotoxicity were determined by chromium assays. Thus, exposure of IVEC for 6 h to 10 micrograms ml-1 HLE resulted in 22 +/- 2.8% lysis and 36.4 +/- 5.4% detachment (mean +/- s.e. mean; n = 4; P < 0.05). 3. WEB 2086, a specific platelet-activating factor (PAF) receptor antagonist, induced a significant concentration-dependent decrease of such a lysis (39.6 +/- 7.7% protection at 100 microM; n = 4). This potential role for PAF was confirmed with two other antagonists of this lipid mediator, i.e., BN 52021 and RP 48740. 4. Finally, we demonstrated that pretreatment of IVEC with WEB 2086 protected significantly against cell lysis induced by stimulated human neutrophils, an experimental model in which HLE participates.

Induction of adherence and degranulation of polymorphonuclear leukocytes: a new expression of the invasive phenotype of Shigella flexneri.

In the present study, the ability of Shigella flexneri to activate polymorphonuclear neutrophils (PMN) was examined. The invasive serotype 5 strain M90T induced strong PMN adherence, which was dependent on both the multiplicity of infection and the duration of incubation. When tested under the same experimental conditions, the noninvasive strain BS176 (cured of the 220-kb virulence plasmid) was less efficient. Indeed, incubation of PMN for 2 h with either M90T or BS176 (multiplicity of infection, 100) induced 51.8% +/- 10.5% and 15.2% +/- 4.2% adherence, respectively (n = 3; P < 0.05). Stronger PMN activation by M90T was confirmed by evaluating PMN degranulation induced by the two strains. Whereas M90T triggered significant PMN secretion, BS176 did not. M90T strains with mutations in ipa genes were then analyzed. When PMN were incubated with these mutants, their activation was of the same intensity as that obtained with BS176. These data provide the first evidence for PMN activation induced by S. flexneri, a process which appears to be mediated by Ipa invasins.