[Biomedical research: the debate on the reduction and emergence concepts]. / Recherche biomédicale: le débat autour des notions de réduction et d'émergence.

The theoretical bases of medical knowledge exert a strong influence on both clinical practice and representations of living and health. In this perspective, reduction and emergence notions play a major role. Microreduction is the predominant analytical strategy used today in biology, as it is usually considered that essential life mechanisms can be reduced to molecular processes. Likewise, macroreduction proposes that parts can be defined in terms of their belonging to wholes, as it is usually assumed, for instance, in genetic epidemiology. With regard to emergence, this notion, which focuses on properties of a whole that cannot be deduced from properties of its parts, is consistent with both nature of living and evolution theory. The apparent success of reduction like analytical modality has generated in scientific community and public opinion an ideological reductionism, which corresponds, ontologically, to both physicalism (things can be entirely understood in terms of their parts), and atomism (things go their own way, independently of other things). Genetic reductionism has generated new cosmological representations of living, where past, present and future of living beings could potentially be deduced from fallacious, simple views of genome sequences. These views may lead to quantitative or qualitative definitions of standard patterns and hierarchies. In practical terms, research activity should integrate limits, strains as well as reductionism advantages. Biologists should also consider risks associated with an ideological, unrestricted reductionism, applied to any existence aspect, a notion with questionable legitimacy and with potential ethical, philosophical, and political involvements that go beyond the simple selection of a research strategy.

Role of ADP-ribosyltransferase activity of pertussis toxin in toxin-adhesin redundancy with filamentous hemagglutinin during Bordetella pertussis infection.

Pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two major virulence factors of Bordetella pertussis. FHA is the main adhesin, whereas PT is a toxin with an A-B structure, in which the A protomer expresses ADP-ribosyltransferase activity and the B moiety is responsible for binding to the target cells. Here, we show redundancy of FHA and PT during infection. Whereas PT-deficient and FHA-deficient mutants colonized the mouse respiratory tract nearly as efficiently as did the isogenic parent strain, a mutant deficient for both factors colonized substantially less well. This was not due to redundant functions of PT and FHA as adhesins, since in vitro studies of epithelial cells and macrophages indicated that FHA, but not PT, acts as an adhesin. An FHA-deficient B. pertussis strain producing enzymatically inactive PT colonized as poorly as did the FHA-deficient, PT-deficient strain, indicating that the ADP-ribosyltransferase activity of PT is required for redundancy with FHA. Only strains producing active PT induced a local transient release of tumor necrosis factor alpha (TNF-alpha), suggesting that the pharmacological effects of PT are the basis of the redundancy with FHA, through the release of TNF-alpha. This may lead to damage of the pulmonary epithelium, allowing the bacteria to colonize even in the absence of FHA.

Genomics of Bordetella pertussis toxins.

Bordetella pertussis, the etiologic agent of whooping cough, produces numerous toxins including pertussis toxin (PTX), adenylate cyclase toxin (AC), dermonecrotic toxin (DNT) and tracheal cytotoxin (TCT). PTX is composed of five different subunits organised in a typical A-B type structure of which the A part possesses an enzymatic ADP-ribosyltransferase activity and the B moiety expresses receptor-binding activity. The secretion of this toxin requires nine other genes (ptl) organised in an operon together with the five structural genes of PTX. To further characterise the genetic locus of this major virulence factor, we analysed the ptx/ptl upstream and downstream sequences. Comparison of these regions between three species of Bordetella (B. pertussis, Bordetella parapertussis and Bordetella bronchiseptica) revealed differences in the upstream region. Analysis of two strains of B. bronchiseptica naturally lacking the ptx genes showed that only the ptx/ptl genes were deleted in these strains, and that the upstream and downstream regions were conserved. Upstream of the PTX structural genes and the promoter, an open reading frame (bugT) was identified, the product of which is homologous with putative proteins from several other Gram-negative organisms. Detailed analysis of the genome of B. pertussis which is currently sequenced at the Sanger Centre revealed the presence of 90 genes coding for proteins homologous to BugT, which qualifies the bug gene family as the most populated one of Bordetella. These bug genes are located in various genetic environments, including the proximities of genes coding for other toxins, such as DNT and AC. The Bug proteins are highly conserved in terms of size and periodicity of predicted secondary structure elements, but have also a high variability in their amino acid composition reflected in their wide range of isoelectric points. The function of these genes which is currently unknown is under investigation. To characterise the expression and regulation of these genes, as well as of novel putative B. pertussis virulence factors, we designed a transcriptional fusion vector to be inserted in precise locations of the B. pertussis chromosome by homologous recombination. The reporter gene present in this vector allowed us to show that at least some of the bug genes are expressed.

Intracellular trafficking and membrane translocation of pertussis toxin into host cells.

The translocation of the pertussis toxin (PTX) S1 subunit into the cytoplasm of host cells was analysed in CHO cells producing S1 fused to a signal peptide. This protein channelled into the endoplasmic reticulum (ER) by the signal peptide, was found to ADP-ribosylate its target G proteins, suggesting that membrane translocation can occur from the ER and does not require the B oligomer. Similar results were obtained with a C-terminally truncated S1 subunit, indicating that this hydrophobic tail is not involved in the translocation mechanism. We also analysed the activity of two PTX mutants in which the S3 and S2 subunits were substituted for each other. The mutant protein containing two S3 subunits (PTXAS2) presented a decreased binding to fetuin or haptoglobin but higher in vivo activity than the wild-type PTX, suggesting that replacement of S2 by S3 favours the targeting of PTX to the compartment where translocation occurs and/or the dissociation of S1 from the B oligomer, thereby leading to a better translocation of S1 into the cytoplasm.

New virulence-activated and virulence-repressed genes identified by systematic gene inactivation and generation of transcriptional fusions in Bordetella pertussis.

An in silico scan of the partially completed genome sequence of Bordetella pertussis and analyses of transcriptional fusions generated with a new integrational vector were used to identify new potential virulence genes. The genes encoding a putative siderophore receptor, adhesins, and an autotransporter protein appeared to be regulated in a manner similar to Bordetella virulence genes by the global virulence regulator BvgAS. In contrast, the gene encoding a putative intimin-like protein appeared to be repressed under conditions of virulence.

The gene encoding the low-affinity penicillin-binding protein 3r in Enterococcus hirae S185R is borne on a plasmid carrying other antibiotic resistance determinants.

Two plasmid-derived NcoI DNA fragments of 14 and 4.5 kb, respectively, have been isolated from the multidrug-resistant strain Enterococcus hirae S185R and analyzed. The 14-kb fragment contains two inverted (L and R) IS1216 insertion modules of the ISS1 family. These modules define a Tn5466 transposon-like structure that contains one copy of the methylase-encoding ermAM conferring erythromycin resistance and one copy of the adenylyl-transferase-encoding aadE conferring streptomycin resistance. Immediately on the left side of IS1216L there occurs a copy of pbp3r encoding the low-affinity penicillin-binding protein (PBP) PBP3r, itself preceded by a psr-like gene (psr3r) that controls the synthesis of PBP3r. ermAM, aadE, and the transposase gene (tnp) of IS1216R have the same polarities, and these are opposite those of psr3r, pbp3r, and the tnp gene of IS1216L. The 4.5-kb fragment is a copy of the 4.5-kb sequence at the 5' end of the 14-kb fragment, although it is not a restriction product of the 14-kb fragment. It contains three genes with the same polarity: psr3r, pbp3r, and tnp in an IS1216 element. Because of the very high degree of identity (99%) with the chromosomal psrfm and pbp5fm genes of Enterococcus faecium D63R, it is proposed that both the psr3r and pbp3r genes were transferred from an E.faecium strain and inserted in a plasmid of E. hirae. E. hirae is the first known bacterial species in which a low-affinity PBP-encoding gene has been found to be plasmid borne.

Structure of the low-affinity penicillin-binding protein 5 PBP5fm in wild-type and highly penicillin-resistant strains of Enterococcus faecium.

Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in beta-lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. faecium D63r containing the pbp5fm gene was cloned and sequenced. Two open reading frames (ORFs) were found. A 2,037-bp ORF encoded the deduced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectively, 99.8, 78.5, and 62% homologous to those of the low-affinity plasmid-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encoded PBP5 of E. hirae R40 and Enterococcus faecalis 56R. A second 597-bp ORF, designated psrfm, was found 2.3 kb upstream of pbp5fm. It appeared to be 285 bp shorter than and 74% homologous with the regulatory gene psr of E. hirae ATCC 9790. Different clinical isolates of E. faecium, for which a wide range of benzylpenicillin MICs were observed, showed that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 micrograms/ml), the increased level of resistance could be explained by the presence of larger quantities of PBP5fm which had an affinity for benzylpenicillin (second-order rate constant of protein acylation [k+2/K] values of 17 to 25 M(-1) s(-1)) that remained unchanged. For the two most highly resistant strains, EFM-1 (MIC, 90 micrograms/ml) and H80721 (MIC, 512 micrograms/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm variants (k+2/K < or = 1.5 M(-1) s(-1)) which were synthesized in small quantities. More specifically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the third position after the conserved SDN triad, by Thr in EFM-1 and by Ala in H80721 were the most likely cause of the decreasing affinity of PBP5fm observed in these strains.

Cloning and sequencing of the low-affinity penicillin-binding protein 3r-encoding gene of Enterococcus hirae S185: modular design and structural organization of the protein.

The clinical isolate Enterococcus hirae S185 has a peculiar mode of resistance to penicillin in that it possesses two low-affinity penicillin-binding proteins (PBPs): the 71-kDa PBP5, also found in other enterococci, and the 77-kDa PBP3r. The two PBPs have the same low affinity for the drug and are immunochemically related to each other. The PBP3r-encoding gene has been cloned and sequenced, and the derived amino acid sequence has been compared by computer-assisted hydrophobic cluster analysis with that of the low-affinity PBP5 of E. hirae R40, the low-affinity PBP2' of Staphylococcus aureus, and the PBP2 of Escherichia coli used as the standard of reference of the high-M(r) PBPs of class B. On the basis of the shapes, sizes, and distributions of the hydrophobic and nonhydrophobic clusters along the sequences and the linear amino acid alignments derived from this analysis, the dyad PBP3r-PBP5 has an identity index of 78.5%, the triad PBP3r-PBP5-PBP2' has an identity index of 29%, and the tetrad PBP3r-PBP5-PBP2'-PBP2 (of E. coli) has an identity index of 13%. In spite of this divergence, the low-affinity PBPs are of identical modular design and possess the nine amino acid groupings (boxes) typical of the N-terminal and C-terminal domains of the high-M(r) PBPs of class B. At variance with the latter PBPs, however, the low-affinity PBPs have an additional approximately 110-amino-acid polypeptide stretch that is inserted between the amino end of the N-terminal domain and the carboxy end of the membrane anchor. While the enterococcal PBP5 gene is chromosome borne, the PBP3r gene appears to be physically linked to the erm gene, which confers resistance to erythromycin and is known to be plasmid borne in almost all the Streptococcus spp. examined.