Multiplicity of Salmonella entry mechanisms, a new paradigm for Salmonella pathogenesis.

The Salmonella enterica species includes about 2600 diverse serotypes, most of which cause a wide range of food- and water-borne diseases ranging from self-limiting gastroenteritis to typhoid fever in both humans and animals. Moreover, some serotypes are restricted to a few animal species, whereas other serotypes are able to infect plants as well as cold- and warm-blooded animals. An essential feature of the pathogenicity of Salmonella is its capacity to cross a number of barriers requiring invasion of a large variety of phagocytic and nonphagocytic cells. The aim of this review is to describe the different entry pathways used by Salmonella serotypes to enter different nonphagocytic cell types. Until recently, it was accepted that Salmonella invasion of eukaryotic cells required only the type III secretion system (T3SS) encoded by the Salmonella pathogenicity island-1. However, recent evidence shows that Salmonella can cause infection in a T3SS-1-independent manner. Currently, two outer membrane proteins Rck and PagN have been clearly identified as Salmonella invasins. As Rck mediates a Zipper-like entry mechanism, Salmonella is therefore the first bacterium shown to be able to induce both Zipper and Trigger mechanisms to invade host cells. In addition to these known entry pathways, recent data have shown that unknown entry routes could be used according to the serotype, the host and the cell type considered, inducing either Zipper-like or Trigger-like entry processes. The new paradigm presented here should change our classic view of Salmonella pathogenicity. It could also modify our understanding of the mechanisms leading to the different Salmonella-induced diseases and to Salmonella-host specificity.

A Listeria monocytogenes strain is still virulent despite nonfunctional major virulence genes.

The low-virulence Listeria monocytogenes strains have been previously assigned to 4 phenotypic groups. This study aimed to characterize the A23 strain, which exhibits a pulsed-field gel electrophoresis profile specific to low-virulence strains. This strain has the same causal mutations as the group III strains and a supplementary mutation in the mpl gene, leading to the absence of internalin A expression and the presence of inactive internalin B, phosphatidyl-inositol phospholipase C, and phosphatidylcholine phospholipase C. Despite these mutations in major virulence genes, the A23 strain formed plaques in cell monolayers and contaminated 100% of inoculated mice, suggesting that it evolved from group III strains by acquiring new virulence genes.

Multiple point mutations in virulence genes explain the low virulence of Listeria monocytogenes field strains.

In order to understand the causes of the low virulence of Listeria monocytogenes field strains, five low-virulence strains were analysed. These five strains showed changes in relation to invasion, phosphatidyl-inositol phospholipase C (PI-PLC) activity, plaque formation and in vivo virulence. Molecular analyses revealed the same mutations in the plcA, inlA and inlB genes in all five strains. The Thr262Ala substitution in the PI-PLC protein was responsible for the absence of PI-PLC activity. This residue, conserved in certain L. monocytogenes species, is located at the outer rim of the active site pocket and could impair the cleavage activity of the enzyme. The low invasion rate of these strains was due to a nonsense codon leading to a lack of InlA protein synthesis, and to an Ala117Thr substitution in the leucine-rich repeat of InlB, which altered the interaction with the Met receptor. Single trans complementation with the inlA(EGDe), inlB(EGDe) or plcA(EGDe) genes restored the capacity of low-virulence strains either to enter epithelial and fibroblastic cells or to express PI-PLC activity. Complementation by allelic exchange of the plcA(EGDe) gene on the chromosome and trans complementation with either the inlA(EGDe) or the inlB(EGDe) gene restored the ability to form plaques, but only partly restored the in vivo virulence, suggesting that there were other gene mutation(s) with consequences that could mainly be observed in vivo. These results indicate that the low virulence of L. monocytogenes strains can be explained by point mutations in a number of virulence genes; these could therefore be important for detecting low-virulence strains. Moreover, the fact that all the strains had the same substitutions suggests that they have a common evolutionary pathway.

Investigation of specific substitutions in virulence genes characterizing phenotypic groups of low-virulence field strains of Listeria monocytogenes.

Several models have shown that virulence varies from one strain of Listeria monocytogenes to another, but little is known about the cause of low virulence. Twenty-six field L. monocytogenes strains were shown to be of low virulence in a plaque-forming assay and in a subcutaneous inoculation test in mice. Using the results of cell infection assays and phospholipase activities, the low-virulence strains were assigned to one of four groups by cluster analysis and then virulence-related genes were sequenced. Group I included 11 strains that did not enter cells and had no phospholipase activity. These strains exhibited a mutated PrfA; eight strains had a single amino acid substitution, PrfAK220T, and the other three had a truncated PrfA, PrfADelta174-237. These genetic modifications could explain the low virulence of group I strains, since mutated PrfA proteins were inactive. Group II and III strains entered cells but did not form plaques. Group II strains had low phosphatidylcholine phospholipase C activity, whereas group III strains had low phosphatidylinositol phospholipase C activity. Several substitutions were observed for five out of six group III strains in the plcA gene and for one out of three group II strains in the plcB gene. Group IV strains poorly colonized spleens of mice and were practically indistinguishable from fully virulent strains on the basis of the above-mentioned in vitro criteria. These results demonstrate a relationship between the phenotypic classification and the genotypic modifications for at least group I and III strains and suggest a common evolution of these strains within a group.

Quantification of ovine cytokine gene expression by a competitive RT-PCR method.

A quantitative competitive RT-PCR method was developed in order to measure IL-1beta, IL-4, IL-12, IFNgamma, TNFalpha and G(3)PDH mRNA from samples of ovine tissue such as lymph node or spleen. The main advantage of the method relies on the use, for each target sequence, of an internal competitor construct similar to the relevant target, but 4-bp different in size. This competitive strategy is validated by the equivalence of the amplification process, observed separately between competitor DNA and target DNA species. Furthermore, the copy number of each cytokine cDNA is normalized to a fixed copy number of G(3)PDH cDNA. The cDNA level of this constitutive gene was effectively shown to remain constant whatever the tissue studied and independently of the experimental conditions used. The accurate and reproducible data obtained permit the application of this quantitative RT-PCR method to measure the sheep cytokine response to Salmonella infection. Early induction of IFNgamma mRNA was observed in the draining lymph node 1 day after infection. At the same time, a strong increase of IL-1beta mRNA was observed in local and systemic lymphoid organs, suggesting the initiation of the inflammatory response. Finally, the overall results demonstrate the efficiency of the method and its suitability for further studies of the immune response in the ovine species.

An IS711 element downstream of the bp26 gene is a specific marker of Brucella spp. isolated from marine mammals.

DNA polymorphism of the bp26 gene, coding for a diagnostic protein antigen for brucellosis, was assessed by PCR and restriction fragment length polymorphism analysis using primers to amplify the bp26 gene with its flanking regions. Surprisingly, whereas PCR performed on DNA of the reference strains of the six recognized Brucella species produced a product of the expected size (1,029 bp), PCR performed on DNA of three representative strains from marine mammals (from a seal, a dolphin, and a porpoise) produced a larger product, of about 1,900 bp. Nucleotide sequencing of the 1,900-bp PCR products revealed the presence of an insertion sequence, IS711, downstream of the bp26 gene and adjacent to a Bru-RS1 element previously described as being a hot spot for IS711 insertion. PCR performed on a large number of field strains from different geographic origins and from marine mammal isolates indicated that the occurrence of an IS711 element downstream of the bp26 gene was a feature specific to the marine mammal Brucella strains. Thus, this PCR assay is able to differentiate Brucella terrestrial isolates from marine mammal isolates and could be applied for diagnostic purposes.

Polymorphism at the dnaK locus of Brucella species and identification of a Brucella melitensis species-specific marker.

The dnaK gene and surrounding sequences from reference strains of the six Brucella species were amplified by the polymerase chain reaction (PCR) with primers chosen according to the published sequence of the B. ovis dnaK gene and studied for polymorphism with nine restriction endonucleases. The restriction patterns were identical for all species with all restriction endonucleases tested except for B. melitensis strain 16M that showed a different pattern with EcoRV, consistent with the presence of a single site instead of two for the other Brucella species. The absence of the second EcoRV site for B. melitensis 16M was confirmed by DNA sequence analysis. The second EcoRV site in other Brucella species was located in a 12-bp segment, which was missing from the published dnaK sequence of B. ovis, between the stop codon of the dnaK gene and its putative transcription terminator sequence. The difference between B. ovis strain 63/290 and B. melitensis 16M was due to an additional base-pair in B. melitensis 16M. Subsequently, 71 other field, vaccinal and reference strains of the six Brucella species and their different biovars were studied for restriction fragment length polymorphism (RFLP) of the dnaK locus with EcoRV. The presence of a unique EcoRV site was specific to B. melitensis strains. Southern blot analysis of whole genomic DNA digested with EcoRV and with the dnaK gene used as probe also detected a distinct pattern for B. melitensis. These results indicate that both PCR-RFLP and Southern blot analysis of the dnaK locus can be used to distinguish B. melitensis strains from the other Brucella species and may be useful for typing and diagnostic purposes as well.

Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: Evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene.

The nucleotide sequences encoding the major 25-kDa outer membrane protein (OMP) (omp25 genes) of Brucella ovis 63/290, Brucella melitensis 16M, Brucella suis 1330, Brucella canis RM6/66, and Brucella neotomae 5K33 (all reference strains) were determined and compared with that of Brucella abortus 544 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The major difference found was between the omp25 gene of B. ovis and those of the other Brucella species; the B. ovis gene had a 36-bp deletion located at the 3' end of the gene. The corresponding regions of other Brucella species contain two 8-bp direct repeats and two 4-bp inverted repeats, which could have been involved in the genesis of the deletion. The mechanism responsible for the genesis of the deletion appears to be related to the "slipped mispairing" mechanism described in the literature. Expression of the 25-kDa outer membrane protein (Omp25) in Brucella spp. or expression from the cloned omp25 gene in Escherichia coli cells was studied with a panel of anti-Omp25 monoclonal antibodies (MAbs). As shown by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy, Omp25 was exported to the outer membrane in E. coli expressing either the truncated omp25 gene of B. ovis or the entire omp25 genes of the other Brucella species. Size and antigenic shifts due to the 36-bp deletion were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and by the differences in binding patterns in ELISA of the anti-Omp25 MAbs at the cell surface of E. coli cells harboring the appropriate gene and of cells of B. ovis and other Brucella species. In particular, MAbs directed against discontinuous epitopes of the entire Omp25 showed the absence of, or a significant reduction in, antibody reactivity with the B. ovis truncated Omp25. The results indicated that, as defined by the MAbs, exported Omp25 probably presents similar topologies in the outer membranes of E. coli and Brucella spp. and that the short deletion found in the omp25 gene of B. ovis has important consequences for the expression of surface B-cell epitopes which should be considered for the development of vaccines against B. ovis infection.

Overproduction of the Brucella melitensis heat shock protein DnaK in Escherichia coli and its localization by use of specific monoclonal antibodies in B. melitensis cells and fractions.

The Brucella melitensis dnaK gene was amplified by the polymerase chain reaction using primers chosen according to the published sequence of B. ovis and cloned in multiple copy plasmids enabling expression under the control of the Plac promoter. Monoclonal antibodies (mAb) obtained by immunizing mice with B. melitensis B115 cell wall (CW) fraction or by infecting mice with virulent B. melitensis strain H38 and recognizing a 73-kDa band in immunoblotting of the B. melitensis CW fraction reacted with the cloned dnaK gene product and were thus shown to be specific for the heat shock protein DnaK. The anti-Dnak protein mAbs did not react with Escherichia coli control cells or cell lysates and could therefore be specific to Brucella DnaK protein epitopes. These mAbs were further used to study overproduction of the DnaK protein. B. melitensis DnaK overproduction in E. coli resulted in a defect in cell septation and formation of cell filaments. Immunogold labelling with the mAbs and electron microscopy localized the DnaK protein inside as well as outside the E. coli cells, probably resulting from lysis due to toxicity of the overproduced DnaK protein. These results indicated that overproduction of the B. melitensis DnaK protein in E. coli had similar physiological consequences as that of E. coli overproduced in E. coli. The DnaK protein localization in B. melitensis cells was essentially cytoplasmic, as shown by immunoelectron microscopy. Heat shock treatment of these cells resulted in increased binding of mAbs and labelling in the cytoplasm. However, in subcellular fractions the DnaK protein was predominantly found in the cell envelope fraction of B. melitensis, which could perhaps be due to interaction of the DnaK protein with membrane proteins.

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection.

Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE) gene of Escherichia coli, a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB-like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium.

Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer-membrane proteins of Brucella.

Seventy-seven Brucella reference and field strains from different geographic origins and hosts representing the six recognized species and their different biovars were analysed for diversity of their genes encoding the major 25 and 36 kDa outer-membrane proteins (OMPs) by PCR-RFLP. The 25 kDa OMP is encoded by a single gene (omp25) whereas two closely related genes (omp2a and omp2b) encode and potentially express the 36 kDa OMP. Analysis of PCR products of the omp25 gene digested with nine restriction enzymes revealed two species-specific markers, i.e. the absence of the EcoRV site in all Brucella melitensis strains and an approximately 50 bp deletion at the 3' terminal end of the gene in all Brucella ovis strains. Analysis of PCR products of the omp2a and omp2b genes digested with 13 restriction enzymes indicated a greater diversity than the omp25 gene among the six Brucella species and within the Brucella abortus, Brucella suis, B. melitensis and B. ovis species. Greater polymorphism was also detected for the omp2b than for the omp2a gene, especially in B. ovis which seemed to carry two similar (but not identical) copies of omp2a instead of one copy each of omp2a and omp2b for the other Brucella species as was previously suggested by Ficht et al. (1990; Mol Microbiol 4, 1135-1142). Results of PCR-RFLP indicated that distinction can be made between Brucellia species and some of their biovars, except between B. canis and B. suis bv. 3 and 4, on the basis of the size and diversity of their major OMP genes, and that it could be of importance for diagnostic, epidemiological and evolutionary study purposes.

Establishment of porcine cell lines producing a murine recombinant retrovirus in order to transfer the nlslacZ gene into porcine cells.

Swine testis (ST) cell lines producing a murine recombinant retrovirus (RRV) were established in order to transfer the bacterial lacZ gene fused to a nuclear location signal (nlslacZ) into animal cells. ST cells were infected with the supernatant of the cat G355.5LacZ2 cell line which produces amphotropic and xenotropic MMuLVSVnlslacZ-defective RRV and wild amphotropic and xenotropic MMuLV. Expression of the nlslacZ reporter gene was under the transcriptional control of both the SV40 early promoter and the retroviral LTR. ST cells expressing the reporter gene were sorted and cloned by limiting dilutions. Fourteen STLacZ-cell lines were isolated and subsequently tested for virus production. Depending on the host range of the retroviruses, two cell lines (STBF11 and STAA3) produced both a xenotropic recombinant pseudotype and wild retroviruses; another (STAB 10) produced both an amphotropic recombinant pseudotype and wild retroviruses. Southern blot analysis of the producer cell lines was carried out to verify proviral integration. The efficiency of the different pseudotypes in the transfer of the nlslacZ reporter gene to cultured animal cells, including porcine cells, was compared to the pseudotyped RRV produced by cat lines. Our results showed that the xenotropic RRV produced by the porcine STBF 11 cell line has a high titre for cells from different species and led to a higher number of porcine endothelial and lymphoblastoid cells expressing the reporter gene than did RRV produced by the cat packaging cell lines.

Purification of Clostridium thermocellum xylanase Z expressed in Escherichia coli and identification of the corresponding product in the culture medium of C. thermocellum.

An endoxylanase encoded by the xynZ gene of Clostridium thermocellum was purified from Escherichia coli harbouring a fragment of the gene cloned in pUC8. The purified enzyme showed two active bands of Mr 41,000 and 39,000, the latter one presumably derived from the former through proteolysis. The enzyme was highly active on xylan and para-nitrophenyl-beta-D-xylobioside. The major end product of xylan hydrolysis was xylobiose. With an antiserum raised against the enzyme purified from E. coli, an immunoreactive polypeptide of Mr 90,000, corresponding to the entire xynZ gene product, was detected in a culture supernatant from C. thermocellum grown on cellulose. By immunological detection, xylanase Z was shown to be associated with a cellulose-binding, high-molecular-weight fraction whose properties coincided with those described previously for the cellulose-degrading complex of C. thermocellum known as the cellulosome.

Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum.

The nucleotide sequence of the xynZ gene, encoding the extracellular xylanase Z of Clostridium thermocellum, was determined. The putative xynZ gene was 2,511 base pairs long and encoded a polypeptide of 837 amino acids. A region of 60 amino acids containing a duplicated segment of 24 amino acids was found between residues 429 and 488 of xylanase Z. This region was strongly similar to the conserved domain found at the carboxy-terminal ends of C. thermocellum endoglucanases A, B, and D. Deletions removing up to 508 codons from the 5' end of the gene did not affect the activity of the encoded polypeptide, showing that the active site was located in the C-terminal half of the protein and that the conserved region was not involved in catalysis. Expression of xylanase activity in Escherichia coli was increased up to 220-fold by fusing fragments containing the 3' end of the gene with the start of lacZ present in pUC19. An internal translational initiation site which was efficiently recognized in E. coli was tentatively identified 470 codons downstream from the actual start codon.

Sequence of the cellulase gene of Clostridium thermocellum coding for endoglucanase B.

The nucleotide sequence of the CelB gene, encoding the extracellular endoglucanase B of Clostridium thermocellum, is reported. The putative start of the 1689 bp coding sequence was assigned to an ATG codon which is preceded by an AGGAGG sequence typical of ribosomal binding sites in Gram-positive bacteria. The amino-terminal end of the deduced protein sequence is similar to signal peptides described for other bacterial secretory proteins. The carboxy-terminal ends of endoglucanases A and B appear to be remarkably homologous. A striking feature of the conserved region is that both proteins contain two reiterated stretches of 23 aminoacids each, separated by 9 residues.