The application of DNA hybridisation methods to a determination of the association of hepatitis B virus with cirrhosis and hepatoma.

Thesis (M. Med.)-University of Natal, Durban, 1987.Autopsy liver material from patients having died of chronic liver disease, cirrhosis, hepatocellular carcinoma (HCC) and causes unrelated to liver diseases was examined by dot blot hybridisation for the presence of HBV DNA. The results indicate that of the patients with chronic liver disease 6/9 were positive for HBV DNA in the liver tissue; of the patients with HCC 3/4 were positive for HBV DNA; of the patients with cirrhosis 4/4 showed the presence of HBV DNA in the liver. Thus by this technique 13/17 (76%) of these patients, all of whom were HBsAg positive, were shown to have HBV DNA present in liver tissue. However, autopsy liver samples were found to be unsuitable for Southern blot hybridisation. Biopsy liver/tumour tissue was examined for the presence of integrated or non-integrated HBV DNA by Southern blot analysis using the enzymes Eco R1 and Hind 111. 5/5 patients who were both HBsAg and HBeAg positive had extrachromosomal HBV DNA and 2/5 also showed the presence of integrated HBV DNA. 3/4 patients who were HBsAg positive and HBeAg negative had extrachromosomal HBV DNA and all three also had integrated HBV DNA. One control patient was negative for both markers and also for Southern blot hybridisation with the HBV DNA probe. These results support the hypothesis that HBV is a factor in the development of HCC, and indicate that the dot blot hybridisation method would be suitable for routine evaluation of patients with chronic liver disease or cirrhosis

clpB, a Novel Member of the Listeria monocytogenes CtsR Regulon, Is Involved in Virulence but Not in General Stress Tolerance

Clp-HSP100 ATPases are a widespread family of ubiquitous proteins that occur in both prokaryotes and eukaryotes and play important roles in the folding of newly synthesized proteins and refolding of aggregated proteins. They have also been shown to participate in the virulence of several pathogens, including Listeria monocytogenes. Here, we describe a member of the Clp-HSP100 family of L. monocytogenes that harbors all the characteristics of the ClpB subclass, which is absent in the closely related gram-positive model organism, Bacillus subtilis. Transcriptional analysis of clpB revealed a heat shock-inducible σ(A)-type promoter. Potential binding sites for the CtsR regulator of stress response were identified in the promoter region. In vivo and in vitro approaches were used to show that expression of clpB is repressed by CtsR, a finding indicating that clpB is a novel member of the L. monocytogenes CtsR regulon. We showed that ClpB is involved in the pathogenicity of L. monocytogenes since the ΔclpB mutant is significantly affected by virulence in a murine model of infection; we also demonstrate that this effect is apparently not due to a defect in general stress resistance. Indeed, ClpB is not involved in tolerance to heat, salt, detergent, puromycin, or cold stress, even though its synthesis is inducible by heat shock. However, ClpB was shown to play a role in induced thermotolerance, allowing increased resistance of L. monocytogenes to lethal temperatures. This work gives the first example of a clpB gene directly controlled by CtsR and describes the first role for a ClpB protein in induced thermotolerance and virulence in a gram-positive organism

CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenes

International audienceStress proteins play an important role in virulence, yet little is known about the regulation of stress response in pathogens. In the facultative intracellular pathogen Listeria monocytogenes, the Clp ATPases, including ClpC, ClpP and ClpE, are required for stress survival and intracellular growth. The first gene of the clpC operon of L. monocytogenes encodes a homologue of the Bacillus subtilis CtsR repressor of stress response genes. An L. monocytogenes ctsR-deleted mutant displayed enhanced survival under stress conditions (growth in the presence of 2% NaCl or at 42 degrees C), but its level of virulence in the mouse was not affected. The virulence of a wild-type strain constitutively expressing CtsR is significantly attenuated, presumably because of repression of the stress response. Regulation of the L. monocytogenes clpC, clpP and clpE genes was investigated using transcriptional fusions in B. subtilis as a host. The L. monocytogenes ctsR gene was placed under the control of an inducible promoter, and regulation by CtsR and heat shock was demonstrated in vivo in B. subtilis. The purified CtsR protein of L. monocytogenes binds specifically to the clpC, clpP and clpE regulatory regions, and the extent of the CtsR binding sites was defined by DNase I footprinting. Our results demonstrate that this human pathogen possesses a CtsR regulon controlling class III heat shock genes, strikingly similar to that of the saprophyte B. subtilis. This is the first description of a stress response regulatory gene in a pathogen

Yersinia pseudotuberculosis Harbors a Type IV Pilus Gene Cluster That Contributes to Pathogenicity

Fimbriae have been shown to play an essential role in the adhesion of pathogenic gram-negative bacteria to host cells. In the enteroinvasive bacterium Yersinia pseudotuberculosis, we characterized a previously unknown 11-kb chromosomal locus involved in the synthesis of type IV pili. The locus consists of 11 open reading frames forming a polycistronic unit and encoding putative Pil proteins, PilLMNOPQRSUVW. When introduced into Escherichia coli, the Y. pseudotuberculosis operon reconstituted bundles of filaments at a pole on the bacterial surface, demonstrating that the pil locus was functional in a heterogenous genetic background. Environmental factors regulated transcription of the Y. pseudotuberculosis operon; in particular, temperature, osmolarity, and oxygen tension were critical cues. Deletion of the type IV pilus gene cluster was associated with a reduction of Y. pseudotuberculosis pathogenicity for mice infected orally. Forty-one percent of Y. pseudotuberculosis strains isolated from human or animal sources harbored the type IV pilus locus. Therefore, the pil locus of Y. pseudotuberculosis might constitute an “adaptation island,” permitting the microorganism to colonize a vast reservoir