Bat-man disease transmission: zoonotic pathogens from wildlife reservoirs to human populations.

Bats are natural reservoir hosts and sources of infection of several microorganisms, many of which cause severe human diseases. Because of contact between bats and other animals, including humans, the possibility exists for additional interspecies transmissions and resulting disease outbreaks. The purpose of this article is to supply an overview on the main pathogens isolated from bats that have the potential to cause disease in humans.

Die for the community: an overview of programmed cell death in bacteria.

Programmed cell death is a process known to have a crucial role in many aspects of eukaryotes physiology and is clearly essential to their life. As a consequence, the underlying molecular mechanisms have been extensively studied in eukaryotes and we now know that different signalling pathways leading to functionally and morphologically different forms of death exist in these organisms. Similarly, mono-cellular organism can activate signalling pathways leading to death of a number of cells within a colony. The reason why a single-cell organism would activate a program leading to its death is apparently counterintuitive and probably for this reason cell death in prokaryotes has received a lot less attention in the past years. However, as summarized in this review there are many reasons leading to prokaryotic cell death, for the benefit of the colony. Indeed, single-celled organism can greatly benefit from multicellular organization. Within this forms of organization, regulation of death becomes an important issue, contributing to important processes such as: stress response, development, genetic transformation, and biofilm formation.

p63/p73 in the control of cell cycle and cell death.

The p53 family apparently derives from a common ancient ancestor that dates back over a billion years, whose function was protecting the germ line from DNA damage. p63 and p73 would maintain this function through evolution while acquiring novel roles in controlling proliferation and differentiation of various tissues. p53 on the other hand would appear in early vertebrates to protect somatic cells from DNA damage with similar mechanism used by its siblings to protect germ line cells. For the predominant role played by p53 mutations in cancer this was the first family member to be identified and soon became one of the most studied genes. Its siblings were identified almost 20 years later and interestingly enough their ancestral function as guardians of the germ-line was one of the last to be identified. In this review we shortly summarize the current knowledge on the structure and function of p63 and p73.

Bacterial peptide methionine sulphoxide reductase: co-induction with glutathione S-transferase during chemical stress conditions.

Peptide methionine sulphoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins, while the glutathione S-transferases (GSTs) are a major family of detoxification enzymes. A gene homologous to MsrA was identified in a chromosomal fragment from the bacterium Ochrobactrum anthropi, and this gene is located just downstream of a GST gene identified previously (OaGST) [Favaloro, Tamburro, Angelucci, De Luca, Melino, Di Ilio and Rotilio (1998) Biochem. J. 335, 573-579]. This raises the question of whether the products of these two genes may be involved in a common cellular protection function. To test this hypothesis, the hypothetical MsrA protein has been overexpressed in Escherichia coli as a functional 51 kDa GST fusion protein. Following cleavage with thrombin and purification, the soluble 24 kDa protein showed MsrA activity with N-acetylmethionine sulphoxide as substrate, as well as with other sulphoxide compounds. Therefore polyclonal antibodies were raised against the recombinant protein, and the modulation of MsrA in this bacterium, grown in the presence of different stimulants simulating several stress conditions, was investigated. The level of expression of MsrA was detected both by measuring the mRNA level and by immunoblotting experiments, in addition to measuring its catalytic activity. MsrA is a constitutive enzyme which is also inducible by chemical stress involving phenolic compounds such as phenol and 4-chlorophenol. Recently we reported that the GST of this bacterium, like MsrA, is only modulated by toxic chemical compounds [Favaloro, Tamburro, Trofino, Bologna, Rotilio and Heipieper (2000) Biochem. J. 346, 553-559]; therefore this is the first indication of a co-induction of the MsrA and GST enzymes during chemical stress.

Evaluation of the role of two conserved active-site residues in beta class glutathione S-transferases.

Glutathione S-transferases (GSTs) normally use hydroxy-group-containing residues in the N-terminal domain of the enzyme for stabilizing the activated form of the co-substrate, glutathione. However, previous mutagenesis studies have shown that this is not true for Beta class GSTs and thus the origin of the stabilization remains a mystery. The recently determined crystal structure of Proteus mirabilis GST B1-1 (PmGST B1-1) suggested that the stabilizing role might be fulfilled in Beta class GSTs by one or more residues in the C-terminal domain of the enzyme. To test this hypothesis we mutated His(106) and Lys(107) of PmGST B1-1 to investigate their possible role in the enzyme's catalytic activity. His(106) was mutated to Ala, Asn and Phe, and Lys(107) to Ala and Arg. The effects of the replacement on the activity, thermal stability and antibiotic-binding capacity of the enzyme were examined. The results are consistent with the involvement of His(106) and Lys(107) in interacting with glutathione at the active site but these residues do not contribute significantly to catalysis, folding or antibiotic binding.

Functional analysis of the evolutionarily conserved proline 53 residue in Proteus mirabilis glutathione transferase B1-1.

The role of the evolutionarily conserved residue Pro-53 in Proteus mirabilis glutathione transferase B1-1 has been examined by replacing it with a serine residue using site-directed mutagenesis. The effect of the replacement on the activity, thermal stability and antibiotic binding capacity of the enzyme was examined. The results presented support the view that Pro-53 participates in the maintenance of the proper conformation of the enzyme fold rather than playing a direct role in the catalytic reaction. Furthermore, this residue appears to be an important determinant of the antibiotic binding to the enzyme. Experiments with wild type and mutated enzymes provide evidence that glutathione transferases may play an important role in antibiotic resistance exhibited by bacteria.

A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications.

BACKGROUND: Glutathione S-transferases (GSTs) are a multifunctional group of enzymes, widely distributed in aerobic organisms, that have a critical role in the cellular detoxification process. Unlike their mammalian counterparts, bacterial GSTs often catalyze quite specific reactions, suggesting that their roles in bacteria might be different. The GST from Proteus mirabilis (PmGST B1-1) is known to bind certain antibiotics tightly and reduce the antimicrobial activity of beta-lactam drugs. Hence, bacterial GSTs may play a part in bacterial resistance towards antibiotics and are the subject of intense interest. RESULTS: Here we present the structure of a bacterial GST, PmGST B1-1, which has been determined from two different crystal forms. The enzyme adopts the canonical GST fold although it shares less than 20% sequence identity with GSTs from higher organisms. The most surprising aspect of the structure is the observation that the substrate, glutathione, is covalently bound to Cys 10 of the enzyme. In addition, the highly structurally conserved N-terminal domain is found to have an additional beta strand. CONCLUSIONS: The crystal structure of PmGST B1-1 has highlighted the importance of a cysteine residue in the catalytic cycle. Sequence analyses suggest that a number of other GSTs share this property, leading us to propose a new class of GSTs - the beta class. The data suggest that the in vivo role of the beta class GSTs could be as metabolic or redox enzymes rather than conjugating enzymes. Compelling evidence is presented that the theta class of GSTs evolved from an ancestral member of the thioredoxin superfamily.

Site-directed mutagenesis of the Proteus mirabilis glutathione transferase B1-1 G-site.

In order to investigate the roles of near N-terminus Tyr, Cys, and Ser residues in the activity of bacterial glutathione transferase (GSTB1-1) site-directed mutagenesis was used to replace the following residues: Tyr-4, Tyr-5, Ser-9, Cys-10, Ser-11, and Ser-13. The results presented here show that, unlike all other alpha, mu, pi, theta and sigma classes of glutathione transferases so far investigated, GSTB1-1 does not utilise any Tyr, Ser or Cys residue to activate glutathione. These results also suggest that the bacterial glutathione transferases may require classification into their own class.

Identification of an N-capping box that affects the alpha 6-helix propensity in glutathione S-transferase superfamily proteins: a role for an invariant aspartic residue.

We have identified an N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) that is strictly conserved, at the beginning of alpha 6 helix, in all glutathione S-transferases (GSTs) and most of the related superfamily proteins. By using CD and peptide modelling we have demonstrated that the capping box residues have an important role in determining the helical conformation adopted by this fragment in the hydrophobic environment of the protein. This is an example in which a local motif, contributing to nucleation of a structural element essential to the global folding of the protein, is strictly conserved in a superfamily of homologous proteins.

Effect of anaerobic environment on the glutathione transferase isoenzymatic pattern in Proteus mirabilis.

When Proteus mirabilis was cultured anaerobically in the presence of nitrate as terminal electron acceptor, a dramatic reduction of glutathione transferase production occurred. The analysis of the glutathione affinity purified materials in terms of substrate specificity, SDS-PAGE pattern, IEF pattern and immunoblotting revealed that a significantly different glutathione transferase pattern also occurred: two new glutathione transferase forms with an isoelectric point at pH 4.8 and 5.0 appeared. Their N-terminal amino acid sequence analysis as well as the ability to bind to a glutathione affinity column indicate that major differences between anaerobic and aerobic glutathione transferase forms are mainly located in the C-terminal region of the primary structure. In contrast, no significant changes occurred in the production of glutathione transferase isoenzymes when P. mirabilis was grown anaerobically in the absence of a terminal electron acceptor. These results support the idea that bacterial glutathione transferase expression is not strictly related to the absence of oxygen stress.

Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis.

The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421-425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli sigma 70 consensus promoter and to promoters of P. mirabilis cat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-beta-D-thiogalactopyranoside and growth at 37 degrees C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.

Helicobacter pylori isolated from stomach corpus and antrum: comparison of DNA patterns.

The genomic DNA of Helicobacter pylori was studied in strains isolated from two different sites of the stomach: the corpus and the antrum. 70 strains of H. pylori were found in 36 patients; 34 out of the 36 patients harboured the strain in both districts analysed. Restriction endonuclease analysis with Hae III and Hind III was used to compare the DNA patterns of strains isolated from the anatomical sites studied. Two pairs of DNA samples were not digested by these enzymes. 27 of the 32 pairs of the digested DNA appeared similar to each other. The analysis of chromosomal DNA in the remaining five pairs showed different electrophoretic patterns. These results indicate that the gastric mucosa can be colonized, at the same time, by strains of H. pylori with different genomic patterns, and this aspect can be important for epidemiological studies.

Inhibition of Helicobacter pylori by garlic extract (Allium sativum).

The antibacterial effect of aqueous garlic extract (AGE) was investigated against Helicobacter pylori. Sixteen clinical isolates and three reference strains of H. pylori were studied. Two different varieties of garlic were used. The concentration of AGE required to inhibit the bacterial growth was between 2-5 mg ml-1. The concentration, for both AGE types, to inhibit 90% (MIC90) of isolates was 5 mg ml-1. The minimum bactericidal concentration (MBC) was usually equal to, or two-fold higher than, minimum inhibitory concentration (MIC). Heat treatment of extracts reduced the inhibitory or bactericidal activity against H. pylori; the boiled garlic extract showed a loss of efficacy from two- to four-fold the values of MIC and the MBC obtained with fresh AGE. The antibacterial activity of garlic was also studied after combination with a proton pump-inhibitor (omeprazole) in a ratio of 250:1. A synergistic effect was found in 47% of strains studied; an antagonistic effect was not observed.

Purification and characterization of a heterodimeric 23/20-kDa proteolytic fragment of bacterial glutathione transferase B1-1.

The proteolytic attack of bacterial glutathione S-transferase (GSTB1-1) by trypsin cleaves and inactivates the enzyme. The polypeptide portion of GSTB1-1 encompassing the cleavage site (Lys35-Lys36) constitutes an exposed and flexible region of the GSTB1-1 G-site. By sequentially using a benzamidine-affinity chromatography and GSH-affinity column, a proteolyzed form of GSTB1-1 (23/20 kDa), in which only one subunit has been cleaved has been purified and characterized. Gel filtration, sequence analysis of subunits separated by HPLC, and CD experiments indicate that the 23/20-kDa GSTB1-1 form is a dimer and maintains its secondary structure. In addition, kinetic determinations reveal that the proteolytic cleavage of one polypeptide chain inactivates one active site but does not influence the catalytic efficiency of the second one. Previous refolding studies on GSTB1-1 have shown that the formation of a correct dimer precedes the recovery of the full activity of the enzyme, indicating that the dimeric structure is essential for catalytic activity of GSTB1-1. Thus, although GSTB1-1 active sites are catalytically independent and, probably, mainly located on each monomer, interactions deriving from the dimeric arrangement of the molecule appear essential for maintaining each active site in a fully active conformation. The catalytic independence of the two active sites, as well as the importance of dimeric structure for catalytic activity, has already been established for other GSTs. Thus, despite the very low sequence identity and kinetic differences between bacterial and other distant members of the GST superfamily, the results reported here indicate that important properties of the GST active site are conserved.

Crystallization and preliminary X-ray analysis of a bacterial glutathione transferase.

Crystals of a bacterial glutathione S-transferase from Proteus mirabilis have been grown from polyethylene glycol by the hanging-drop vapour-diffusion method. Successful crystallization required the presence of the substrate glutathione. The crystals belong to the tetragonal space group P4 with cell dimensions a = b = 90.9 and c = 117.3 A. They contain between three and six monomers in the asymmetric unit and diffract to beyond 2.3 A resolution.

Failure to detect Helicobacter pylori in nasal mucus in H pylori positive dyspeptic patients.

Stomach biopsies and samples of nasal mucus were cultured in patients with dyspeptic symptoms who underwent endoscopy to evaluate the possible route of transmission of Helicobacter pylori (H pylori). 42 patients were examined. For each patient two biopsies from the stomach corpus and antrum were taken and, before endoscopy, one nasal swab was obtained. Biopsy samples were tested for urease test, microbiological culture, and histological examination. The nasal swab was processed for microbiological examination. H pylori was not found in the nasal mucus of any of the patients, including the 36 who had H pylori in gastric biopsies.

Analysis by limited proteolysis of domain organization and GSH-site arrangement of bacterial glutathione transferase B1-1.

Limited proteolysis method has been used to study the structure-function relationship of bacterial glutathione transferase (GSTB1-1). In absence of three-dimensional structural data of prokaryote GST, the results represent the first information concerning the G-site and domains organization of GSTB1-1. The tryptic cleavages occur mainly at the peptide bonds Lys35-Lys36 and Phe43-Leu44, generating two major molecular species of 20-kDa, 3-kDa and traces of 10-kDa. 1-chloro-2,4-dinitrobenzene favoured the proteolysis of the 20-kDa fragment markedly enhancing the production of the 10-kDa peptide by cleaving the chemical bonds Lys87-Ala88 and Arg91-Tyr92. The tryptic cleavage sites of GSTB1-1 was found to be located close to those previously found for the mammalian GSTP1-1 isozyme. It was concluded that despite their low sequence homology (18%), GSTB1-1 and GSTP1-1 displayed similar structural features in their G-site regions and probably a common organization in structural domains.

Microbiological evidence of Helicobacter pylori from dental plaque in dyspeptic patients.

To evaluate the possible route of transmission of Helicobacter pylori, stomach biopsies and dental plaques were cultured from patients with dyspeptic symptoms who underwent endoscopy. A total of 31 patients were examined. Twenty patients out of thirty one (64%) were H. pylori positive in gastric biopsy. Among the microorganisms isolated in dental plaque only one sample (corresponding to a patients with duodenal ulcer H. pylori positive) showed colonies morphologically and biochemically compatible with H. pylori. Proteic patterns of whole cells and restriction endonuclease analysis with Hind III and Hae III endonucleases of DNA extracted from the strain subcultured from a stomach biopsy and from dental plaque of the same patient indicated that both sites were infected with the same strain of H. pylori.

Immunogold localization of glutathione transferase B1-1 in Proteus mirabilis.

By using the immunolabelling technique, the cellular localization of glutathione transferase in Proteus mirabilis was investigated. Evidence was obtained indicating a significant higher content of glutathione transferase in the periplasmic than cytoplasmic space. This result further support the idea that bacterial glutathione transferase is involved in xenobiotic detoxication.

Helicobacter pylori: a fickle germ.

The morphologic changes from bacillary to coccoid forms of Helicobacter pylori were studied. These form changes were analyzed by bacterial growth in Brucella broth plus 2% fetal calf serum. The coccoid forms were observed at five days of incubation and a rapid decrease of CFU/ml was recorded. At two weeks of microaerophilic incubation, all coccoid forms observed were not culturable in vitro. The coccoid morphology was observed earlier when the culture of H. pylori was incubated in aerobic conditions and with subinhibitory concentrations of omeprazole and roxithromycin. To evaluate the possibility of resistance of coccal forms, before plating, the cultures were heated to 80 C for 10 min and sonicated. In the absence of these treatments the cultures did not show growth in vitro. The proteic patterns of the same strains of two different morphologies were studied revealing significant differences.