Biomedical subjects

C Guidi-Rontani

Publications and source records attributed to C Guidi-Rontani.

At least 19 recordsLinked to original sources

PubMed2002

Macrophage interactions.

B. anthracis virulence is the sum of the contributions of factors involved in toxicity, growth and persistence in the host. Recent data has revealed that the interactions between B. anthracis and macrophage is central to the B. anthracis pathogenesis. This review presents and describes tactics by which B. anthracis not only overcomes and avoids macrophages but also perverts the host defense immune system and defense-related products to its advantage. The understanding of the complex network of such interactions is likely to allow new therapeutic and preventative strategies to be developed.

Animals↗

PubMed2001

Fate of germinated Bacillus anthracis spores in primary murine macrophages.

We investigated the fate of germinated Bacillus anthracis spores after their germination in Swiss murine peritoneal macrophages and in the cell line RAW264.7. We found that the lethal toxin and the oedema toxin are germ-associated factors that are essential for the survival of the vegetative form in host cells. We also found that pX02 is not involved in this complex pathogenic process. By transmission electron microscopy, we showed the tight interaction between the exosporium of the spore and the phagosomal membrane of the macrophage. Our data strongly suggest that the B. anthracis toxinogenic, unencapsulated Sterne strain (7702) does not multiply within macrophages. These results contributed to reveal the strategies used by B. anthracis to survive within the host and to reach the external medium where they proliferate.

Adenylyl Cyclases↗

PubMed2000

Characterization of a plasmid region involved in Bacillus anthracis toxin production and pathogenesis.

The germination of spores within the host is the initial step of anthrax infection. We have shown, using immunofluorescence staining, confocal scanning laser microscopy and image cytometry analysis, that the alveolar macrophage is the primary site of B. anthracis germination in a murine inhalation infection model. B. anthracis germinated inside macrophages, in vesicles derived from the phagosomal compartment. We have demonstrated that the toxin genes and their trans-activator, AtxA, are expressed within the macrophages after germination. It was also shown that the pXO1 plasmid strongly enhanced capsule formation and that this influence is mediated by AtxA. This indicates the existence of a regulon where AtxA is the regulatory protein acting on genes located on different plasmids. We identified a tricistronic germination operon gerX located between the pag and atxA genes on the 40-kb toxin-encoding fragment of pXO1 . Analysis of a gerX null mutant indicated that gerX-encoded proteins are involved in the virulence of B. anthracis.

Antigens, Bacterial↗

PubMed2000

Lethal factor of Bacillus anthracis cleaves the N-terminus of MAPKKs: analysis of the intracellular consequences in macrophages.

The lethal toxin of Bacillus anthracis consists of two proteins, PA and LF, which together induce lethal effects in some animal species and cause macrophage lysis. LF is a zinc-binding protein with metalloprotease activity. With a two-hybrid system approach we identified MAP kinase kinases (MAPKKs) Mekl and Mek2 as proteins interacting with LF. LF was shown to cleave Mek1 and Mek2 and an additional MAPKK family member MKK3, within their N-terminal region. We examined macrophage cell lines and primary peritoneal cells with different sensitivities to LF but did not find a direct correlation between MAPKKs cleavage and cell death. On the other hand, sublytic doses of LF cleave MAPKKs and cause a reduction in the LPS/IFNgamma-induced production of proinflammatory mediators. These findings are discussed with respect to the possible role of LF in the initial phase of infection.

Animals↗

PubMed2000

Translocation of Bacillus anthracis lethal and oedema factors across endosome membranes.

The two exotoxins of Bacillus anthracis, the causative agent of anthrax, are the oedema toxin (PA-EF) and the lethal toxin (PA-LF). They exert their catalytic activities within the cytosol. The internalization process requires receptor-mediated endocytosis and passage through acidic vesicles. We investigated the translocation of EF and LF enzymatic moieties across the target cell membrane. By selective permeabilization of the plasma membrane with Clostridium perfringens delta-toxin, we observed free full-size lethal factor (LF) within the cytosol, resulting from specific translocation from early endosomes. In contrast, oedema factor (EF) remained associated with the membranes of vesicles.

Animals↗

PubMed1999

Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNgamma-induced release of NO and TNFalpha.

The lethal toxin of Bacillus anthracis consists of two proteins, PA and LF, which together induce lethal effects in animals and cause macrophage lysis. LF is a zinc-endopeptidase which cleaves two mitogen-activated protein kinase kinases (MAPKKs), Mek1 and Mek2, within the cytosol. Here, we show that also MKK3, another dual-specificity kinase that phosphorylates and activates p38 MAP kinase, is cleaved by LF in macrophages. No direct correlation between LF-induced cell death and cleavage of these MAPKKs was found in macrophage cell lines and primary peritoneal cells exhibiting different sensitivity to LF. However, we present the first evidence that sublytic doses of LF cleave Meks and cause a substantial reduction in the production of NO and tumour necrosis factor-alpha induced by lipopolysaccharide/interferon gamma. We suggest that this effect of LF is relevant during the first stages of B. anthracis infection, when a reduction of the inflammatory response would permit growth and diffusion of the bacterium.

Animals↗

PubMed1999

Germination of Bacillus anthracis spores within alveolar macrophages.

The fatal character of the infection caused by inhalation of Bacillus anthracis spores results from a complex pathogenic cycle involving the synthesis of toxins by the bacterium. We have shown using immunofluorescent staining, confocal scanning laser microscopy and image cytometry analysis that the alveolar macrophage was the primary site of B. anthracis germination in a murine inhalation infection model. Bacillus anthracis germinated inside murine macrophage-like RAW264.7 cells and murine alveolar macrophages. Germination occurred in vesicles derived from the phagosomal compartment. We have also demonstrated that the toxin genes and their trans-activator, AtxA, were expressed within the macrophages after germination.

Animals↗

PubMed1999

Identification and characterization of a germination operon on the virulence plasmid pXO1 of Bacillus anthracis.

The spores of Bacillus anthracis, the agent of anthrax disease, germinate within professional phagocytes, such as murine macrophage-like RAW264.7 cells and alveolar macrophages. We identified a cluster of germination genes extending for 3608 nucleotides between the pag and atxA genes on the B. anthracis virulence plasmid pXO1. The three predicted proteins (40, 55 and 37 kDa in size) have significant sequence similarities to B. subtilis, B. cereus and B. megaterium germination proteins. Northern blot analysis of total RNA from sporulating cells indicated that the gerX locus was organized as a tricistronic operon (gerXB, gerXA and gerXC). Primer extension analysis identified a major potential transcriptional start site 31 bp upstream from the translation initiation codon of gerXB. Expression of the gerX operon was studied using a gerXB-lacZ transcriptional fusion. Expression began 2.5-3 h after the initiation of sporulation and was detected exclusively in the forespore compartment. A gerX null mutant was constructed. It was less virulent than the parental strain and did not germinate efficiently in vivo or in vitro within phagocytic cells. These data strongly suggest that gerX-encoded proteins are involved in the virulence of B. anthracis.

Animals↗

PubMed1999

Functional analysis of the carboxy-terminal domain of Bacillus anthracis protective antigen.

Protective antigen (PA) is the common receptor-binding component of the two anthrax toxins. We investigated the involvement of the PA carboxy-terminal domain in the interaction of the protein with cells. A deletion resulting in removal of the entire carboxy-terminal domain of PA (PA608) or part of an exposed loop of 19 amino acids (703 to 722) present within this domain was introduced into the pag gene. PA608 did not induce the lethal-factor (LF)-mediated cytotoxic effect on macrophages because it did not bind to the receptor. In contrast, PA711- and PA705-harboring lethal toxins (9- and 16-amino-acid deletions in the loop, starting after positions 711 and 705, respectively) were 10 times less cytotoxic than wild-type PA. After cleavage by trypsin, the mutant PA proteins formed heptamers and bound LF. The capacity of PA711 and PA705 to interact with cells was 1/10 that of wild-type PA. In conclusion, truncation of the carboxy-terminal domain or deletions in the exposed loop resulted in PA that was less cytotoxic or nontoxic because the mutated proteins did not efficiently bind to the receptor.

Animals↗

PubMed1998

[Anthrax toxins].

Bacillus anthracis, a Gram positive bacterium, is the causative agent of anthrax. This organism is capsulogen and toxinogenic. It secretes two toxins which are composed of three proteins: the protective antigen (PA), the lethal factor (LF) and the edema factor (EF). The lethal toxin (PA + LF) provokes a subite death in animals, the edema toxin (PA + EF) induces edema. The edema and the lethal factors are internalised into the target cells via the protective antigen. EF and LF exert an adenylate cyclase and a metalloprotease activity respectively. The structure-function relationship of these three proteins were defined using in vitro and in vivo approaches.

Animals↗

PubMed1997

Anthrax lethal toxin-induced mitogenic response of human T-cells.

Bacillus anthracis lethal toxin (PALF) stimulated the proliferation of human peripheral blood T-cells in vitro. Activation of T-lymphocytes by PALF required the presence of monocytes and did not result from a collaborative effect between T-cells and B-cells. PALF acted directly on monocytes and independently of T-cells. The monocytes contributed to the proliferation of T-cells by secretion of mediator(s). The mitogenic activity of the lethal toxin was dependent on its metalloprotease activity.

Antigens, Bacterial↗

PubMed1996

Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system.

In an attempt to introduce a large peptide that is not normally translocated across membranes into the cytosol of eukaryotic cells, we created a new chimeric protein termed CEDH between Pseudomonas aeruginosa exotoxin A (ETA) and a variant enzyme of Mus musculus dihydrofolate reductase (DHFR) with reduced affinity for antifolates, ETA(1-413).DHFR(1-187).ETA(609-613). We have defined, genetically constructed and expressed the chimeric protein in Escherichia coli. We showed that the CEDH chimeric protein, purified to homogeneity on an immunoaffinity resin, confers a methotrexate-resistant phenotype to Chinese hamster ovary cells. Furthermore, the chimeric protein allowed the growth of dihydrofolate reductase-deficient Chinese hamster ovary cells in the absence of hypoxanthine and thymidine. These results demonstrated that the chimeric protein exhibited enzyme activity and possessed the tightly folded native structure, and that the DHFR protein can be selectively internalized and translocated via domains of exotoxin A. These data show that the ETA system is an efficient system for the delivery of a variety of large polypeptides into the cytosol without stress to the target cells, and extends the use of this delivery system to proteins that are not normally translocated across membranes.

ADP Ribose Transferases↗

PubMed1994

Bifactorial versus monofactorial molecular status of Staphylococcus aureus gamma-toxin.

The extracellular Staphylococcus aureus gamma-toxin (hemolysin) released by the Smith 5R strain has been purified (M(r) 38 kDa, pl 9.55). We established that this cytolysin is a single polypeptide fully lytic on rabbit erythrocytes. In contrast, this toxin alone was unable to lyse other cells and was required to act jointly with an accessory 58 kDa protein released by the same strain. This protein, named sensitizing protein (SP), was required in order to damage the cytoplasmic membranes of other red blood cells including human erythrocytes as well as that of other eukaryotic cells (Jurkat and Hep-2). The lytic process can be referred to as conditional synergistic or cooperative lysis. gamma-toxin, and SP were also found able to disrupt phospholipid/cholesterol containing liposomes. We demonstrated that a minor membrane phospholipid, phosphatidylinositol, is crucial for gamma-toxin binding to cells and/or channel formation through membrane lipid bilayer.

Bacterial Proteins↗

PubMed1992

Cytotoxic activity of a recombinant chimaeric protein between Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin.

A segment of the exotoxin A gene of Pseudomonas aeruginosa, coding for the N-terminal end of domain I and domain II of the toxin (ETA), was genetically fused to the diphtheria toxin gene of Corynebacterium diphtheriae, coding for the N-terminal end of A fragment of diphtheria toxin (DT). The resulting hybrid protein (termed CED1) was produced in large amounts and exported to the periplasm in Escherichia coli. This chimaeric protein reacted with both anti-ETA and anti-DT antisera. Furthermore, the chimaeric protein displayed ADP-ribosylation activity and exhibited cytotoxicity to mouse 3T6 fibroblasts. These results demonstrated that the chimaeric protein is cytotoxic, and that the toxic potential of DTA can be selectively internalized and translocated via domains I and II of exotoxin A, which are thus sufficient to direct and translocate an enzymatically active heterologous polypeptide segment into the cytosol of sensitive cells.

3T3 Cells↗

PubMed1992

Localization of alg, opr, phn, pho, 4.5S RNA, 6S RNA, tox, trp, and xcp genes, rrn operons, and the chromosomal origin on the physical genome map of Pseudomonas aeruginosa PAO.

The genes encoding the rrn operons, the 4.5S and 6S RNAs, elements of protein secretion, and outer membrane proteins F and I, and regulatory as well as structural genes for exotoxin A, alkaline phosphatase, and alginate and tryptophan biosynthesis, were assigned on the SpeI/DpnI macrorestriction map of the Pseudomonas aeruginosa PAO chromosome. The zero point of the map was relocated to the chromosomal origin of replication.

Chromosome Mapping↗

PubMed1991

Functional analysis of exotoxin A-related protein of Pseudomonas aeruginosa lacking residues 225-412.

The crystal structure of the exotoxin A (ETA) of Pseudomonas aeruginosa showed that this protein is folded into three distinct domains. Domain I (Ia and Ib), the amino-terminal domain, is the receptor-binding domain of ETA and domain III, the carboxy-terminal domain, is responsible for the ADP-ribosyl transferase activity of the toxin. To elucidate the function(s) of domains 1b and II in the intoxication process and to define the region of the domain III necessary for ADP-ribosylating activity, a defined deletion in the structural gene of P. aeruginosa ETA encompassing residues 225-412 was constructed and an ETA-related product DeID, (from which all of domains II and Ib were deleted) was expressed. The ETA-related protein did not penetrate sensitive cells, but retained the same specific activity to ADP-ribosylate elongation factor-2 as wild-type toxin. This suggests that domain II is necessary to allow toxin internalization by sensitive cells and that the absence of domain Ib does not interfere with enzymic activity. The domain strictly involved in ADP-ribosylation activity encompasses residues 412-613.

ADP Ribose Transferases↗

PubMed1987

Exotoxin A of Pseudomonas aeruginosa: evidence that domain I functions in receptor binding.

We have constructed defined deletions in the structural gene of Pseudomonas aeruginosa exotoxin A (ETA) in order to probe the function of Domain I of this protein. Three forms of the gene containing specific deletions were expressed in a strain of Escherichia coli K12 with lesions in the htpR and Ion genes; extracts containing the gene products were tested for ADP-ribosylation activity, cytotoxicity, and ability to protect sensitive cells from the cytotoxic action of authentic ETA. Two of the mutant ETAs gave concentration-dependent protection against authentic ETA, and protection correlated with the presence of the bulk of Domain I. The results support the notion that Domain I functions in binding the toxin to specific cell-surface receptors.

ADP Ribose Transferases↗

PubMed1987

Exotoxin A of Pseudomonas aeruginosa: active, cloned toxin is secreted into the periplasmic space of Escherichia coli.

We subcloned the structural gene for exotoxin A (ETA) of Pseudomonas aeruginosa in front of the tac promoter in an Escherichia coli expression vector and studied the intracellular location and properties of the protein product. The E. coli K-12 strain that carried this recombinant plasmid produced an immunoreactive protein that was identical to authentic ETA in size and in cytotoxic and ADP-ribosyl transferase activities per unit of immunoreactive material. The protein was predominantly in the periplasmic fraction; and a mutation in the secA gene blocked secretion, processing, and conversion of the protein to a fully toxic conformation. The results indicate that expression of the ETA gene in E. coli yields native ETA, which is localized within the periplasmic space. This organism may therefore serve as a useful host for studying structure and function in ETA.

ADP Ribose Transferases↗