Biomedical subjects

G Domínguez-Bernal

Publications and source records attributed to G Domínguez-Bernal.

9 recordsLinked to original sources

PubMed2001

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Adaptation, Physiological↗

PubMed2001

Pathogenicity islands and virulence evolution in Listeria.

As in other bacterial pathogens, the virulence determinants of Listeria species are clustered in genomic islands scattered along the chromosome. This review summarizes current knowledge about the structure, distribution and role in pathogenesis of Listeria virulence loci. Hypotheses about the mode of acquisition and evolution of these loci in this group of Gram-positive bacteria are presented and discussed.

Amino Acid Sequence↗

PubMed2001

Listeria pathogenesis and molecular virulence determinants.

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal individuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.

Animals↗

PubMed2000

SmcL, a novel membrane-damaging virulence factor in Listeria.

We describe here the fourth listerial membrane-damaging virulence factor, a sphingomyelinase C (SMase) that is produced specifically by the ruminant pathogen Listeria ivanovii. Its coding gene, smcL, is a monocistron expressed independently of PrfA. The smcL product, SmcL, is highly similar to the staphylococcal beta-toxin and is responsible for the differential hemolytic properties of L. ivanovii (bizonal hemolysis and CAMP-like reaction with R. equi). The role of SmcL in virulence was assessed by gene disruption and complementation. Our data show that SmcL mediates disruption of the membrane of primary phagosomes, thereby promoting bacterial intracellular proliferation. They also suggest that SmcL may play a role in host tropism. smcL is located in LIPI-2, a novel 18-kb pathogenicity island which also contains a cluster of internalin genes. LIPI-2 is unstable, L. ivanovii-specific and required for full virulence in mice and lambs.

Amino Acid Sequence↗

PubMed1999

The smcL gene of Listeria ivanovii encodes a sphingomyelinase C that mediates bacterial escape from the phagocytic vacuole.

The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel-shaped co-operative haemolytic ('CAMP-like') reaction with Rhodococcus equi. We cloned the gene responsible for the differential haemolytic properties of L. ivanovii, smcL. It encodes a sphingomyelinase C (SMase) highly similar (> 50% identity) to the SMases from Staphylococcus aureus (beta-toxin), Bacillus cereus and Leptospira interrogans. smcL was transcribed monocistronically and was expressed independently of PrfA. Low-stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L. ivanovii. We constructed an smcL knock-out mutant. Its phenotype on blood agar was identical to that of L. monocytogenes (i.e. weak haemolysis and no shovel-shaped CAMP-like reaction with R. equi ). This mutant was less virulent for mice, and its intracellular proliferation was impaired in the bovine epithelial-like cell line MDBK. The role of SmcL in intracellular survival was investigated using an L. monocytogenes mutant lacking the membrane-damaging determinants hly, plcA and plcB, being thus unable to grow intracellularly. Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells. Transmission electron microscopy showed that SmcL mediates the disruption of the phagocytic vacuole and the release of bacteria into the cytosol. Therefore, L. ivanovii possesses a third phospholipase with membrane-damaging activity that, together with PlcA and PlcB, may act in concert with the pore-forming toxin Hly to mediate efficient escape from the vacuolar compartment. The 5' end of smcL is contiguous with the internalin locus i-inlFE, which is also specific to L. ivanovii and is required for full virulence in mice. Thus, smcL forms part of a novel virulence gene cluster in Listeria that is species specific.

Amino Acid Sequence↗

PubMed1998

A novel PrfA-regulated chromosomal locus, which is specific for Listeria ivanovii, encodes two small, secreted internalins and contributes to virulence in mice.

Several large, cell wall-associated internalins and one small, secreted internalin (InlC) have been described previously in Listeria monocytogenes. Using degenerate primers derived from sequenced peptides of an L. ivanovii major secreted protein, we identified a new 4.25 kb internalin locus of L. ivanovii, termed i-inlFE. The two proteins encoded by this locus, i-InlE and i-InlF, belong to the group of small, secreted internalins. Southern blot analyses show that the i-inlFE locus does not occur in L. monocytogenes. These data also indicate that six genes encoding small, secreted internalins are present in L. ivanovii, in contrast to L. monocytogenes, in which inlC encodes the only small internalin. The mature i-InlE protein (198 amino acids) is secreted in large amounts into the brain-heart infusion (BHI) culture medium in the stationary growth phase. In minimum essential medium (MEM), which has been used previously to induce PrfA-dependent gene transcription, i-inlE mRNA and i-InlE protein are expressed at high levels. As shown by Northern blot analysis and primer extension, transcription of the tandemly arranged i-inlF and i-inlE genes is dependent on the virulence regulator PrfA, and characteristic palindromic sequences ('PrfA-boxes') were identified in the promoter regions of i-inlF and i-inlE. Non-polar i-inlE and i-inlF deletion mutants and an i-inlFE double deletion mutant were constructed and tested in the mouse infection model. After intravenous infection, all three mutants entirely failed to kill C57BL/6 mice even at high infectious doses of 109 bacteria per mouse, whereas the LD50 for the parental strain was determined as 4 x 107 bacteria per mouse. These data suggest an important role for i-InlE and i-InlF in L. ivanovii virulence.

Amino Acid Sequence↗

PubMed1998

Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA.

Most Listeria monocytogenes virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cyclic AMP (cAMP) receptor protein (CRP). Its coding gene, prfA, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-dependent plcA promoter. We have recently characterized prfA* mutants from L. monocytogenes which, as a result of a single amino acid substitution in PrfA, Gly145Ser, constitutively overexpress prfA and the genes of the PrfA virulence regulon. Here, we show that about 10 times more PrfA protein is produced in a prfA* strain than in the wild type. Thus, the phenotype of prfA* mutants is presumably due to the synthesis of a PrfA protein with higher promoter-activating activity (PrfA*), which keeps its intracellular levels constantly elevated by positive feedback. We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA. Gel retardation assays performed with a DNA fragment carrying the PrfA binding site of the plcA promoter demonstrated that the PrfA* mutant form is much more efficient than wild-type PrfA at forming specific DNA-protein complexes. In footprinting experiments, the two purified PrfA forms interacted with the same nucleotides at the target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA*. These results show that the primary functional consequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence. Interestingly, similar mutations at the equivalent position in CRP result in a transcriptionally active, CRP* mutant form which binds with high affinity to target DNA in the absence of the activating cofactor, cAMP. Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a cofactor.

Bacterial Proteins↗

PubMed1997

A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes.

Virulence genes in Listeria monocytogenes are coordinately expressed under the control of the transcriptional activator PrfA, encoded by prfA, a member of the cyclic AMP (cAMP) receptor protein (CRP)/FNR family of bacterial regulators. Strain P14-A is a spontaneous mutant of L. monocytogenes serovar 4b which produces elevated levels of virulence factors (M. T. Ripio, G. Domínguez-Bernal, M. Suárez, K. Brehm, P. Berche, and J. A. Vázquez-Boland, Res. Microbiol. 147:371-384, 1996). Here we report that P14-A and other variant strains with the same phenotype carry a point mutation in codon 145 of prfA, leading to a Gly-->Ser substitution. trans-complementation experiments with PrfA-deficient mutants demonstrated that the Gly145Ser prfA allele causes overexpression of virulence factors in L. monocytogenes, to the levels found in the virulence factor-overexpressing variants. In strain P14-A with a chromosomal Glyl45Ser prfA background, transcription of prfA and of PrfA-dependent virulence genes remained constitutively high under culture conditions in which virulence factor expression is downregulated in wild-type L. monocytogenes. The Glyl45Ser substitution is located in a PrfA stretch (residues 141 to 151) showing high sequence similarity to the D alpha-helix of CRP. Interestingly, well-characterized crp* mutations, which make CRP functionally active in the absence of cAMP, map in this region (i.e., Gly141Ser and Ala144Thr substitutions). By analogy with the CRP model, the phenotype conferred to L. monocytogenes by the Gly145Ser substitution in PrfA could be due to the mutant regulatory protein being locked in a transcriptionally active, cofactor-independent conformational state. Our observations allow the construction of a model for PrfA-dependent virulence gene regulation in which the levels of virulence factor expression depend primarily on the conformational state of the PrfA protein, which alternates between active and inactive forms according to its interaction with an environmentally regulated signal molecule or cofactor.

Amino Acid Sequence↗

PubMed1996

Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition.

A panel of 103 Listeria monocytogenes strains of different origins was examined for haemolysin and lecithinase production in brain-heart infusion (BHI). Three distinct phenotypes were observed. Phenotype 1 was characterized by low to undetectable levels of expression and was exhibited by almost all strains tested. Phenotype 2 expressed high levels of haemolysin and lecithinase and was displayed by five strains: one (P14-A) was a spontaneous mutant derived from a type 1 isolate (P14); the four others (EGD-A, NCTC 7973, SLCC 2373 and CLIP 545) were all laboratory strains kept under in vitro conditions for a long period. Phenotype 3 was intermediate and was exhibited by another laboratory strain (L028). We therefore concluded that phenotype 1 corresponded to the wild type, whereas phenotypes 2 and 3 represented mutant or variant phenotypes. Interestingly, wild-type strains were able to dramatically increase the expression of virulence factors when cultured in BHI treated with activated charcoal (BHIC), up to levels similar to those constitutively expressed by the hyperhaemolytic/lecithinase variants in BHI. Experiments with P14 and P14-A demonstrated that both charcoal and the hyperhaemolytic/lecithinase mutation exerted their effect by inducing (or derepressing) transcription of prfA, the pleiotropic transcriptional activator of the L. monocytogenes virulence regulon. Moreover, P14 and P14-A were equally virulent for mice despite the different levels of virulence factor expression in BHI. Taken together, these observations indicate that L. monocytogenes turns off virulence gene expression when growing in vitro in a rich medium, and suggest that the increased levels of virulence factors in the hyperhaemolytic/lecithinase mutants and in wild-type strains grown in BHIC might represent the levels of expression needed in vivo by L. monocytogenes for infecting host tissues.

Animals↗