Biomedical subjects

A M Marciel

Publications and source records attributed to A M Marciel.

4 recordsLinked to original sources

PubMed2001

Use of operon fusions in Mannheimia haemolytica to identify environmental and cis-acting regulators of leukotoxin transcription.

The leukotoxin of Mannheimia haemolytica is an important virulence factor that contributes to much of the pathology observed in the lungs of animals with bovine shipping fever pneumonia. We believe that identification of factors that regulate leukotoxin expression may provide insight into M. haemolytica pathogenicity. The DNA sequence upstream of the leukotoxin operon is divergently shared by P(lapT), which transcribes an arginine permease gene. The intergenic region contains several elements that are potential sites for transcriptional modulation of the promoters. We have developed plasmid-borne chloramphenicol acetyltransferase (cat) operon fusions, as well as lktC::cat chromosomal fusions, to study transcription initiation in M. haemolytica. Using these genetic tools, we have identified cis-acting sequences and environmental conditions that modulate transcription of the leukotoxin and lapT promoters. By deletion analysis, promoters were shown to rely on sequences upstream of their -10 and -35 regions for full activity. Direct repeats of the sequence TGT-N(11)-ACA and a static bend region caused by phased adenine tracts were necessary for full activation of P(lkt). A computer-generated model of the promoter's structure shows how DNA bending brings the repeat sequences within close proximity to the P(lkt) RNA polymerase, and we hypothesize that these repeats are a binding site for an activator of leukotoxin transcription. The lktC::cat operon fusion was also used to demonstrate that, like that of other RTX toxins, leukotoxin transcription is environmentally regulated. Roles for iron deprivation and temperature change were identified.

Bacterial Toxins↗

PubMed1997

Molecular population genetic analysis of a Streptococcus pyogenes bacteriophage-encoded hyaluronidase gene: recombination contributes to allelic variation.

Many strains of the human pathogenic bacterium Streptococcus pyogenes produce hyaluronidase, an enzyme that degrades hyaluronic acid, a major component of the extracellular matrix. Degradation of hyaluronic acid is thought to aid in host tissue invasion and dissemination of S. pyogenes. The molecular population genetics of the bacteriophage-encoded hyaluronidase gene (hyl) was analysed by sequencing the gene from 13 streptococcal strains representing seven well-differentiated multilocus enzyme electrophoretic types and eight M or T protein serotypes. Substantial levels of allelic polymorphism were identified, and the analysis found strong statistical evidence that recombinational processes have contributed to the generation of molecular variation in this gene. A 111 base pair segment of hyl encoding a collagenous motif, that may bind collagen, was absent in a serotype M14 isolate and 13 serotype M18 multilocus enzyme electrophoretic type 20 strains examined. The analysis provides a molecular population genetics framework for studies examining the role of naturally occurring hyaluronidase variation in host-pathogen interactions.

Alleles↗

PubMed1997

Structure-function and pathogenesis studies of Streptococcus pyogenes extracellular cysteine protease.

Replacement of the single cysteine residue (C192) with serine in the Streptococcus pyogenes extracellular cysteine protease (SCP) prevented auto-catalytic processing of the 40-kDa zymogen to the 28-kDa mature form and eliminated proteolytic activity. SCP incubated with human endothelial cells induced a time- and concentration-dependent increase in a 66-kDa gelatinase/type IV collagenase in culture supernatants. Activation of this gelatinase/collagenase may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive S. pyogenes infection.

Bacterial Proteins↗

PubMed1996

Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease.

Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection.

Cells, Cultured↗