HBD Gene Research Articles

P0063 PP THE ROLE OF HUMAN BETA-DEFENSINS DURING CAMPYLOBACTER JEJUNI INFECTION

Introduction: Campylobacter jejuni (C. jejuni) is one of the commonest causes of infective diarrhoea worldwide effecting mainly young children (<5 years) both in developing countries and the western world (1). The clinical spectrum varies from mild watery to bloody, inflammatory diarrhoea and is frequently associated with post infectious extraintestinal complications such as Guillain-Barré Syndrome (2). Despite the serious health problem caused by the bacterium, disease pathogenesis remains poorly understood. Bacterial adhesion and invasion of the intestinal epithelium is known to be a critical feature of infection (3). Human beta defensins (hBDs), a family of epithelial antimicrobial peptides are a major component of innate host defence (4). We and others have highlighted the role of these peptides during gastrointestinal infection and inflammation (5). In the present study we have investigated the role of hBDs during C.jejuni infection. Methods: Human colonic intestinal cell line (Caco-2) was infected with virulent strain (NCTC 11168) of C. jejuni. Both time and dose dependent studies of hBD gene expression were assessed by RT-PCR. The bactericidal activity of recombinant hBD-1,-2,-3 against wild type and isogenic mutants of C. jejuni was evaluated by broth-dilution assay. Results: A marked time-dependent induction of hBD3 was observed with maximal expression at 8 hours post-infection. In contrast hBD2 gene expression was modest. Importantly the bacterium was found to be highly susceptible to the antimicrobial action of hBD-3 amongst the peptides tested. Conclusion: The present study provides evidence for dynamic cross talk between the bacterium and host epithelial innate defence. The potent bactericidal activity of hBD3 may contribute to the self-limiting nature of the infection via enhanced bacterial clearance in a healthy host.

Expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection.

BackgroundConstitutive expression and localization of antimicrobial human β-defensin-1 (HBD-1) in human kidneys as a potential mechanism of antimicrobial defense has been previously reported. Inducible expression of human β-defensin-2 (HBD-2) has been described in various epithelial organs but not for the urogenital tract.MethodsWe investigated the gene- and protein expression of HBD-1 and HBD-2 by reverse transcriptase-polymerase chain reaction, and immunohistochemistry in 15 normal human kidney samples and 15 renal tissues with chronic bacterial infection. Additionally, cell culture experiments were performed to study HBD gene expression by real-time RT-PCR in response to inflammatory cytokines TNFα and IL-1β as well as lipopolysaccharide from Gram-negative bacteria.ResultsConstitutive HBD-1 gene- and protein expression was detected in normal renal tissue and kidneys with chronic infection. As a novel finding, inducible HBD-2 gene- and protein expression was demonstrated in tubulus epithelia with chronic infection but not in normal renal tissue. In pyelonephritic kidneys HBD-1 and HBD-2 expression showed a similar pattern of localizaton in distal tubules, loops of Henle and in collecting ducts of the kidney. Furthermore, real-time RT-PCR of kidney derived cell lines stimulated with inflammatory agents TNF-α, IL-1β and LPS revealed a strong increase in relative HBD-2 transcription level and also a slight increase in relative HBD-1 transcription level.ConclusionsUpregulated HBD-2 expression in renal tubulus epithelium indicates a role of a wider range of human defensins for antimicrobial host defense in the urogenital tract than previously recognized.

Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824.

The enzymes beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase (BHBD), crotonase, and butyryl-CoA dehydrogenase (BCD) from Clostridium acetobutylicum are responsible for the formation of butyryl-CoA from acetoacetyl-CoA. These enzymes are essential to both acid formation and solvent formation by clostridia. Clustered genes encoding BHBD, crotonase, BCD, and putative electron transfer flavoprotein alpha and beta subunits have been cloned and sequenced. The nucleotide sequence of the crt gene indicates that it encodes crotonase, a protein with 261 amino acid residues and a calculated molecular mass of 28.2 kDa; the hbd gene encodes BHBD, with 282 residues and a molecular mass of 30.5 kDa. Three open reading frames (bcd, etfB, and etfA) are located between crt and hbd. The nucleotide sequence of bcd indicates that it encodes BCD, which consists of 379 amino acid residues and has high levels of homology with various acyl-CoA dehydrogenases. Open reading frames etfB and etfA, located downstream of bcd, encode 27.2- and 36.1-kDa proteins, respectively, and show homology with the fixAB genes and the alpha and beta subunits of the electron transfer flavoprotein. These findings suggest that BCD in clostridia might interact with the electron transfer flavoprotein in its redox function. Primer extension analysis identified a promoter consensus sequence upstream of the crt gene, suggesting that the clustered genes are transcribed as a transcriptional unit and form a BCS (butyryl-CoA synthesis) operon. A DNA fragment containing the entire BCS operon was subcloned into an Escherichia coli-C. acetobutylicum shuttle vector. Enzyme activity assays showed that crotonase and BHBD were highly overproduced in cell extracts from E. coli harboring the subclone. In C. acetobutylicum harboring the subclone, the activities of the enzymes crotonase, BHBD, and BCD were elevated.

Structure and transcription of genes within the β- hbd-adh1 region of Clostridium acetobutylicum P262

The 1.2-kb DNA fragment upstream of the linked β- hbd (3-hydroxybutyryl-CoA dehydrogenase) and adh1 (NADPH-dependent alcohol dehydrogenase) genes from Clostridium acetobutylicum P262 was sequenced. The upstream region contained an open reading frame (ORFB) which was found to have 44% amino acid identity to the fixB gene products of yRhizobium and Azorhizobium. The β- hbd and ORFB genes were expressed during the acidogenic and solventogenic phases. The β- hbd gene was transcribed on a single mRNA species of 2.0 kb, whereas the ORFB gene was transcribed on two species of mRNA of 2.0 and 3.5 kb, respectively. The adh1 gene was induced or derepressed at the pH breakpoint before the onset of solventogenesis and was transcribed on a single species of mRNA of 2.4 kb.