14,076 research outputs found
A host-vector system for heterologous gene expression in Streptococcus gordonii
No full textWe have developed a host-vector system for heterologous expression in Streptococcus gordonii (Sg) Challis (formerly Streptococcus sanguis), a commensal bacterium of the human oral cavity. The system is based on (i) integration of plasmid insertion vectors into the chromosome of specially engineered recipient hosts, and (ii) the use of the M6-protein-encoding gene (emm6) as a partner for construction of translational gene fusions. M6 is a streptococcal surface protein already proven useful as a fusion partner for the delivery of foreign antigens to the surface of Sg [Pozzi et al., Infect. Immun. 60 (1992) 1902-1907]. Insertion vectors carry a drug-resistance marker, different portions of emm6 and a multiple cloning site to allow construction of a variety of emm6-based fusions. Upon transformation of a recipient host with an insertion vector, 100% of transformants acquire both the drug-resistance marker and the capacity of displaying the M6 molecule on the cell surface. Chromosomal integration occurred at high frequency in recipient host GP1221. Transformation with 1 microgram of insertion vector DNA yielded 8.1 X 10(5) transformants per ml of competent cells
Response (letter) to Radiologic evaluation of autoimmune pancreatitis
No full textResponse to a comment on:
Radiology. 2011 Aug;260(2):428-36. Epub 2011 May 25.
Autoimmune pancreatitis: pancreatic and extrapancreatic MR imaging-MR cholangiopancreatography findings at diagnosis, after steroid therapy, and at recurrence.
Manfredi R, Frulloni L, Mantovani W, Bonatti M, Graziani R, Pozzi Mucelli R
Comparative genomics for identification of clone-specific sequence blocks in Streptococcus pneumoniae
No full textThe partial genome sequences of a serotype 3 and a serotype 2 pneumococcal strain were compared to the complete type 4 pneumococcal genome. Over 500000 and 150000 base pairs of the partial genome data, obtained from published patents, were analysed respectively. Global alignment showed that nearly the whole genome is highly conserved in accordance with data of multilocus sequence typing of housekeeping genes. The search for clone-specific genes revealed 17 new open reading frames in the type 3 strain, while no new open reading frame was detected in the type 2 strain. Allelic variation of genes was restricted by the use of crude sequence data, but still permitted identification of some new alleles and the observation that all surface proteins present in the partial genome data were highly conserved. In both strains we observed also a variety of chromosomal rearrangements and variations due to mobile genetic elements. All together, this comparative genomic approach gives a genome-based overview of strain relatedness and a prospective on what could be expected when sequencing other pneumococcal strains
Conjugative mobilization of the cloned M6 protein gene from Streptococcus pneumoniae to Streptococcus pyogenes
No full textThe host-vector system omega 6001-pDP36 was used to transfer the M6 protein gene (emm-6.1) of Streptococcus pyogenes to other S. pyogenes strains, isogenic and nonisogenic to D471, the strain from which emm-6.1 was originally cloned. The first step was to subclone emm-6.1 into the insertion vector pDP36. The resulting plasmid, pRMB20, was used as donor in transformation to insert emm-6.1 into the conjugative transposon omega 6001. Streptococcus pneumoniae DP1322, carrying omega 6001 integrated into the chromosome, was the recipient in the transformation experiment. omega 6001 containing emm-6.1 was then transferred by conjugation from S. pneumoniae to the chromosomes of M+ and M- S. pyogenes strains. S. pyogenes transconjugants contained one intact copy of emm-6.1 integrated into the chromosome, but no expression of M6 protein could be detected by Western blot analysis. We found no evidence of the positive transacting regulation of emm gene expression postulated by other authors. In fact, the cloned emm-6.1 was not expressed in three strains expressing their own M proteins (M5, M17 and a shorter M6). In these partial diploids M protein genes were expressed only when present in the original chromosomal locus
Omega6001-Mediated conjugative mobilisation of the cloned M6 protein gene to the chromosomes of different strains of Streptococcus pyogenes
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Secretin-enhanced MR imaging of the pancreas
No full textSecretin is a hormone that stimulates the exocrine pancreatic secretion of bicarbonate-rich fluid from the acinar cells of the pancreas that accumulates within the pancreatic ductal lumen. The exogenous administration of secretin improves the visualization of pancreatic ducts at magnetic resonance (MR) cholangiopancreatography (MRCP), because of an enlargement of the pancreatic duct system and an increase of the fluid content within the lumen of the pancreatic ducts, responsible of an increase of MR signal. In this review, the technique of secretin-enhanced MRCP, which has the aim to depict the whole pancreatic duct system, the biliary tree, the major and minor papillae, and the duodenum, will be described. Because of the anatomic contiguity between the pancreas and the gastrointestinal tract, the presence of fluid within the stomach may overlap with the pancreatic duct system and therefore the pancreatic duct may be difficult to visualize, representing a potential source of diagnostic pitfalls. The technique to reduce the signal intensity of the static fluid present within the stomach and in the duodenal lumen is also described. The technique of secretin administration will be illustrated, with emphasis on the synchronization of secretin administration and MR image acquisition. Furthermore, the frequency and number of MRCP images necessary to achieve a temporal resolution adequate to visualize the physiologic changes in the pancreatic gland, induced by the administration of secretin, is described. The assessment of pancreatic, morphologic, and functional response to the administration of secretin, as depicted on MRCP images, will be illustrated. Finally, the indications for secretin-enhanced MRCP will be discussed to define which patients will benefit from secretin-enhanced MR imaging for their treatment planning
Recombinant Streptococcus gordonii as live vehicle for vaccine antigens
No full textAll recombinant strains, including the parent recombinant S. gordonii GP231 expressing whole M6, do produce in liquid culture the cloned proteins from mid mid exponential phase of growth to the late stationary phase. The maximum protein quantity per cell is detected about one generation after the beginning of the stationary phase (Medaglini et al., 1993). This characteristic enables to collect the bacterial cells for protein analysis and purification purposes at the highest possible cell density. Recombinant proteins are detected in media containing no dextrose (Tryptic Soy Broth without dextrose) and in standard media containing 0.2% of glucose, while the protein quantity diminishes, in stationary phase of growth, when high glucose containing media (1, 2, 5 10% glucose) are used possibly due to proteolytic degradation. On solid media the recombinant proteins are produced and are detectable since the colony becomes visible on the plate and thus making protein detection through colony blot screening possible. Cell fractioning, immunofluorescence and electron microscopy on bacteria grown to late exponential phase did confirm surface display of the M6-based fusion proteins
Allelic variation in the highly polymorphic locus pspC of Streptococcus pneumoniae
No full textPspC, also called SpsA, CbpA, PbcA, and Hic, is a surface protein of Streptococcus pneumoniae studied for its antigenic properties, its capability to bind secretory IgA, C3 and complement factor H, and its activity as an adhesin. In this work we characterized the pspC locus of 43 pneumococcal strains by DNA sequencing of PCR fragments. Using PCR primers designed on two unrelated open reading frames, flanking the pspC locus, it was possible to amplify the pspC locus of each of the 43 strains of S. pneumoniae. In 37 out of 43 strains there was a single copy of the pspC gene, while two tandem copies of pspC were found in the other six strains. The sequence of the pspC locus was different in each of the 43 strains. Insertion sequences were found in the pspC locus of 11 out of 43 strains. Analysis of the deduced amino acid sequence of the PspC variants showed a common organization of the molecules: (i) a 37 amino acid leader peptide which is conserved in all proteins, (ii) an N-terminal portion which is essentially alpha-helical, and is the result of assembly of eight major sequence blocks, (iii) a proline-rich region, and (iv) a C-terminal anchor responsible for the cell surface attachment. By sequence comparison we identified 11 major groups of PspC proteins. Proteins within one group displayed only minor variations of the amino acid sequence. An unexpected finding was that PspC variants could differ in the anchor sequence. While 32 of the PspC proteins displayed the typical choline binding domain of pneumococcal surface proteins, 17 other PspCs showed the LPXTG motif, which is typical of surface proteins of other gram-positive bacteria. This major difference in the anchor region was also observed in the adjacent proline-rich regions which differed considerably in size and composition
DNA probe for identification of Streptococcus pneumoniae
No full textA total of 287 clinical isolates of Streptococcus pneumoniae (pneumococcus) were tested for their ability to undergo autolysis when treated with sodium deoxycholate. The test was positive for all but one isolate, strain DOC-1. This autolysis required the activity of an enzyme which is unique and characteristic of S. pneumoniae: a choline-dependent N-acetylmuramoyl-L-alanine amidase, the gene product of the lytA gene. We used lytA as a DNA probe to test the distribution of the autolysin gene among clinical isolates of S. pneumoniae. In dot blot hybridization experiments our probe reacted with the DNA of 60 of 60 strains tested, including the autolysis-deficient clinical isolate DOC-1. No hybridization occurred when strains of Streptococcus sanguis, Streptococcus mutans, Streptococcus pyogenes, Streptococcus (Enterococcus) faecalis, Streptococcus (Enterococcus) faecium, Streptococcus agalactiae, and Streptococcus bovis were tested. The lytA gene appears to be an ideal candidate for use as a DNA probe for the identification of S. pneumoniae
Vaginal immunization with recombinant gram-positive bacteria
No full textPROBLEM:
Many viral and bacterial pathogens enter the body through the genital mucosa. Therefore, one of the major goals of a vaccine against sexually transmitted diseases (STDs) should be to induce an immune response in the genital mucosa capable of controlling the entry of the pathogen. Our approach for the development of vaccines against STDs is based on the use of nonpathogenic Gram-positive bacteria as live vaccine vectors.
METHOD OF STUDY:
Recombinant Gram-positive bacteria expressing vaccine antigens were constructed using genetic systems developed in our laboratory. Balb/c mice and Cynomolgus monkeys were inoculated by the vaginal route and vaginal samples were collected using absorbent wicks. Colonization was evaluated by the presence of recombinant bacteria in the vaginal samples. Local and systemic immune responses were studied.
RESULTS:
We have developed genetic systems for the expression of heterologous antigens on the surface of the human commensals Streptococcus gordonii and Lactobacillus spp. Both S. gordonii and L. casei stably colonized the murine vagina after a single inoculum. Vaginal colonization of mice with recombinant strains of S. gordonii, expressing human papillomavirus (HPV) and human immunodeficiency virus (HIV) antigens, induced antigen-specific vaginal immunoglobulin A (IgA) and serum IgG. Local and systemic immune responses also were detected in monkeys immunized intravaginally with recombinant S. gordonii.
CONCLUSION:
The results obtained indicated that the approach of using colonizing Gram-positive bacteria as live vectors has a great potential for the development of vaccines against STDs
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