Detection and subtyping of Actinobacillus pleuropneumoniae strains by PCR-RFLP analysis of the tbpA and tbpB genes

A PCR-based procedure for detection and serotype identification of Actinobacillus pleuropneumoniae strains was developed and evaluated. The A. pleuropneumoniae tbpA and tbpB genes were used as targets for amplification of DNA fragments, with a pair of specific primers for each gene. Amplification with tbpA primers rendered a 2.8-kb PCR product from all 12 A. pleuropneumoniae reference strains as well as from Actinobacillus suis strain CCM 5586, while amplification of a 1.9-kb PCR product was observed when testing ten Haemophilus parasuis strains of different serovars. Amplification of the tbpB gene from A. pleuropneumoniae serotypes 1, 6, 8 and 12, and A. suis CCM 5586 rendered an identical 1.8-kb fragment, while from A. pleuropneumoniae serotypes 2, 3, 4, 7, 9, 10 and 11, and H. parasuis strains it produced a 1.7-kb fragment. No PCR amplification product was observed when examining strains of 19 other swine pathogens or closely related species. The minimal detection limit for whole-cell A. pleuropneumoniae templates was between 5-50 and 3 x 10(2)-3 x 10(3) CFU when tbpA and tbpB specific primers, respectively, were used. Restriction fragment length polymorphism (RFLP) analysis of the PCR-generated products rendered different patterns, easily allowing us to discriminate between A. pleuropneumoniae, H. parasuis and A. suis and, more importantly, to distinguish ten RFLP A. pleuropneumoniae groups (the highest discrimination reported so far for a PCR assay with A. pleuropneumoniae), in such a way that the only serotypes with profiles identical to each other were 4 to 11 and 7 to 9. Moreover, the PCR-RFLP analysis was assayed in 36 A. pleuropneumoniae field isolates and in porcine samples (lungs and nasal swabs from experimentally infected animals). In both cases the system proved to be very efficient in A. pleuropneumoniae identification and serotype discrimination.     

Mouse staufen genes are expressed in germ cells during oogenesis and spermatogenesis

The Drosophila melanogaster staufen gene encodes an RNA binding protein (Dm Stau) required for the localization and translational repression of mRNAs within the Drosophila oocyte. In mammals translational repression is important for normal spermatogenesis in males and storage of mRNAs in the oocytes of females. In the present study we identified two mouse cDNA expressed sequence tags (ESTs), encoding proteins with significant homology to Dm Stau and used these firstly to screen a mouse kidney cDNA library and secondly to determine whether staufen mRNAs are expressed in the ovaries and testes of mice and rats. Sequence analysis of the cDNAs revealed that they originated from two different genes. Using Northern blots of RNAs from kidneys, ovaries and testes, both cDNAs hybridized to mRNA species of approximately 3 kb in all three tissues. On sections of mouse ovaries, staufen mRNA was localized specifically to oocytes. On sections of mouse testes, staufen mRNA was expressed in spermatocytes found in seminiferous tubules at stages VI-XII of the spermatogenic cycle. In conclusion, we have shown that the mammalian homologues of Dm stau are expressed in germ cells in both male and female mice, consistent with a role for these RNA binding proteins in mammalian gametogenesis.     

Emilin genes are duplicated and dynamically expressed during zebrafish embryonic development.

Emilins are a family of extracellular matrix proteins with common structural organization and containing a characteristic N-terminal cysteine-rich domain. The prototype of this family, Emilin-1, is found in human and murine organs in association with elastic fibers, and other emilins were recently isolated in mammals. To gain insight into these proteins in lower vertebrates, we investigated the expression of emilins in the fish Danio rerio. Using sequence similarity tools, we identified eight members of this family in zebrafish. Each emilin gene has two paralogs in zebrafish, showing conserved structure with the human ortholog. In situ hybridization revealed that expression of zebrafish emilin genes is regulated in a spatiotemporal manner during embryonic development, with overlapping and site-specific patterns mostly including mesenchymal structures. Expression of certain emilin genes in peculiar areas, such as the central nervous system or the posterior notochord, suggests that they may play a role in key morphogenetic processes.     

EXT genes are differentially expressed in bone and cartilage during mouse embryogenesis.

Hereditary multiple exostoses (HME) is a genetically heterogeneous disease characterized by the development of bony protuberances at the ends of all long bones. Genetic analyses have revealed HME to be a multigenic disorder linked to three loci on chromosomes 8q24 (EXT1), 11p11-13 (EXT2), and 19p (EXT3). The EXT1 and EXT2 genes have been cloned and defined as glycosyltransferases involved in the synthesis of heparan sulfate. EST database analysis has demonstrated additional gene family members, EXT-like genes (EXTL1, EXTL2, and EXTL3), not associated with a HME locus. The mouse homologs of EXT1 and EXT2 have also been cloned and shown to be 99% and 95% identical to their human counterparts, respectively. Here, we report the identification of the mouse EXTL1 gene and show it is 74% identical to the human EXTL1 gene. Expression studies of all three mouse EXT genes throughout various stages of embryonic development were carried out and whole-mount in situ hybridization in the developing limb buds showed high levels of expression of all three EXT genes. However, in situ hybridization of sectioned embryos revealed remarkable differences in expression profiles of EXT1, EXT2, and EXTL1. The identical expression patterns found for the EXT1 and EXT2 genes support the recent observation that both proteins form a glycosyltransferase complex. We suggest a model for exostoses formation based on the involvement of EXT1 and EXT2 in the Indian hedgehog/parathyroid hormone-related peptide (PTHrP) signaling pathway, an important regulator of the chondrocyte maturation process.     

Mammalian cell-entry proteins encoded by the mce3 operon of Mycobacterium tuberculosis are expressed during natural infection in humans

The mammalian cell-entry (mce)3 operon is one of four homologous mce operons on Mycobacterium tuberculosis genome that encodes six putative invasin/ adhesin-like proteins (Mce3A-F) possibly involved in the entry and survival of this bacterium inside macrophages. To study the in vivo expression of the mce3 operon-encoded proteins during natural human infection, the genes encoding Mce3A-F were cloned and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST) at the N-terminal and a x6 histidine (His) tag at the C-terminal end. The recombinant proteins appeared as major cellular proteins in SDS-PAGE gels and reacted with anti-GST and antipenta-His antibodies at the expected molecular mass of 70, 61, 68, 71, 66 and 72 [corrected] kDa for GST-Mce3A, GST-Mce3B, GST-Mce3C, GST-Mce3D, GST-Mce3E and GST-Mce3F, respectively. In Western immunoblots, all the six fusion proteins, particularly GST-Mce3A, GST-Mce3C, GST-Mce3D and GST-Mce3E, reacted with antibodies in combined human serum from 11 tuberculosis (TB) patients. Pure Mce3A, Mce3D and Mce3E could be isolated by specific proteolytic cleavage by thrombin protease of the respective purified fusion protein followed by preparative SDS-PAGE. The pure Mce3A, Mce3D and Mce3E reacted to various extents with antibodies in serum samples from TB patients. The Mce3E reacted with 51 of 55 (93%) and all the three proteins reacted with 34 of 55 (62%) serum samples. The Mce3A, Mce3D and Mce3E proteins also reacted, albeit at lower frequency, with one of 23 (4%) serum sample obtained from M. bovis bacillus Calmette-Guérin-vaccinated healthy subjects and four of 18 (22%) serum samples from long-term contacts of TB patients showing reactivity with all the three Mce3 proteins. The data show that Mce3A, Mce3D and Mce3E encoded by mce3 operon of M. tuberculosis are expressed and elicit antibody responses in humans during natural infection with this pathogen.     

Immunogenicity of gonococcal transferrin binding proteins during natural infections

In this study, we examined the immune response during gonococcal infection to the individual transferrin binding proteins by using a quantitative enzyme-linked immunosorbent assay (ELISA). Recombinant transferrin binding protein A (rTbpA) and rTbpB were purified under nondenaturing conditions for use as ELISA antigens. Sera and secretions from culture-positive individuals were analyzed for antibodies to rTbpA and rTbpB and compared to samples from individuals with no history of gonococcal infection. Although antibodies to both rTbpA and rTbpB were detected in serum, in most cases the antibody levels were not significantly different from those measured in the control population. Also, previous history of gonococcal infection did not increase antibody levels in serum, suggesting the lack of an anamnestic response. Analysis of secretion samples revealed antibody levels that were generally below the limits of detection in our assay. Overall, this study demonstrated a paucity of systemic and local antibody responses to rTbps as a result of natural infection and represents a baseline over which a protective antibody response will have to be generated in order to develop an efficacious gonococcal vaccine.     

Vibrio cholerae expresses cell surface antigens during intestinal infection which are not expressed during in vitro culture

Vibrio cholerae O1 bacteria harvested directly from ligated or nonligated intestines of rabbits with experimental cholera expressed at least 7 to 8 novel, in vivo-specific cell envelope (env) proteins that were not found on vibrios after in vitro culture in various ordinary liquid media. At the same time, several of the env proteins ordinarily expressed in vitro had disappeared or become much reduced. The infection-induced novel env protein were immunogenic. In immunoblot analyses, antisera raised against in vivo-grown vibrios and then absorbed with in vitro-grown bacteria of the same strain specifically stained at least eight infection-induced antigens ranging from 62 to approximately 200 kilodaltons; absorption with washed in vivo-grown bacteria, on the other hand, removed the antibodies reacting with these antigens, indicating that the antigens were present on the bacterial cell surface. Conversely, antiserum against in vitro-grown bacteria reacted with several env antigens in in vitro-grown bacteria that were missing in the infection-derived vibrios. These adaptational changes were strikingly similar for different strains of cholera vibrios of both classical and El Tor biotypes. Most of the in vivo-specific proteins (with apparent molecular masses of approximately 200, approximately 150, approximately 140, 92, 68, 62, 43, and 29 kilodaltons) were not induced during cultivation of bacteria in iron-depleted medium and are probably not related to the iron-regulated env proteins known to be involved in iron transport systems.     

The evolutionary watershed of susceptibility to gonococcal infection

Gonococci do not cause genital infection in any convenient experimental animal, but all too easily cause genital infection in humans. To determine the 'evolutionary watershed' of gonococcal infections (the point on the evolutionary tree at which susceptibility to gonococcal infection begins) we extended previous studies of the interaction of gonococci with animal oviduct mucosa to include chimpanzees and baboons. Gonococci attached to, damaged, and invaded the oviduct (fallopian tube) mucosa of chimpanzees (which are apes) but not the oviduct mucosa of baboons (which are monkeys). Thus, the pattern of gonococcal infection in chimpanzees was identical to that in humans, whereas the pattern in baboons was like that in other animals. These studies indicate that the point in evolution at which susceptibility to gonococcal infection commences is between baboons and chimpanzees (or between monkeys and apes). Susceptibility to gonococcal disease appears to require the presence on genital epithelial cells of receptors for gonococcal ligands such as pili, receptors for gonococcal lipopolysaccharide, or both. The physiological role of these receptors may be to interact with more useful, as yet unidentified molecules.     

Identification of Actinobacillus pleuropneumoniae genes important for survival during infection in its natural host

Actinobacillus pleuropneumoniae is a strict respiratory tract pathogen of swine and is the causative agent of porcine pleuropneumonia. We have used signature-tagged mutagenesis (STM) to identify genes required for survival of the organism within the pig. A total of 2,064 signature-tagged Tn10 transposon mutants were assembled into pools of 48 each, and used to inoculate pigs by the endotracheal route. Out of 105 mutants that were consistently attenuated in vivo, only 11 mutants showed a >2-fold reduction in growth in vitro compared to the wild type, whereas 8 of 14 mutants tested showed significant levels of attenuation in pig as evidenced from competitive index experiments. Inverse PCR was used to generate DNA sequence of the chromosomal domains flanking each transposon insertion. Only one sibling pair of mutants was identified, but three apparent transposon insertion hot spots were found--an anticipated consequence of the use of a Tn10-based system. Transposon insertions were found within 55 different loci, and similarity (BLAST) searching identified possible analogues or homologues for all but four of these. Matches included proteins putatively involved in metabolism and transport of various nutrients or unknown substances, in stress responses, in gene regulation, and in the production of cell surface components. Ten of the sequences have homology with genes involved in lipopolysaccharide and capsule production. The results highlight the importance of genes involved in energy metabolism, nutrient uptake and stress responses for the survival of A. pleuropneumoniae in its natural host: the pig.