Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains.

AIMS: Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen-like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium. METHODS AND RESULTS: The tetrasaccharide consists of three rhamnose residues and an unique monosaccharide--anthrose. As anthrose was not found in spores of related strains of bacteria, we envisioned the detection of B. anthracis spores based on antibodies against anthrose-containing polysaccharides. Carbohydrate-protein conjugates containing the synthetic tetrasaccharide, an anthrose-rhamnose disaccharide or anthrose alone were employed to immunize mice. All three formulations were immunogenic and elicited IgG responses with different fine specificities. All sera and monoclonal antibodies derived from tetrasaccharide immunized mice cross-reacted not only with spore lysates of a panel of virulent B. anthracis strains, but also with some of the B. cereus strains tested. CONCLUSIONS: Our results demonstrate that antibodies to synthetic carbohydrates are useful tools for epitope analyses of complex carbohydrate antigens and for the detection of particular target structures in biological specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: Although not strictly specific for B. anthracis spores, antibodies against the tetrasaccharide may have potential as immuno-capturing components for a highly sensitive spore detection system.

Contributions of the procyclin 3' untranslated region and coding region to the regulation of expression in bloodstream forms of Trypanosoma brucei.

When bloodstream forms of Trypanosoma brucei differentiate into procyclic forms they rapidly synthesise a new surface coat composed of procyclins. Procyclin genes are transcribed in bloodstream forms at approximately one-tenth of the rate in procyclic forms, but little, if any, mRNA can be detected, indicating that further down-regulation must occur post-transcriptionally. We have examined the role of the 297 bp procyclin 3' untranslated region (UTR) in regulating expression in bloodstream forms and have identified three discrete elements: a dominant, negative element between positions 101 and 173, and two positive elements. When chloramphenicol acetyl transferase (CAT) was used as the reporter gene, deletion of the negative element caused a approximately 6-fold increase in the level of steady state mRNA and > 30-fold increase in CAT activity, suggesting that both RNA stability and translation were affected. Similar results were obtained with glutamic acid/alanine-rich protein (GARP), the T. congolense analogue of procyclin, indicating that the 3' UTR acts independently of the coding region. In contrast, when trypanosomes were stably transformed with a construct in which the procyclin coding region was linked to a truncated form of the 3' UTR which lacked the negative element, they expressed high levels of mRNA, but no protein could be detected in cell lysates or culture supernatants. These results imply that the procyclin coding region exerts yet another layer of control which prevents inappropriate expression of the protein in the mammalian host.

Elements in the 3' untranslated region of procyclin mRNA regulate expression in insect forms of Trypanosoma brucei by modulating RNA stability and translation.

Procyclins are the major surface glycoproteins of insect forms of Trypanosoma brucei. We have previously shown that a conserved 16-mer in the 3' untranslated region (UTR) of procyclin transcripts functions as a positive element in procyclic-form trypanosomes. A systematic analysis of the entire 297-base 3' UTR has now revealed additional elements which are involved in posttranscriptional regulation: a positive element which requires the first 40 bases of the 3' UTR and at least one negative element between nucleotides 101 and 173 (the LII domain). Deletion of either positive element resulted in a >8-fold reduction in the amount of protein but only an approximately 2-fold decrease in the steady-state level of mRNA, suggesting that regulation also occurred at the level of translation. In contrast, deletion of LII caused a threefold increase in the steady-state levels of both the mRNA and protein. LII-16-mer double deletions also gave high levels of expression, suggesting that the 16-mer functions as an antirepressor of the negative element rather than as an independent activator. All three elements have an effect on RNA turnover. When either positive element was deleted, the half-life (t(1/2)) of the mRNA was reduced from approximately 50 min (the t(1/2) of the wild-type 3' UTR) to < 15 min, whereas removal of the LII element resulted in an increased t(1/2) of approximately 100 min. We present a model of posttranscriptional regulation in which the negative domain is counteracted by two positive elements which shield it from nucleases and/or translational repressors.

Accurate polyadenylation of procyclin mRNAs in Trypanosoma brucei is determined by pyrimidine-rich elements in the intergenic regions.

Polycistronic precursor RNAs from trypanosomes are processed into monocistronic mRNAs by the excision of intergenic sequences and the addition of a 39-nucleotide spliced leader by trans splicing. These mRNAs are also polyadenylated, yet they do not contain the hexamer AAUAAA within their 3' untranslated regions (UTRs). To identify the signals required for the accurate polyadenylation of mRNAs, we tested the effects of deletions in either the procyclin 3' UTR or the downstream intergenic region on the polyadenylation of transcripts from a reporter gene. Deletion of the entire 3' UTR does not affect polyadenylation, but a crucial element is located in the intergenic region and includes a pyrimidine-rich sequence from positions 79 to 112 followed by an AG dinucleotide. Related motifs are also found a similar distance downstream of other genes in both the procyclin and the variant surface glycoprotein expression sites. These sequences bear a strong resemblance to splice acceptor sites, but they are generally several hundred base pairs upstream of the major splice acceptor site of the next gene in the transcription unit. There is evidence, however, that some of them can give rise to alternatively spliced transcripts with unusually long 5' UTRs.