Cotrimoxazole resistance of Streptococcus pneumoniae and commensal streptococci from Kampala, Uganda.

Trimethoprim sulfamethoxazole (cotrimoxazole, CTX) is used frequently as part of standard medical care for people living with HIV/AIDS in Africa. The mechanisms of resistance to sulfonamides and trimethoprim in commensal streptococci from Uganda were determined and compared to S. pneumoniae. Commensal streptococci showing high-level resistance to cotrimoxazole were cultured and analysed for species identity and polymorphisms in the genes coding for dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). Seven isolates of S. pneumoniae from blood and cerebrospinal fluid (CSF) were similarly examined. There was considerable polymorphism in both DHPS and DHFR. In DHFR, the mutations E20D and I100L were present in all sequenced isolates. Other mutations such as L135F, and different substitutions in D92, were frequent. The most common DHPS variants had 2 serine residues added after amino acid 60, or arginine and proline added after amino acid 59. In addition, 3 new insertions/substitutions were found. There were no obvious differences between the mutation patterns in S. pneumoniae and commensal streptococci, suggesting that the chromosomal mutations have been spread by transformational interchanges of DNA among related organisms.

Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation.

Heparan sulfate (HS) proteoglycans influence embryonic development and adult physiology through interactions with protein ligands. The interactions depend on HS structure, which is determined largely during biosynthesis by Golgi enzymes. How biosynthesis is regulated is more or less unknown. During polymerization of the HS chain, carried out by a complex of the exostosin proteins EXT1 and EXT2, the first modification enzyme, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), introduces N-sulfate groups into the growing polymer. Unexpectedly, we found that the level of expression of EXT1 and EXT2 affected the amount of NDST1 present in the cell, which, in turn, greatly influenced HS structure. Whereas overexpression of EXT2 in HEK 293 cells enhanced NDST1 expression, increased NDST1 N-glycosylation, and resulted in elevated HS sulfation, overexpression of EXT1 had opposite effects. Accordingly, heart tissue from transgenic mice overexpressing EXT2 showed increased NDST activity. Immunoprecipitaion experiments suggested an interaction between EXT2 and NDST1. We speculate that NDST1 competes with EXT1 for binding to EXT2. Increased NDST activity in fibroblasts with a gene trap mutation in EXT1 supports this notion. These results support a model in which the enzymes of HS biosynthesis form a complex, or a GAGosome.

Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation.

The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant.

Enzymatically active N-deacetylase/N-sulfotransferase-2 is present in liver but does not contribute to heparan sulfate N-sulfation.

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing.

Heparin-deficient mice, generated by gene targeting of N-deacetylase/N-sulfotransferase-2 (NDST-2), display severe mast cell defects, including an absence of stored mast cell proteases. However, the mechanism behind these observations is not clear. Here we show that NDST-2+/+ bone marrow-derived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparin-like polysaccharide. The corresponding NDST-2-/- cells produced highly sulphated CS only. Carboxypeptidase A (CPA) activity was detected in NDST+/+ cells but was almost absent in the NDST-/- cells, whereas tryptase (mouse mast cell protease 6; mMCP-6) activity and antigen was detected in both cell types. Antigen for the chymase mMCP-5 was detected in NDST-2+/+ cells but not in the heparin-deficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wild-type and NDST-2-/- cells. A approximately 36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a approximately 50 kDa pro-CPA band was present in NDST-2+/+ cells. The NDST-2-/- mast cells contained similar levels of pro-CPA as the wild-type mast cells, but the approximately 36 kDa band was totally absent. This indicates that the processing of pro-CPA to its active form may require the presence of heparin and provides the first insight into a mechanism by which the absence of heparin may cause disturbed secretory granule organisation in mast cells.