No abstract

We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-toalanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all ␤-lactams. Affinity for penicillin generally correlated with ␤-lactam MICs for the mutants, but these associations were not strictly proportional.Ampicillin resistance in Enterococcus faecium is due to expression of the low-affinity class B penicillin-binding protein 5 (PBP 5) (15). Early studies suggested that higher levels of ampicillin resistance in Enterococcus hirae (similar to those in E. faecium) were achieved by increasing levels of PBP 5 expression (5). However, higher levels of resistance in clinical isolates are only rarely associated with increased levels of PBP 5 expression (12, 16). More commonly, mutations that are presumed to lower the affinity for ␤-lactam antibiotics have been identified within pbp5 genes of highly resistant clinical isolates (1,12,16). It has been presumed that these mutations serve to lower the affinity of the PBP 5 molecule for ␤-lactam antibiotics, yielding higher MICs as a result.The specific impacts of individual or multiple pbp5 mutations on the resistance level have been difficult to determine because most analyses have been performed with clinical isolates, in which factors other than the PBP 5 amino acid sequence may contribute to resistance. In a previous study (14) a single mutation (M485A) was introduced into a cloned pbp5, and the impact of this mutation was relatively minor. However, the plasmid used in those experiments contained only pbp5 and its promoter, and expression of resistance prior to introduction of the mutation was minimal (ampicillin MIC, 6 g/ml) (14). The role of PBP 5 in expression of ampicillin resistance in E. faecium C68, a clinical isolate resistant to high levels of ampicillin (MICs, 256 to 512 g/ml), was reported recently (11). pbp5 of C68 is located downstream of two open reading frames, designated ftsW Efm and psr. It was observed that expression of ampicillin resistance from a cloned version of the C68 pbp5 was higher when pbp5 was located downstream of ftsW Efm and psr (ampicillin MICs, 64 to 128 g/ml) than when it was cloned with only its own promoter (ampicillin MICs, 8 to 16 g/ml) (data not shown) (11). The role of the putative ftsW Efm gene product is unknown, although recent work suggests that its homologue in Escherichia coli may serve as a chaperone protei...

T cell clones obtained from a human volunteer immunized with Plasmodium falciparum sporozoites specifically recognized the native circumsporozoite (CS) antigen expressed on P. falciparum sporozoites, as well as bacteria- and yeast-derived recombinant falciparum CS proteins. The response of these CD4+ CD8- cells was species-specific, since the clones did not proliferate or secrete gamma interferon when challenged with sporozoites or recombinant CS proteins of other human, simian, or rodent malarias. The epitope recognized by the sporozoite-specific human T cell clones mapped to the 5' repeat region of the CS protein and was contained in the NANPNVDPNANP sequence.

A restriction site-free cloning method has been developed for inserting a PCR product into a vector flexibly and precisely at any desired location with high efficiency. The method uses a pair of DNA integration primers with two portions. The 3' portion isolates the inserts by PCR, and the 5' portion integrates the PCR products into the homologous region of the vector. For mutagenesis, a third portion of mutation-generating sequences can be placed in between the 3' and 5' portions. This method has been used to clone the E. coli gene that codes for peptidyl-tRNA hydrolase, expressing it as a native protein and as a glutathione S-transferase fusion protein. It was also applied to convert a construct of the E. coli fatty acid biosynthesis protein with an N-terminal hexa-histidine tag into a construct with a C-terminal hexa-histidine tag.

To circumvent problems associated with polymorphic vaccine candidates for Plasmodium falciparum malaria, we evaluated recombinant proteins representing sequences from relatively highly conserved regions of the precursor to the major merozoite surface proteins, gp190, for their ability to protect Saimiri monkeys against malaria challenge. Recombinant proteins represented amino acid residues 147 to 321 (p190-1) or 147 to 321 and 1060 to 1195 (p190-3), and their efficacy was compared with that of native gpl90 and its processed products. All antigens were derived from P. falciparum Ki, a Thai isolate, while the challenge strain was Palo Alto (from Uganda, Africa), which contains, with the exception of the N-terminal 375 amino acids, which are almost identical to the Kl sequence, essentially the MAD-20 allelic form of gpl90. By 12 days following challenge, each control monkey required drug treatment. Three monkeys injected with p190-3 required therapy, while one cleared the parasites without therapy. Two monkeys injected with p190-1 received therapy on day 14, while the remaining two cleared the parasites without therapy. Of four animals injected with native gpl90, because of health reasons unrelated to malaria, one was not challenged with parasites and one was removed from the study 8 days after challenge when its parasitemia was 1.1% (parasitemias in control animals ranged from 4.3 to 9%); the remaining two cleared the parasites after maximum parasitemias of 0.45 and 0.53%. The highest levels of antiparasite antibody were produced by animals immunized with native gpl90. There was a significant correlation between monkeys which did not require drug treatment and antiparasite antibody. These results may suggest that native gpl90 and/or its processed products can provide excellent protection against heterologous challenge and that antibody is important for protection. The challenge for vaccine development is to identify the protective sequence(s).