Molecular characterization of two sub-family specific monoclonal antibodies to meningococcal Factor H binding protein.

Factor H binding protein (FHbp) is a component of two licensed vaccines for prevention of sepsis and meningitis caused by serogroup B meningococci. FHbp binds human Factor H (FH), which contributes to evasion of host immunity and FHbp sequence variants can be classified into two sub-families. Antibodies against FHbp elicit complement-mediated killing and can inhibit recruitment of FH to the bacterial surface. We report epitope mapping studies of two murine IgG mAbs, designated JAR 31 and JAR 36, isolated from a mouse immunized with FHbp in sub-family A, which is present in ∼30-40% of invasive isolates. In the present study, we tested the reactivity of mAbs JAR 31 and JAR 36 with seven natural FHbp sequence variants from different phylogenic groups. We screened bacteriophage-displayed peptide libraries to identify amino acid residues contributing to the JAR 36 epitope. Based on the reactivities of mAbs JAR 31 and JAR 36 with the seven FHbp variants, and the frequent occurrences of aspartate (D) and lysine (K) residues in the JAR 36-bound phage peptides, we selected six residues in the carboxyl-terminal region of FHbp for replacement with alanine (A). The D201A and K203A substitutions respectively eliminated and decreased binding of mAbs JAR 31 and JAR 36 to FHbp. These substitutions did not affect binding of the control mAb JAR 33 or of human FH. JAR 31 or JAR 36 mediated cooperative complement-mediated bactericidal activity with other anti-FHbp mAbs. The identification of two amino acid residues involved in the epitopes recognized by these anti-FHbp mAbs may contribute to a more complete understanding of the spatial requirements for cooperative anti-FHbp mAb bactericidal activity.

Potential association of specific Candida parapsilosis genotypes, bloodstream infections and colonization of health workers' hands.

Fungal nosocomial infections continue to be a serious problem among hospitalized patients, decreasing quality of life and adding millions of euros to healthcare costs. The aim of this study was to describe the pattern of fungi associated with the hands of healthcare workers and to genotype Candida parapsilosis isolates in order to understand whether their high clinical prevalence stems from endemic nosocomial genotypes or from the real emergence of epidemiologically-unrelated strains. Approximately 39% (50/129) of healthcare workers were positive for yeasts and among 77 different fungal isolates recovered, C. parapsilosis was the most frequent (44/77; 57%). Twenty-seven diverse genotypes were obtained by microsatellite analysis of 42 selected blood and hand isolates. Most of the isolates from hands showed a new, unrelated, genotype, whereas a particular group of closely related genotypes prevailed in blood samples. Some of the latter genotypes were also found on the hands of healthcare workers, indicating a persistence of these clones within our hospital. C. parapsilosis genotypes from the hands were much more heterogeneous than clinical ones, thus reflecting a high genetic diversity among isolates, which is notably unusual and unexpected for this species.

Transmission of Trichosporon asahii oesophagitis by a contaminated endoscope.

Two cases of oesophageal trichosporonosis due to a suspected nosocomial infection are reported. Both the patients were immunocompetent and had undergone an endoscopic examination on the same day. Six strains of Trichosporon were isolated: three strains from the oesophageal biopsy of the first patient, one strain from the endoscopic forceps, one from the air in the endoscopy room, and one from the oesophageal biopsy of the second patient. The nosocomial nature of the infection and the role of the endoscopic forceps in transporting the micro-organism was suspected, but the morphology and physiology of the isolated strains did not confirm such hypothesis. To elucidate the nature of the infection and the genetic similarities of the strains isolated, all strains were typed with RFLPs of the rDNA fragment and with RAPD. The results of RAPD using primer (GTG)5 (GACA)4, M13 core sequence, and the 15-mer oligonucleotide GAGGGTGGXGGXTCT indicated the molecular identity of three strains supporting the hypothesis concerning a transport of the aetiological agent from the first patient to the second and that the carrier was the forceps of the endoscopic device.

Molecular subtyping of clinical and environmental strains of Cryptococcus neoformans variety neoformans serotype A isolated from southern Italy.

Analysis of ribosomal DNA (rDNA) restriction fragment-length polymorphism (RFLP) and random amplification of polymorphic DNA (RAPD) was used to investigate the genetic variability and biogeographic distribution of clinical and environmental strains of Cryptococcus neoformans isolated from a limited area of southern Italy, where the selection of a predominant cryptococcal genotype could be expected. All isolates belonged to the species Cr. neoformans variety neoformans serotype A. RFLP analysis of a specific rDNA fragment allowed the distinction of strains of Cr. neoformans from closely related fungal reference species, but neither intraspecies nor intravarieties polymorphism was detected. On the contrary, RAPD fingerprints produced by priming with four different primers [(GTG)5, (GACA)4, M13 core sequence and the 8-mer oligonucleotide (GCGGACGG)] were able to characterize the isolates up to the individual level, indicating the presence of marked heterogeneity among Cr. neoformans serotype A strains in southern Italy.

Genetic relatedness and diversity of Cryptococcus neoformans strains in the Maltese Islands.

The genetic relatedness of clinical and environmental Cryptococcus neoformans strains in the Maltese Islands was investigated by randomly amplified polymorphic DNA fingerprinting with four primers. The clinical strains isolate over the course of 1 year from AIDS patients showed identical fingerprints. The electrophoretic patterns of the two clinical strains were also the most common patterns among the environmental strains, but the patterns among the environmental strains showed a wide variability and no correlation with the site of isolation.

Loop mutations can cause a substantial conformational change in the carboxy terminus of the ferritin protein.

Although some protein folding theories sustain that the peptides (loops) that connect elements of more compact secondary structure may be important in the folding process, most of the data accumulated until now seems to contradict this notion. To approach this problem we have isolated and characterized a number of mutants in which the amino acid sequence of the peptide that connects helix D and helix E in the H-chain of human ferritin has been randomized. Our results indicate that, though no single loop residue is absolutely required for ferritin to attain the native conformation, most of the mutants that we have obtained by random regional mutagenesis, affect its folding/assembly process. This conclusion was reached utilizing a sensitive test that associates the color formed by a colony synthesizing a hybrid ferritin-beta-galactosidase protein to the ability of the ferritin domain to fold and assemble as the native protein. The characterization of the folding/assembly properties of our collection of mutants and the comparison of the mutant loop sequences, have allowed us to draw the following conclusions. Mutants that have positively charged residues at position 159, 160 or 161 fail to assemble into the native protein shell and form an insoluble aggregate. Interestingly some loop amino acid sequences cause the E-helix to reverse direction and to expose its COOH group, normally hidden inside the protein cavity, to the solvent. The propensity of a given ferritin mutant to fold into this "non-native" conformation can be attenuated by the introduction of Gly at position 159 and 164, as in the natural ferritin.

Fibrosclerotic lymphedema: pathophysiology and therapy.

We describe our experience with 12 patients with severe fibrotic lymphedema treated between 1979 and 1987. Each patient initially underwent nonoperative treatment (postural drainage and pneumatic compression) and in 10 patients who required operation, these measures were continued postoperatively. Operation included excision of subcutaneous tissue (debulking), which was extensive in 8 and limited in 2 patients. Only 2 patients were satisfactorily managed by nonoperative treatment alone. Based on the extensive pathophysiologic changes that occur in the tissue microenvironment with lymph stasis, it is unlikely that at this advanced stage of lymphedema that nonoperative treatment alone or "physiologic" operations such as lymphatic-venous shunt or lymphatic collector reconstruction is satisfactory. Rather, nearly all such patients require limited or extensive excision of the fibrotic-edematous subcutaneous tissue.

A strategy to optimize translation initiation in recombinant mRNA: application to the Rop gene.

Using the Escherichia coli Rop gene, we demonstrate a strategy that could be applied generally to optimize the initiation of translation of recombinant genes in E. coli. This involves cloning of the gene encoding the protein of interest in a suitable expression vector between an "efficient" ribosome binding site and the gene for the alpha-peptide of beta-galactosidase. By oligonucleotide-directed deletion mutagenesis, the two coding sequences are then fused in the correct frame. A second oligonucleotide is then used to place the initiator AUG (or GUG) at the correct distance from the Shine-Dalgarno sequence. In this step, however, we use an oligonucleotide that has a degenerate sequence. That is, on the basis of the "efficient" ribosome binding site sequence, we introduce random substitutions at various positions, both upstream and downstream from the initiator ATG, to obtain, after the mutagenesis experiment, a collection of random ribosome binding sites fused to the coding sequence. The development of blue colonies on indicator plates permits selection of clones in which an efficient ribosome binding site has been created for the specific gene of interest. We discuss the results obtained by applying the method to the Rop gene.