Bartonella bacilliformis: dangerous pathogen slowly emerging from deep background.

Bartonella bacilliformis was perhaps the most lethal bacterial human pathogen in the pre-antibiotic era, but infections were and are limited to a specific geographical area, largely in Peru, corresponding to the range of its sand fly vector. B. bacilliformis targets both red cells and endothelial cells. Recent phylogenetic realignments have revealed a close genetic relationship to other bacteria which cause human diseases, including bacterial angiomatosis, to the former Grahamella species which infect red cells in other mammals, and to plant pathogens and symbionts including Agrobacterium tumefaciens and Rhizobium meliloti. Features of B. bacilliformis that contribute to its pathogenesis are slowly coming into view, and are here reviewed.

Purification of deformin, an extracellular protein synthesized by Bartonella bacilliformis which causes deformation of erythrocyte membranes.

A factor capable of deforming erythrocyte membranes, found in the culture supernatants of Bartonella bacilliformis, was purified 1840-fold using hydrophobic, ion exchange and gel exclusion chromatography. The final fractions contained a single detectable polypeptide species, referred to as deformin, having a molecular weight of 67000 by SDS-PAGE and a native molecular weight of 130,000 by gel exclusion chromatography or velocity sedimentation in a glycerol gradient. Erythrocytes treated with deformin acquire trenches, indentations, and invaginations which could be reversed by vanadate, dilauroylphosphatidylcholine (DLPC), or by raising the internal Ca2+ concentrations with the inophore A23187. Internal vacuoles also form. Erythrocytes treated with trypsin or neuraminidase are much more sensitive to deformin than untreated erythrocytes; erythrocytes treated with phospholipase D are less sensitive to deformin. This protein may play a role in causing the severe anemia which can result as a consequence of infection by B. bacilliformis.

Beta-galactosidase fused to the hydrophobic domain of cytochrome b5 spontaneously associates with liposomes.

Since liver microsomal cytochrome b5 spontaneously associates with liposomes and membranes by means of its C-terminal hydrophobic domain (HP), chimeric proteins containing HP prepared by genetic fusion might also spontaneously associate with liposomes or cellular membranes. Synthetic DNA corresponding to the hydrophobic domain of cytochrome b5 was enzymatically fused in-frame to cloned DNA corresponding to the C-terminus of the Escherichia coli enzyme, beta-galactosidase. This protein, LacZ:HP, synthesized in E. coli and purified from a crude E. coli membrane extract, was shown to spontaneously associated with liposomes, as does cytochrome b5. Association is rapid and stable in the presence of salt and high pH and the fusion protein behaves as an integral membrane protein. LacZ:HP can be readily and extensively purified from crude extracts by association with liposomes and this procedure may provide a convenient purification scheme for proteins not otherwise readily purified, for example polypeptides from cloned gene fragments to be used for antibody production. These hybrid proteins may represent a new potentially useful class of polypeptides capable of hydrophobic interactions with membranes.

The hydrophobic domain of cytochrome b5 is capable of anchoring beta-galactosidase in Escherichia coli membranes.

Cytochrome b5 is inserted posttranslationally into membranes in vivo and spontaneously into liposomes in vitro by a short carboxyl-terminal hydrophobic membrane-anchoring sequence. DNA corresponding to this hydrophobic sequence has been synthesized, and two gene fusions with the Escherichia coli enzyme beta-galactosidase have been constructed by locating the hydrophobic domain in one case at the EcoRI site near the C terminus and in the other at the normal C terminus of the enzyme. The latter fusion protein was enzymatically active, having approximately 50% of the specific activity of beta-galactosidase, and cells expressing this protein grew normally with lactose as the sole carbon source. Both fusion proteins were localized to the E. coli inner membrane, converting beta-galactosidase from a cytoplasmic enzyme to a membrane-associated enzyme. The hydrophobic domain of cytochrome b5 therefore contains the information required to target polypeptides containing this domain to the membrane. Use of the cytochrome b5 hydrophobic peptide, either alone or in conjunction with other localizing sequences such as signal sequences, provides a general procedure for associating proteins with membranes. Polypeptides bearing this hydrophobic peptide may have considerable use as pharmaceuticals when associated with liposomes or cellular membranes.

Formation of transmembrane channels in liposomes during injection of lambda DNA.

Bacteriophage lambda binds to unilamellar liposomes bearing its receptor protein, LamB, and the lambda DNA can be injected into the internal aqueous space. During this process, transmembrane channels are formed in the liposomes which permit the entry and escape of small molecules, but not proteins. The channels are stable and persist after DNA injection.

Long range base-pairing in the leftward transcription unit of bacteriophage lambda. Characterization by electron microscopy and computer-aided sequence analysis.

Restriction fragments of bacteriophage lambda DNA corresponding to the major leftward transcription unit were purified, denatured to form single-stranded DNA, self-annealed, and examined by electron microscopy. Three intrastrand stem and loop secondary structures were observed reproducibly and the locations of the paired regions were determined. A method for computer-aided sequence analysis of these regions is presented and used to identify sets of base-pairings likely to account for the observed structures. One loop observed within gene Ea47 is postulated to involve pairing of sequences which include the polypeptide initiation and termination codons. Another loop is postulated to involve pairing of sequences in gene int with sequences located in the gam-cIII region. A third loop appears to involve sequences in and to the right of gene Ea22 paired with sequences located in the bet-gam region. A general discussion of base-pairing which gives rise to long range interactions is presented along with possible effects of the postulated models on gene expression.

Proteinase sensitivity of bacteriophage lambda tail proteins gpJ and pH in complexes with the lambda receptor.

Previous studies have shown that bacteriophage lambda initially binds to liposomes bearing its receptor protein by the tip of the tail fiber (type 1 complex). It then associates more directly so that the hollow tail tube is in direct contact with the membrane (type 2 complex). DNA can be injected across the lipid bilayer into the liposome from type 2 complexes. We show here that gpJ, the tail fiber protein, becomes more sensitive to proteolytic degradation in type 2 complexes, indicating that the tail fiber does not pass into the liposome and that the tail fiber may undergo a conformational change in type 2 complexes. Another bacteriophage protein, pH, is sensitive to proteolytic degradation in free bacteriophage, type 1 complexes, or type 2 complexes formed with free receptor, but is resistant to proteinases in type 2 complexes formed with liposomes. This finding suggests that pH associates with the membrane. We suggest that this association is part of the mechanism by which a transmembrane hole for DNA entry is formed.