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Visceral leishmaniasis caused by the intracellular parasiteLeishmania donovaniis a significant public health problem in many regions of the world. Because of its large genome and complex biology, developing a vaccine for this pathogen has proved to be a challenging task and, to date, protective recombinant vaccine candidates have not been identified. To tackle this difficult problem, we adopted a reductionist approach with the intention of identifying cDNA sequences in anL. donovaniamastigote cDNA library that collectively or singly conferred protection against parasite challenge in a murine model of visceral leishmaniasis. We immunized BALB/c mice with plasmid DNA isolated and pooled from 15 cDNA sublibraries (∼2,000 cDNAs/sublibrary). Following systemic challenge withL. donovani, mice immunized with 6 of these 15 sublibraries showed a significantly reduced (35- to 1,000-fold) hepatic parasite burden. Because of the complexity and magnitude of the sequential fractionation-immunization-challenge approach, we restricted our attention to the two sublibraries that conferred the greatest in vivo protection. From one of these two sublibraries, we identified several groups of cDNAs that afforded protection, including a set of nine novel cDNAs and, surprisingly, a group of five cDNAs that encodedL. donovanihistone proteins. At each fractionation step, the cDNA sublibraries or the smaller DNA fractions that afforded in vivo protection against the parasite also induced in vitro parasite-specific T helper 1 immune responses. Our studies demonstrate that immunization with sequential fractions of a cDNA library is a powerful strategy for identifying anti-infective vaccine candidates.

Rhodospirillum rubrum is a spiral anoxygenic photosynthetic bacterium that can exist under either aerobic or anaerobic conditions. The organism thrives in the presence of light or complete darkness and represents one of the oldest species of living organisms, possibly 2-3.5 billion years old. The success of this prokaryotic species may be attributed to the evolution of certain indole compounds that offer protection against life-threatening oxygen radicals produced by an evolutionary harsh environment. Melatonin, N-acetyl-5-methoxytryptamine, is an indolic highly conserved molecule that exists in protists, plants, and animals. This study was undertaken to determine the presence of an immunoreactive melatonin in the kingdom Monera and particularly in the photosynthetic bacterium, R. rubrum, under conditions of prolonged darkness or prolonged light. Immunoreactive melatonin was measured during both the extended day and extended night. Significantly more melatonin was observed during the scotophase than the photophase. This study marks the first demonstration of melatonin in a bacterium. The high level of melatonin observed in bacteria may provide on-site protection of bacterial DNA against free radical attack.

DNA from the E. coli replicative origin binds with high affinity to outer membrane preparations. Specific binding regions are contained within a 463 bp stretch of origin DNA between positions −46 and +417 on the oriC map. This region of DNA contains an unusually high number of GATC sites, the recognition sequence for the E. coli DNA adenine methylase. We show here that oriC DNA binds to membrane only when it is hemimethylated. The E. coli chromosomal origin is hemimethylated for 8–10 min after initiation of replication, and origin DNA binds to membranes only during this time period. Based on these results, we propose a speculative model for chromosome segregation in E. coli.

The notion that the bacterial chromosome is attached to the membrane is by now firmly embedded in the folklore of bacterial structure. It is derived from a series of experiments on the segregation of the Escherichia coli chromosome and its plasmids, which Jacob et al. (1963) used for their original postulation of the replicon model. Unfortunately, in the subsequent years this notion remains more compelling for its logical appeal than for the rigorousness of the experimental evidence. In any case, this aspect of the replicon model has not contributed very much to our understanding of genome segregation or of the regulation of its synthesis. Despite this, no one so far has presented a serious alternative for the physical basis for chromosome segregation.

Resistance to complement-mediated lysis in Trypanosoma cruzi is due to the expression of complement-regulatory factors by the virulent developmental forms of this protozoan parasite. An 87- to 93-kDa molecule, which we have termed T-DAF (trypomastigote decay-accelerating factor), is present on the surface of the parasite and inhibits complement activation in a manner functionally similar to the mammalian complement regulatory component, decay-accelerating factor. In this report, we characterized monospecific polyclonal and monoclonal antibodies which were obtained from mice and rabbits immunized with fast protein liquid chromatography-purified T-DAF. These polyclonal antibodies were shown to inhibit T-DAF activity and were capable of inducing lysis of the parasites. Both the polyclonal and monoclonal antibodies were used to screen a cDNA expression library prepared from T. cruzi trypomastigote mRNA. From this library, we obtained a partial lambda gt11 cDNA clone which showed genetic and functional similarity to the human C3 convertase inhibitor DAF (A. Nicholson-Weller, J. Burge, D. T. Fearon, P. F. Weller, and K. F. Austen, J. Immunol. 129:184-189, 1982).