A protein linkage map of Escherichia coli bacteriophage T7.

Genome sequencing projects are predicting large numbers of novel proteins, whose interactions with other proteins must mediate the function of cellular processes. To analyse these networks, we used the yeast two-hybrid system on a genome-wide scale to identify 25 interactions among the proteins of Escherichia coli bacteriophage T7. Among these is a set of six interactions connecting proteins that function in DNA replication and DNA packaging. Remarkably, two genes, arranged such that one entirely overlaps the other and uses a different reading frame, encode interacting proteins. Several of the interactions reflect intramolecular associations of different domains of the same polypeptide, suggesting that the two-hybrid assay may be useful in the analysis of protein folding. This global approach to protein-protein interactions may be applicable to the analysis of more complex genomes whose sequences are becoming available.

Altered in vitro handling of Mycobacterium avium complex by monocytes and serum from HIV(+) patients.

In patients with acquired immunodeficiency syndrome (AIDS), mycobacterial diseases are leading opportunistic infections. The reasons for the peculiar propensity for disseminated infection with Mycobacterium avium complex (MAC) remain unclear. We have previously examined, in detail, the ability of monocytes from healthy donors to take up and kill MAC under both nonopsonic and opsonic conditions. We have now evaluated the in vitro ability of peripheral blood monocytes from HIV(+) patients to take up and kill MAC organisms, and have discovered a reduced ability under both nonopsonic and opsonic conditions. This reduction is due to: 1) apparent defect(s) in the phagocytes themselves, and 2) substance(s) in the HIV(+) serum which actively suppresses phagocyte activity.

Nonopsonic uptake of Mycobacterium avium complex by human monocytes and alveolar macrophages.

The uptake of Mycobacterium avium complex (MAC) microorganisms by human peripheral blood monocytes (PBMs) and alveolar macrophages (AMs) is not well understood. We have previously shown, under opsonic conditions, that humoral factors are important in mediating the uptake of MAC by PBMs. However, the receptor-ligand interactions occurring under nonopsonic conditions remain unclear. We compared the uptake of untreated human PBMs and AMs in a serum-free medium with phagocytes treated to remove surface receptors. Removal of complement receptors CR1 and CR3, the Fc receptor (FcR), and the transferrin receptor (TfR) resulted in significantly lower levels of MAC uptake in serum-free medium by both PBMs and AMs. The addition of barley beta-glucan or mannan from Saccharomyces cerevisiae inhibited MAC uptake by untreated phagocytes in a dose-dependent manner. MAC uptake by PBMs or AMs was never completely abrogated by combining treatments (removal of CR1, CR3, FcR, and TfR and adding mannan or beta-glucan), indicating still-unknown mechanisms of uptake under nonopsonic conditions. We conclude that CR1, CR3, FcR, TfR, the mannose receptor, and possibly a separate beta-glucan-inhibitable receptor all may be involved in nonopsonic uptake of MAC by both PBMs and AMs.

Natural killer cell-mediated lysis of Mycobacterium-avium complex-infected monocytes.

Since the precise mechanism of host responses to infection with Mycobacterium-avium complex (MAC) is unclear and since cytotoxic lymphocytes may be involved in the destruction of cells infected with intracellular pathogens, we investigated the ability of normal peripheral blood lymphocytes to kill MAC-infected monocytes in a short-term isotope release assay. Nylon wool-passed lymphocytes lysed MAC-infected but not uninfected monocytes during a 4-hr assay. Infected monocytes were less sensitive to cell-mediated killing than the standard natural killer (NK) cell-sensitive cell line K562, although the kinetics of lysis were similar. The release of lymphocyte-derived mediators such as tumor necrosis factor, interleukin-2 (IL-2), and interferon-alpha and -gamma could not be implicated as a cause of monocyte death. Through the use of cell-specific monoclonal antibodies plus complement, the phenotype of the effector cell was that of an NK cell (CD3 negative, partially CD8 negative, and CD16 positive). The use of highly purified, negatively selected NK cells confirmed these results. NK cell-mediated lysis of infected monocytes decreased MAC viability, indicating that this cytotoxic activity would not favor dissemination of the organism. The killing of MAC-infected monocytes was reduced by K562 cells, suggesting that these targets shared common recognition/binding structures. These results suggest that NK-cell function may be important in the prevention of or response to MAC infection and may help explain the predilection of AIDS patients to develop widespread disease.