Intracellular infection by L pneumophila

L. pneumophila may be ingested by macrophages without opsonization, although they are ingested much more efficiently after exposure to serum and fixation of complement via the alternate pathway. In either case, the bacteria are able to alter the normal phagocytic pathway, avoid intracellular microbiological mechanisms and establish productive intracellular infection.

The uptake of the bacterium into macrophages often involves a novel mechanism of ingestion known as 'coiling phagocytosis', so named because macrophage pseudopods encircle and eventually spiral around the bacterium several times during engulfment (Figure 1, step 2). In these membrane coils, macrophage surface molecules are selectively sorted so that the nascent phagosomal membrane is different from the plasma membrane from which it was derived. Specifically, major histocompatibility complex (MHC) class I and class II molecules and alkaline phosphatase are excluded during uptake, while the CR3 receptor and 5'-nucleotidase are retained in the coiled phagocytic membranes and subsequently eliminated within an hour following bacterial uptake. Ingestion of L. pneumophila by amoebae also has novel ultrastructural features. The bacterium is often engaged at the tip of an extended filipod (Figure 1, step 1) and then retracted toward the body of the cell and engulfed. It is not known whether membrane sorting occurs in protozoa; however, Abu Kuwaik and colleagues demonstrated that uptake by the amoeba, Hartmannella vermiformis, is a more prolonged process than macrophage uptake, and it requires new protein synthesis. In addition, virulent and avirulent L. pneumophila induce distinct sets of amoebal proteins during ingestion.

Once virulent L. pneumophila are ingested by either macrophages or protozoa, the phagosomes evade events that normally follow phagocytosis, and the bacteria-containing endosomes become permissive for bacterial replication. In macrophages, normal acidification of the phagosome is attenuated, and the oxidative burst is minimized. During the first 15-60 minutes after uptake, the bacterial phagosome associates with smooth vcsicles (Figure 1, step 3). Later, they make intimate contact first with mito-

Figure 1 Events associated with intracellular infection by L. pneumophila. (1) Bacteria make contact with filipodia (in amoebae) or (2) are taken up by coiling phagocytosis (in macrophages). (3) The bacterium is ingested within a phagosome, which associated transiently with mitochondria and smooth vesicles. Some avirulent mutants are directed to the degradative endocytic compartments (Ly, lysosome) (4a), but most virulent bacteria are encircled by rough endoplasmic reticulum at ~4 hours after uptake (4b). (5) Bacteria begin to divide in this endosome 4-8 hours after uptake. They double in 2 hours, and grow to large numbers within the endosome over the next 24—48 hours. (6) When the internal bacterial load becomes very large, the cell is lysed.

Figure 1 Events associated with intracellular infection by L. pneumophila. (1) Bacteria make contact with filipodia (in amoebae) or (2) are taken up by coiling phagocytosis (in macrophages). (3) The bacterium is ingested within a phagosome, which associated transiently with mitochondria and smooth vesicles. Some avirulent mutants are directed to the degradative endocytic compartments (Ly, lysosome) (4a), but most virulent bacteria are encircled by rough endoplasmic reticulum at ~4 hours after uptake (4b). (5) Bacteria begin to divide in this endosome 4-8 hours after uptake. They double in 2 hours, and grow to large numbers within the endosome over the next 24—48 hours. (6) When the internal bacterial load becomes very large, the cell is lysed.

chondria and then with rough endoplasmic reticulum (ER). Viable L. pneumophila-comammg phagosomes were shown by Horwitz to evade fusion with thorium-labeled lysosomes (Figure 1, step 4b). More recent immunofluorescence microscopy studies confirm that the late endosomal and lysosomal proteins CD63, LAMP-1, LAMP-2 and cathepsin D are frequently associated with L. pneumophila-containing phagosomes. The mechanisms by which lysosomal fusion is avoided is not known; however, Berger and Isberg have identified and cloned a bacterial gene (designated dotA) that is required for this pathologic process.

By 4 hours after ingestion, phagosomes containing live L. pneumophila appear studded with ribosomes as a result of the association with the ER. This pathologic process resembles autophagocytosis, a cellular process that occurs in response to limitation of nutrients. During autophagocytosis, host cells demarcate sections of cytoplasm by encirclement with smooth endoplasmic reticulum. Proteins within the encircled area are degraded and recycled to the viable parts of the cell. The notion that the L. pneumopbila-ER

association might represent a form of autophagocytosis was proposed in 1995 by Swanson and Isberg. These investigators showed that amino acid restriction of host cells increases the association of ingested L. pneumophila with the ER and facilitates intracellular bacterial growth. Since /.. pneumophila utilize amino acids as their primary carbon source, direct stimulation of host cell autophagv may account for the ER association. Autophagocvtic degradation may make simple nutrients available to the growing bacteria, or, alternatively, the enzymes or channels contained in the ER may facilitate bacterial nutrition.

Between 4 and 8 hours after ingestion, I., pneumophila begin to multiply. By 24 to 48 hours, endo-somes packed with bacteria fill the cell. Cell lysis occurs, and the released bacteria can reinitiate infection in a neighbouring cell (Figure 1, steps 5 and 6).

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