Roles of Ca2+ and F-actin in intracellular aggregation of Chlamydia trachomatis in eucaryotic cells

Abstract

The effect of intracellular free Ca2+ ([Ca2]11) on the intracellular aggregation of Chlamydia trachomatis serovars L2 and E in McCoy and HeLa cells is investigated. Loading the cells with the Ca2+ chelator M.APT/AM (1,2-bis-5-methyl-amino-phenoxylethane-N,N,n'-tetra-acetoxymethyl acetate), thereby decreasing the [Ca2+], from 67 to 19 nM, decreased the number of cells with a local aggregation of chlamydiae in a dose-dependent manner. Neither the attachment nor the uptake of elementary bodies (EBs) was, however, affected after depletion of Ca2+ from the cells. There was no significant difference in the level of measured [Ca211i between infected and uninfected cells. Reducing the [Ca2+]i also significantly inhibited chlamydial inclusion formation. Differences in the organization of the actin filament network were observed in response to [Ca2+], depletion. In Ca2+-depleted cells, where few EB aggregates were formed, few local accumulations of F-actin were observed in the cytosol. These results suggest that the aggregation of EBs in eucaryotic cells requires a normal homeostasis of intracellular Ca2+. By affecting F-actin reorganization and putatively certain Ca2+-binding proteins, [Ca2]i1 plays a vital role in the infectious process of chlamydiae.Chlamydiae are obligate intracellular parasites of eucaryotic cells and cause a wide spectrum of human diseases (33). As intracellular parasites, chlamydiae have developed the morphologically distinct infectious elementary body (EB) which is metabolically inert and resistant to the inimical extracellular environment. The noninfectious reticulate body is the metabolically active and replicating form (28). The developmental cycle starts when the EB attaches to and enters the host cell. It remains within an endosome, where it changes to the reticulate body, which divides by binary fission within the endosome. After a number of divisions, the reticulate bodies reorganize into EBs, producing the characteristic inclusion. It is not clear how and why the chlamydial endosome avoids fusion with host cell lysosomes (11). Earlier studies have suggested that multiple Chlamydia trachomatis endosomes fuse at some time after infection (4, 6). These findings were supported by ultrastructural studies (19,41) indicating that fusion occurs very shortly after infection. Ridderhof and Barnes (31) have shown that C. trachomatis-containing phagosomes fuse with each other in a microfilament-dependent process. However, how this fusion is controlled is unclear.The responsiveness of cells to a wide range of stimuli depends upon internal signaling systems such as cyclic nucleotides (cAMP, cGMP) (22) and intracellular free Ca2+

Calcium signalling during cell interactions with bacterial pathogens

“…C. trachomatis exists under two distinct forms : an infectious non-metabolically active form EB (elementary body), an a non-infectious metabolically active form RB (replicating body) that replicates inside host cells. After internalisation by host cells, EBs form aggregates within intracellular vacuole in a process involving actin polymerisation and Ca 2+ (Majeed et al, 1993), and these vacuoles do not mature to fuse with lysosomes. The mechanism of phagolysosome inhibition by C. trachomatis is still undefined but appears to implicate Ca 2+ signalling.…”

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection

“…Both microfilaments (MFs) and microtubules (MTs) have been proposed to be involved in the chlamydial redistribution from the host cell surface to the perinuclear region. Majeed and co-workers (Majeed and Kihlströ m, 1991;Majeed et al, 1993) showed that aggregated F-actin and clathrin collocate correspondingly in time and position with the perinuclear aggregates of C. trachomatis serovars E and L2, indicating an MF-dependent mechanism of redistribution. However, in a study by Schramm and Wyrick (1995) on the effect of cytoskeletal disruption on C. trachomatis infection, it was demonstrated that C.…”

Type III secretion genes identify a putative virulence locus of Chlamydia

“…A number of recent studies have indicated that chlamydiae do indeed exploit host intracellular trafficking. Soon after infection, the host cytoskeleton is involved in the redistribution of internalized chlamydiae from the cellular to the nuclear periphery (Majeed et al, 1993;Schramm and Wyrick, 1995), and calcium-dependent annexins are found collocalized with the Chlamydia vacuole (Majeed et al, 1994). Dependent on the multiplicity of infection and on time elapsed after infection, development of the Chlamydia vacuole may involve fusogenicity with both early and late endosomes (van Ooij et al, 1997), and with Golgi-derived exosomes from which sphingolipids are redirected to the parasitophorous vacuole membrane and to membranes of growing reticulate bodies (Hackstadt et al, 1996).…”

“…Within 15 min after infection of cultured HeLa cells by C. trachomatis serovar L2, several host proteins are found to be tyrosine phosphorylated (Birkelund et al, 1994). During the course of infection, Chlamydia-containing vacuoles redistribute from the cellular to the nuclear periphery in a F actin-dependent mechanism (Majeed and Kihlströ m, 1991) which is regulated by intracellular Ca 2þ (Majeed et al, 1993). Later, elementary bodies differentiate to reticulate bodies, which multiply, and eventually (40þ h after infection) the intracellular chlamydial vacuole(s) expand(s) to occupy nearly all intracellular space not already claimed by the nucleus or cellular organelles.…”