Endocytosis of chlamydiae by McCoy cells: measurement and effects of centrifugation

Abstract Chlamydia trachomatis strain 434 and C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) were compared for entry into McCoy cells and expression of productive infection (inclusion body formation). Entry was measured as the difference between extracellular cell-associated organisms, determined directly after fluorescence staining of live cells, and total cell-associated organisms (intracellular and extracellular); the latter were evaluated from radioactivity measurement and known particle: radioactivity ratios for stock radiolabelled suspensions. Under inoculation conditions of natural (spontaneous) infection, 69–82% of cell-associated organisms of both strains were internalised and entry was not enhanced by centrifugation of inocula with monolayers. For 434, inclusion bodies were seen in 10–20% of cells containing organisms and numbers were little influenced by mode of infection. For GPIC, productive infection initiated by centrifugation was comparable with that of 434 but some 15-fold reduced in spontaneous infection. The results suggest that unproductive infection by GPIC occurs, not because of defective entry, but from inhibition at an intracellular step which is circumvented when infection is initiated by centrifugation.     

Interaction of L Cells and Chlamydia psittaci: Entry of the Parasite and Host Responses to Its Development

The entry and development of Chlamydia psittaci in the L cell was studied by using purified, infectious parasites at high multiplicity. Entry of the parasite was accomplished by an act of phagocytosis by the host which was independent of an adsorption stage but was temperature-dependent. Kinetic studies of phagocytosis performed with (14)C-amino acid-labeled, purified parasites indicated that the rate of phagocytosis was directly proportional to the multiplicity of inoculation. Electron microscopy of cells infected at high multiplicity with purified infectious C. psittaci showed that phagocytosed chlamydiae were segregated in a host phagocytic vacuole throughout their developmental cycle which consisted of the transition of infecting elementary bodies to reticulate bodies dividing by binary fission, followed by the reemergence of a population of elementary bodies. The process of the transition was examined and a proposed sequence of intermediate bodies is presented. In isopycnic gradients of fractionated, infected L cells, chlamydial phagocytic vacuoles were apparent as a dense band distinct from lysosome and mitochondrion peaks, as indicated by acid phosphatase and cytochrome oxidase activities. Chlamydiae inactivated by heat or neutralized by antiserum were phagocytosed and appeared in lysosomes within 12 hr after infection according to electron microscopy; however, chlamydiae which were continuously inhibited in their development by chloramphenicol were retained intact in the cell for 24 hr without lysosomal response. The possibility of a lysosomal inhibitor on the native parasite is discussed.     

Effect of trypsinization on susceptibility of primary human amniotic cells toChlamydia trachomatis TW-3 strain

The effect of trypsinization of human amnion membranes on the susceptibility of amnion cells toChlamydia trachomatis TW-3 infection was examined by infectivity titrations using standard procedures of chlamydial inoculation, and detection of chlamydial inclusions. Epithelial cells derived from freshly trypsinized membranes as well as primary and secondary cultured cells that were freshly removed from monolayers by trypsin treatment were not susceptible to infection at 30 min and at 2 and 6 h after trypsinization. Monolayers grown 18 h and up to 5 or more days after trypsinization were susceptible to infection. Primary 5-day monolayers derived from each of nine placentas inoculated with chlamydiae showed a range of titers from 10⁻³ to 10^−6.5 (SD=1.2 logarithm). Primary monolayers supported the multiplication of chlamydiae to consistently higher titers than secondary and tertiary monolayers from the same amnion.     

The developmental cycle of Chlamydia trachomatis in McCoy cells treated with cytochalasin B.

The growth of a genital trachoma-inclusion conjunctivitis agent strain of Chlamydia trachomatis in McCoy cells treated with cytochalasin B was studied by quantitative infectivity estimations and by light and electron microscopy. Provided that infection of the monolayer was initiated by centrifuging the infectious particles on to the cells before incubation, this chlamydial strain grew as fast and to as high a titre [approximately 10(7) inclusion-forming units (i.f.u.) per culture] as those chlamydiae which infect cell cultures in vitro without centrifugation. Each i.f.u. inoculated yielded approximately 600 i.f.u., and extracellular infectivity was detected soon after intracellular infectivity appeared. Inclusions were recognized by fluorescent antibody staining techniques early in the developmental cycle when cultures were not infectious and when only reticulate bodies were seen by electron microscopy. Inclusions were recognized in Giemsa-stained preparations examined by dark ground microscopy only when elementary bodies appeared in the inclusions. Iodine staining was not a reliable indicator either of the number of inclusions present or of their infectivity.     

Type III secretion genes identify a putative virulence locus of Chlamydia

Four genes of Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), whose predicted products are highly homologous to structural and regulatory components of a contact-dependent or type III secretion apparatus, were isolated. Related to genes present in several animal and plant bacterial pathogens, these genes may represent a section of a previously undetected chromosomal virulence locus analogous to several recently described virulence-associated type III secretion loci. The existence of contact-dependent secretion in Chlamydia strongly suggests that these bacteria use pathogenic mechanisms that are similar to those of other intracellular bacterial pathogens. Unlike other intracellular bacteria, however, chlamydiae are metabolically inactive extracellularly and only become capable of global protein synthesis several hours after infection. This implies that chlamydial contact-dependent secretion is only active from within, uniquely after the bacteria have been internalized by eukaryotic cells. The possible role(s) of this pathway in chlamydial pathogenesis are discussed.     

Factors affecting the adhesion and uptake of an oculo-genital strain of Chlamydia trachomatis to McCoy cells

Abstract We have studied adhesion and uptake of C. trachomatis serovar E in McCoy cells under various infection conditions. Adhesion and uptake of chlamydiae was completed about 3 h after the initiation of stationary infection at 37°C, but ingestion of cell membrane-attached organisms was finished within 0.5 h at 37°C. Reincubated chlamydiae, not attached after 3 h at 37°C, attached readily to fresh McCoy cell monolayers, but to a lesser extent than the original inoculum. Our results indicate that the lack of further attachment after 3 h incubation at 37°C under stationary infection conditions has complex causes, involving both host cell and parasite. Centrifugation did not affect the uptake of chlamydiae already bound to the cell membrane, suggesting that the uptake phase of C. trachomatis serovar E by McCoy cells is unaffected by centrifugation.     

Trifluoperazine inhibits the infectivity of Chlamydia trachomatis for McCoy cells

Abstract Trifluoperazine (TFP), an inhibitor of the Ca²⁺-binding protein calmodulin, was used to study the infectivity of Chlamydia trachomatis for McCoy cells. TFP inhibited the number of chlamydial inclusions and the chlamydia-dependent amino acid incorporation when added within 9 h after inoculation with chlamydiae. However, TFP did not affect the attachment of chlamydiae to the cells or the protease-removable fraction of cell-bound chlamydiae.
These results suggest that an early step in the intracellular development of chlamydiae, partly coinciding with the elementary body-reticulate body conversion, is sensitive to TFP and that clathrin coats are not crucial in the ingestion of chlamydiae by McCoy cells.     

Invasiveness of Salmonella typhi strains in HeLa S3 monolayer cells

The internalization and intracellular multiplication, i.e., the invasiveness, of Salmonella typhi strains recently isolated from typhoid fever patients were confirmed in HeLa cell monolayers. When stained with Giemsa solution, intracellular bacteria were 0.6 X 1.2 micron in size and stained purple, whereas extracellular bacteria associated or not with the HeLa cell surface were 1.0 X 3.0 micron and stained deep blue. Strain GIFU 10007 was internalized into 23% of the HeLa cells within 10 min after inoculation. About 90% of the HeLa cells were infected after 24 hr incubation in kanamycin (KM)-containing medium. Intracellular multiplication of the challenge organism was verified by a large number of intracellular bacteria after 24 hr incubation in KM-containing medium by both light-microscopy of the Giemsa stained preparation and viable counts of intracellular bacteria. The viable counts of strain 10007 showed an increase of more than 40-fold within 24 hr after inoculation, whereas in the four other less or non-infective strains, recovery of viable bacteria was poor or nil. Strains which were highly invasive usually failed to show strong adhesion. The contribution of Vi antigen to the internalization of challenge organisms was not proved. Infective strains, when killed by formalin were still adhesive, but were not internalized. The same strains, when killed by boiling, were neither adhesive nor internalized. From these findings it was concluded that the internalization and multiplication of infective S. typhi strains in cultured HeLa cells should be regarded as an invasion rather than phagocytosis by host cells, and such invasiveness could be an indicator to estimate the virulence of S. typhi strains.     

Pinocytosis and intracellular degradation of exogenous protein: modulation by amino acids

Intracellular degradation of exogenous (serum) proteins provides a source of amino acids for cellular protein synthesis. Pinocytosis serves as the mechanism for delivering exogenous protein to the lysosomes, the major site of intracellular degradation of exogenous protein. To determine whether the availability of extracellular free amino acids altered pinocytic function, we incubated monolayers of pulmonary alveolar macrophages with the fluid-phase marker, [14C]sucrose, and we dissected the pinocytic process by kinetic analysis. Additionally, intracellular degradation of endogenous and exogenous protein was monitored by measuring phenylalanine released from the cell monolayers in the presence of cycloheximide. Results revealed that in response to a subphysiological level of essential amino acids or to amino acid deprivation, (a) the rate of fluid-phase pinocytosis increased in such a manner as to preferentially increase both delivery to and size of an intracellular compartment believed to be the lysosomes, (b) the degradation of exogenously supplied albumin increased, and (c) the fraction of phenylalanine derived from degradation of exogenous albumin and reutilized for de novo protein synthesis increased. Thus, modulation of the pinosome-lysosome pathway may represent a homeostatic mechanism sensitive to the availability of extracellular free amino acids.     

Low-nutrient induction of abnormal chlamydial development: A novel component of chlamydial pathogenesis?

Abstract The intracellular development of chlamydiae in McCoy cells incubated in Eagle's minimal essential medium lacking all 13 amino acids was examined both by fluorescence and electron microscopy and by infectivity titration. Aberrant development occurred in almost all inclusions of strains of Chlamydia trachomatis and C. psittaci with the production of abnormal forms which differed in size, shape and internal structure from normal reticulate and elementary body forms. Detailed analysis of the response of C. trachomatis L2 strain 434 to graded reductions in amino acid level showed that infectivity was reduced and morphological abnormality increased as amino acid concentrations were lowered from 33 to 0% of amino acids present in minimal essential medium. Reversion of inclusions to normal and reappearance of infectious forms occured on restoration of amino acids and further incubation. It is suggested that aberrant development may account for the presence in vivo of non-cultivable chlamydiae and that such development can arise via tryptophan deprivation mediated by local release of interferon gamma.     

Host modification of chlamydiae: differential infectivity for cell monolayers of chlamydiae grown in eggs and monolayers.

Cell monolayer-grown chlamydiae (CGO) differed from egg-grown organisms (EGO) in their increased spontaneous infectivity relative to centrifuge-assisted infectivity for monolayers. For each population spontaneous: centrifuge-assisted infectivity ratios were constant over a wide dose range. Spontaneous infection increased linearly with time and could not be exhausted from either population by prolonged adsorption; there was no change in infectivity ratios in residual supernatants. Further, one passage of EGO through monolayers gave CGO with stable infectivity properties not increased by further cell passage yet reverting on a single passage in eggs. Spontaneous infection of monolayers with EGO gave progeny with the same infectivity ratios as monolayers infected with EGO by centrifugation. The change in properties following EGO infection of monolayers occurred prior to natural release from cells. We conclude that EGO and CGO are two phenotypically distinct, homogeneous populations. The two infection modes are not properties of subpopulations within EGO and CGO. The relationship of these observations on chlamydiae to other possible host-imposed phenomena is considered.     

Endocytosis of an antibody ricin A-chain conjugate (immuno-A-toxin) adsorbed on colloidal gold. Effects of ammonium chloride and monensin

An immunotoxin (IT) formed by a specific antibody coupled to the ricin A chain was adsorbed on colloidal gold particles (IT-Au). Binding and internalization of IT-Au in human lymphoblastic CEM cells were studied using electron microscopy. IT-Au showed specific cytotoxic activity toward the target cells. After 1 h at 4 degrees C, IT-Au were linked diffusely to the plasma membrane with 45% of the particles regrouped in clusters. Upon transfer to 37 degrees C, the particles carrying the ligand were regrouped more frequently and internalized into the cell by endocytosis through smooth microinvaginations or coated pits of the plasma membrane. After 15 min, IT-Au was observed in endocytic vacuoles, or receptosomes, in tubular structure near the Golgi apparatus and in lysosomes. Entry of IT-Au into lysosomes was rapid (around 50% of intracellular IT-Au particles after 30 min). NH4Cl or monensin, well-known potentiators of immunotoxin activity, when present in incubation medium, altered neither the processes nor the rate of IT-Au endocytosis. In the presence of either of these substances, IT-Au accumulated in the normal or often enlarged endocytic vacuoles, and entry into the lysosomes was slowed down (50% of particles after 2 h 15 min). We conclude that this intense slowing-down in the speed of IT-Au transportation into lysosomes and the functional modifications of these organelles help to explain the increased efficacy of immunotoxins in the presence of potentiators.     

Lipid raft-dependent uptake, signalling and intracellular fate of Porphyromonas gingivalis in mouse macrophages

Lipid rafts are cholesterol-enriched microdomains involved in cellular trafficking and implicated as portals for certain pathogens. We sought to determine whether the oral pathogen Porphyromonas gingivalis enters macrophages via lipid rafts, and if so, to examine the impact of raft entry on its intracellular fate. Using J774A.1 mouse macrophages, we found that P. gingivalis colocalizes with lipid rafts in a cholesterol-dependent way. Depletion of cellular cholesterol using methyl-beta-cyclodextrin resulted in about 50% inhibition of P. gingivalis uptake, although this effect was reversed by cholesterol reconstitution. The intracellular survival of P. gingivalis was dramatically inhibited in cholesterol-depleted cells relative to untreated or cholesterol-reconstituted cells, even when infections were adjusted to allow equilibration of the initial intracellular bacterial load. P. gingivalis thus appeared to exploit raft-mediated uptake for promoting its survival. Consistent with this, lipid raft disruption enhanced the colocalization of internalized P. gingivalis with lysosomes. In contrast, raft disruption did not affect the expression of host receptors interacting with P. gingivalis, although it significantly inhibited signal transduction. In summary, P. gingivalis uses macrophage lipid rafts as signalling and entry platforms, which determine its intracellular fate to the pathogen's own advantage.     

Regulation of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) growth in McCoy cells by amino acid antagonism

Chlamydiae have amino acid requirements for growth in tissue culture as defined by those amino acids whose individual omission from the growth medium prevents chlamydial multiplication. We have tested the hypothesis that this inhibition of growth arises as a result of antagonism between particular amino acids such that inhibition occurs when the concentration of one amino acid is reduced in the presence of the antagonist amino acid at high concentration. Using the Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), in the presence of cycloheximide, the requirement for valine was abrogated by the simultaneous omission of isoleucine, that for phenylalanine by simultaneous omission of tryptophan and that for leucine by simultaneous omission of isoleucine plus valine. The antagonism shown between leucine and isoleucine plus valine appears to be unique among bacteria. In the absence of cycloheximide, GPIC had an additional need for tryptophan, tyrosine and isoleucine; these amino acid requirements were shown for both infected McCoy, HeLa and BHK cells. The results are consistent with a mechanism for regulation of parasite growth which depends on the balance of amino acid concentrations in the extracellular environment.     

Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts

We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.     

Unique ultrastructure in the elementary body of Chlamydia sp. strain TWAR.

The ultrastructure of two prototype strains (TW-183 and AR-39) of Chlamydia sp. strain TWAR was described. The TWAR elementary body (EB) demonstrated a unique morphology and structure distinct from those of other chlamydial organisms. It was pleomorphic but typically pear shaped. The average size was 0.38 micron, with a long axis of 0.44 micron, a short axis of 0.31 micron, and a ratio of the long to the short axes of 1.42. The cytoplasmic mass was round, with an average diameter of 0.24 micron. There was a large periplasmic space. Small, round electron-dense bodies (0.05 micron in diameter), which were attached to the cytoplasm by a stringlike structure, were seen in the periplasmic space. These features are in contrast to those of other chlamydiae, which are typically round with a narrow or barely discernible periplasmic space. The TWAR reticulate body (RB) was morphologically and structurally similar to those of other Chlamydia species, having an average diameter of 0.51 micron and being circular in shape. The ultrastructural observations of the intracellular growth of TWAR in HeLa cells revealed that TWAR underwent the same developmental cycle as do other chlamydiae, i.e., transformation of EB to RB, multiplication by binary fission, and maturation by transformation of RB to EB via the intermediate-form stage.     

Chlamydia species-dependent differences in the growth requirement for lysosomes

Genome reduction is a hallmark of obligate intracellular pathogens such as Chlamydia, where adaptation to intracellular growth has resulted in the elimination of genes encoding biosynthetic enzymes. Accordingly, chlamydiae rely heavily on the host cell for nutrients yet their specific source is unclear. Interestingly, chlamydiae grow within a pathogen-defined vacuole that is in close apposition to lysosomes. Metabolically-labeled uninfected host cell proteins were provided as an exogenous nutrient source to chlamydiae-infected cells, and uptake and subsequent labeling of chlamydiae suggested lysosomal degradation as a source of amino acids for the pathogen. Indeed, Bafilomycin A1 (BafA1), an inhibitor of the vacuolar H(+)/ATPase that blocks lysosomal acidification and functions, impairs the growth of C. trachomatis and C. pneumoniae, and these effects are especially profound in C. pneumoniae. BafA1 induced the marked accumulation of material within the lysosomal lumen, which was due to the inhibition of proteolytic activities, and this response inhibits chlamydiae rather than changes in lysosomal acidification per se, as cathepsin inhibitors also inhibit the growth of chlamydiae. Finally, the addition of cycloheximide, an inhibitor of eukaryotic protein synthesis, compromises the ability of lysosomal inhibitors to block chlamydial growth, suggesting chlamydiae directly access free amino acids in the host cytosol as a preferred source of these nutrients. Thus, chlamydiae co-opt the functions of lysosomes to acquire essential amino acids.     

Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain guinea pig inclusion conjunctivitis

The infectious cycle of phiCPG1, a bacteriophage that infects the obligate intracellular pathogen, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis, was observed using transmission electron microscopy of phage-hyperinfected, Chlamydia-infected HeLa cells. Phage attachment to extracellular, metabolically dormant, infectious elementary bodies and cointernalisation are demonstrated. Following entry, phage infection takes place as soon as elementary bodies differentiate into metabolically active reticulate bodies. Phage-infected bacteria follow an altered developmental path whereby cell division is inhibited, producing abnormally large reticulate bodies, termed maxi-reticulate bodies, which do not mature to elementary bodies. These forms eventually lyse late in the chlamydial developmental cycle, releasing abundant phage progeny in the inclusion and, upon lysis of the inclusion membrane, into the cytosol of the host cell. Structural integrity of the hyperinfected HeLa cell is markedly compromised at late stages. Released phage particles attach avidly to the outer leaflet of the outer membranes of lysed and unlysed Chlamydiae at different stages of development, suggesting the presence of specific phage receptors in the outer membrane uniformly during the chlamydial developmental cycle. A mechanism for phage infection is proposed, whereby phage gains access to replicating chlamydiae by attaching to the infectious elementary body, subsequently subverting the chlamydial developmental cycle to its own replicative needs. The implications of phage infection in the context of chlamydial infection and disease are discussed.     

Binding, internalization, and intracellular localization of interleukin-1 beta in human diploid fibroblasts

This study demonstrates internalization of interleukin-1 (IL-1) via its cell surface receptor on human diploid fibroblasts and shows intracellular localization of IL-1 beta. Binding experiments at 8 degrees C using confluent fibroblast monolayers revealed 5,000-15,000 IL-1 receptors/cell that bound both IL-1 alpha and IL-1 beta. Incubation of monolayers with 125I-IL-1 beta (10(-9) M) at 8 degrees C and then at 37 degrees C for various times up to 8 h revealed a t1/2 for internalization of receptor-bound IL-1 beta of about 1.5 h. In addition, it was shown that IL-1 beta internalized via receptors was undegraded and retained binding activity. Electron microscopic autoradiography of monolayers incubated with 125I-IL-1 beta, as above, showed a progressive increase in the ratio of cytoplasmic to cell surface-associated grains. Grains at the cell surface were primarily localized at cell processes or attachment sites, frequently close to intra- and extracellular filamentous material. During incubation at 37 degrees C, most grains were free in the cytoplasm, with few present in lysosomes or vesicles. After 1 h, approximately 15% of the grains were over nuclei. Control cultures incubated at 37 degrees C with 125I-IL-1 beta and 100-fold excess unlabeled IL-1 beta showed increased uptake of label into lysosomes and little into nuclei. This study shows that IL-1 receptors are primarily located at fibroblast processes and that receptor-mediated internalization of the ligand is slow. Nuclear localization apparently requires IL-1 receptor-specific internalization of IL-1 beta, suggesting a possible role for this process in eliciting the IL-1 signal.     

Differential neutralization of spontaneous and centrifuge-assisted chlamydial infectivity

Neutralization by specific antibody of a fast-killing variant strain of Chlamydia trachomatis, which showed high spontaneous infectivity for cell monolayers, was examined. It appeared that in spontaneous infection antibody-treated chlamydiae were neutralized by inhibition of attachment to cells. Centrifugation imposed a different effect: infection was inhibited at some step at or subsequent to attachment.