Purification of Rickettsia tsutsugamushi by Percoll density gradient centrifugation

Purification of Rickettsia tsutsugamushi has been achieved by Percoll density gradient centrifugation. The microorganisms purified showed good retention of infectivity and intracellular morphology. Budding rickettsiae in the egressing stage and intracellular rickettsiae in the multiplying process were harvested separately and purified by this technique. In electron microscopic observations, the intracellular rickettsiae obtained were surrounded with double membrane-layers of cell wall and cell membrane, and the budding rickettsiae were enveloped with an additional outermost membrane which may have originated from host cell membrane obtained in the budding process.     

Growth pattern of Rickettsia tsutsugamushi in irradiated L cells.

Irradiated L cells infected with Rickettsia tsutsugamushi were studied under the electron microscope to define the morphological growth pattern of the organism. For 2 days after inoculation, no rickettsiae were found either extra- or intracellularly; after 2 days multiple rickettsiae appeared within the host cells without morphological evidence of their entry. These observations showed that the rickettsiae within the cell were assembled in situ by segregation of portions of the granular cytoplasm and subsequent internal differentiation and surface membrane assembly of the segregated bodies. The protoplasmic (P) bodies, which seemed to be formed by shedding infected-cell granular cytoplasm, consistently appeared on the surface and within the phagosomes of the host cells. Rickettsiae were occasionally seen entering host cells in the later phase of infection; these were apparently the ones assembled within the P bodies. This suggested that the P bodies, and not the rickettsiae, were the major infectious particles that transmitted the rickettsial genetic substance among the host cells. On the basis of the present morphological observations, viral-type multiplication for R. tsutsugamushi is proposed.     

Penetration of Rickettsia tsutsugamushi into cultured mouse fibroblasts (L cells): an electron microscopic observation

The mechanism of penetration of purified Rickettsia tsutsugamushi (Gilliam strain) into cultured mouse fibroblasts (L cells) was examined by electron microscopy. After 10-40 min of infection, rickettsiae in the process of being phagocytized were often seen on the cell surface. These were restricted to the rickettsiae which seemed to be intact in morphology, while heavy plasmolyzed ones were never phagocytized. Additionally, rickettsiae were taken up individually into a phagosome, and phagocytosis of several rickettsiae together was rarely observed, except in the case of heat-inactivated microorganisms. In the cells, phagosomes whose membranes enclosed rickettsiae either tightly or loosely were seen. Rickettsiae in the loose phagosomes often showed signs of plasmolysis and were rarely released into the cell cytoplasm. Partial disintegration of phagosomal membranes and the escape of rickettsiae from the phagosomes were seen only in tight phagosomes. Large phagosomes containing a clump of several rickettsiae were observed occasionally, in which case the microorganisms were deformed and seemed to be denatured. From the above observations and the frequency of appearance of these different penetration stages in the specimens 10, 20, and 40 min after infection, it was concluded that the rickettsiae enter initially into a tight phagosome by phagocytosis and are then released into the cell cytoplasm by disruption of the phagosomal membrane. No other mechanisms of penetration were found. On the other hand, rickettsiae inactivated by trypsin did not attach to host cells. Inactivation by heat or UV irradiation resulted in reduction of phagocytosis, and rickettsiae treated with rifamycin could penetrate into the host cell cytoplasm to the same extent as in the case of infection with intact rickettsiae.     

Interaction of Rickettsia akari with the host cell in vitro: multiplication, formation of spheroplast-like forms and its destruction in phagolyosomes

The electron microscopic study of the interaction of R. akari, strain CK, with the monolayer culture of L-cells was made 4 days after inoculation. Rickettsiae multiplied by transverse binary fission immediately in the cytoplasm of the cells and left the cells by gemmation, surrounded with plasmolemma and a fragment of the host cytoplasm. Alongside with multiplying rickettsiae, spheroplast-like rickettsiae and rickettsiae at the stage of destruction were regularly observed in phagolysosomes. The authors suggest that the normal interaction of rickettsiae with the host cell may be realized by three following routes (1) reproduction, (2) destruction in phagolysosomes and (3) formation of altered (anomalous) forms. The ability of the vegetative forms of rickettsiae and chlamydiae to yield spheroplast-like forms (the initial phase of bacterial L-transformation) indicates that these organisms are similar to bacteria and cannot be themselves regarded as L-forms.     

Epitheliocystis disease in the striped bass Morone saxatilis from the Chesapeake Bay.

Epitheliocystis disease in the gills of the striped bass Morone saxatilis from Chesapeake Bay was studied using light and electron microscopy. The epitheliocystis infection appeared synchronous in that all capsules on a single host were at the same stage of development. The disease appeared to begin in a single cell on the gill lamella, which gradually enlarged to form a large cyst, encapsulated by a thick cellular capsule of epithelial origin. The epitheliocystis inclusion was filled with cells of the general morphology and size of rickettsiae; however, the infection was atypical for rickettsiae in that the cells had a dense central nucleoid region and they developed within an inclusion separated from the host cytoplasm.     

Dynamic remodeling of the endosomal system during formation of Salmonella-induced filaments by intracellular Salmonella enterica

The infection by Salmonella enterica results in the massive remodeling of the endosomal system of eukaryotic host cells. One unique consequence is the formation of long tubular endosomal compartments, so-called Salmonella-induced filaments (SIF). Formation of SIF requires the function of type III secretion system and is a requirement of efficient intracellular proliferation of Salmonella. Using high-resolution live cell imaging approaches and electron microscopy, we report for the first time the highly dynamic characteristics of SIF and their ultrastructural properties. In the early phase of infection (4-5 h), SIF display highly dynamic properties in various types of host cells. SIF extend, branch and contract rapidly, and a stabilized network of SIF is formed later (>or=8 h after infection). The velocities of SIF extension and contraction in the different phases of infection were quantified. Our observations lead to novel models for the modification of host cell transport processes by virulence factors of intracellular Salmonella.     

Characterization and growth of polymorphic Rickettsia felis in a tick cell line

Morphological differentiation in some arthropod-borne bacteria is correlated with increased bacterial virulence, transmission potential, and/or as a response to environmental stress. In the current study, we utilized an in vitro model to examine Rickettsia felis morphology and growth under various culture conditions and bacterial densities to identify potential factors that contribute to polymorphism in rickettsiae. We utilized microscopy (electron microscopy and immunofluorescence), genomic (PCR amplification and DNA sequencing of rickettsial genes), and proteomic (Western blotting and liquid chromatography-tandem mass spectrometry) techniques to identify and characterize morphologically distinct, long-form R. felis. Without exchange of host cell growth medium, polymorphic R. felis was detected at 12 days postinoculation when rickettsiae were seeded at a multiplicity of infection (MOI) of 5 and 50. Compared to short-form R. felis organisms, no change in membrane ultrastructure in long-form polymorphic rickettsiae was observed, and rickettsiae were up to six times the length of typical short-form rickettsiae. In vitro assays demonstrated that short-form R. felis entered into and replicated in host cells faster than long-form R. felis. However, when both short- and long-form R. felis organisms were maintained in cell-free medium for 12 days, the infectivity of short-form R. felis was decreased compared to long-form R. felis organisms, which were capable of entering host cells, suggesting that long-form R. felis is more stable outside the host cell. The relationship between rickettsial polymorphism and rickettsial survivorship should be examined further as the yet undetermined route of horizontal transmission of R. felis may utilize metabolically and morphologically distinct forms for successful transmission.     

Experimental selection of long‐term intracellular mycobacteria

Some intracellular bacteria are known to cause long‐term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long‐term intracellular lifestyle. Selected Mycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re‐infection and also with the specific expression of stress‐ and survival‐related genes. Our findings identify bacterial traits implicated in the establishment of long‐term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments.     

Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity

Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.     

Susceptibility of Rickettsia monacensis and Rickettsia peacockii to Cecropin A, Ceratotoxin A, and Lysozyme

Ticks host obligate intracellular bacteria that range from benign symbiotes to virulent human pathogens. The effects on those bacteria of antimicrobial peptides (AMPs) involved in arthropod innate immunity to microbial infections are largely unknown. We evaluated effects of AMPs and a c-type lysozyme on host cell-free suspensions of the tick symbiotes Rickettsia monacensis and Rickettsia peacockii with stain-based infectivity and viability assays. Cecropin A at a concentration of 8 μM had a lethal effect on both rickettsiae while ceratotoxin A was approximately 20-fold less effective. Toxicity of both AMPs was synergized by lysozyme, an enzyme expressed by ticks. Lactoferrin, a transferrin, had no effect on R. monacensis at up to 110 μM. The rickettsiae were less sensitive to the AMPs than is typical of bacteria that grow extracellularly. Our assays may be useful in the study of AMP activity against other obligate intracellular bacteria.     

Agglutination of Trypanosoma cruzi in infected cells treated with serum from chronically infected mice

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. The chronic stage of infection is characterized by a production of neutralizing antibodies in the vertebrate host. A polyclonal antibody, anti-egressin, has been found to inhibit egress of parasites from the host cell late in the intracellular cycle, after the parasites have transformed from the replicative amastigote into the trypomastigote. It has also been found that BALB/c mouse fibroblasts in the late stages of parasite infection become permeable to molecules as large as antibodies, leading to the possibility that anti-egressin affects the intracellular parasites. This project addresses the fate of the intracellular trypomastigotes that have been inhibited from egressing the host cell. Extended cultures of infected fibroblasts treated with chronic mouse serum reduced parasite egress at all time points measured. Parasites released from infected fibroblasts treated with chronic serum had a reduced ability to infect fibroblasts in culture, yet did not lose infectivity entirely. Absorption of chronic serum with living trypomastigotes removed the anti-egressin effect. The possibility that the target of anti-egressin is a parasite surface component is further indicated by the agglutination of extracellular trypomastigotes by chronic serum. The possibility that cross-linking by antibody occurs intracellularly, thus inhibiting egress, was reinforced by cleaving purified IgG into Fab fragments, which did not inhibit egress when added to infected cultures. From this work, it is proposed that the current, best explanation of the mechanism of egress inhibition by anti-egressin is intracellular agglutination, preventing normal parasite-driven egress.     

Chlamydia attachment to mammalian cells requires protein disulfide isomerase

For Chlamydia, an intracellular pathogen of humans, host cell invasion is obligatory for survival, growth and pathogenesis. At the molecular level, little is known about the binding and entry of Chlamydia into the mammalian host cell. Chlamydia are genetically intractable therefore experimental approaches targeting the host are often necessary. CHO6 is a mutagenized cell line resistant to attachment and infection by Chlamydia. In this study, CHO6 was shown using proteomic methods to have a defect in processing of the leader sequence for protein disulfide isomerase (PDI). Complementation by expression of full-length PDI restored C. trachomatis binding and infectivity in the CHO6 mutant cell line. The cell line was also resistant to diphtheria toxin and required complemented cell-surface PDI for toxin entry. These data demonstrate that native PDI at the cell surface is required for effective chlamydial attachment and infectivity.     

Life Cycle of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Plasmodiphora brassicae is a soil-borne obligate parasite. The pathogen has three stages in its life cycle: survival in soil, root hair infection, and cortical infection. Resting spores of P. brassicae have a great ability to survive in soil. These resting spores release primary zoospores. When a zoospore reaches the surface of a root hair, it penetrates through the cell wall. This stage is termed the root hair infection stage. Inside root hairs the pathogen forms primary plasmodia. A number of nuclear divisions occur synchronously in the plasmodia, followed by cleavage into zoosporangia. Later, 4–16 secondary zoospores are formed in each zoosporangium and released into the soil. Secondary zoospores penetrate the cortical tissues of the main roots, a process called cortical infection. Inside invaded roots cells, the pathogen develops into secondary plasmodia which are associated with cellular hypertrophy, followed by gall formation in the tissues. The plasmodia finally develop into a new generation of resting spores, followed by their release back into soil as survival structures. In vitro dual cultures of P. brassicae with hairy root culture and suspension cultures have been developed to provide a way to nondestructively observe the growth of this pathogen within host cells. The development of P. brassicae in the hairy roots was similar to that found in intact plants. The observations of the cortical infection stage suggest that swelling of P. brassicae-infected cells and abnormal cell division of P. brassicae-infected and adjacent cells will induce hypertrophy and that movement of plasmodia by cytoplasmic streaming increases the number of P. brassicae-infected cells during cell division.     

Early development ofRhizobium-induced root nodules ofParasponia rigida. I. Infection and early nodule initiation

Summary The first of two major steps in the infection process in roots ofParasponia rigida (Ulmaceae) following inoculation byRhizobium strain RP501 involves the invasion ofRhizobium into the intercellular space system of the root cortex. The earliest sign of root nodule initiation is the presence of clumps of multicellular root hairs (MCRH), a response apparently unique amongRhizobium-root associations. At the same time or shortly after MCRH are first visible, cell divisions are initiated in the outer root cortex of the host plant, always subjacent to the MCRH. No infection threads were observed in root hairs or cortical cells in early stages. Rhizobial entry through the epidermis and into the root cortex was shown to occur via intercellular invasion at the bases of MCRH. The second major step in the infection process is the actual infectionper se of host cells by the rhizobia and formation of typical intracellular infection threads with host cell accommodation. This infection step is probably the beginning of the truly symbiotic relationship in these nodules. Rhizobial invasion and infection are accompanied by host cortical cell divisions which result in a callus-like mass of cortical cells. In addition to infection thread formation in some of these host cortical cells, another type of rhizobial proliferation was observed in which large accumulations of rhizobia in intercellular spaces are associated with host cell wall distortion, deposition of electron-dense material in the walls, and occasional deleterious effects on host cell cytoplasm.     

Ultrastructural morphology suggesting a new hypothesis for development of microsporidians seen in Loma salmonae infecting the gills of rainbow and brook trout

In two variants of Loma salmonae that have specificity for rainbow trout Oncorhynchus mykiss (OA variant) and specificity to brook trout Salvelinus fontinalis (SV variant), the parasitophorous vacuole forms at the onset of sporogony. In the OA variant the merogonial stage is bound by a single plasma membrane in direct contact with host cytoplasm. The parasitophorous vacuole formation is initiated by the host cell surrounding the merogonial stages with endoplasmic reticulum (ER) as occurs in autophagy. Of the two host ER membranes surrounding the parasite, one remains in close association with the plasma membrane of the meront, while the other forms the limiting membrane of the vacuole. The sporogonial stage is bounded by two closely apposed membranes, giving the appearance of a thick electron dense plasmalemma. The observations from this study support the novel hypothesis that this microsporidian uses the intracellular process of autophagy to aid formation of a parasitophorous vacuole. The morphology of the SV‐variant is consistent with that of the OA‐variant suggesting that it uses the same mechanism for development.     

Scanning Electron Microscopic Observation of Differentiation from the Reproductive Short Form to the Infectious Long Form of Holospora obtusa

To identify the surface features of Holospora obtusa during its differentiation from the reproductive short form to the infectious long form, bacteria of four different buoyant densities were isolated by Percoll density gradient centrifugation of homogenates of host cells or isolated macronuclei, and examined with a scanning electron microscope. Bacteria of buoyant density 1.09 g/ml were reproductive short forms as well as cells at various stages in the elongation process including fully elongated ones. Bacteria of buoyant densities 1.11 g/ml and 1.13 g/ml were premature long forms and those of 1.16 g/ml were mature infectious long forms. Bacteria of buoyant density 1.09 g/ml had an entirely rough surface while those of buoyant densities 1.11 g/ml and 1.13 g/ml were smooth and had wale-like stripes on their surface. A small tapered tip was observed at one end of the bacteria of buoyant density 1.13 g/ml. Bacteria of buoyant density 1.16 g/ml had an entirely smooth surface, but one end always showed a rough surface; this locally differentiated surface of the special tip of the infectious long form may be responsible for both the nuclear and species specificities of the infectivity of H. obtusa. These observations indicate that the surface of H. obtusa changes during differentiation and the special tip develops in bacteria of buoyant density 1.13 g/ml.     

Electron microscope studies on the host cell energy requirement for injection of PL-1 phage DNA intoLactobacillus casei

The process of genome DNA injection, after adsorption, by phage PL-1 intoLactobacillus casei ATCC 27092 was monitored by electron microscopy. The DNA injection depended on the incubation temperature, and the apparent activation energy was about 11 kcal. It was inhibited when the cells had been previously starved, where their intracellular ATP contents was lowered less than one-hundredth that of the unstarved cells. There was a good correlation between the ATP contents of cells and the extent of the phage DNA injection. Dicyclohexyl carbodiimide inhibited the process with little effect both on the viability of cells and the infectivity of phages. These results agreed with the view that a high energy level of the host cells would be required for the formation of blender-resistant phage-cell complexes to complete injection of phage DNA into host cells.     

Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity

In response to virus infection, cells can alter protein expression to modify cellular functions and limit viral replication. To examine host protein expression during infection with human cytomegalovirus (HCMV), an enveloped DNA virus, we performed a semiquantitative, temporal analysis of the cell surface proteome in infected fibroblasts. We determined that resident low density lipoprotein related receptor 1 (LRP1), a plasma membrane receptor that regulates lipid metabolism, is elevated early after HCMV infection, resulting in decreased intracellular cholesterol. siRNA knockdown or antibody-mediated inhibition of LRP1 increased intracellular cholesterol and concomitantly increased the infectious virus yield. Virions produced under these conditions contained elevated cholesterol, resulting in increased infectivity. Depleting cholesterol from virions reduced their infectivity by blocking fusion of the virion envelope with the cell membrane. Thus, LRP1 restricts HCMV infectivity by controlling the availability of cholesterol for the virion envelope, and increased LRP1 expression is likely a defense response to infection.     

Macrophages in resistance to rickettsial infection: macrophage activation in vitro for killing of Rickettsia tsutsugamushi.

Rickettsia tsutsugamushi, strain Gilliam, replicates in cultures of resident peritoneal macrophages from BALB/c mice. Macrophage cultures treated with culture supernatants of spleen cells from rickettsial-infected mice stimulated with heat-killed rickettsiae markedly suppressed macrophage infection by rickettsiae. Rickettsiacidal activity of activated macrophages was dependent upon both lymphokine concentration and time of incubation in lymphokines. Treatment of macrophage cultures with lymphokines before exposure to viable rickettsiae resulted in an immediate decrease in percent macrophages infected and numbers of viable intracellular rickettsiae. In these cultures, enhanced intracellular killing was also apparent with further incubation (24 hr). The immediate effect of lymphokine-pretreated macrophages was dissociated from intracellular killing by infecting macrophage cultures first and adding lymphokines after infection. In these cultures, both percent macrophages infected and titers of viable intracellular rickettsiae were dramatically reduced as well.     

Fluorescence Activated Cell Sorting of Rickettsia prowazekii-Infected Host Cells Based on Bacterial Burden and Early Detection of Fluorescent Rickettsial Transformants

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens.