The endosymbiotic bacterium Holospora obtusa enhances heat-shock gene expression of the host Paramecium caudatum

The bacterium Holospora obtusa is a macronuclear-specific symbiont of the ciliate Paramecium caudatum. H. obtusa-bearing paramecia could survive even after the cells were quickly heated from 25 degrees C to 35 degrees C. To determine whether infection with H. obtusa confers heat shock resistance on its host, we isolated genes homologous to the heat shock protein genes hsp60 and hsp70 from P. caudatum. The deduced amino acid sequences of both cDNAs were highly homologous to hsp family sequences from other eukaryotes. Competitive PCR showed that H. obtusa-free paramecia expressed only trace amounts of hsp60 and hsp70 mRNA at 25 degrees C, but that expression of hsp70 was enhanced immediately after the cells were transferred to 35 degrees C. H. obtusa-bearing paramecia expressed high levels of hsp7O mRNA even at 25 degrees C and the level was further enhanced when the cells were incubated at 35 degrees C. In contrast, the expression pattern of hsp60 mRNA was the same in H. obtusa-bearing as in H. obtusa-free paramecia. These results indicate that infection with its endosymbiont can confer a heat-shock resistant nature on its host cells.     

Co-infection of the macronucleus of Paramecium caudatum by free-living bacteria together with the infectious Holospora obtusa

Infection experiments were performed incubating Paramecium caudatum with non-infectious free-living bacteria or weakly infectious intracellular bacteria together with the infectious Holospora obtusa. Two of four non-infectious free-living bacteria (Enterobacter aerogenes and Klebsiella pneumoniae) were found to get into the nuclei when added to Paramecium together with H. obtusa. The endonuclear bacterium Nonospora macronucleata that is weakly infectious by itself increases its infectivity when presented together with the infectious holosporas. The results provide evidence that H. obtusa may facilitate entry of other, non-infectious bacteria into the nuclei of Paramecium.     

Fate of the 63-kDa periplasmic protein of the infectious form of the endonuclear symbiotic bacterium Holospora obtusa during the infection process

Holospora obtusa is a macronucleus-specific endosymbiotic bacterium of the ciliate Paramecium caudatum. We report the secretion of a 63-kDa periplasmic protein of an infectious form of the bacterium into the macronucleus of its host. Indirect immunofluorescence microscopy with five monoclonal antibodies against the 63-kDa protein demonstrated that, soon after the bacterial invasion into the host macronucleus, the protein was detected in the infected macronucleus and that levels of the protein increased dramatically within one day of infection. The use of inhibitors for host and bacterial protein synthesis illustrated that, in early infection of H. obtusa, not only the pre-existing but also a newly synthesized 63-kDa protein was secreted into the host macronucleus. A partial amino acid sequence of the protein was determined, and a gene encoding the 63-kDa protein was cloned. The deduced amino acid sequence shows that this protein is a novel protein.     

Changes in fine structure and polypeptide pattern during development of Holospora obtusa, a bacterium infecting the macronucleus of Paramecium caudatum.

The development of the bacterium Holospora obtusa, which infects the macronucleus of Paramecium caudatum, was investigated in the course of a new infection from the infectious form into the reproductive form and vice versa. In parallel with a complete structural reorganization of the bacterium, the protein pattern changed gradually in this development. During the differentiation of the infectious form into the reproductive form, the voluminous periplasm was gradually reduced and the cytoplasm expanded, until the entire bacterium was filled by the cytoplasm. At this stage the long cell divided into five to seven short cells and thereby established the reproductive form, the main stage of the bacterium being maintained and multiplying in the host nucleus. In parallel with the reduction of the periplasm, some of the main proteins of the infectious form gradually disappeared in the electrophoresis pattern; some proteins disappeared earlier than others. Simultaneously, other proteins appeared and gradually became more prominent in the pattern of the developing reproductive form. In the reverse development, when the reproductive form differentiated into the infectious form, the bacterium grew longer, the cytoplasm was condensed, and electron-dense material was deposited in the extending periplasmic space. In parallel with this morphological development, the polypeptide pattern reverted to that of the infectious form.     

Monoclonal Antibody to a Bacterial Endonuclear Symbiont Holospora Cross Reacts with Proteins of Contractile Vacuole Radial Canals of Paramecium Species

ABSTRACT A monoclonal antibody (mAb) IR-2-1 was raised against a 67-kDa protein purified from the macronucleus-specific bacterial symbiont Holospora obtusa of Paramecium caudatum. The mAb was found to react with two bands (31 and 67-kDa) on gels of H. obtusa. Indirect immunofluorescence microscopy showed that these antigens were distributed inside the cells. However, unexpectedly, this mAb also cross reacted with the radial arms of the contractile vacuole in P. caudatum, P. tetraurelia, P. multimicronucleatum, P. jenningsi and P. bursaria as well as with their cytoplasm. Immunoelectron microscopy showed that the antigens were located on the decorated spongiome of the radial arms. In immunoblots, mAb IR-2-1 reacted with a band of 67 kDa in all Paramecium species examined. However, no band appeared in the immunoblot of isolated macronuclei of H. obtusa-free P. caudatum and no label was seen in the nuclear matrix of the macronucleus of air-dried P. caudatum. These results suggest that the 67-kDa antigen found in H. obtusa was not imported from the host macronucleus and the same antigen in the host contractile vacuoles and cytoplasm were not derived from the symbiont. These results also showed that an epitope on the decorated spongiome of the Paramecium species is shared by its bacterial symbiont. In contrast to the decorated tubule-specific mAb, DS-1, the antigens for IR-2-1 appeared to be loosely membrane bound as they were lost in paraformaldehyde fixed and acetone permeabilized Paramecium. Supplementary key words. Contractile vacuole complexes, Holospora obtusa, monoclonal antibody, Paramecium.     

The Release of Holospora obtusa from Paramecium caudatum Observed with a New Device for Extended In Vivo Microscopy

The macronudeus-specilic bacterium Holospora obtusa is released from its host cell Paramecium caudatum generally after a preceding cell division. Before division, most infectious forms of the bacteria can be found in a connecting strand of the dividing macronucleus. This connecting strand is deformed and finally expelled as a whole from the daughter cells. This process was observed and photographed with a simple device allowing observations for a long lime under a microscope without damage to the living cells. Key words. Bacterium, cytoproct, defecation, infection, macronucleus, symbiosis.     

Initial steps of infection of the ciliate Paramecium with bacteria Holospora sp

New light and electron microscope data on the initial steps of endocytobiosis establishment between the ciliate Paramecium and specific intranuclear bacteria Holospora are provided. At the cytoplasmic step of infection bacteria of all Holospora species are found in a vesicle originating from the membrane of the host cell phagosome. The association between host cell microfilaments and the bacterium bearing vesicle may suggest a possible involvement of the ciliate cytoskeleton in the transportation of bacteria to the host cell nucleus. The authors subdivide the process of infection into 6 steps. Some strains of P. caudatum never take up infectious Holospora bacteria in the course of phagocytosis.     

Effects of antibiotics on the early infection process of a macronuclear endosymbiotic bacterium Holospora obtusa of Paramecium caudatum

We examined the effects of antibiotics involved in bacterial DNA, RNA and protein synthesis and host protein synthesis on the early infection process of the bacterium Holospora obtusa, a macronucleus-specific symbiont of the ciliate Paramecium caudatum. Infection of the host macronucleus by the bacterium was not inhibited by mitomycin C, rifampicin and chloramphenicol. However, ingestion of the bacterium into the host digestive vacuoles and escape of the bacterium from the vacuoles to the host cytoplasm were significantly arrested with emetine. The results suggest that newly synthesized host proteins play an important role in the early infection process.     

Temperature-dependent transmission and latency of Holospora undulata, a micronucleus-specific parasite of the ciliate Paramecium caudatum

Transmission of parasites to new hosts crucially depends on the timing of production of transmission stages and their capacity to start an infection. These parameters may be influenced by genetic factors, but also by the environment. We tested the effects of temperature and host genotype on infection probability and latency in experimental populations of the ciliate Paramecium caudatum, after exposure to infectious forms of its bacterial parasite Holospora undulata. Temperature had a significant effect on the expression of genetic variation for transmission and maintenance of infection. Overall, low temperature (10 degrees C) increased levels of (multiple) infection, but arrested parasite development; higher temperatures (23 and 30 degrees C) accelerated the onset of production of infectious forms, but limited transmission success. Viability of infectious forms declined rapidly at 23 and 30 degrees C, thereby narrowing the time window for transmission. Thus, environmental conditions can generate trade-offs between transmission relevant parameters and alter levels of multiple infection or parasite-mediated selection, which may affect evolutionary trajectories of parasite life history or virulence.     

Identification and localization of major stage-specific polypeptides of infectious Holospora obtusa with monoclonal antibodies.

With the help of monoclonal antibodies (MAbs) we investigated the occurrence of six polypeptides throughout parts of the life cycle of Holospora obtusa, a bacterium infecting the macronucleus of the ciliate Paramecium caudatum. The polypeptides of interest formed major bands in the protein pattern of the infectious form (IF) of H. obtusa. All MAbs used recognized individual polypeptide bands of the IF proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides were also detected in the reproductive form in trace amounts. Two-dimensional electrophoresis revealed that the 33,000-, 28,000-, and 14,000-Mr polypeptides wre acidic and exhibited multiple isoelectric points under native conditions. Four polypeptides (Mrs of 50,000, 33,000, 28,000, and 20,000) were no longer detected or became drastically reduced within the first 30 min of invasion. Concomitantly, a loss of electron-dense periplasmic material occurred, which is typical for invading IFs (H.-D. G?rtz and M. Wiemann, J. Protistol. 24:101-109, 1989). In an attempt to clarify the subcellular localization of the six polypeptides, we performed chloroform extraction studies and identified four of the released polypeptides with MAbs. A 14,000-Mr polypeptide was immunocytochemically localized in the periplasm of the IF. The results showed that the six major polypeptides of the IF were stage specific or stage specifically enriched and are likely to contribute to the electron-dense periplasmic material of the IF.     

Analysis of natural diversity of symbiotic relationships in the Paramecium bursaria--Holospora curviuscula system

Bacteria of the genus Holospora belong to obligatory endonucleobionts of ciliates of the genus Paramecium. The bacteria show specificity towards the particular host species and the types of nuclei they infect: macro- or micronuclei. During a long-term screening of P. bursaria clones, belonging to three different syngens, Holospora inhibited cells of two syngens only. Using the number of host clones and symbiont isolates, it was shown that H. curviuscula was unable to pass successfully through the syngen barrier even under experimental infection. Considering the species level of specificity in Holospora associations of P. caudatum we suggest the existence of a greater evolutionary divergence in P. bursaria syngens than in syngens of P. caudatum. We have revealed that in incompatible combinations "host clone--symbionts isolate" the complicated bacterial life cycle may be blocked at definite stages depending on genetic features of both partners. Thus, the recognition of the full block spectrum could break the continuous infection process down to independently controlled steps. The block spectrum revealed in the system of P. bursaria--H. curviuscula demonstrates its significant similarity to block spectra of other systems within the Holospora--Paramecium complex. A block of transverse binding formation has been first revealed in Holospora dividing in the nucleus.     

Structure and Expression of a GroE-homologous Operon of a Macronucleus-Specific Symbiont Holospora obtusa of the Ciliate Paramecium caudatum

ABSTRACT The reproductive form of a macronucleus-specific symbiont Holospora obtuse , when harbored by the macronucleus of the ciliate Paramecium caudatum , selectively synthesized a 63-kDa protein which is immunologically related to GroEL, or HSP60, of Escherichia coli. Heat shock treatment of isolated cells of the reproductive and infectious form of the bacterium also induced the synthesis of the GroEL homolog. Immunoblotting showed that the amount of this protein per cell, whether the reproductive or infectious form, is roughly constant. Cloning and sequencing of a gene coding for the GroEL homolog suggested that the protein is 55.2% identical to GroEL of E. coli at the amino acid sequence level, and that the gene is preceded by an open reading frame which encodes a protein 39.6% identical to GroES of E. coli. Northern blot hybridization showed that the GroEL homologous gene is highly expressed in the reproductive form, but only in a trace amount in the intermediate and infectious form. Immunoelectron microscopy revealed that the GroEL homolog is localized in the cytoplasm of the reproductive and infectious form.     

Quantitative Changes in Periplasmic Proteins of the Macronucleus-Specific Bacterium Holospora obtusa in the Infection Process of the Ciliate Paramecium caudatum

ABSTRACT. The Gram-negative bacterium Holospora obrusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum. The infectious form of this bacterium infects the host macronucleus through digestive vacuoles and differentiates into the reproductive form two days after the infection in the nucleus. The monoclonal antibodies IF-3–1 and IF-3–2 reacted with 39 and 1S kDa periplasmic proteins, respectively, that were specific for the infectious form of H. obrusa. Because the antigens were not detected in the reproductive form of the bacterium, it appears that expression of the proteins decreases during or soon after the infection. Using these antibodies, quantitative changes in the antigens in the early infection process were examined by immunoblotting and immunogold electron microscopy. Immunoblotting showed that the amounts of both antigens were reduced within 1 h after the bacteria were engulfed into the digestive vacuoles of the paramecia, but that the amounts of IF-3–2 antigens declined earlier than the IF-3–1 antigen. Immunogold labeling showed that the level of IF-3–2 antigens became very low in the bacteria in the host digestive vacuoles, whereas there was no similar decrease in amount of IF-3–1 antigens. Possible functions of the antigens are discussed. The IF-3–1 antigens decrease in concentration in parallel with the decrease in the periplasmic region.     

Host growth conditions regulate the plasticity of horizontal and vertical transmission in Holospora undulata,a bacterial parasite of the protozoan Paramecium caudatum

Abstract.— A parasite might be prohibited from investing simultaneously in horizontal (infection of new hosts) and vertical (infection of the current host's offspring) transmission because of developmental, physiological, or evolutionary costs and constraints. Rather, these constraints may select for adaptive phenotypic plasticity, where the parasite uses the transmission pathway that maximizes transmission in the current ecological and epidemiological conditions. By varying environmental conditions for the host's replication, we investigated the plasticity of vertical and horizontal transmission of Holospora undulata , a micronucleus-specific bacterial parasite of the protozoan Paramecium caudatum . We observed a negative correlation between the host's growth rate and the parasite's investment in horizontal transmission. In rapidly dividing hosts, the parasite remained in the reproductive stage and was passed on vertically to the daughter nuclei during mitotic division of the Paramecium . In contrast, at low or negative growth rates of the host, the parasite's reproductive forms differentiated into infectious forms, the agents of horizontal transmission. Furthermore, in treatments that were initiated with a high proportion of individuals harboring horizontally transmitted infectious forms, rapid replication resulted in a switch back from predominantly horizontal to almost exclusively vertical transmission. These results suggest a trade-off between the efficacies of vertical and horizontal transmission, with the parasite switching to horizontal transmission only if conditions for host replication, and thus vertical transmission, deteriorate.     

Cryopreservation of Holospora undulata, a bacterial parasite of the ciliate Paramecium caudatum

We investigated cryopreservation of horizontal transmission stages of Holospora undulata, a micronucleus-specific bacterial parasite of Paramecium caudatum. Unlike in previous studies on related Holospora species, protocols using glycerol as cryoprotectant failed entirely. In contrast, freezing with dimethyl sulfoxide (Me2SO) conserved infectiousness of nearly all replicate inocula, although infection success was considerably lower than that of fresh inocula. Infection probability was enhanced by increasing the Me2SO concentration from 5 to 10%, and by freezing at -196 degrees C rather than -80 degrees C. Prolonged storage of up to 3 months had no significant effect on the viability of the inocula.     

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.     

Micronucleus‐Specific Bacterium Holospora elegans Irreversibly Enhances Stress Gene Expression of the Host Paramecium caudatum

ABSTRACT. The bacterium Holospora is an endonuclear symbiont of the ciliate Paramecium. Previously, we reported that paramecia bearing the macronuclear‐specific symbiont Holospora obtusa survived better than symbiont‐free paramecia, even under high temperatures unsuitable for growth. The paramecia with symbionts expressed high levels of hsp70 mRNAs even at 25 °C, a usual growth temperature. We report herein that paramecia bearing the micronuclear‐specific symbiont Holospora elegans also acquire the heat‐shock resistance. Even after the removal of the bacteria from the hosts by treatment with penicillin, the resulting aposymbiotic paramecia nevertheless maintained their heat shock‐resistant nature for over 1 yr. Like symbiotic paramecia, these aposymbiotic paramecia also expressed high levels of both hsp60 and hsp70 mRNAs even at 25 °C. Moreover, analysis by fluorescent in situ hybridization with a probe specific for Holospora 16S rRNA revealed that the 16S rRNA of H. elegans was expressed around the nucleoli of the macronucleus in the aposymbiotic cells. This result suggests the possible transfer of Holospora genomic DNA from the micronucleus into the macronucleus in symbiotic paramecia. Perhaps this exogenous DNA could trigger the aposymbiotic paramecia to induce a stress response, inducing higher expression of Hsp60 and Hsp70, and thus conferring heat‐shock resistance.     

Consequences of herpes simplex virus type 2 and human cell interaction at supraoptimal temperatures.

The consequences of herpes simplex virus type 2 (HSV-2) and human embryonic fibroblast cell interaction at different temperatures (37, 40, and 42 degrees C) were investigated. Incubation at 37 or 40 degrees C was permissive for HSV-2 inhibition of host DNA synthesis, induction of virus-specific DNA replication, and infectious virus production. The amount of [methyl-3H]thymidine incorporated into viral DNA and the final yield of new infectious virus were significantly reduced at 40 degrees C compared to 37 degrees C. At 42 degrees C, detectable virus-specific DNA synthesis was totally blocked. Maximum stimulation of host cell DNA synthesis at 42 degrees C was measured after a multiplicity of infection of 0.5 to 1.0 PFU/cell. By autoradiography, data indicated that HSV-2 stimulates host cell chromosomal DNA synthesis. Stimulation of thymidine kinase activity with thermostability properties in common with a virus enzyme was detected during the first 24 h of infection at 42 degrees C, after 24 h the enhanced thymidine kinase activity had properties in common with host cell isozymes. The data obtained during this investigation indicated that stimulation of host cell DNA synthesis does not require viral DNA synthesis.     

Temperature-dependent changes in the swimming behaviour of Tetrahymena pyriformis-NT1 and their interrelationships with electrophysiology and the state of membrane lipids

The swimming velocity and the amplitude of the helical swimming path of T. pyriformis-NT1 cells grown at 20 degrees C (Tg 20 degrees C) and 38 degrees C (Tg 38 degrees C) were monitored between 0 and 40 degrees C in the presence and absence of electric fields. Within physiological limits the swimming velocity increased and the amplitude decreased as temperature was raised. The temperature profiles of these properties were not linear, and showed discontinuities at different temperatures for the different cultures. The break points in Arrhenius plots of the resting potential, regenerative spike magnitude, repolarization time, swimming velocity and swimming amplitude are tabulated and compared. The initial breakpoints upon cooling were clustered about the breakpoints in fluorescence polarization of D.P.H. in extracted phospholipids, and around the transition temperatures estimated from the literature for the pellicular membrane of these cells. The average of the initial breakpoints on cooling was 22.9 degrees C for Tg 38 degrees C cells and 13.7 degrees C for Tg 20 degrees C cells, a shift of 9.2 degrees C. Unlike Paramecium there is no depolarizing receptor potential in Tetrahymena upon warming. It is suggested that this may be the basis of a behavioural difference between Tetrahymena and Paramecium--namely that in Tetrahymena maximum swimming velocity occurs above growth temperature whereas in Paramecium the two points coincide. Swimming velocity and resting potential were correlated with membrane fluidity within physiological limits, but for other parameters the relationship with fluidity was more complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal variation in temperature determines disease spread and maintenance in Paramecium microcosm populations

The environment is rarely constant and organisms are exposed to temporal and spatial variations that impact their life histories and inter-species interactions. It is important to understand how such variations affect epidemiological dynamics in host-parasite systems. We explored effects of temporal variation in temperature on experimental microcosm populations of the ciliate Paramecium caudatum and its bacterial parasite Holospora undulata. Infected and uninfected populations of two P. caudatum genotypes were created and four constant temperature treatments (26°C, 28°C, 30°C and 32°C) compared with four variable treatments with the same mean temperatures. Variable temperature treatments were achieved by alternating populations between permissive (23°C) and restrictive (35°C) conditions daily over 30 days. Variable conditions and high temperatures caused greater declines in Paramecium populations, greater fluctuations in population size and higher incidence of extinction. The additional effect of parasite infection was additive and enhanced the negative effects of the variable environment and higher temperatures by up to 50 per cent. The variable environment and high temperatures also caused a decrease in parasite prevalence (up to 40%) and an increase in extinction (absence of detection) (up to 30%). The host genotypes responded similarly to the different environmental stresses and their effect on parasite traits were generally in the same direction. This work provides, to our knowledge, the first experimental demonstration that epidemiological dynamics are influenced by environmental variation. We also emphasize the need to consider environmental variance, as well as means, when trying to understand, or predict population dynamics or range.