Survival and Growth of Francisella tularensis in Acanthamoeba castellanii

Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.     

Interaction of Pasteurella multocida with free-living amoebae

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.     

Interaction of Pasteurella multocida with Free-Living Amoebae

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.     

Larval exposure to Francisella tularensis LVS affects fitness of the mosquito Culex quinquefasciatus

Francisella tularensis is an environmental bacterium capable of infecting a wide spectrum of species from mammals and birds to reptiles. It has been demonstrated that F. tularensis can invade and survive within protozoa, but an association with aquatic insects has not been thoroughly investigated. We examined the interaction of F. tularensis LVS biofilms and Culex quinquefasciatus larvae to determine the effects on larvae and adults. Our results demonstrate that F. tularensis LVS can form and persist as biofilms in natural water and that the mosquito larvae of C. quinquefasciatus readily feed on biofilm and planktonic forms of F. tularensis LVS. Larvae raised in both bacteria-only cultures suffered significant delays in pupation. Adults resulting from larvae continuously exposed to the bacteria had significantly reduced wing lengths in males and fecundity of both sexes. The bacteria may be exerting these effects through localization and persistence within the midgut and Malpighian tubule cells of the larvae. The study of oral acquisition of pathogens by insect larvae can significantly contribute to the study of environmental persistence of pathogens. We show that oral uptake of F. tularensis LVS by C. quinquefasciatus larvae results in not only larval effects but also has effects on adult mosquitoes. These effects are important in understanding both the ecology of tularemia as well as bacterial interactions with aquatic invertebrates.     

Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions

Legionella pneumophila persists for a long time in aquatic habitats, where the bacteria associate with biofilms and replicate within protozoan predators. While L. pneumophila serves as a paradigm for intracellular growth within protozoa, it is less clear whether the bacteria form or replicate within biofilms in the absence of protozoa. In this study, we analyzed surface adherence of and biofilm formation by L. pneumophila in a rich medium that supported axenic replication. Biofilm formation by the virulent L. pneumophila strain JR32 and by clinical and environmental isolates was analyzed by confocal microscopy and crystal violet staining. Strain JR32 formed biofilms on glass surfaces and upright polystyrene wells, as well as on pins of "inverse" microtiter plates, indicating that biofilm formation was not simply due to sedimentation of the bacteria. Biofilm formation by an L. pneumophila fliA mutant lacking the alternative sigma factor sigma(28) was reduced, which demonstrated that bacterial factors are required. Accumulation of biomass coincided with an increase in the optical density at 600 nm and ceased when the bacteria reached the stationary growth phase. L. pneumophila neither grew nor formed biofilms in the inverse system if the medium was exchanged twice a day. However, after addition of Acanthamoeba castellanii, the bacteria proliferated and adhered to surfaces. Sessile (surface-attached) and planktonic (free-swimming) L. pneumophila expressed beta-galactosidase activity to similar extents, and therefore, the observed lack of proliferation of surface-attached bacteria was not due to impaired protein synthesis or metabolic activity. Cocultivation of green fluorescent protein (GFP)- and DsRed-labeled L. pneumophila led to randomly interspersed cells on the substratum and in aggregates, and no sizeable patches of clonally growing bacteria were observed. Our findings indicate that biofilm formation by L. pneumophila in a rich medium is due to growth of planktonic bacteria rather than to growth of sessile bacteria. In agreement with this conclusion, GFP-labeled L. pneumophila initially adhered in a continuous-flow chamber system but detached over time; the detachment correlated with the flow rate, and there was no accumulation of biomass. Under these conditions, L. pneumophila persisted in biofilms formed by Empedobacter breve or Microbacterium sp. but not in biofilms formed by Klebsiella pneumoniae or other environmental bacteria, suggesting that specific interactions between the bacteria modulate adherence.     

Francisella tularensis inhibits the intrinsic and extrinsic pathways to delay constitutive apoptosis and prolong human neutrophil lifespan

Francisella tularensis is a facultative intracellular bacterium that infects many cell types, including neutrophils. We demonstrated previously that F. tularensis inhibits NADPH oxidase assembly and activity and then escapes the phagosome to the cytosol, but effects on other aspects of neutrophil function are unknown. Neutrophils are short-lived cells that undergo constitutive apoptosis, and phagocytosis typically accelerates this process. We now demonstrate that F. tularensis significantly inhibited neutrophil apoptosis as indicated by morphologic analysis as well as annexin V and TUNEL staining. Thus, ～80% of infected neutrophils remained viable at 48 h compared with ～50% of control cells, and ～40% of neutrophils that ingested opsonized zymosan. In keeping with this finding, processing and activation of procaspases-8, -9, and -3 were markedly diminished and delayed. F. tularensis also significantly impaired apoptosis triggered by Fas crosslinking. Of note, these effects were dose dependent and could be conferred by either intracellular or extracellular live bacteria, but not by formalin-killed organisms or isolated LPS and capsule, and were not affected by disruption of wbtA2 or FTT1236/FTL0708-genes required for LPS O-antigen and capsule biosynthesis. In summary, we demonstrate that F. tularensis profoundly impairs constitutive neutrophil apoptosis via effects on the intrinsic and extrinsic pathways, and thereby define a new aspect of innate immune evasion by this organism. As defects in neutrophil turnover prevent resolution of inflammation, our findings also suggest a mechanism that may in part account for the neutrophil accumulation, granuloma formation, and severe tissue damage that characterizes lethal pneumonic tularemia.     

Green fluorescent protein-expressing Escherichia coli as a selective probe for HOCl generation within neutrophils

Escherichia coli were transformed by electroporation to introduce a plasmid harboring a GFP gene-containing vector. The fluorescence of the purified GFP isolated from the transformant was quenched by myeloperoxidase (MPO)-generated HOCl, by peroxynitrous acid (ONOOH) and by enzymatically or radiolytically generated NO(2)(.) but not by other putative neutrophil-generated oxidants. Fluorescence from the bacterium was effectively quenched by HOCl but not peroxynitrite, oxidizing radicals derived from its O-O bond homolysis, or the other oxidants under study. Exposure of serum-opsonized bacteria to human neutrophils resulted in extensive loss of GFP fluorescence; fluorescence microscopy revealed that phagocytosed bacteria were completely quenched but that bacteria remaining in the external media were unquenched. Addition of sodium azide to the medium to inhibit MPO prevented neutrophil-mediated fluorescence quenching. Because the amount of HOCl required to inhibit bacterial fluorescence was an order of magnitude greater than required to inhibit colonial growth, these results imply that sufficient HOCl was formed within the neutrophil phagosome to kill the microbe.     

Increased persistence of Salmonella enterica serovar Typhi in the presence of Acanthamoeba castellanii

Salmonella enterica serovar Typhi (S. Typhi) is the etiological agent of the systemic disease typhoid fever. Transmission occurs via ingestion of contaminated food or water. S. Typhi is specific to humans, and no animal or environmental reservoirs are known. As the free-living amoeba Acanthamoeba castellanii is an environmental host for many pathogenic bacteria, this study investigates interactions between S. Typhi and A. castellanii by using cocultures. Growth of both organisms was estimated by cell count, viable count, flow cytometry, and fluorescence microscopy. Results indicate that S. Typhi can survive at least 3 weeks when grown with A. castellanii, as opposed to less than 10 days when grown as singly cultured bacteria under the same conditions. Interestingly, growth rates of amoebae after 14 days were similar in cocultures or when amoebae were singly cultured, suggesting that S. Typhi is not cytotoxic to A. castellanii. Bacteria surviving in coculture were not intracellular and did not require a physical contact with amoebae for their survival. These results suggest that S. Typhi may have a selective advantage when it is associated with A. castellanii and that amoebae may contribute to S. Typhi persistence in the environment.     

Activation of the inflammasome upon Francisella tularensis infection: interplay of innate immune pathways and virulence factors

Tularaemia is a zoonotic disease caused by the facultative intracellular bacterium Francisella tularensis. The virulence of this pathogen depends on its ability to escape into the cytosol of host cells. Pathogens are detected by the innate immune system's pattern recognition receptors which are activated in response to conserved microbial molecules (pathogen-associated molecular patterns). Cytosolic bacteria are sensed intracellularly, often leading to activation of the cysteine protease caspase-1 within a multimolecular complex called the inflammasome. Caspase-1 activation leads to both host cell death and release of pro-inflammatory cytokines in a process called pyroptosis. Here we review the pathway leading to, and the consequences of, inflammasome activation upon F. tularensis infection both in vitro and in vivo. Finally, we discuss recent data on how other innate immune pathways and F. tularensis virulence factors control the activation of the inflammasome during infection.     

Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living amoeba Acanthamoeba castellanii

Vibrio cholerae species are extracellular, waterborne, gram-negative bacteria that are overwhelmed by predators in aquatic environments. The unencapsulated serogroup V. cholerae O1 and encapsulated V. cholerae O139 cause epidemic and pandemic outbreaks of cholera. It has recently been shown that the aquatic and free-living amoeba Acanthamoeba castellanii is not a predator to V. cholerae O139; rather, V. cholerae O139 has shown an intracellular compatibility with this host. The aim of this study was to examine the ability of V. cholerae O1 classical and El Tor strains to grow and survive in A. castellanii. The interaction between A. castellanii and V. cholerae O1 strains was studied by means of amoeba cell counts and viable counts of the bacteria in the absence or presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Confocal microscopy and electron microscopy were used to determine the intracellular localization of V. cholerae in A. castellanii. The results showed that V. cholerae O1 classical and El Tor strains grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were also found in the cysts of A. castellanii. The interaction showed a facultative intracellular behaviour of V. cholerae O1 classical and El Tor strains and a possible role of A. castellanii as an environmental host of V. cholerae species.     

土拉弗朗西斯菌与巨噬细胞膜的早期相互作用

评估土拉弗朗西斯菌LVS在感染鼠巨噬细胞早期与细胞膜的相互作用。用表达GFP的土拉弗朗西斯菌LVS感染鼠巨噬细胞1774A1。结合单抗的小窝蛋白-1或转铁蛋白受体-1分别用键合了Alexa594的羊抗鼠二抗显色。土拉弗朗西斯菌疫苗株LVS可以诱导宿主细胞膜伸出伪足,将细菌吸收进入巨噬细胞。分布在细胞膜上的小窝蛋白-1和转铁蛋白受体-1参与巨噬细胞对弗朗西斯菌的摄入。这些发现说明,弗朗西斯菌进入巨噬细胞需要细胞膜微结构域和小窝蛋白;在感染早期转铁蛋白受体-1参与了细菌的摄入,这可能与弗朗西斯菌获取铁以利在胞内生存有关。     

Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection.     

Construction of a virulent, green fluorescent protein-tagged Yersinia ruckeri and detection in trout tissues after intraperitoneal and immersion challenge

A green fluorescent protein (GFP) expressing strain of Yersinia ruckeri was created by the transposition of a Tn10-GFP-kan cassette into the genome of Y. ruckeri Strain YRNC10. The derivative, YRNC10-gfp, was highly GFP fluorescent, retained the gfp-km marker in the absence of kanamycin selection, and exhibited in vitro growth kinetics similar to those of the wild type strain. YRNC10-gfp colonized and caused mortality in immersion and intraperitoneally challenged rainbow trout Oncorhynchus mykiss, although it was modestly attenuated compared to the wild type strain. The distribution and location of YRNC10-gfp in infected fish was visualized by epifluorescence microscopy. Abundant extracellular bacteria and a small number of intracellular bacteria were observed in the kidney, spleen and peripheral blood. To determine the percentage of trout cells containing intracellular bacteria, GFP fluorescence was measured by flow cytometry. A small population of GFP positive leukocytes was detected in peripheral blood (1.6%), spleen (1.1%) and anterior kidney (0.4%) tissues. In summary, this is the first report of the construction of a virulent, GFP-tagged Y. ruckeri, which may be a useful model for detecting and imaging the interactions between an aquatic pathogen and the natural salmonid host.     

Feasibility of using GFP-expressing Escherichia coli, coupled with fluorimetry, to determine protozoan ingestion rates

The feasibility of using a live Escherichia coli population, which had been engineered to express the green fluorescent protein (GFP), coupled with fluorimetry, was tested as a means for determining protozoan ingestion rates. Its potential use was based on evidence that once cells are acidified, e.g. in a food vacuole, the fluorescence is lost. Of the 29 protozoa tested, over 85% ingested the GFP-expressing E. coli and a detailed experiment with the ciliate Tetrahymena pyriformis was carried out, principally to assess the performance of the live bacterium against two commonly used surrogate prey, i.e. fluorescently labelled bacteria (FLB) and fluorescently labelled microspheres (FLMs). A decrease in GFP-expressing E. coli fluorescence and, hence, concentration, was recorded by fluorimetry and epifluorescence microscopy, with calculated ingestion rates being equivalent. A higher ingestion rate was determined by counting the number of fluorescent E. coli within the ciliate over 120 s, but this was equivalent to that obtained for the stained E. coli using the same direct method of analysis. However, the ciliate was shown to process the stained and unstained E. coli cells differently, with only the latter resulting in an increase in ciliate abundance.     

Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. Although expression of GFP in plants has been widely reported, research on the use of GFP in plant cell cultures for bioprocess applications has been limited. In this study, the suitability of GFP as a secretory reporter and a useful tool in plant cell bioprocess optimization was demonstrated. GFP was produced and secreted from suspension cells derived from tobacco that was transformed with a binary vector containing mgfp5-ER cDNA, a modified GFP for efficient sorting to the endoplasmic reticulum, under control of the CaMV 35S promoter. For cell line gfp-13, extracellular and intracellular GFP accumulated to 15.4 and 29.4 mg x 1(-1), respectively. Extracellular GFP accounted for 30.9% of the total extracellular protein. The molecular mass of extracellular GFP was nearly identical to that of a recombinant GFP standard, indicating cleavage of the signal sequence. Neomycin phosphotransferase II, a cytosolic selection marker, was found almost exclusively in cellular extracts with less than 2% in the extracellular medium. These results suggest that extracellular GFP is most likely the result of secretion rather than nonspecific leakage from cells. Furthermore, medium fluorescence intensity correlated nicely with extracellular GFP concentration supporting the use of GFP as a quantitative secretory reporter. During the batch cultivation, culture GFP fluorescence also followed closely with cell growth. A medium feeding strategy was then developed based on culture GFP fluorescence that resulted in improved biomass as well as GFP production in a fed-batch culture.     

Survival of coliforms and bacterial pathogens within protozoa during chlorination.

The susceptibility of coliform bacteria and bacterial pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa. Viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory strains of Acanthamoeba castellanii and Tetrahymena pyriformis. Cocultures of bacteria and protozoa were incubated to facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated to release intracellular bacteria. Qualitative susceptibility of protozoan strains to free chlorine was also assessed. Protozoa were shown to survive and grow after exposure to levels of free chlorine residuals that killed free-living bacteria. Ingested coliforms Escherichia coli, Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella oxytoca and bacterial pathogens Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, Legionella gormanii, and Campylobacter jejuni had increased resistance to free chlorine residuals. Bacteria could be cultured from within treated protozoans well after the time required for 99% inactivation of free-living cells. All bacterial pathogens were greater than 50-fold more resistant to free chlorine when ingested by T. pyriformis. Escherichia coli ingested by a Cyclidium sp., a ciliate isolated from a drinking water reservoir, were also shown to be more resistant to free chlorine. The mechanism that increased resistance appeared to be survival within protozoan cells. This study indicates that bacteria can survive ingestion by protozoa. This bacterium-protozoan association provides bacteria with increased resistance to free chlorine residuals which can lead to persistence of bacteria in chlorine-treated water. We propose that resistance to digestion by predatory protozoa was an evolutionary precursor of pathogenicity in bacteria and that today it is a mechanism for survival of fastidious bacteria in dilute and inhospitable aquatic environments.     

The effect of the osmotic pressure of the culture medium on cells of Francisella tularensis vaccinal strain 15

The dependence of the growth rate of F. tularensis on the osmotic properties of the medium can be presented as a curve with the maximum in the area of 500-600 mOsm. Under these circumstances the intracellular osmotic pressure exceeds the extracellular one by 50-100 mOsm. With the rise of the osmotic pressure in the medium the increase of the concentration of K+ in the cells occurs. The energy-dependent accumulation of K+ in the cells at rest is activated by the rise of the osmotic pressure in the medium. F. tularensis are probably capable of osmoregulation, ensured by the energy-dependent osmosensitive K(+)-transporting system.