Effects of tobacco smoke on the expression of virulence genes in Escherichia coli.

It is widely acknowledged that smoking exacerbates the severity of infectious diseases. A presumed mechanism involves the damage inflicted by tobacco smoke on the organs of host organisms. In this study, an alternative hypothesis was explored: smoking enhances the virulence of bacteria. This possibility was investigated using Escherichia coli as the model bacteria and Drosophila as the host organism. Our inquiry focused on the potential gene expression changes in E. coli subsequent to exposure to tobacco smoke extracts. Analysis of the transcription promoter activity of genes encoding proteins within the E. coli two-component system, a regulatory machinery governing gene expression, revealed the suppression of thirteen out of 23 promoters in response to tobacco smoke extracts. Subsequently, Drosophila was infected with E. coli exposed to tobacco smoke extracts or left untreated. Interestingly, there were no significant differences observed in the survival periods of Drosophila following infection with E. coli, whether treated or untreated with tobacco smoke extracts. Contrary to the initial hypothesis, the findings suggest that while tobacco smoke extracts alter gene expression in E. coli, these changes do not appear to impact bacterial virulence. Although this study has illuminated the influence of tobacco smoke extracts on the gene expression of E. coli, further analyses are necessary to elucidate the implications of these changes. Nevertheless, the results imply that smoking affects not only host organisms but may also exert influence on invading bacteria.

Apoptosis and Phagocytosis as Antiviral Mechanisms.

Viruses are infectious entities that make use of the replication machinery of their hosts to produce more progenies, causing disease and sometimes death. To counter viral infection, metazoan hosts are equipped with various defense mechanisms, from the rapid-evoking innate immune responses to the most advanced adaptive immune responses. Previous research demonstrated that cells in fruit flies and mice infected with Drosophila C virus and influenza, respectively, undergo apoptosis, which triggers the engulfment of apoptotic virus-infected cells by phagocytes. This process involves the recognition of eat-me signals on the surface of virus-infected cells by receptors of specialized phagocytes, such as macrophages and neutrophils in mice and hemocytes in fruit flies, to facilitate the phagocytic elimination of virus-infected cells. Inhibition of phagocytosis led to severe pathologies and death in both species, indicating that apoptosis-dependent phagocytosis of virus-infected cells is a conserved antiviral mechanism in multicellular organisms. Indeed, our understanding of the mechanisms underlying apoptosis-dependent phagocytosis of virus-infected cells has shed a new perspective on how hosts defend themselves against viral infection. This chapter explores the mechanisms of this process and its potential for developing new treatments for viral diseases.

Role for phagocytosis in the prevention of neoplastic transformation in Drosophila.

Immunity is considered to be involved in the prevention of cancer. Although both humoral and cellular immune reactions may participate, underlying mechanisms have yet to be clarified. The present study was conducted to clarify this issue using a Drosophila model, in which neoplastic transformation was induced through the simultaneous inhibition of cell-cycle checkpoints and apoptosis. We first determined the location of hemocytes, blood cells of Drosophila playing a role of immune cells, in neoplasia-induced and normal larvae, but there was no significant difference between the two groups. When gene expression pattern in larval hemocytes was determined, the expression of immunity-related genes including those necessary for phagocytosis was reduced in the neoplasia model. We then asked the involvement of phagocytosis in the prevention of neoplasia examining animals where the expression of engulfment receptors instead of apoptosis was retarded. We found that the inhibition of engulfment receptor expression augmented the occurrence of neoplasia induced by a defect in cell-cycle checkpoints. This suggested a role for phagocytosis in the prevention of neoplastic transformation in Drosophila.

Transcription repressor-mediated control of engulfment receptor expression in Drosophila phagocytes.

We previously reported that Drosophila phagocytes enhance their phagocytic activity after apoptotic cell engulfment accompanied by the activation of the transcription repressor Tailless and an increase in the levels of engulfment receptors. We herein investigated the underlying mechanisms. We found that Tailless phosphorylation levels decreased in Drosophila phagocytes following the stimulation with apoptotic cells. Anticipating the involvement of another transcription repressor, we examined the possible involvement of Krüppel, a bibliographically identified repressor whose expression is controlled by Tailless. The level of Krüppel in phagocytes decreased after the stimulation in a Tailless-dependent manner. The RNAi knockdown of Krüppel abrogated increases in the levels of engulfment receptors and phagocytic activity in stimulated phagocytes. The binding of Krüppel to the 5'-upstream regions of genes coding for engulfment receptors was demonstrated. These results suggest the following pathway: Tailless is activated by de-phosphorylation; Krüppel expression is inhibited by Tailless; the transcription of engulfment receptors-encoding genes is augmented due to a decrease of inhibition by Krüppel; and finally phagocytic activity is enhanced.

Inhibitory effects of viral infection on cancer development.

Immune responses evoked on viral infections prevent the dissemination of infection that otherwise leads to the development of diseases in host organisms. In the present study, we investigated whether viral infection influences tumorigenesis in cancer-bearing animals using a Drosophila model of cancer. Cancer was induced in the posterior part of wing imaginal discs through the simultaneous inhibition of apoptosis and cell-cycle checkpoints. The larvae and embryos of cancer-induced flies were infected with Drosophila C virus, a natural pathogen to Drosophila, and larval wing discs and adult wings were morphologically examined for cancer characteristics relative to uninfected controls. We found that viral infections brought about an approximately 30% reduction in the rate of cancer development in both wing discs and wings. These inhibitory effects were not observed when growth-defective virus was used to infect animals. These results indicate that productive viral infections repress tumorigenesis in Drosophila.

Characterization of Spz5 as a novel ligand for Drosophila Toll-1 receptor.

The Drosophila Toll-1 receptor is involved in embryonic development, innate immunity, and tissue homeostasis. Currently, as a ligand for the Toll-1 receptor, only Spätzle (Spz) has been identified and characterized. We previously reported that Drosophila larva-derived tissue extract contains ligand activity for the Toll-1 receptor, which differs from Spz based on the observation that larval extract prepared from spz mutants possessed full ligand activity. Here, we demonstrate that Spz5, a member of the Spz family of proteins, functions as a ligand for the Toll-1 receptor. Processing of Spz5 by Furin protease, which is known to be important for ligand activity of Spz5 to Toll-6, is not required for its function to the Toll-1 receptor. By generating a spz5 null mutant, we further showed that the Toll-1 ligand activity of larva-derived extract is mainly derived from Spz5. Finally, we found a genetic interaction between spz and spz5 in terms of developmental processes. This study identified a novel ligand for the Drosophila Toll-1 receptor, providing evidence that Toll-1 is a multi-ligand receptor, similar to the mammalian Toll-like receptor.

Mechanisms and Significance of Phagocytic Elimination of Cells Undergoing Apoptotic Death.

Cells that have become unwanted by the body need to be selectively, rapidly, and safely removed. The removal of these cells is achieved by apoptosis-dependent phagocytosis: unwanted cells are induced to undergo apoptosis and given susceptibility to phagocytosis. Phagocytes recognize these cells using engulfment receptors that bind substances expressed on the surface of target cells during the apoptotic process. The phagocytic elimination of cells undergoing apoptosis is a mechanism that is conserved among multicellular organisms. Malfunctions in this process may lead to structural and functional defects in morphogenesis and tissue homeostasis. Therefore, molecules involved in this phenomenon may be targeted in medical treatments. The mechanisms responsible for the apoptosis-dependent phagocytosis of unwanted cells as well as its physiological and pathological consequences are described herein.

Induction of Apoptosis and Subsequent Phagocytosis of Virus-Infected Cells As an Antiviral Mechanism.

Viruses are infectious entities that hijack host replication machineries to produce their progeny, resulting, in most cases, in disease and, sometimes, in death in infected host organisms. Hosts are equipped with an array of defense mechanisms that span from innate to adaptive as well as from humoral to cellular immune responses. We previously demonstrated that mouse cells underwent apoptosis in response to influenza virus infection. These apoptotic, virus-infected cells were then targeted for engulfment by macrophages and neutrophils. We more recently reported similar findings in the fruit fly Drosophila melanogaster, which lacks adaptive immunity, after an infection with Drosophila C virus. In these experiments, the inhibition of phagocytosis led to severe influenza pathologies in mice and early death in Drosophila. Therefore, the induction of apoptosis and subsequent phagocytosis of virus-infected cells appear to be an antiviral innate immune mechanism that is conserved among multicellular organisms. We herein discuss the underlying mechanisms and significance of the apoptosis-dependent phagocytosis of virus-infected cells. Investigations on the molecular and cellular features responsible for this underrepresented virus-host interaction may provide a promising avenue for the discovery of novel substances that are targeted in medical treatments against virus-induced intractable diseases.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos.

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Signaling pathway for phagocyte priming upon encounter with apoptotic cells.

The phagocytic elimination of cells undergoing apoptosis is an evolutionarily conserved innate immune mechanism for eliminating unnecessary cells. Previous studies showed an increase in the level of engulfment receptors in phagocytes after the phagocytosis of apoptotic cells, which leads to the enhancement of their phagocytic activity. However, precise mechanisms underlying this phenomenon require further clarification. We found that the pre-incubation of a Drosophila phagocyte cell line with the fragments of apoptotic cells enhanced the subsequent phagocytosis of apoptotic cells, accompanied by an augmented expression of the engulfment receptors Draper and integrin αPS3. The DNA-binding activity of the transcription repressor Tailless was transiently raised in those phagocytes, depending on two partially overlapping signal-transduction pathways for the induction of phagocytosis as well as the occurrence of engulfment. The RNAi knockdown of tailless in phagocytes abrogated the enhancement of both phagocytosis and engulfment receptor expression. Furthermore, the hemocyte-specific RNAi of tailless reduced apoptotic cell clearance in Drosophila embryos. Taken together, we propose the following mechanism for the activation of Drosophila phagocytes after an encounter with apoptotic cells: two partially overlapping signal-transduction pathways for phagocytosis are initiated; transcription repressor Tailless is activated; expression of engulfment receptors is stimulated; and phagocytic activity is enhanced. This phenomenon most likely ensures the phagocytic elimination of apoptotic cells by stimulated phagocytes and is thus considered as a mechanism to prime phagocytes in innate immunity.

Protective effects of Phaseolus vulgaris lectin against viral infection in Drosophila.

Phytohemagglutinin (PHA) isolated from the family of Phaseolus vulgaris beans is a promising agent against viral infection; however, it has not yet been demonstrated in vivo. We herein investigated this issue using Drosophila as a host. Adult flies were fed lectin approximately 12 h before they were subjected to a systemic viral infection. After a fatal infection with Drosophila C virus, death was delayed and survival was longer in flies fed PHA-P, a mixture of L4, L3E1, and L2E2, than in control unfed flies. We then examined PHA-L4, anticipating subunit L as the active form, and confirmed the protective effects of this lectin at markedly lower concentrations than PHA-P. In both experiments, lectin feeding reduced the viral load prior to the onset of fly death. Furthermore, we found a dramatic increase in the levels of the mRNAs of phagocytosis receptors in flies after feeding with PHA-L4 while a change in the levels of the mRNAs of antimicrobial peptides was marginal. We concluded that P. vulgaris PHA protects Drosophila against viral infection by augmenting the level of host immunity.

Inhibition of Phagocytic Killing of Escherichia coli in Drosophila Hemocytes by RNA Chaperone Hfq.

An RNA chaperone of Escherichia coli, called host factor required for phage Qß RNA replication (Hfq), forms a complex with small noncoding RNAs to facilitate their binding to target mRNA for the alteration of translation efficiency and stability. Although the role of Hfq in the virulence and drug resistance of bacteria has been suggested, how this RNA chaperone controls the infectious state remains unknown. In the present study, we addressed this issue using Drosophila melanogaster as a host for bacterial infection. In an assay for abdominal infection using adult flies, an E. coli strain with mutation in hfq was eliminated earlier, whereas flies survived longer compared with infection with a parental strain. The same was true with flies deficient in humoral responses, but the mutant phenotypes were not observed when a fly line with impaired hemocyte phagocytosis was infected. The results from an assay for phagocytosis in vitro revealed that Hfq inhibits the killing of E. coli by Drosophila phagocytes after engulfment. Furthermore, Hfq seemed to exert this action partly through enhancing the expression of σ(38), a stress-responsive σ factor that was previously shown to be involved in the inhibition of phagocytic killing of E. coli, by a posttranscriptional mechanism. Our study indicates that the RNA chaperone Hfq contributes to the persistent infection of E. coli by maintaining the expression of bacterial genes, including one coding for σ(38), that help bacteria evade host immunity.

Protection of Insects against Viral Infection by Apoptosis-Dependent Phagocytosis.

We investigated whether phagocytosis participates in the protection of insects from viral infection using the natural host-virus interaction between Drosophila melanogaster and Drosophila C virus (DCV). Drosophila S2 cells were induced to undergo apoptotic cell death upon DCV infection. However, UV-inactivated virus was unable to cause apoptosis, indicating the need for productive infection for apoptosis induction. S2 cells became susceptible to phagocytosis by hemocyte-derived l(2)mbn cells after viral infection, and the presence of phagocytes in S2 cell cultures reduced viral proliferation. Phagocytosis depended, in part, on caspase activity in S2 cells, as well as the engulfment receptors Draper and integrin ßν in phagocytes. To validate the in vivo situation, adult flies were abdominally infected with DCV, followed by the analysis of fly death and viral growth. DCV infection killed flies in a dose-responding manner, and the activation of effector caspases was evident, as revealed by the cleavage of a target protein ectopically expressed in flies. Furthermore, hemocytes isolated from infected flies contained DCV-infected cells, and preinjection of latex beads to inhibit the phagocytic activity of hemocytes accelerated fly death after viral infection. Likewise, viral virulence was exaggerated in flies lacking the engulfment receptors, and was accompanied by the augmented proliferation of virus. Finally, phagocytosis of DCV-infected cells in vitro was inhibited by phosphatidylserine-containing liposome, and virus-infected flies died early when a phosphatidylserine-binding protein was ectopically expressed. Collectively, our study demonstrates that the apoptosis-dependent, phosphatidylserine-mediated phagocytosis of virus-infected cells plays an important role in innate immune responses against viral infection in Drosophila.

Necrotic Cells Actively Attract Phagocytes through the Collaborative Action of Two Distinct PS-Exposure Mechanisms.

Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells are swiftly removed from animal bodies to prevent harmful inflammatory and autoimmune responses. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in the excitotoxic necrosis of six touch neurons and their subsequent engulfment and degradation inside engulfing cells. How necrotic cells are recognized by engulfing cells is unclear. Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells. Here we observed PS exposure on the surface of necrotic touch neurons. In addition, the phagocytic receptor CED-1 clusters around necrotic cells and promotes their engulfment. The extracellular domain of CED-1 associates with PS in vitro. We further identified a necrotic cell-specific function of CED-7, a member of the ATP-binding cassette (ABC) transporter family, in promoting PS exposure. In addition to CED-7, anoctamin homolog-1 (ANOH-1), the C. elegans homolog of the mammalian Ca(2+)-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure efficient exposure of PS on necrotic cells to attract their phagocytes. In addition, CED-8, the C. elegans homolog of mammalian Xk-related protein 8 also makes a contribution to necrotic cell-removal at the first larval stage. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common "eat me" signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through at least two distinct molecular mechanisms rather than leaking out PS passively.

Peptidoglycan recognition protein-triggered induction of Escherichia coli gene in Drosophila melanogaster.

Interaction between the host and pathogen determines the fate of both organisms during the infectious state. The host is equipped with a battery of immune reactions, while the pathogen displays a variety of mechanisms to compromise host immunity. Although bacteria alter their pattern of gene expression in host organisms, studies to elucidate the mechanism behind this are only in their infancy. We here examined the possibility that host immune proteins directly participate in the change of gene expression in bacteria. Escherichia coli was treated with a mixture of the extracellular region of peptidoglycan recognition protein (PGRP)-LC and the antimicrobial peptide attacin of Drosophila, and subjected to DNA microarray analysis for mRNA repertoire. We identified 133 annotated genes whose mRNA increased after the treatment, and at least four of them were induced in response to PGRP-LC. One such gene, lipoprotein-encoding nlpI, showed a transient increase of mRNA in adult flies depending on PGRP-LC but not PGRP-LE. NlpI-lacking E. coli had a lowered growth rate and/or viability in flies than the parental strain. These results suggest that a host immune receptor triggers a change of gene expression in bacteria simultaneously with their recognition and induction of immune responses.

Role for σ38 in prolonged survival of Escherichia coli in Drosophila melanogaster.

Bacteria adapt themselves to host environments by altering the pattern of gene expression. The promoter-recognizing subunit σ of bacterial RNA polymerase plays a major role in the selection of genes to be transcribed. Among seven σ factors of Escherichia coli, σ(38) is responsible for the transcription of genes in the stationary phase and under stressful conditions. We found a transient increase of σ(38) when E. coli was injected into the hemocoel of Drosophila melanogaster. The loss of σ(38) made E. coli rapidly eliminated in flies, and flies infected with σ(38)-lacking E. coli stayed alive longer than those infected with the parental strain. This was also observed in fly lines defective in humoral immune responses, but not in flies in which phagocytosis was impaired. The lack of σ(38) did not influence the susceptibility of E. coli to phagocytosis, but made them vulnerable to killing after engulfment. The changes caused by the loss of σ(38) were recovered by the forced expression of σ(38)-encoding rpoS as well as σ(38)-regulated katE and katG coding for enzymes that detoxify reactive oxygen species. These results collectively suggested that σ(38) contributes to the prolonged survival of E. coli in Drosophila by inducing the production of enzymes that protect bacteria from killing in phagocytes. Considering the similarity in the mechanism of innate immunity against invading bacteria between fruit flies and humans, the products of these genes could be new targets for the development of more effective antibacterial remedies.

Involvement of EnvZ-OmpR two-component system in virulence control of Escherichia coli in Drosophila melanogaster.

Bacteria adapt to environmental changes by altering gene expression patterns with the aid of signal transduction machinery called the two-component regulatory system (TCS), which consists of two protein components, a sensor kinase and response regulator. We examined the role of the TCS in bacterial adaptation to host environments using genetically tractable organisms, Escherichia coli as a pathogen and Drosophila melanogaster as a host. To determine the strength of the transcription promoters of TCS-encoding genes in Drosophila, adult flies were infected with a series of E. coli strains that expressed GFP driven by the promoters of genes coding for 27 sensor kinases and 32 response regulators of E. coli TCS followed by the measurement of fluorescence intensities. We further analyzed EnvZ-OmpR among the TCS encoded by genes having stronger promoters. A mutant E. coli strain lacking EnvZ-OmpR had a higher pathogenic effect on fly survival than that of the parental strain, and the forced expression of envZ and ompR in the mutant strain lowered its pathogenicity. The lack of EnvZ-OmpR did not affect the growth of E. coli in a culture medium as well as the level of colony-formable E. coli in flies. An increase in E. coli virulence with the loss of EnvZ-OmpR was observed in flies defective in an Imd-mediated humoral response, and both the mutant and parental strains were equally engulfed by hemocytes in vitro. These results suggest that EnvZ-OmpR mitigated the virulence of E. coli in Drosophila by a mechanism not accompanied by a change of bacterial burden. This behavior of E. coli is most likely a bacterial strategy to achieve persistent infection.

Differences in the mode of phagocytosis of bacteria between macrophages and testicular Sertoli cells.

Sertoli cells, the sole somatic cell type in the seminiferous epithelium, play an essential role in spermatogenesis and spermiogenesis by nursing germ cells for their survival and differentiation as well as physically inhibiting the entrance of harmful substances into the seminiferous tubules. Sertoli cells possess the characteristics of immune cells; they express pattern recognition receptors, secrete antimicrobial proteins, and engulf dead or dying cells. In this study, we determined the mechanism by which Sertoli cells engulf and kill bacteria compared to that of macrophages. When the primary cultured Sertoli cells of rats were incubated with Staphylococcus aureus, they produced the mRNA of neutrophil protein 3, an antimicrobial peptide of the α-defensin family, but not superoxide or nitric oxide, in contrast to mouse peritoneal macrophages. Sertoli cells effectively phagocytosed S. aureus in a manner that was accompanied by cytoskeleton rearrangement and dependent on phosphatidylinositol 3-kinase. Engulfed bacteria appeared to stay alive in Sertoli cells, while they were rapidly killed in macrophages. These results collectively suggest that Sertoli cells eliminate bacteria that have invaded the seminiferous epithelium without evoking inflammation, unlike macrophages.