RESUMEN
Actinetobacter baumannii is an important nosocomial pathogen that can cause a wide range of serious conditions including pneumonia, meningitis, necrotizing fasciitis and sepsis. It is also a major cause of wound infections in military personnel injured during the conflicts in Afghanistan and Iraq, leading to its popular nickname of 'Iraqibacter'. Contributing to its success in clinical settings is resistance to environmental stresses such as desiccation and disinfectants. Moreover, in recent years there has been a dramatic increase in the number of A. baumannii strains with resistance to multiple antibiotic classes. Acinetobacter baumannii is an inhabitant of oral biofilms, which can act as a reservoir for pneumonia and chronic obstructive pulmonary disease. Subgingival colonization by A. baumannii increases the risk of refractory periodontitis. Pathogenesis of the organism involves adherence, biofilm formation and iron acquisition. In addition, A. baumannii can induce apoptotic cell death in epithelial cells and kill hyphal forms of Candida albicans. Virulence factors that have been identified include pili, the outer membrane protein OmpA, phospholipases and extracellular polysaccharide. Acinetobacter baumannii can sense blue light through a blue-light sensing using flavin (BLUF) domain protein, BlsA. The resulting conformational change in BlsA leads to changes in gene expression, including virulence genes.
Asunto(s)
Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii , Infección Hospitalaria/microbiología , Boca/microbiología , Periodontitis/microbiología , Factores de Virulencia/fisiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/patogenicidad , Acinetobacter baumannii/fisiología , Apoptosis , Adhesión Bacteriana , Biopelículas/crecimiento & desarrollo , Farmacorresistencia Bacteriana , Humanos , Hierro/metabolismo , Luz , Interacciones Microbianas , Virulencia/genética , Virulencia/fisiologíaRESUMEN
Legionella pneumophila replicates within alveolar macrophages, and possibly, alveolar epithelial cells and also within protozoa in the aquatic environment. Here we characterize an L. pneumophila mutant defective in the HtrA/DegP stress-induced protease/chaperone homologue and show that HtrA is indispensable for intracellular replication within mammalian macrophages and alveolar epithelial cells and for intrapulmonary replication in A/J mice. Importantly, amino acid substitutions of two conserved residues in the catalytic domain of (H103mapstoR and S212mapstoA) and in-frame deletions of either or both of the two conserved PDZ domains of HtrA abolish its function. Interestingly, the htrA mutant exhibits a parental-type phenotype in intracellular replication within the protozoan host Acanthamoeba polyphaga. We used a promoterless lacZ fusion to the htrA promoter to probe the phagosomal microenvironment harboring L. pneumophila within macrophages and within A. polyphaga for the exposure to stress stimuli. The data show that expression through the htrA promoter is induced by 12,000- to 20,000-fold throughout the intracellular infection of macrophages but its induction is by 120- to 500-fold within protozoa compared to in vitro expression. Data derived from confocal laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the htrA mutant within U937 macrophages colocalize with the late endosomal-lysosomal marker LAMP-2, similar to killed L. pneumophila. Coinfection experiments examined by confocal laser scanning microscopy show that in communal phagosomes harboring both the parental strain and the htrA mutant, replication of the mutant is not rescued, while replication of a dotA mutant control, which is normally trafficked into a phagolysosome, is rescued by the parental strain. Our data show, for the first time, that the stress response by L. pneumophila (mediated, at least in part, by HtrA) is indispensable for intracellular replication within mammalian but not protozoan cells.
Asunto(s)
Eucariontes/microbiología , Proteínas de Choque Térmico , Legionella pneumophila/crecimiento & desarrollo , Proteínas Periplasmáticas , Serina Endopeptidasas/fisiología , Animales , Línea Celular , Humanos , Legionella pneumophila/genética , Pulmón/microbiología , Macrófagos/microbiología , Ratones , Sistemas de Lectura Abierta , Serina Endopeptidasas/genéticaRESUMEN
We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar to rep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in the rep helicase homologue mutant, rendering it defective in intracellular replication.
Asunto(s)
Adenosina Trifosfatasas/fisiología , ADN Helicasas , Legionella pneumophila/fisiología , Adenosina Trifosfatasas/genética , Línea Celular , Mapeo Cromosómico , Daño del ADN , Retículo Endoplásmico Rugoso/fisiología , Proteínas de Escherichia coli , Humanos , Legionella pneumophila/enzimología , Macrófagos/microbiología , Sistemas de Lectura Abierta , Fagosomas/fisiología , Alveolos Pulmonares/microbiología , Timina/metabolismo , Timina/farmacología , Células U937RESUMEN
Legionella pneumophila does not induce apoptosis in the protozoan host, but induces pore formation-mediated cytolysis after termination of intracellular replication (L.-Y. Gao and Y. Abu Kwaik, Environ. Microbiol. 2:79-90, 2000). In contrast to this single mode of killing of protozoa, we have recently proposed a biphasic model by which L. pneumophila kills macrophages, in which the first phase is manifested through the induction of apoptosis during early stages of the infection, followed by an independent and temporal induction of necrosis during late stages of intracellular replication. Here we show that, similar to the protozoan host, the induction of necrosis and cytolysis of macrophages by L. pneumophila is mediated by the pore-forming toxin or activity. This activity is temporally and maximally expressed only upon termination of bacterial replication and correlates with cytolysis of macrophages and alveolar epithelial cells in vitro. We have identified five L. pneumophila mutants defective in the pore-forming activity. The phagosomes harboring the mutants do not colocalize with the late endosomal or lysosomal marker Lamp-1, and the mutants replicate intracellularly similar to the parental strain. Interestingly, despite their prolific intracellular replication, the mutants are defective in cytotoxicity and are "trapped" within and fail to lyse and egress from macrophages and alveolar epithelial cells upon termination of intracellular replication. However, the mutants are subsequently released from the host cell, most likely due to apoptotic death of the host cell. Data derived from cytotoxicity assays, confocal laser scanning microscopy, and electron microscopy confirm the defect in the mutants to induce necrosis of macrophages and the failure to egress from the host cell. Importantly, the mutants are completely defective in acute lethality (24 to 48 h) to intratracheally inoculated A/J mice. We conclude that the pore-forming activity of L. pneumophila is not required for phagosomal trafficking or for intracellular replication. This activity is expressed upon termination of bacterial replication and is essential to induce cytolysis of infected macrophages to allow egress of intracellular bacteria. In addition, this activity plays a major role in pulmonary immunopathology in vivo.
Asunto(s)
Antígenos Bacterianos , Legionella pneumophila/patogenicidad , Macrófagos/patología , Alveolos Pulmonares/patología , Animales , Apoptosis , Proteínas Bacterianas/fisiología , Femenino , Humanos , Proteínas de la Membrana/fisiología , Ratones , Necrosis , Células U937RESUMEN
It is becoming apparent that several intracellular bacterial pathogens of humans can also survive within protozoa. This interaction with protozoa may protect these pathogens from harsh conditions in the extracellular environment and enhance their infectivity in mammals. This relationship has been clearly established in the case of the interaction between Legionella pneumophila and its protozoan hosts. In addition, the adaptation of bacterial pathogens to the intracellular life within the primitive eukaryotic protozoa may have provided them with the means to infect the more evolved mammalian cells. This is evident from the existence of several similarities, at both the phenotypic and the molecular levels, between the infection of mammalian and protozoan cells by L. pneumophila. Thus, protozoa appear to play a central role in the transition of bacteria from the environment to mammals. In essence, protozoa may be viewed as a 'biological gym', within which intracellular bacterial pathogens train for their encounters with the more evolved mammalian cells. Thus, intracellular bacterial pathogens have benefited from the structural and biochemical conservation of cellular processes in eukaryotes. The interaction of intracellular bacterial pathogens and protozoa highlights this conservation and may constitute a simplified model for the study of these pathogens and the evolution of cellular processes in eukaryotes. Furthermore, in addition to being environmental reservoirs for known intracellular pathogens of humans and animals, protozoa may be sources of emerging pathogenic bacteria. It is thus critical to re-examine the relationship between bacteria and protozoa to further our understanding of current human bacterial pathogenesis and, possibly, to predict the appearance of emerging pathogens.
Asunto(s)
Bacterias/crecimiento & desarrollo , Eucariontes/microbiología , Mamíferos/microbiología , Amoeba/microbiología , Animales , Bacterias/genética , Humanos , Legionella pneumophila/genética , Legionella pneumophila/crecimiento & desarrolloRESUMEN
Increasing evidence indicates that apoptosis of the host cell may constitute a defense mechanism to confine the infection by bacterial pathogens. Certain pathogens have developed elegant mechanisms to modulate the fate of the host cell, which include induction or blockage of apoptosis. These studies will promote our understanding of the pathogenesis of infectious diseases and aid the development of means for therapeutic intervention.
Asunto(s)
Apoptosis , Bacterias/patogenicidad , Proteínas Bacterianas , Proteínas de la Membrana , Animales , Toxinas Bacterianas , Caspasas/metabolismo , Exotoxinas , Proteínas de Choque Térmico , Proteínas Hemolisinas , Humanos , Modelos Biológicos , PirógenosRESUMEN
Legionella pneumophila has been shown to possess multiple genetic loci that play roles in its ability to survive within host cells. The mil (macrophage-specific infectivity loci) mutants of L. pneumophila exhibit a spectrum of defects in intracellular survival in and cytopathogenicity to macrophages and alveolar epithelial cells. This study characterizes one of the mil mutants (GB111). Intracellular growth of GB111 in macrophages was approximately 100- to 1,000-fold less than that of AA100, the parental strain, at 24 and 48 h postinfection. This defect in turn corresponded to a defect in cytopathogenicity. Sequence analysis of the affected GB111 open reading frame (ORF) revealed it to encode a putative transport protein, and the ORF was designated milA. The phenotypic defect of the milA mutant was complemented with a PCR fragment containing only milA, indicating that the defect in GB111 was due to the disruption of milA. Intracellular trafficking of the mutant was examined by laser scanning confocal microscopy. The data showed that 50% of the GB111 phagosomes colocalized with the late endosomal/lysosomal marker LAMP-2 (2 and 4 h postinfection), while less than 10% of the AA100 phagosomes colocalized with this marker. On the other hand, over 80% of the GB111 phagosomes were similar to the AA100 phagosome in that they were devoid of LAMP-1 and cathepsin D, and they were colocalized with the endoplasmic reticulum (ER) marker BiP. However, the number of GB111 phagosomes that colocalized with BiP decreased to 50% 6 h postinfection compared to that of AA100, which remained constant (80% colocalization). Thus, compared to AA100, the milA mutation caused a defect in intracellular replication, which was associated with colocalization of the phagosome with LAMP-2 and BiP, while colocalization with LAMP-1 and cathepsin D was not affected.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Inmunofilinas/genética , Legionella pneumophila/genética , Legionella pneumophila/patogenicidad , Macrófagos/microbiología , Proteínas de la Membrana/genética , Isomerasa de Peptidilprolil , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , División Celular , Prueba de Complementación Genética , Hemólisis , Humanos , Inmunofilinas/química , Cinética , Legionella pneumophila/crecimiento & desarrollo , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutación , Fagosomas/microbiología , Homología de Secuencia de Aminoácido , Ovinos , Células U937 , Virulencia/genéticaRESUMEN
The identification of bacterial genes regulated in response to the intracellular environment is crucial to the understanding of host-pathogen interactions. Several techniques have been developed to identify and characterize bacterial genes that are induced during the intracellular infection and, potentially, may play a role in pathogenesis. This review discusses the strategies that have been utilized to examine differential gene expression by bacterial pathogens during the intracellular infection. Furthermore, a number of the differentially expressed genes are described.
Asunto(s)
Bacterias/genética , Bacterias/patogenicidad , Regulación Bacteriana de la Expresión Génica , Animales , Infecciones Bacterianas/microbiología , Ratones , Fagosomas/microbiología , Fagosomas/fisiología , Reacción en Cadena de la Polimerasa/métodos , Virulencia/genéticaRESUMEN
In contrast to Legionella pneumophila, little is known about the pathogenesis of other legionellae species that are capable of causing Legionnaires' disease. In this report, we contrast L. pneumophila and L. micdadei for their cytopathogenicity and intracellular replication within mammalian and protozoan cells. We show by transmission electron microscopy that L. micdadei replicates within an endoplasmic reticulum (RER)-free phagosome within human macrophages, alveolar epithelial cells, and within the protozoan Hartmannella vermiformis. In contrast, L. pneumophila replicates within a RER-surrounded phagosome within the same host cells. In contrast to replication of L. pneumophila within Acanthamoebae polyphaga, L. micdadei does not replicate within this protozoan host. Despite the prolific intracellular replication, L. micdadei is less cytopathogenic to all host cells than L. pneumophila. Since both species replicate intracellularly to a similar level, we have examined whether the reduced cytopathogenicity of L. micdadei is due to a reduced capacity to induce apoptosis or pore formation-mediated necrosis, both of which contribute to killing of the host cell by L. pneumophila. The data show that both species induced apoptosis-mediated killing of mammalian cells to a similar level. In contrast to L. pneumophila, expression of the pore-forming toxin by L. micdadei and its necrotic effect on macrophages and alveolar epithelial cells is undetectable. This has been further confirmed showing that L. micdadei is completely defective in contact-dependent haemolysis of RBCs, an activity mediated by the pore-forming toxin. Finally, in contrast to L. pneumophila, there was no significant intrapulmonary replication of L. micdadei in the A/J mice animal model. Our data show dramatic differences between L. pneumophila and L. micdadei in intracellular replication, cytopathogenicity, and infectivity to mammalian and protozoan cells.
Asunto(s)
Amébidos/microbiología , Legionella/crecimiento & desarrollo , Legionella/patogenicidad , Macrófagos/microbiología , Alveolos Pulmonares/microbiología , Acanthamoeba/microbiología , Acanthamoeba/ultraestructura , Animales , Apoptosis , Células Epiteliales/microbiología , Femenino , Hartmannella/microbiología , Hartmannella/ultraestructura , Humanos , Etiquetado Corte-Fin in Situ , Legionella pneumophila/crecimiento & desarrollo , Legionella pneumophila/patogenicidad , Pulmón/microbiología , Macrófagos/ultraestructura , Ratones , Microscopía Electrónica , Alveolos Pulmonares/citología , Alveolos Pulmonares/ultraestructura , VirulenciaRESUMEN
The hallmark of Legionnaires' disease is replication of Legionella pneumophila within cells in the alveolar spaces. The mechanisms by which L. pneumophila replicates intracellularly and kills the host cell are largely not understood. We have recently shown that within 3 h of initiation of the infection and prior to intracellular replication, L. pneumophila induces apoptosis in macrophages, alveolar epithelial cells, and peripheral blood monocytes, which correlates with cytopathogenicity (L.-Y. Gao and Y. Abu Kwaik, Infect. Immun. 67:862-870, 1999). In this report, we show that the ability of L. pneumophila to induce apoptosis is, largely, not growth phase regulated. We demonstrate that the induction of apoptosis by L. pneumophila in macrophages is mediated through the activation of caspase 3. The enzymatic activity of caspase 3 to cleave a specific synthetic substrate in vitro is detected in L. pneumophila-infected macrophages at 2 h after infection and is maximal at 3 h, with over 900% increase in activity. The activity of caspase 3 to cleave a specific substrate [poly(ADP-ribose) polymerase, or PARP] in vivo is also detected at 2 h and is maximal at 3 h postinfection. The activity of caspase 3 to cleave the synthetic substrate in vitro and PARP in vivo is blocked by a specific inhibitor of caspase 3. The kinetics of caspase 3 activation correlates with that of L. pneumophila-induced nuclear apoptosis. Inhibition of caspase 3 activity blocks L. pneumophila-induced nuclear apoptosis and cytopathogenicity during early stages of the infection. Consistent with the ability to induce apoptosis, extracellular L. pneumophila also activates caspase 3. Three dotA/icmWXYZ mutants of L. pneumophila that are defective in inducing apoptosis do not induce caspase 3 activation, suggesting that expression and/or export of the apoptosis-inducing factor(s) is regulated by the dot/icm virulence system. This is the first description of the role of caspase 3 activation in induction of nuclear apoptosis in the host cell infected by a bacterial pathogen.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Legionella pneumophila/enzimología , Caspasa 3 , Núcleo Celular , Fragmentación del ADN , Activación Enzimática , Humanos , Legionella pneumophila/crecimiento & desarrollo , Legionella pneumophila/fisiología , Macrófagos/citología , Macrófagos/microbiología , Especificidad por Sustrato , Células U937RESUMEN
The hallmark of Legionnaires' disease is intracellular replication of Legionella pneumophila within cells in the alveolar spaces. Cytopathogenicity of this bacterium to the host cell has been well demonstrated, but the mechanisms of host cell death due to infection by L. pneumophila are not well understood. In this study, induction of apoptosis in macrophages and alveolar epithelial cells by L. pneumophila during early stages of infection was confirmed by using multiple criteria, including DNA fragmentation by agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, surface exposure of phosphatidylserine, and cellular morphology by transmission electron microscopy. Induction of nuclear apoptosis in L. pneumophila-infected macrophages is mediated by activation of the caspase cascade death machinery. We provide genetic and biochemical evidence that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells does not require intracellular bacterial replication or new protein synthesis. In addition, extracellular L. pneumophila is capable of inducing apoptosis. Furthermore, induction of apoptosis by L. pneumophila correlates with cytopathogenicity. We conclude that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells plays an important role in cytopathogenicity to the host cell during early stages of infection.
Asunto(s)
Apoptosis , Legionella pneumophila/fisiología , Macrófagos/microbiología , Clorometilcetonas de Aminoácidos/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Células Epiteliales/microbiología , Células Epiteliales/patología , Espacio Extracelular , Humanos , Líquido Intracelular , Macrófagos/patología , Biosíntesis de Proteínas , Células U937RESUMEN
The intracellular pathogens Legionella micdadei and Legionella pneumophila are the two most common Legionella species that cause Legionnaires' disease. Intracellular replication within pulmonary cells is the hallmark of Legionnaires' disease. In the environment, legionellae are parasites of protozoans, and intracellular bacterial replication within protozoans plays a major role in the transmission of Legionnaires' disease. In this study, we characterized the initial host signal transduction mechanisms involved during attachment to and invasion of the protozoan host Hartmannella vermiformis by L. micdadei. Bacterial attachment prior to invasion of H. vermiformis by L. micdadei is associated with tyrosine dephosphorylation of multiple host cell proteins, including a 170-kDa protein. We have previously shown that this 170-kDa protein is the galactose N-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin receptor that mediates attachment to and invasion of H. vermiformis by L. pneumophila. Subsequent bacterial entry targets L. micdadei into a phagosome that is not surrounded by the rough endoplasmic reticulum (RER). In contrast, uptake of L. pneumophila mediated by attachment to the Gal/GalNAc lectin is followed by targeting of the bacterium into an RER-surrounded phagosome. These results indicate that despite similarities in the L. micdadei and L. pneumophila attachment-mediated signal transduction mechanisms in H. vermiformis, the two bacterial species are targeted into morphologically distinct phagosomes in their natural protozoan host.
Asunto(s)
Hartmannella/metabolismo , Hartmannella/microbiología , Legionella/fisiología , Fagosomas/microbiología , Transducción de Señal , Acetilgalactosamina/farmacología , Animales , Adhesión Bacteriana , Retículo Endoplásmico/ultraestructura , Galactosa/farmacología , Hartmannella/genética , Hartmannella/ultraestructura , Lectinas/metabolismo , Legionella/ultraestructura , Microscopía Electrónica , Fagosomas/ultraestructura , Fosforilación , Proteínas Protozoarias/metabolismo , Tirosina/metabolismoRESUMEN
The ability of Legionella pneumophila to cause Legionnaires' disease is dependent on its capacity to survive in the intracellular environment of its host cells. Furthermore, outbreaks of this disease have been associated with contaminated water sources where L. pneumophila survives as a parasite of protozoa. In this study, we determined the effect of nutritional auxotrophy on the ability of L. pneumophila to survive in the intracellular environment of its host cells. We generated a diaminopimelic acid (DAP) auxotroph (AA400) of L. pneumophila by disruption of the aspartate-beta-semialdehyde (asd) gene. The ability of AA400 to survive within macrophages and protozoa was found to be defective. This defect was due solely to the asd disruption since complementation of the mutant with the wild-type asd gene restored its capacity for intracellular survival. Furthermore, the defect was not completely complemented by DAP supplementation to the culture media. Thus, our results suggest that disruption of the asd gene may prove to be useful in the design of attenuated vaccines against Legionnaires' disease.
Asunto(s)
Aspartato-Semialdehído Deshidrogenasa/genética , Genes Bacterianos , Legionella pneumophila/genética , Secuencia de Aminoácidos , Animales , Vacunas Bacterianas/inmunología , Secuencia de Bases , Línea Celular , Clonación Molecular , Legionella pneumophila/enzimología , Legionella pneumophila/inmunología , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Mutación , Vacunas Atenuadas/inmunologíaRESUMEN
Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 microm) or short (0.1 to 0.6 microm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL). An insertion mutation was constructed in pilEL and introduced into the L. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that the pilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires' disease and invasion of amoebas in the environment.
Asunto(s)
Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/genética , Eucariontes/microbiología , Fimbrias Bacterianas/fisiología , Genes Bacterianos , Legionella pneumophila/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/fisiología , Secuencia de Bases , Proteínas Fimbrias , Células HeLa , Humanos , Legionella pneumophila/genética , Datos de Secuencia Molecular , MutaciónRESUMEN
The eukaryotic protein synthesis inhibitor cycloheximid has been used by many investigators to selectively radiolabel intracellular bacteria. Although cycloheximide has no direct effect on bacterial gene expression, there are concerns that long-term inhibition of the host cell protein synthesis may have secondary effects on bacterial gene expression. Therefore, prior to further identification and cloning of the macrophage-induced (MI) genes of Legionella pneumophila, the effects of cycloheximide on L. pneumophila-infected U937 cells were evaluated by transmission electron microscopy. Inhibition of protein synthesis of the host cell for 6 h had no major effect on the ultrastructure of the host cell, on the formation of rough endoplasmic reticulum-surrounded replicative phagosome, or on initiation of intracellular bacterial replication. In contrast, by 15 h of cycloheximide treatment, there was profound deterioration in the host cell as well as in the phagosome. To examine protein synthesis by L. pneumophila during the intracellular infection, U937 macrophage-like cells were infected with L. pneumophila, and intracellular bacteria were radiolabeled during a 2-h cycloheximide treatment or following 12 h of cycloheximide treatment. Comparison by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein profile of radiolabeled in vitro-grown L. pneumophila to that of intracellularly radiolabeled bacteria showed that 23 proteins were induced in response to the intracellular environment during 2 h of inhibition of host cell protein biosynthesis. Twelve MI proteins of L. pneumophila were artifactually induced due to prolonged inhibition of the host cell protein synthesis. The gene encoding a 20-kDa MI protein was cloned by a reverse genetics technique. Sequence analysis showed that the cloned gene encoded a protein that was 80% similar to the enzyme inorganic pyrophosphatase. Studies of promoter fusion to a promoterless lacZ gene showed that compared to in vitro-grown bacteria, expression of the pyrophosphatase gene (ppa) was induced fourfold throughout the intracellular infection. There was no detectable induction in transcription of the ppa promoter during exposure to stress stimuli in vitro. The ppa gene of L. pneumophila is the first example of a regulated ppa gene which is selectively induced during intracellular infection and which may reflect enhanced capabilities of macromolecular biosynthesis by intracellular L. pneumophila. The data indicate caution in the long-term use of inhibition of host cell protein synthesis to selectively examine gene expression by intracellular bacteria.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Legionella pneumophila/genética , Enfermedad de los Legionarios/genética , Macrófagos/microbiología , Secuencia de Aminoácidos , Fusión Artificial Génica , Proteínas Bacterianas/análisis , Proteínas Bacterianas/biosíntesis , Células Cultivadas , Clonación Molecular , Cicloheximida/farmacología , Elementos Transponibles de ADN , ADN Bacteriano/análisis , ADN Bacteriano/genética , Retículo Endoplásmico Rugoso/efectos de los fármacos , Retículo Endoplásmico Rugoso/ultraestructura , Operón Lac , Legionella pneumophila/ultraestructura , Macrófagos/efectos de los fármacos , Macrófagos/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular , Mutagénesis Insercional , Fagosomas/efectos de los fármacos , Fagosomas/ultraestructura , Plásmidos , Regiones Promotoras Genéticas , Inhibidores de la Síntesis de la Proteína/farmacología , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Recombinación Genética , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Alveolar epithelial cells, which constitute the majority of the alveolar surface, may represent a potential niche for intracellular replication of Legionella pneumophila that has been largely overlooked. We examined the phenotypes of a bank of 121 macrophage-defective mutants of L. pneumophila (designated as pmi and mil) for their cytopathogenicity to and intracellular survival and replication within human alveolar epithelial cells. Our data showed that 91 of 121 mutants that were defective (modest-severe) in macrophages exhibited wild type-like phenotypes in human type I alveolar epithelial cells. In contrast, the other 30 mutants were defective in both macrophages and alveolar epithelial cells. Transmission electron microscopy of the intracellular infection by three mutants showed that the defect in intracellular replication in macrophages and epithelial cells was associated with a defect in recruitment of the RER around the phagosome. Differences in attachment to macrophages and epithelial cells were also exhibited by some of the mutants. Pulmonary infection studies of A/J mice showed that a mutant defective in macrophages but not in alveolar epithelial cells replicated like the wild type strain in the lungs of A/J mice. In contrast, a mutant defective in both macrophages and alveolar epithelial cells failed to replicate and was killed. We conclude that certain distinct genetic loci of L. pneumophila are uniquely required for intracellular survival and replication within phagocytic but not epithelial cells, which may be important in vivo.
Asunto(s)
Células Epiteliales/microbiología , Legionella pneumophila/patogenicidad , Macrófagos Alveolares/microbiología , Alveolos Pulmonares/microbiología , Animales , Adhesión Bacteriana , Línea Celular , Células HeLa , Humanos , Legionella pneumophila/genética , Legionella pneumophila/crecimiento & desarrollo , Ratones , Ratones Endogámicos A , Ratones Noqueados , Microscopía Electrónica , Mutación , Fenotipo , Alveolos Pulmonares/citología , Células U937 , VirulenciaRESUMEN
The Legionnaires' disease bacterium, Legionella pneumophila, is an intracellular pathogen of humans that is amplified in the environment by intracellular multiplication within protozoa. Within both evolutionarily distant hosts, the bacterium multiplies in a rough endoplasmic reticulum-surrounded phagosome that is retarded from maturation through the endosomal-lysosomal degradation pathway. To gain an understanding of the mechanisms utilized by L. pneumophila to invade and replicate within two evolutionarily distant hosts, we isolated a collection of 89 mini-Tn10::kan insertion mutants that exhibited defects in cytotoxicity, intracellular survival, and replication within both U937 macrophage-like cells and Acanthamoeba polyphaga. Interestingly, the patterns of defects in intracellular survival and replication of the mutants within both host cells were highly similar, and thus we designated the defective loci in these mutants pmi (for protozoan and macrophage infectivity loci). On the basis of their ability to attach to host cells and their growth kinetics during the intracellular infection, the mutants were grouped into five groups. Groups 1 and 2 included 41 mutants that were severely defective in intracellular survival and were completely or substantially killed during the first 4 h of infection in both host cells. Three members of group 1 were severely defective in attachment to both U937 cells and A. polyphaga, and another four mutants of group 1 exhibited severe defects in attachment to A. polyphaga but only a mild reduction in their attachment to U937 cells. Four members of groups 1 and 2 were serum sensitive. Intracellular replication of mutants of the other three groups was less defective than that of mutants of groups 1 and 2, and their growth kinetics within both host cells were similar. The mutants were tested for several other phenotypes in vitro, revealing that 14 of the pmi mutants were resistant to NaCl, 3 had insertions in dot or icm, 3 were aflagellar, 12 were highly intolerant to a hyperosmotic medium, and one failed to grow in a minimal medium. Our data indicated that similar mechanisms are utilized by L. pneumophila to replicate within two evolutionarily distant hosts. Although some mechanisms of attachment to both host cells were similar, other distinct mechanisms were utilized by L. pneumophila to attach to A. polyphaga. Our data supported the hypothesis that preadaptation of L. pneumophila to infection of protozoa may play a major role in its ability to replicate within mammalian cells and cause Legionnaires' disease.
Asunto(s)
Acanthamoeba/microbiología , Legionella pneumophila/fisiología , Macrófagos/microbiología , Animales , Adhesión Bacteriana , Actividad Bactericida de la Sangre , Línea Celular , Humanos , Hierro/metabolismo , Mutación , Cloruro de Sodio/farmacologíaRESUMEN
The Legionnaire's disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen which invades and replicates within two evolutionarily distant hosts, free-living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaire's disease. Although attachment and invasion of human macrophages by L. pneumophila is mediated in part by the complement receptors CR1 and CR3, the protozoan receptor involved in bacterial attachment and invasion has not been identified. To define the molecular events involved in invasion of protozoa by L. pneumophila, we examined the role of protein tyrosine phosphorylation of the protozoan host Hartmannella vermiformis upon attachment and invasion by L. pneumophila. Bacterial attachment and invasion were associated with a time-dependent tyrosine dephosphorylation of multiple host cell proteins. This host cell response was highly specific for live L. pneumophila, required contact with viable bacteria, and was completely reversible following washing off the bacteria from the host cell surface. Tyrosine dephosphorylation of host proteins was blocked by a tyrosine phosphatase inhibitor but not by tyrosine kinase inhibitors. One of the tyrosine dephosphorylated proteins was identified as the 170-kD galactose/N-acetylgalactosamine-inhibitable lectin (Gal/GalNAc) using immunoprecipitation and immunoblotting by antibodies generated against the Gal/GalNAc lectin of the protozoan Entamoeba histolytica. This Gal/GalNAc-inhibitable lectin has been shown previously to mediate adherence of E. histolytica to mammalian epithelial cells. Uptake of L. pneumophila by H. vermiformis was specifically inhibited by two monovalent sugars, Gal and GalNAc, and by mABs generated against the 170-kD lectin of E. histolytica. Interestingly, inhibition of invasion by Gal and GalNAc was associated with inhibition of bacterial-induced tyrosine dephosphorylation of H. vermiformis proteins. High stringency DNA hybridization confirmed the presence of the 170-kD lectin gene in H. vermiformis. We conclude that attachment of L. pneumophila to the H. vermiformis 170-kD lectin is required for invasion and is associated with tyrosine dephosphorylation of the Gal lectin and other host proteins. This is the first demonstration of a potential receptor used by L. pneumophila to invade protozoa.
Asunto(s)
Acetilgalactosamina/farmacología , Adhesión Bacteriana , Galactosa/farmacología , Hartmannella/microbiología , Lectinas/fisiología , Legionella pneumophila/fisiología , Proteínas Protozoarias/fisiología , Animales , Genisteína , Isoflavonas/farmacología , Lectinas/análisis , Lectinas/antagonistas & inhibidores , Peso Molecular , Fosforilación , Tirosina/metabolismoRESUMEN
Expression of the global stress protein gene (gspA) is induced during the intracellular infection of macrophages and upon exposure of Legionella pneumophila to in vitro stress stimuli. Transcription of gspA is regulated by two promoters, one of which is regulated by the sigma 32 heat-shock transcription factor. We utilized a gspA promoter fusion to a promoter less lacZ to probe the phagososmal 'microenvironment' for the kinetics of exposure of intracellular L. pneumophila to stress stimuli. Expression through the gspA promoter was constitutively induced by approx. 16-fold throughout the intracellular infection, and occurred predominantly through the sigma 32-regulated promoter. Expression of the gspA promoter was induced approx. 4.5-fold, 5-, 11- and 9-fold upon exposure of L. pneumophila to heat shock, oxidative stress, acid shock, and osmotic shock, respectively. An isogenic insertion mutant of L. pneumophila in gspA (strain AA224) was constructed by allelic exchange in the wild-type strain AA200. Compared to in vitro-grown wild-type strain AA200, AA224 was more susceptible to all four in vitro stress stimuli. The wild-type phenotypes were restored to strain AA224 by complementation with a plasmid containing wild-type gspA. There was no difference between the wild-type strain and the gspA mutant in cytopathogenicity to U937 cells or in their kinetics of intracellular replication within macrophages and amoebae. However, compared to in vitro-grown bacteria, macrophage-grown and amoebae-grown AA200 and AA224 showed an equal and dramatic increase in resistance to in vitro stress stimuli. Our data showed that regardless of the capacity of L. pneumophila to subvert the microbicidal mechanisms of the macrophage, intracellular L. pneumophila is exposed to a high level of stress stimuli throughout the intracellular infection. Although the GspA protein is required for protection of the bacteria against in vitro stress stimuli, and is induced during intracellular multiplication, the loss of its function is probably compensated for by other macrophage-induced and stress-induced proteins within the intracellular environment.