JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Genetic Manipulation of Acinetobacter baumannii.
Curr Protoc Microbiol
PUBLISHED: 11-05-2014
Show Abstract
Hide Abstract
Acinetobacter baumannii is a Gram-negative nosocomial pathogen of clinical importance. A lack of genetic tools has hindered the research of this organism in the past; however, recently, various methods have been designed, modified, and optimized to facilitate the genetic manipulation of A. baumannii. This unit describes some of the recent genetic advances and new recombinant tools developed for this pathogen, including standard transformation and conjugation techniques specifically developed for the bacteria. As the need to understand the basic biology of A. baumannii increases with the prospect of developing new therapeutics, the use of the basic genetic methods herein can provide the critical first step to identify genes required for infection. © 2014 by John Wiley & Sons, Inc.
Related JoVE Video
Host-directed antimicrobial drugs with broad-spectrum efficacy against intracellular bacterial pathogens.
MBio
PUBLISHED: 07-31-2014
Show Abstract
Hide Abstract
We sought a new approach to treating infections by intracellular bacteria, namely, by altering host cell functions that support their growth. We screened a library of 640 Food and Drug Administration (FDA)-approved compounds for agents that render THP-1 cells resistant to infection by four intracellular pathogens. We identified numerous drugs that are not antibiotics but were highly effective in inhibiting intracellular bacterial growth with limited toxicity to host cells. These compounds are likely to target three kinds of host functions: (i) G protein-coupled receptors, (ii) intracellular calcium signals, and (iii) membrane cholesterol distribution. The compounds that targeted G protein receptor signaling and calcium fluxes broadly inhibited Coxiella burnetii, Legionella pneumophila, Brucella abortus, and Rickettsia conorii, while those directed against cholesterol traffic strongly attenuated the intracellular growth of C. burnetii and L. pneumophila. These pathways probably support intracellular pathogen growth so that drugs that perturb them may be therapeutic candidates. Combining host- and pathogen-directed treatments is a strategy to decrease the emergence of drug-resistant intracellular bacterial pathogens. Importance: Although antibiotic treatment is often successful, it is becoming clear that alternatives to conventional pathogen-directed therapy must be developed in the face of increasing antibiotic resistance. Moreover, the costs and timing associated with the development of novel antimicrobials make repurposed FDA-approved drugs attractive host-targeted therapeutics. This paper describes a novel approach of identifying such host-targeted therapeutics against intracellular bacterial pathogens. We identified several FDA-approved drugs that inhibit the growth of intracellular bacteria, thereby implicating host intracellular pathways presumably utilized by bacteria during infection.
Related JoVE Video
Identification of novel Coxiella burnetii Icm/Dot effectors and genetic analysis of their involvement in modulating a mitogen-activated protein kinase pathway.
Infect. Immun.
PUBLISHED: 06-23-2014
Show Abstract
Hide Abstract
Coxiella burnetii, the causative agent of Q fever, is a human intracellular pathogen that utilizes the Icm/Dot type IVB secretion system to translocate effector proteins into host cells. To identify novel C. burnetii effectors, we applied a machine-learning approach to predict C. burnetii effectors, and examination of 20 such proteins resulted in the identification of 13 novel effectors. To determine whether these effectors, as well as several previously identified effectors, modulate conserved eukaryotic pathways, they were expressed in Saccharomyces cerevisiae. The effects on yeast growth were examined under regular growth conditions and in the presence of caffeine, a known modulator of the yeast cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway. In the presence of caffeine, expression of the effectors CBU0885 and CBU1676 caused an enhanced inhibition of yeast growth, and the growth inhibition of CBU0388 was suppressed. Furthermore, analysis of synthetic lethality effects and examination of the activity of the CWI MAP kinase transcription factor Rlm1 indicated that CBU0388 enhances the activation of this MAP kinase pathway in yeast, while CBU0885 and CBU1676 abolish this activation. Additionally, coexpression of CBU1676 and CBU0388 resulted in mutual suppression of their inhibition of yeast growth. These results strongly indicate that these three effectors modulate the CWI MAP kinase pathway in yeast. Moreover, both CBU1676 and CBU0885 were found to contain a conserved haloacid dehalogenase (HAD) domain, which was found to be required for their activity. Collectively, our results demonstrate that MAP kinase pathways are most likely targeted by C. burnetii Icm/Dot effectors.
Related JoVE Video
Analysis of the transcriptome of Legionella pneumophila hfq mutant reveals a new mobile genetic element.
Microbiology (Reading, Engl.)
PUBLISHED: 05-31-2013
Show Abstract
Hide Abstract
Hfq is a small RNA-binding protein involved in the post-transcriptional regulation of gene expression by affecting the stability of the mRNA and by mediating efficient pairing between small regulatory RNAs and their target mRNAs. In Legionella pneumophila, the aetiological agent of Legionnaires disease, mutation of hfq results in increased duration of the lag phase and reduced growth in low-iron medium. In an effort to uncover genes potentially regulated by Hfq, the transcriptome of an hfq mutant strain was compared to that of the wild-type. Unexpectedly, many genes located within a 100 kb genomic island, including a section of the previously identified efflux island, were overexpressed in the hfq mutant strain. Since this island contains a putative conjugative system and an integrase, it was postulated that it could be a new integrated mobile genetic element. PCR analysis revealed that this region exists both as an integrated and as an episomal form in the cell population and that it undergoes differential excision in the hfq mutant background, which was further confirmed by trans-complementation of the hfq mutation. This new plasmid-like element was named pLP100. Differential excision did not affect the copy number of pLP100 at the population level. This region contains a copper efflux pump encoded by copA, and increased resistance to copper was observed for the hfq mutant strain that was abrogated in the complemented strain. A strain carrying a mutation of hfq and a deletion of the right side recombination site, attR, showed that overexpression of pLP100 genes and increased copper resistance in the hfq mutant strain were dependent upon excision of pLP100.
Related JoVE Video
Methods to study legionella transcriptome in vitro and in vivo.
Methods Mol. Biol.
PUBLISHED: 04-23-2013
Show Abstract
Hide Abstract
The study of transcriptome responses can provide insight into the regulatory pathways and genetic factors that contribute to a specific phenotype. For bacterial pathogens, it can identify putative new virulence systems and shed light on the mechanisms underlying the regulation of virulence factors. Microarrays have been previously used to study gene regulation in Legionella pneumophila. In the past few years a sharp reduction of the costs associated with microarray experiments together with the availability of relatively inexpensive custom-designed commercial microarrays has made microarray technology an accessible tool for the majority of researchers. Here we describe the methodologies to conduct microarray experiments from in vitro and in vivo samples.
Related JoVE Video
Legionella pneumophila transcriptional response following exposure to CuO nanoparticles.
Appl. Environ. Microbiol.
PUBLISHED: 02-15-2013
Show Abstract
Hide Abstract
Copper ions are an effective antimicrobial agent used to control Legionnaires disease and Pontiac fever arising from institutional drinking water systems. Here, we present data on an alternative bactericidal agent, copper oxide nanoparticles (CuO-NPs), and its efficacy on Legionella pneumophila. In broth cultures, the CuO-NPs caused growth inhibition, which appeared to be concentration and exposure time dependent. The transcriptomic response of L. pneumophila to CuO-NP exposure was investigated by using a whole-genome microarray. The expression of genes involved in metabolism, transcription, translation, DNA replication and repair, and unknown/hypothetical proteins was significantly affected by exposure to CuO-NPs. In addition, expression of 21 virulence genes was also affected by exposure to CuO-NP and further evaluated by quantitative reverse transcription-PCR (qRT-PCR). Some virulence gene responses occurred immediately and transiently after addition of CuO-NPs to the cells and faded rapidly (icmV, icmW, lepA), while expression of other genes increased within 6 h (ceg29, legLC8, legP, lem19, lem24, lpg1689, and rtxA), 12 h (cegC1, dotA, enhC, htpX, icmE, pvcA, and sidF), and 24 h (legP, lem19, and ceg19), but for most of the genes tested, expression was reduced after 24 h of exposure. Genes like ceg29 and rtxA appeared to be the most responsive to CuO-NP exposures and along with other genes identified in this study may prove useful to monitor and manage the impact of drinking water disinfection on L. pneumophila.
Related JoVE Video
Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 02-04-2013
Show Abstract
Hide Abstract
Legionella and Coxiella are intracellular pathogens that use the virulence-related Icm/Dot type-IVB secretion system to translocate effector proteins into host cells during infection. These effectors were previously shown to contain a C-terminal secretion signal required for their translocation. In this research, we implemented a hidden semi-Markov model to characterize the amino acid composition of the signal, thus providing a comprehensive computational model for the secretion signal. This model accounts for dependencies among sites and captures spatial variation in amino acid composition along the secretion signal. To validate our model, we predicted and synthetically constructed an optimal secretion signal whose sequence is different from that of any known effector. We show that this signal efficiently translocates into host cells in an Icm/Dot-dependent manner. Additionally, we predicted in silico and experimentally examined the effects of mutations in the secretion signal, which provided innovative insights into its characteristics. Some effectors were found to lack a strong secretion signal according to our model. We demonstrated that these effectors were highly dependent on the IcmS-IcmW chaperons for their translocation, unlike effectors that harbor a strong secretion signal. Furthermore, our model is innovative because it enables searching ORFs for secretion signals on a genomic scale, which led to the identification and experimental validation of 20 effectors from Legionella pneumophila, Legionella longbeachae, and Coxiella burnetii. Our combined computational and experimental methodology is general and can be applied to the identification of a wide spectrum of protein features that lack sequence conservation but have similar amino acid characteristics.
Related JoVE Video
A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy.
Eur. J. Immunol.
PUBLISHED: 01-09-2013
Show Abstract
Hide Abstract
Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin-containing protein with unknown role. We show that the legA9 mutant replicate in WT mice and their bone marrow-derived macrophages. This is the first L. pneumophila mutant to be found to replicate in WT bone marrow-derived macrophages other than the Fla mutant. Less legA9 mutant-containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila-containing vacuole for autophagy uptake.
Related JoVE Video
In vitro derivation of macrophage from guinea pig bone marrow with human M-CSF.
J. Immunol. Methods
PUBLISHED: 01-02-2013
Show Abstract
Hide Abstract
The guinea pig has a storied history as a model in the study of infectious disease and immunology. Because of reproducibility of data and availability of various reagents, inbred mice have since supplanted the guinea pig as the animal model-of-choice in these fields. However, several clinically-significant microorganisms do not cause the same pathology in mice, or mice may not be susceptible to these infections. These demonstrate the utility of other animal models - either as the primary method to study a particular infection, or to confirm or refute findings in the mouse before translating basic science into clinical practice. The mononuclear phagocyte, or macrophage (M?), plays a key role in antigen presentation and the pathogenesis of intracellular bacteria, such as Mycobacterium tuberculosis and Legionella pneumophila. Because of variable yield and difficult extraction from tissue, the preferred method of producing M? for in vitro studies is to expand murine bone marrow (BM) precursors with mouse macrophage colony-stimulating factor (M-CSF). This has not been shown in the guinea pig. Here, we report the empiric observation that human M-CSF - but not mouse M-CSF, nor human granulocyte/macrophage colony-stimulating factor - can be used to induce BM precursor differentiation into bonafide M?. The differentiated cells appeared as enlarged adherent cells, capable of both pinocytosis and large particle phagocytosis. Furthermore, we showed that these guinea pig BM-derived M?, similar to human monocyte/M? lines but unlike most murine BM M?, support growth of wild type L. pneumophila. This method may prove useful for in vitro studies of M? in the guinea pig, as well as in the translation of results found using mouse BM-derived M? towards studies in human immunology and infectious disease.
Related JoVE Video
The overlapping host responses to bacterial cyclic dinucleotides.
Microbes Infect.
PUBLISHED: 08-25-2011
Show Abstract
Hide Abstract
Macrophages respond to infection with Legionella pneumophila by the induction of inflammatory mediators, including type I Interferons (IFN-Is). To explore whether the bacterial second messenger cyclic 3-5 diguanylate (c-diGMP) activates some of these mediators, macrophages were infected with L. pneumophila strains in which the levels of bacterial c-diGMP had been altered. Intriguingly, there was a positive correlation between c-diGMP levels and IFN-I expression. Subsequent studies with synthetic derivatives of c-diGMP, and newly described cyclic 3-5 diadenylate (c-diAMP), determined that these molecules activate overlapping inflammatory responses in human and murine macrophages. Moreover, UV crosslinking studies determined that both dinucleotides physically associate with a shared set of host proteins. Fractionation of macrophage extracts on a biotin-c-diGMP affinity matrix led to the identification of a set of candidate host binding proteins. These studies suggest that mammalian macrophages can sense and mount a specific inflammatory response to bacterial dinucleotides.
Related JoVE Video
Chemical synthesis enables biochemical and antibacterial evaluation of streptolydigin antibiotics.
J. Am. Chem. Soc.
PUBLISHED: 07-20-2011
Show Abstract
Hide Abstract
Inhibition of bacterial transcription represents an effective and clinically validated anti-infective chemotherapeutic strategy. We describe the evolution of our approach to the streptolydigin class of antibiotics that target bacterial RNA polymerases (RNAPs). This effort resulted in the synthesis and biological evaluation of streptolydigin, streptolydiginone, streptolic acid, and a series of new streptolydigin-based agents. Subsequent biochemical evaluation of RNAP inhibition demonstrated that the presence of both streptolic acid and tetramic acid subunits was required for activity of this class of antibiotics. In addition, we identified 10,11-dihydrostreptolydigin as a new RNAP-targeting agent, which was assembled with high synthetic efficiency of 15 steps in the longest linear sequence. Dihydrostreptolydigin inhibited three representative bacterial RNAPs and displayed in vitro antibacterial activity against S. salivarius . The overall increase in synthetic efficiency combined with substantial antibacterial activity of this fully synthetic antibiotic demonstrates the power of organic synthesis in enabling design and comprehensive in vitro pharmacological evaluation of new chemical agents that target bacterial transcription.
Related JoVE Video
A new heterolobosean amoeba Solumitrus palustris n. g., n. sp. isolated from freshwater marsh soil.
J. Eukaryot. Microbiol.
PUBLISHED: 04-19-2011
Show Abstract
Hide Abstract
During the course of research on the bacterial feeding behavior and resistance of amoebae to virulent pathogens, we isolated a new strain of amoeba from organic rich soil at the margin of freshwater swamp in the northeastern United States. Light microscopic morphology is characteristically heterolobosean, resembling vahlkampfiids, including a broadened, limax shape, and eruptive locomotion, but occasionally becoming more contracted and less elongated with lateral or anterior bulges and somewhat branching sparse, uroidal filaments. Electron microscopic evidence, including mitochondria with flattened cristae surrounded by rough endoplasmic reticulum, further indicates a heterolobosean affinity. The solitary nucleus contains a centrally located nucleolus. Cysts are rounded with occasionally an eccentrically located nucleus. The cyst walls are relatively thin, becoming crenated, and loosely enclosing the cyst when mature. Molecular genetic evidence places this isolate among the Heterolobosea, branching most closely in a clade including Allovahlkampfia spelaea and previously isolated, un-named strains of soil amoebae. Based on differentiated features, including morphology of the uroid, cyst wall structure, and molecular genetic evidence that distinguish it from A. spelaea, a new genus and species, Solumitrus palustris, is proposed for this new heterolobosean.
Related JoVE Video
Legionella Pneumophila Transcriptome during Intracellular Multiplication in Human Macrophages.
Front Microbiol
PUBLISHED: 03-18-2011
Show Abstract
Hide Abstract
Legionella pneumophila is the causative agent of Legionnaires disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot type IVB secretion system, which is used to translocate more than 150 effector proteins into host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called selective capture of transcribed sequences to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to l-arginine, l-histidine, and l-proline as well as many transport systems involved in amino acid and iron uptake. Genes involved in catabolism of glycerol are also induced during intracellular growth, suggesting that glycerol could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.
Related JoVE Video
Small Regulatory RNA and Legionella pneumophila.
Front Microbiol
PUBLISHED: 01-15-2011
Show Abstract
Hide Abstract
Legionella pneumophila is a gram-negative bacterial species that is ubiquitous in almost any aqueous environment. It is the agent of Legionnaires disease, an acute and often under-reported form of pneumonia. In mammals, L. pneumophila replicates inside macrophages within a modified vacuole. Many protein regulators have been identified that control virulence-related properties, including RpoS, LetA/LetS, and PmrA/PmrB. In the past few years, the importance of regulation of virulence factors by small regulatory RNA (sRNAs) has been increasingly appreciated. This is also the case in L. pneumophila where three sRNAs (RsmY, RsmZ, and 6S RNA) were recently shown to be important determinants of virulence regulation and 79 actively transcribed sRNAs were identified. In this review we describe current knowledge about sRNAs and their regulatory properties and how this relates to the known regulatory systems of L. pneumophila. We also provide a model for sRNA-mediated control of gene expression that serves as a framework for understanding the regulation of virulence-related properties of L. pneumophila.
Related JoVE Video
Cyclic diguanylate signaling proteins control intracellular growth of Legionella pneumophila.
MBio
PUBLISHED: 01-11-2011
Show Abstract
Hide Abstract
Proteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle of Legionella pneumophila, the causative agent of Legionnaires disease, has not previously been examined. The L. pneumophila genome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes as cdgS (cyclic diguanylate signaling) genes. Strains of L. pneumophila containing deletions of all individual cdgS genes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, several cdgS genes strongly decreased the ability of L. pneumophila to grow inside host cells. Expression of these cdgS genes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth in L. pneumophila. L. pneumophila strains overexpressing these cdgS genes were less cytotoxic to THP-1 macrophages than wild-type L. pneumophila but retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused by cdgS gene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of the cdgS genes in a Salmonella enterica serovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that several cdgS genes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability of L. pneumophila to grow in host cells.
Related JoVE Video
Antibiotics and UV radiation induce competence for natural transformation in Legionella pneumophila.
J. Bacteriol.
PUBLISHED: 12-17-2010
Show Abstract
Hide Abstract
Natural transformation by competence is a major mechanism of horizontal gene transfer in bacteria. Competence is defined as the genetically programmed physiological state that enables bacteria to actively take up DNA from the environment. The conditions that signal competence development are multiple and elusive, complicating the understanding of its evolutionary significance. We used expression of the competence gene comEA as a reporter of competence development and screened several hundred molecules for their ability to induce competence in the freshwater living pathogen Legionella pneumophila. We found that comEA expression is induced by chronic exposure to genotoxic molecules such as mitomycin C and antibiotics of the fluoroquinolone family. These results indicated that, in L. pneumophila, competence may be a response to genotoxic stress. Sunlight-emitted UV light represents a major source of genotoxic stress in the environment and we found that exposure to UV radiation effectively induces competence development. For the first time, we show that genetic exchanges by natural transformation occur within an UV-stressed population. Genotoxic stress induces the RecA-dependent SOS response in many bacteria. However, genetic and phenotypic evidence suggest that L. pneumophila lacks a prototypic SOS response and competence development in response to genotoxic stress is RecA independent. Our results strengthen the hypothesis that competence may have evolved as a DNA damage response in SOS-deficient bacteria. This parasexual response to DNA damage may have enabled L. pneumophila to acquire and propagate foreign genes, contributing to the emergence of this human pathogen.
Related JoVE Video
ArgR-regulated genes are derepressed in the Legionella-containing vacuole.
J. Bacteriol.
PUBLISHED: 07-09-2010
Show Abstract
Hide Abstract
Legionella pneumophila is an intracellular pathogen that infects protozoa in aquatic environments and when inhaled by susceptible human hosts replicates in alveolar macrophages and can result in the often fatal pneumonia called Legionnaires disease. The ability of L. pneumophila to replicate within host cells requires the establishment of a specialized compartment that evades normal phagolysosome fusion called the Legionella-containing vacuole (LCV). Elucidation of the biochemical composition of the LCV and the identification of the regulatory signals sensed during intracellular replication are inherently challenging. L-Arginine is a critical nutrient in the metabolism of both prokaryotic and eukaryotic organisms. We showed that the L. pneumophila arginine repressor homolog, ArgR, is required for maximal intracellular growth in the unicellular host Acanthamoeba castellanii. In this study, we present evidence that the concentration of L-arginine in the LCV is sensed by ArgR to produce an intracellular transcriptional response. We characterized the L. pneumophila ArgR regulon by global gene expression analysis, identified genes highly affected by ArgR, showed that ArgR repression is dependent upon the presence of L-arginine, and demonstrated that ArgR-regulated genes are derepressed during intracellular growth. Additional targets of ArgR that may account for the argR mutants intracellular multiplication defect are discussed. These results suggest that L-arginine availability functions as a regulatory signal during Legionella intracellular growth.
Related JoVE Video
Legionella pneumophila 6S RNA optimizes intracellular multiplication.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-05-2010
Show Abstract
Hide Abstract
Legionella pneumophila is a Gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to identify sRNAs expressed by L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which 6 were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed in postexponential phase, and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of sigma(70)-containing RNA polymerase. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA, as well as many genes involved in acquisition of nutrients and genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media.
Related JoVE Video
Interferons direct an effective innate response to Legionella pneumophila infection.
J. Biol. Chem.
PUBLISHED: 08-31-2009
Show Abstract
Hide Abstract
Legionella pneumophila remains an important opportunistic pathogen of human macrophages. Its more limited ability to replicate in murine macrophages has been attributed to redundant innate sensor systems that detect and effectively respond to this infection. The current studies evaluate the role of one of these innate response systems, the type I interferon (IFN-I) autocrine loop. The ability of L. pneumophila to induce IFN-I expression was found to be dependent on IRF-3, but not NF-kappaB. Secreted IFN-Is then in turn suppress the intracellular replication of L. pneumophila. Surprisingly, this suppression is mediated by a pathway that is independent of Stat1, Stat2, Stat3, but correlates with the polarization of macrophages toward the M1 or classically activated phenotype.
Related JoVE Video
Intracellular bacteria encode inhibitory SNARE-like proteins.
PLoS ONE
PUBLISHED: 07-31-2009
Show Abstract
Hide Abstract
Pathogens use diverse molecular machines to penetrate host cells and manipulate intracellular vesicular trafficking. Viruses employ glycoproteins, functionally and structurally similar to the SNARE proteins, to induce eukaryotic membrane fusion. Intracellular pathogens, on the other hand, need to block fusion of their infectious phagosomes with various endocytic compartments to escape from the degradative pathway. The molecular details concerning the mechanisms underlying this process are lacking. Using both an in vitro liposome fusion assay and a cellular assay, we showed that SNARE-like bacterial proteins block membrane fusion in eukaryotic cells by directly inhibiting SNARE-mediated membrane fusion. More specifically, we showed that IncA and IcmG/DotF, two SNARE-like proteins respectively expressed by Chlamydia and Legionella, inhibit the endocytic SNARE machinery. Furthermore, we identified that the SNARE-like motif present in these bacterial proteins encodes the inhibitory function. This finding suggests that SNARE-like motifs are capable of specifically manipulating membrane fusion in a wide variety of biological environments. Ultimately, this motif may have been selected during evolution because it is an efficient structural motif for modifying eukaryotic membrane fusion and thus contribute to pathogen survival.
Related JoVE Video
The perplexing functions and surprising origins of Legionella pneumophila type IV secretion effectors.
Cell. Microbiol.
PUBLISHED: 06-26-2009
Show Abstract
Hide Abstract
Only a limited number of bacterial pathogens evade destruction by phagocytic cells such as macrophages. Legionella pneumophila is a Gram-negative gamma-proteobacterial species that can infect and replicate in alveolar macrophages, causing Legionnaires disease, a severe pneumonia. L. pneumophila uses a complex secretion system to inject host cells with effector proteins capable of disrupting or altering the host cell processes. The L. pneumophila effectors target multiple processes but are essentially aimed at modifying the properties of the L. pneumophila phagosome by altering vesicular trafficking, gradually creating a specialized vacuole in which the bacteria replicate robustly. In nature, L. pneumophila is thought to parasitize free-living protists, which may have selected for traits that promote virulence of L. pneumophila in humans. Indeed, many effector genes encode proteins with eukaryotic domains and are likely to be of protozoan origin. Sustained horizontal gene transfer events within the protozoan niche may have allowed L. pneumophila to become a professional parasite of phagocytes, simultaneously giving rise to its ability to infect macrophages, cells that constitute the first line of cellular defence against bacterial infections.
Related JoVE Video
Chemical genetics reveals bacterial and host cell functions critical for type IV effector translocation by Legionella pneumophila.
PLoS Pathog.
PUBLISHED: 06-05-2009
Show Abstract
Hide Abstract
Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host-pathogen interactions.
Related JoVE Video
Gemfibrozil inhibits Legionella pneumophila and Mycobacterium tuberculosis enoyl coenzyme A reductases and blocks intracellular growth of these bacteria in macrophages.
J. Bacteriol.
PUBLISHED: 05-08-2009
Show Abstract
Hide Abstract
We report here that gemfibrozil (GFZ) inhibits axenic and intracellular growth of Legionella pneumophila and of 27 strains of wild-type and multidrug-resistant Mycobacterium tuberculosis in bacteriological medium and in human and mouse macrophages, respectively. At a concentration of 0.4 mM, GFZ completely inhibited L. pneumophila fatty acid synthesis, while at 0.12 mM it promoted cytoplasmic accumulation of polyhydroxybutyrate. To assess the mechanism(s) of these effects, we cloned an L. pneumophila FabI enoyl reductase homolog that complemented for growth an Escherichia coli strain carrying a temperature-sensitive enoyl reductase and rendered the complemented E. coli strain sensitive to GFZ at the nonpermissive temperature. GFZ noncompetitively inhibited this L. pneumophila FabI homolog, as well as M. tuberculosis InhA and E. coli FabI.
Related JoVE Video
SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila.
J. Bacteriol.
PUBLISHED: 02-13-2009
Show Abstract
Hide Abstract
Legionella pneumophila is the causative agent of the severe and potentially fatal pneumonia Legionnaires disease. L. pneumophila is able to replicate within macrophages and protozoa by establishing a replicative compartment in a process that requires the Icm/Dot type IVB secretion system. The signals and regulatory pathways required for Legionella infection and intracellular replication are poorly understood. Mutation of the rpoS gene, which encodes sigma(S), does not affect growth in rich medium but severely decreases L. pneumophila intracellular multiplication within protozoan hosts. To gain insight into the intracellular multiplication defect of an rpoS mutant, we examined its pattern of gene expression during exponential and postexponential growth. We found that sigma(S) affects distinct groups of genes that contribute to Legionella intracellular multiplication. We demonstrate that rpoS mutants have a functional Icm/Dot system yet are defective for the expression of many genes encoding Icm/Dot-translocated substrates. We also show that sigma(S) affects the transcription of the cpxR and pmrA genes, which encode two-component response regulators that directly affect the transcription of Icm/Dot substrates. Our characterization of the L. pneumophila small RNA csrB homologs, rsmY and rsmZ, introduces a link between sigma(S) and the posttranscriptional regulator CsrA. We analyzed the network of sigma(S)-controlled genes by mutational analysis of transcriptional regulators affected by sigma(S). One of these, encoding the L. pneumophila arginine repressor homolog gene, argR, is required for maximal intracellular growth in amoebae. These data show that sigma(S) is a key regulator of multiple pathways required for L. pneumophila intracellular multiplication.
Related JoVE Video
A pathogens journey in the host cell: Bridges between actin and traffic.
Bioarchitecture
Show Abstract
Hide Abstract
Manipulation of the actin cytoskeleton is a commonly used process by which bacterial pathogens and viruses are able to neutralize host defense mechanisms and subvert them in order to replicate in a hostile environment. Diverse bacteria display a wide array of mechanisms of regulation of microfilaments to enter, move within or exit the host cell. A less studied subject is how pathogens may co-opt the actin cytoskeleton to disturb vesicle trafficking pathways, namely phagolysosomal fusion, and avoid degradation. In fact, although actin plays a role in endosomal trafficking and phagosome maturation, the knowledge on the exact mechanisms and additional players is still scarce. Recently, we found that the Legionella pneumophila virulence factor VipA is an actin nucleator, associates with actin filaments and early endosomes during infection, and interferes in yeast organelle trafficking pathways, suggesting it may be linking actin dynamics to endosome biogenesis. Further studies on this protein, together with work on other bacterial effectors, may help shed light in the role of actin in endosomal maturation.
Related JoVE Video
The Legionella pneumophila effector VipA is an actin nucleator that alters host cell organelle trafficking.
PLoS Pathog.
Show Abstract
Hide Abstract
Legionella pneumophila, the causative agent of Legionnaires disease, invades and replicates within macrophages and protozoan cells inside a vacuole. The type IVB Icm/Dot secretion system is necessary for the translocation of effector proteins that modulate vesicle trafficking pathways in the host cell, thus avoiding phagosome-lysosome fusion. The Legionella VipA effector was previously identified by its ability to interfere with organelle trafficking in the Multivesicular Body (MVB) pathway when ectopically expressed in yeast. In this study, we show that VipA binds actin in vitro and directly polymerizes microfilaments without the requirement of additional proteins, displaying properties distinct from other bacterial actin nucleators. Microscopy studies revealed that fluorescently tagged VipA variants localize to puncta in eukaryotic cells. In yeast these puncta are associated with actin-rich regions and components of the Multivesicular Body pathway such as endosomes and the MVB-associated protein Bro1. During macrophage infection, native translocated VipA associated with actin patches and early endosomes. When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets. Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization. These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways. VipA constitutes a novel type of actin nucleator that may contribute to the intracellular lifestyle of Legionella by altering cytoskeleton dynamics to target host cell pathways.
Related JoVE Video
Whole-genome sequence of the human pathogen Legionella pneumophila serogroup 12 strain 570-CO-H.
J. Bacteriol.
Show Abstract
Hide Abstract
We present the genomic sequence of the human pathogen Legionella pneumophila serogroup 12 strain 570-CO-H (ATCC 43290), a clinical isolate from the Colorado Department of Health, Denver, CO. This is the first example of a genome sequence of L. pneumophila from a serogroup other than serogroup 1. We highlight the similarities and differences relative to six genome sequences that have been reported for serogroup 1 strains.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.