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Find video protocols related to scientific articles indexed in Pubmed.
A Toxin-Antitoxin Module of Salmonella Promotes Virulence in Mice.
PLoS Pathog.
PUBLISHED: 12-01-2013
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Toxin-antitoxin (TA) modules are widely prevalent in both bacteria and archaea. Originally described as stabilizing elements of plasmids, TA modules are also widespread on bacterial chromosomes. These modules promote bacterial persistence in response to specific environmental stresses. So far, the possibility that TA modules could be involved in bacterial virulence has been largely neglected, but recent comparative genomic studies have shown that the presence of TA modules is significantly associated with the pathogenicity of bacteria. Using Salmonella as a model, we investigated whether TA modules help bacteria to overcome the stress conditions encountered during colonization, thereby supporting virulence in the host. By bioinformatics analyses, we found that the genome of the pathogenic bacterium Salmonella Typhimurium encodes at least 11 type II TA modules. Several of these are conserved in other pathogenic strains but absent from non-pathogenic species indicating that certain TA modules might play a role in Salmonella pathogenicity. We show that one TA module, hereafter referred to as sehAB, plays a transient role in virulence in perorally inoculated mice. The use of a transcriptional reporter demonstrated that bacteria in which sehAB is strongly activated are predominantly localized in the mesenteric lymph nodes. In addition, sehAB was shown to be important for the survival of Salmonella in these peripheral lymphoid organs. These data indicate that the transient activation of a type II TA module can bring a selective advantage favouring virulence and demonstrate that TA modules are engaged in Salmonella pathogenesis.
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Salmonella T3SSs: successful mission of the secret(ion) agents.
Curr. Opin. Microbiol.
PUBLISHED: 01-05-2013
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Bacteria of the genus Salmonella express nanosyringe-like organelles called type three secretion systems (T3SSs). These systems promote the secretion of bacterial compounds and their translocation into host cells. Pathogenic Salmonella use two distinct T3SSs, with specialized functions, having the purpose to modify the biology of the host organism and to ensure a successful infection. The bacterial proteins translocated through the first T3SS (T3SS-1) facilitate the entry of Salmonella into host cells, whereas T3SS-2 is an important factor for shaping the intracellular replication niche. In addition both T3SSs have a strong impact on the host inflammation. For a long time the two T3SSs were thought to act separately. However, there is increasing evidence that their regulation depends not only on separate but also shared regulatory mechanisms and that their time of action during infection overlaps. Here, we review the current understanding of the structure and of the regulation of expression and activity of both T3SSs. The output image is multifaceted, as recent studies show that subpopulations of Salmonella present diverging patterns of expression and activity of T3SSs during important steps of infection. These diversities may advance the chances of Salmonella to outpace competitors and to well establish itself in its niche in the host.
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In Vivo Identification and Characterization of CD4(+) Cytotoxic T Cells Induced by Virulent Brucella abortus Infection.
PLoS ONE
PUBLISHED: 01-01-2013
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CD4(+) T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4(+) T cells (CD4(+) CTL) during Brucella abortus infection. These CD4(+) CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-?. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4(+) CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4(+) T cell population appears at early stages of the infection concomitantly with high levels of IFN-? and granzyme B expression. CD4(+) CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.
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Peyers patch dendritic cells sample antigens by extending dendrites through M cell-specific transcellular pores.
Gastroenterology
PUBLISHED: 09-28-2011
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Peyers patches (PPs) of the small intestine are antigen sampling and inductive sites that help establish mucosal immunity. Luminal antigens are transported from the mucosal surface of PPs to the subepithelial dome (SED), through the specialized epithelial M cells of the follicle-associated epithelium. Among the SED resident dendritic cells (DCs), which are situated ideally for taking up these antigens, some express high levels of lysozyme (LysoDC) and have strong phagocytic activity. We investigated the mechanisms by which LysoDCs capture luminal antigens in vivo.
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Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella.
PLoS ONE
PUBLISHED: 03-29-2011
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During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.
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Salmonella detoxifying enzymes are sufficient to cope with the host oxidative burst.
Mol. Microbiol.
PUBLISHED: 03-21-2011
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The oxidative burst produced by the NADPH oxidase (Phox) is an essential weapon used by host cells to eradicate engulfed pathogens. In Salmonella typhimurium, oxidative stress resistance has been previously proposed to be mediated by the pathogenicity island 2 type III secretion system (T3SS-2), periplasmic superoxide dismutases and cytoplasmic catalases/peroxidases. Here, we fused an OxyR-dependent promoter to the gfp to build the ahpC-gfp transcriptional fusion. This reporter was used to monitor hydrogen peroxide levels as sensed by Salmonella during the course of an infection. We showed that the expression of this fusion was under the exclusive control of reactive oxygen species produced by the host. The ahpC-gfp expression was noticeably modified in the absence of bacterial periplasmic superoxide dismutases or cytoplasmic catalases/peroxidases. Surprisingly, inactivation of the T3SS-2 had no effect on the ahpC-gfp expression. All together, these results led to a model in which Salmonella resistance relies on its arsenal of detoxifying enzymes to cope with Phox-mediated oxidative stress.
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Salmonella-induced tubular networks.
Trends Microbiol.
PUBLISHED: 01-12-2011
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Salmonella virulence relies on its capacity to replicate inside various cell types in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). A unique feature of Salmonella-infected cells is the presence of tubular structures originating from and connected to the SCV, which often extend throughout the cell cytoplasm. These tubules include the well-studied Salmonella-induced filaments (SIFs), enriched in lysosomal membrane proteins. However, recent studies revealed that the Salmonella-induced tubular network is more extensive than previously thought and includes three types of tubules distinct from SIFs: sorting nexin tubules, Salmonella-induced secretory carrier membrane protein 3 (SCAMP3) tubules and lysosome-associated membrane protein 1 (LAMP1)-negative tubules. In this review, we examine the molecular mechanisms involved in the formation of Salmonella-induced tubular networks and discuss the importance of the tubules for Salmonella virulence and establishment of a Salmonella intracellular replicative niche.
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Kinesin regulation by Salmonella.
Virulence
PUBLISHED: 01-01-2011
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As the result of their adaptation to the host, intracellular pathogens have evolved mechanisms to usurp and take the control of eukaryotic processes. In the case of Salmonella, this is in part achieved through the cytoplasmic translocation of bacterial effectors capable of acting on the biology of infected cells. These bacterial effectors might have enzymatic activities or target eukaryotic proteins. We have identified two Salmonella effectors that target the plus-end directed microtubule motor kinesin-1. PipB2 is a Salmonella vacuole-specific cargo adaptor for kinesin-1 while SifA binds the host protein SKIP, which interacts with the microtubule motor. SKIP is a large, multi domain protein of unknown function. Our recent investigations show that SKIP regulates the positioning of late endosomal compartments in a microtubules and kinesin-1 dependent manner. Moreover they indicate that SKIP activates the microtubule motor both in the context of infected and non-infected cells. Here we review these recent results and propose a model for the Salmonella effector-mediated regulation of kinesin-1 activity.
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The virulence protein SopD2 regulates membrane dynamics of Salmonella-containing vacuoles.
PLoS Pathog.
PUBLISHED: 06-16-2010
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Salmonella enterica serovar Typhimurium is a Gram-negative bacterial pathogen causing gastroenteritis in humans and a systemic typhoid-like illness in mice. The capacity of Salmonella to cause diseases relies on the establishment of its intracellular replication niche, a membrane-bound compartment named the Salmonella-containing vacuole (SCV). This requires the translocation of bacterial effector proteins into the host cell by type three secretion systems. Among these effectors, SifA is required for the SCV stability, the formation of Salmonella-induced filaments (SIFs) and plays an important role in the virulence of Salmonella. Here we show that the effector SopD2 is responsible for the SCV instability that triggers the cytoplasmic release of a sifA(-) mutant. Deletion of sopD2 also rescued intra-macrophagic replication and increased virulence of sifA(-) mutants in mice. Membrane tubular structures that extend from the SCV are the hallmark of Salmonella-infected cells. Until now, these unique structures have not been observed in the absence of SifA. The deletion of sopD2 in a sifA(-) mutant strain re-established membrane trafficking from the SCV and led to the formation of new membrane tubular structures, the formation of which is dependent on other Salmonella effector(s). Taken together, our data demonstrate that SopD2 inhibits the vesicular transport and the formation of tubules that extend outward from the SCV and thereby contributes to the sifA(-) associated phenotypes. These results also highlight the antagonistic roles played by SopD2 and SifA in the membrane dynamics of the vacuole, and the complex actions of SopD2, SifA, PipB2 and other unidentified effector(s) in the biogenesis and maintenance of the Salmonella replicative niche.
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SKIP, the host target of the Salmonella virulence factor SifA, promotes kinesin-1-dependent vacuolar membrane exchanges.
Traffic
PUBLISHED: 04-06-2010
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In Salmonella-infected cells, the bacterial effector SifA forms a functional complex with the eukaryotic protein SKIP (SifA and kinesin-interacting protein). The lack of either partner has important consequences on the intracellular fate and on the virulence of this pathogen. In addition to SifA, SKIP binds the microtubule-based motor kinesin-1. Yet the absence of SifA or SKIP results in an unusual accumulation of kinesin-1 on the bacterial vacuolar membrane. To understand this apparent contradiction, we investigated the interaction between SKIP and kinesin-1 and the function of this complex. We show that the C-terminal RUN (RPIP8, UNC-14 and NESCA) domain of SKIP interacted specifically with the tetratricopeptide repeat (TPR) domain of the kinesin light chain. Overexpression of SKIP induced a microtubule- and kinesin-1-dependent anterograde movement of late endosomal/lysosomal compartments. In infected cells, SifA contributed to the fission of vesicles from the bacterial vacuole and the SifA/SKIP complex was required for the formation and/or the anterograde transport of kinesin-1-enriched vesicles. These observations reflect the role of SKIP as a linker and/or an activator for kinesin-1.
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Interaction between the SifA virulence factor and its host target SKIP is essential for Salmonella pathogenesis.
J. Biol. Chem.
PUBLISHED: 09-28-2009
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SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a DeltasifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.
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Pathogenic bacteria and dead cells are internalized by a unique subset of Peyers patch dendritic cells that express lysozyme.
Gastroenterology
PUBLISHED: 09-07-2009
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Lysozyme has an important role in preventing bacterial infection. In the gastrointestinal tract, lysozyme is thought to be mainly expressed by Paneth cells of the crypt epithelium. We investigated its expression in the Peyers patch, a major intestinal site of antigen sampling and pathogen entry.
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Salmonella regulates polyubiquitination and surface expression of MHC class II antigens.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-05-2009
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Salmonella typhimurium is a facultative pathogen capable of entering and replicating in both professional and non-professional antigen presenting cells. Control of infection requires MHC class II restricted CD4 T-helper cell responses. Here we show that Salmonella infection induced polyubiquitination of HLA-DR, a post-translational modification that led to removal of mature, peptide loaded, alphabeta dimers from the cell surface. Immature alphabetaIi complexes were unaffected. Surface expression of all class II isotypes, HLA-DP, -DQ, and -DR, was reduced in infected cells, but other cell-surface molecules that traffic through class II peptide loading compartments were unaffected. A Salmonella strain carrying a mutation in ssaV did not induce ubiquitination of class II, implicating Salmonella T3SS-2 effector proteins in the process. T3SS-2 effectors, with established or proposed roles in ubiquitination, were not required for class II down-regulation, suggesting that an additional T3SS-2 effector is involved in regulating MHC class II ubiquitination. Although recognized as a viral immune evasion strategy, here, we demonstrate that bacteria can control surface MHC expression through ubiquitination.
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Redundant hydrogen peroxide scavengers contribute to Salmonella virulence and oxidative stress resistance.
J. Bacteriol.
PUBLISHED: 05-15-2009
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Salmonella enterica serovar Typhimurium is an intracellular pathogen that can survive and replicate within macrophages. One of the host defense mechanisms that Salmonella encounters during infection is the production of reactive oxygen species by the phagocyte NADPH oxidase. Among them, hydrogen peroxide (H(2)O(2)) can diffuse across bacterial membranes and damage biomolecules. Genome analysis allowed us to identify five genes encoding H(2)O(2) degrading enzymes: three catalases (KatE, KatG, and KatN) and two alkyl hydroperoxide reductases (AhpC and TsaA). Inactivation of the five cognate structural genes yielded the HpxF(-) mutant, which exhibited a high sensitivity to exogenous H(2)O(2) and a severe survival defect within macrophages. When the phagocyte NADPH oxidase was inhibited, its proliferation index increased 3.7-fold. Moreover, the overexpression of katG or tsaA in the HpxF(-) background was sufficient to confer a proliferation index similar to that of the wild type in macrophages and a resistance to millimolar H(2)O(2) in rich medium. The HpxF(-) mutant also showed an attenuated virulence in a mouse model. These data indicate that Salmonella catalases and alkyl hydroperoxide reductases are required to degrade H(2)O(2) and contribute to the virulence. This enzymatic redundancy highlights the evolutionary strategies developed by bacterial pathogens to survive within hostile environments.
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Interactions between human NK cells and macrophages in response to Salmonella infection.
J. Immunol.
PUBLISHED: 03-21-2009
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NK cells play a key role in host resistance to a range of pathogenic microorganisms, particularly during the initial stages of infection. NK cell interactions with cells infected with viruses and parasites have been studied extensively, but human bacterial infections have not been given the same attention. We studied crosstalk between human NK cells and macrophages infected with intracellular Salmonella. These macrophages activated NK cells, resulting in secretion of IFN-gamma and degranulation. Reciprocally, NK cell activation led to a dramatic reduction in numbers of intramacrophagic live bacteria. We identified three elements in the interaction of NK cells with infected macrophages. First, communication between NK cells and infected macrophages was contact-dependent. The second requirement was IL-2- and/or IL-15-dependent priming of NK cells to produce IFN-gamma. The third was activation of NK cells by IL-12 and IL-18, which were secreted by the Salmonella-infected macrophages. Adhesion molecules and IL-12Rbeta2 were enriched in the contact zone between NK cells and macrophages, consistent with contact- and IL-12/IL-18-dependent NK activation. Our results suggest that, in humans, bacterial clearance is consistent with a model invoking a "ménage à trois" involving NK cells, IL-2/IL-15-secreting cells, and infected macrophages.
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A hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia.
Eur. J. Immunol.
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Using N-ethyl-N-nitrosourea-induced mutagenesis, we established a mouse model with a novel form of neutropenia resulting from a point mutation in the transcriptional repressor Growth Factor Independence 1 (Gfi1). These mice, called Genista, had normal viability and no weight loss, in contrast to mice expressing null alleles of the Gfi1 gene. Furthermore, the Genista mutation had a very limited impact on lymphopoiesis or on T- and B-cell function. Within the bone marrow (BM), the Genista mutation resulted in a slight increase of monopoiesis and in a block of terminal granulopoiesis. This block occurred just after the metamyelocytic stage and resulted in the generation of small numbers of atypical CD11b(+) Ly-6G(int) neutrophils, the nuclear morphology of which resembled that of mature WT neutrophils. Unexpectedly, once released from the BM, these atypical neutrophils contributed to induce mild forms of autoantibody-induced arthritis and of immune complex-mediated lung alveolitis. They additionally failed to provide resistance to acute bacterial infection. Our study demonstrates that a hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia characterized by a split pattern of functional responses, reflecting the distinct thresholds required for eliciting neutrophil-mediated inflammatory and anti-infectious responses.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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

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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.