- Email: [email protected]
Roundtrip explorations of bacterial infection: from single cells to the entire host and back
Review
TRENDS in Microbiology
Vol.15 No.11
Roundtrip explorations of bacterial infection: from single cells to the entire host and back Jost Enninga1,2, Philippe Sansonetti1,2 and Regis Tournebize1,2 1 2
Unite´ de Pathoge´nie Microbienne, Institut Pasteur, 28, rue du Dr Roux, 75724 Paris cedex 15, France Unite´ INSERM 786, France
Host–pathogen interactions are highly regulated, dynamic processes that take place at the molecular, cellular and organ level. Innovative imaging technologies have emerged recently to investigate the underlying mechanisms of host–pathogen interactions. Innovations in fluorescence microscopy enable functional studies on the single-cell level. New light microscopes have been developed that improve the resolution to less than 100 nm. At the other extreme, intravital microscopy enables the correlation of cellular events on the organ level. This is also achieved by alternatives to microscopy such as bioluminescence, positron-emission tomography and magnetic resonance imaging. The methodologies described here will have a tremendous effect on our understanding of host–pathogen interactions. Pathogen invasion of single cells and organs During infection, bacterial pathogens surmount various host epithelial barriers in different tissues and host cell types [1]. The microenvironment of the tissues varies enormously, and different host cells react in multiple ways on contact with pathogens. Thus, pathogens have to adapt their lifestyle to successfully disseminate and propagate inside the host. To achieve this, pathogens have developed molecular mechanisms to subvert the host immune response and major host signaling pathways [2,3]. Emerging technologies using fluorescence imaging have been adapted for the study of host–pathogen interactions to delineate their molecular mechanisms in the cellular context [4]. These technologies enable highly sensitive and dynamic studies owing to the accessibility of state-of-theart microscope hardware and development of fluorescence markers and reporters (Box 1) [5]. This has enabled investigation of the molecular mechanisms on both the host and pathogen with high temporal and/or spatial resolution. Furthermore, physicists are currently developing microscopes that overcome the spatial resolution limit of visible light [6]. Such microscopes have the potential to revolutionize the analysis of host–pathogen interactions at the cellular level. On the organ level, multiphoton microscopy of living tissue enables the tracking of the infection process, whereas bioluminescence (BLI), positron-emission Corresponding authors: Enninga, J. ([email protected]). Available online 5 November 2007. www.sciencedirect.com
([email protected]); Tournebize, R.
tomography (PET) and magnetic resonance imaging (MRI) techniques yield the specific visualization of up to a few cells in the whole body. We will outline below how these approaches have enriched our understanding of the progression on infectious processes inside the host. Innovative fluorescent assays Tracking pathogen invasion Bacterial pathogens invade host cells or attach to their surface [7]. When pathogens are recognized and subsequently ingested by phagocytic cells of the host immune system, they use specific strategies to prevent their destruction and to undermine the host immune response [8]. During all these events specific gene- expression programs are triggered inside the intruding pathogen preparing it for combat with the host [9]. Fluorescence methods have been used for studying gene expression of the single bacterium level and on the protein level for host–pathogen interactions. During the past decade, the study of gene-expression profiles in individual living cells has become possible through the use of genetically encoded fluorescent reporters, such as the green fluorescent protein (GFP). Cells containing GFP-encoding sequences engineered downstream of specific promoters light up after promoter induction. For bacteria, this approach, termed differential fluorescence induction (DFI), has been generally combined with fluorescence-activated cell sorting (FACS). However, DFI is also readily applicable using fluorescence microscopy to track individual bacteria [10]. Initially, DFI was developed to investigate differential pathogen gene expression during macrophage uptake of Salmonella typhimurium [11]. Similar approaches have been adopted to study other host–pathogen interactions, for example, to identify virulence factors involved in cell-to-cell spread of intracellularly growing Shigella flexneri [12]. Furthermore, DFI revealed that the pathogen type III secretion (T3S) machinery of Pseudomonas aeruginosa is expressed in a metabolism-dependent fashion [13]. Importantly, DFI approaches have been performed in the context of the entire host organism and, in the case of Salmonella, have identified pathogen promoters that are highly induced at the site of infection [14,15]. Data from such studies can now be merged with proteomic results from systematic analyses of metabolic Salmonella networks [16].
0966-842X/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2007.10.006
484
Review
TRENDS in Microbiology Vol.15 No.11
Box 1. Accessing state-of-the-art microscopes and interpreting your images correctly Even though microbiologists have been teaming up with cell biologists recently, many microbiology laboratories still lack cutting-edge lightmicroscopy equipment. This does not mean that such laboratories cannot perform projects similar to the ones described here. Three possibilities exist. First, a specific microscope can be purchased by a laboratory. This is often expensive, and the choice of instrument has to be balanced between the various projects that the microscope is supposed to be used for. Furthermore, space has to be considered owing to the size of a microscope. The second option is to work together with light-microscopy core facilities that are now established at a large number of research institutions. Sometimes, core facilities acquire equipment in collaboration with research laboratories to the benefit of both groups. This way, the core facility attracts a more diverse group of users, and the research group is helped with equipment maintenance and upgrading. Third, it is
Gene-expression analyses have indicated that bacterial secretion machineries are expressed by pathogens at increased levels during infections. These machineries translocate a cocktail of toxins, or effector proteins, into host cells [17]. New approaches have been developed to reveal the molecular mechanism of effector-protein secretion through the various secretion devices (Figure 1). For example, the
possible to collaborate with research groups or core facilities working with a particular piece of equipment. Working with core facilities or addressing questions with expert groups (for example, bioinformaticians) helps tremendously with one of the most difficult problems in imaging — the correct, unbiased and quantitative interpretation of the imaging data. During recent years, the amount of imaging data presented in research papers has exploded, and with it the amount of artifacts. Working together with imaging experts who are data-processing specialists is of highest importance to interpret raw imaging data correctly. Such personnel should be present at each imaging core facility. In the coming years, it will be important to increase the stringency of imaging-data interpretation to profit from the strength of imaging as a method, and to keep imaging and the scientific advances it provides credible to the research community.
T3S apparatus that functions as a molecular syringe has been a major research focus [18,19]. However, studies have been hampered because secreted effectors cannot readily be rendered fluorescent through GFP fusion because GFP cannot pass through the T3S syringes. This difficulty has been overcome using various small protein tags in conjunction with indirect immunofluorescence. Such studies
Figure 1. Fluorescent assays to monitor the dynamics of bacterial effector secretion. (a) Translocation of the effectors into host cells is monitored through a FRET assay. Effectors are fused to b-lactamase that cleaves a FRET probe inside the host cells. If FRET takes placde, the cells fluoresce green (no effector injection), if no FRET takes place (effector injection), cells fluoresce blue. This approach is well suited for screenings. Symbols: A, FRET acceptor; D, FRET donor. (b) Arrival of the translocated effector proteins in the host cell is measured through its binding to an effector-specific chaperone (InvB) that has been fluorescently labeled with GFP yielding GFP-InvB (in green). This is the only approach that does not alter the effectors; however, this approach requires their specific localization at the entry site. (c) The effectors are tagged with a small tetracysteine motif that binds with high affinity the small metallo-organic FlAsH compound. Depletion of the effector–4Cys–FlAsH molecules inside the bacterium is directly observed during effector secretion. (d) Effectors are fused to the Cre recombinase. After translocation into cell lines containing a LoxP reporter plasmid, the LoxP sites are cleaved and transcription of the reporter (for example, GFP) is triggered (geen). This approach does not yield real-time resolution owing to GFP maturation. This approach has been used to track type IV secretion. T1 indicates a time point before secretion, and T2 a time point during the secretion process. www.sciencedirect.com
Review
TRENDS in Microbiology
showed localization of secreted Salmonella T3S effectors upon translocation in single cells using microscopy on fixed samples [20]. Novel reporters using fluorescence resonance energy transfer (FRET) carry the potential to track the translocation of T3S effectors into host cells dynamically (Figure 1a) [21]. FRET describes the ability of one fluorophor (the FRET donor, D) to transfer light directly without emission to another nearby fluorophor (the FRET acceptor, A) [22]. FRET from D to A leads to an emission spectrum with the characteristics of A [22]. Often, FRET is used to study protein–protein interactions through live cell imaging or in fixed samples using confocal or epifluorescent set-ups [23]. However, FRET probes can also be used to study various cellular phenomena as discussed below [23]. One FRET-based approach used enteropathogenic Escherichia coli (EPEC) expressing chimeras that consist of a T3S signal sequence fused to b-lactamase (Figure 1a). These bacteria were then used to challenge host cells that had been loaded with a b-lactamase-cleavable FRET substrate. Upon secretion of the T3S effector fusion, the FRET substrate was cleaved, and the fluorescence signal changed to enable the identification of novel T3S signal sequences. This approach has also been adapted to investigate which immune cells are attacked by Yersinia pestis in living rodents [24]. Recently, tracking the type III effector secretion of Salmonella and Shigella has been tackled with different strategies (Figure 1b and c) [25,26]. Translocation of the T3S effectors from both pathogens took place with similar kinetics, emphasizing that this process is functionally conserved. The secretion of the Salmonella T3S effectors SipA took 100 to 600 s [25]. SipA binds within bacteria to the chaperone InvB, and translocation into host cells was measured using host cells expressing GFP–InvB fusions. Upon translocation, the chaperone–GFP chimera accumulated at the bacterial entry site representing the amount of secreted SipA [25]. In the case of Shigella, effectors IpaB and IpaC within bacteria were directly visualized through tetracysteine–FlAsH, which does not impede the secretion of the labelled effectors [5,26]. This indicated their concomitant secretion with half of the effectors secreted 240 s after host cellular contact [26]. The type IV secretion apparatus is another machinery employed by pathogens to secrete various effector substrates from the bacterium into host cells [27]. Various approaches based on fluorescence microscopy have been developed to monitor the translocation of the type IV effectors. For the plant pathogen Agrobacterium, a Cre reporter has been used to track the arrival of the reporter–effector chimera into the host cell containing plasmids with lox sites for recombination [28]. The type IV secretion substrates VirE2 and VirF were fused to the Cre recombinase, and their arrival inside the host cells could be detected through recombination. The sensitivity of this approach was improved through a GFP reporter that was expressed after Cre/Lox recombination as a direct result of translocation (Figure 1d) [29]. Recently, this approach has been adapted for various pathogens, such as Bartonella, to identify novel substrates for the type IV secretion machinery [30]. Furthermore, this reporter enabled the characterization of the type IV secretion signal www.sciencedirect.com
Vol.15 No.11
485
sequence at the C-terminus of the secreted effector proteins. Tracking host responses The contact between pathogenic bacteria and host cells triggers particular morphological changes at the pathogen contact site and leads to a pathogen-specific immune response. Innovative approaches have been used to monitor induced signaling cascades as well as new methods to study how invasive pathogens alter the endocytic pathway. The activation of specific signaling cascades can be measured in single cells using methods that are based on FRET [31]. In recent years, multiple FRET-based assays have been developed to measure the active state and the sites of activation of various signaling molecules in fixed and living cells, such as small GTPases and kinases [31–33]. Increasingly, these approaches are being adopted for the study of host–pathogen interactions. For example, the localization and the active state of the small GTPase Rac1 were investigated during the invasion of Yersinia pseudotuberculosis [34]. Another important signaling pathway during pathogen invasion involves the regulation of phosphatidyl-inositol (PtdIns). During the invasion by Listeria monocytogenes, activation of Rac1 takes place in concert with the PtdIns pathway [34]. Using a PtdIns FRET-based reporter, the PtdIns-3-kinase was activated upstream and independently of Rac1 at the site of bacterial entry [35]. Interpreting FRET data is not simple, and multiple quantification procedures, for example, ratiometric analysis, have been derived to yield reliable data [31]. Another way to overcome the difficulty of FRET data analysis is fluorescence lifetime imaging (FLIM), which measures the lifetime of the donor fluorophor of a FRET pair [22]. When FRET takes place, the electrons of the donor stay in an excited state for a shorter time period than without FRET. Therefore, the fluorescence lifetime of the donor fluorophor is reduced and can be correlated with the proximity of the interacting FRET acceptor. FLIM measurements require either a pulsed excitation light source that is expensive, or they can be performed during phase shifts of the coherent light source during FRET [36]. A great advantage of FLIM is that it relies only on the fluorescence of the FRET donor. Innovative imaging technologies have not only been used to investigate signaling cascades and the orchestration of the activation of host factors at the site of bacterial entry, but also to follow the dynamics of intracellular trafficking events [8]. In this regard, David Russell’s group has implemented interesting approaches to follow the spatiotemporal coordination of enzymatic activities and pH changes in the endosomal pathway [37,38]. Their approaches measure the hydrolytic activity inside the endosomal pathway with specifically designed fluorescent probes linked to phagocytosed beads [37]. One probe profiles the activity of cysteine proteases inside maturing phagosomes, another measures the activity of triglyceride lipases [37]. Such methodologies have the potential to be adopted for the study of invasive bacteria that interfere with membrane trafficking, such as Salmonella or Mycobacterium.
486
Review
TRENDS in Microbiology Vol.15 No.11
Microscope innovations Microscope hardware has been improved tremendously over recent years to study host–pathogen interactions (as reviewed in [4]). In particular, laser scanning confocal microscopy (LSCM) yields images of host–pathogen interactions in three dimensions with a spatial resolution down to a few hundred nanometers. Furthermore, rapid confocal microscopic systems equipped with ‘Nipkow Discs’, so called spinning-disc confocal (SDCM) microscopes, yield threedimensional information and have a temporal resolution to track the dynamics of host–pathogen interactions. New detectors, such as back-illuminated electron multiplying CCD cameras, lower the acquisition times of SDCM, giving up to 102–103 images per second. Recently, improved LSCM technology also enables fast image acquisition. The spatial resolution that can be measured with conventional microscopes is limited by the wavelength of visual light (400 to 800 nm) and by the numerical aperture of the optical lenses used. Resolution (r) is defined by the equation r = l/2*NA, where wavelength is l and the numerical aperture is NA. Under optimal conditions, two points of similar color in the red range of the spectrum that are 250 nm apart can still be resolved with an objective with a NA of 1.3. Physicists have started to tackle this barrier [6,39]. The physics and optics basis of the new approaches have been reviewed elsewhere in detail [39]. One promising way to break the perceived resolution limit of diffraction of a general light microscope is to use the 4Pi method that uses a specialized lens system with two opposing lenses on both sides of the specimen [40]. If light is illuminating the specimen coherently from both lenses, the illuminating light almost perfectly mimics a spherical wavefront that does not result in a distorted focal spot in the z-axis. Such a system yields up to a sevenfold increase in resolution along the z-axis. This technique has already resolved organelles in living cells, such as mitochondria in yeast or the mammalian Golgi apparatus with an equilateral resolution of 100 nm [41,42]. However, this technique has not been adopted for the study of host–pathogen interactions yet. Another way to obtain images with the resolution of individual biological macromolecules is through photoactivated localization microscopy (PALM) or through stochastic optical reconstruction microscopy (STORM) [43,44]. These techniques take into account that fluorescence activation takes place stochastically. Using short laser pulses to activate the fluorescence of some molecules, and then repeating this process up to 10,000 times gives datasets that can be used to calculate the exact location of individual macromolecules. Very recently, STORM has also been adopted for multicolor imaging [45]. Although, these methodologies yield very high resolution below 100 nm, they require repeated imaging of one sample; they can therefore currently be used only on fixed samples. Another pulsed approach that has been used to overcome the perceived resolution limit is stimulated emission depletion (STED). This form of microscopy uses a second red-shifted light beam that impinges on the specimen with a time lag after initial excitation of the fluorophor. The second beam is overlaid over the first beam in a donut shape depleting the fluorophors in the excited state. The www.sciencedirect.com
donut shape of the second beam leads to a specific regional depletion, and increasing its intensity improves the resolution of the objects that can be resolved [46]. STED can be performed with multiple fluorophors and possibly also in living cells. Importantly, STED resolved the clustering of vesicular components during the process of exocytosis with a resolution below 100 nm [47]. Potentially, STED could easily be adopted for studies on host-pathogen interactions. Going beyond single cells: infection in the context of living organs Bacterial pathogens do not encounter isolated cells during their challenge of the host. Depending on the organ of infection and the spread of the bacterial pathogen, the intruding bacteria have to circumvent multiple defense lines to succeed. Therefore, a major aim in the study of host–pathogen interaction has been to follow the dynamics of infection in the context of living organs. Multiphoton microscopy is changing this research area. The main advantage of multiphoton imaging is the depth of tissue that can be imaged (approximately 400 mm), and this is crucial for studies of intact organs [48]. Single photon microscopy does not penetrate deep into the specimen, and is therefore limited to events in cell culture, or in the distal zones of an organ. Nevertheless, there are difficulties that need to be addressed with intravital multiphoton microscopy, such developing fluorescent labeling of specific cellular structures, speeding up the relatively slow pace of image acquisition, accessing the organ of interest, and keeping the organ still throughout image acquisition. During recent years, intravital multiphoton microscopy has been used to study the activation of the immune system. Thus far, studies have been carried out on mouse popliteal lymph node cortex, providing interesting evidence of the dynamics of interactions between dendritic and T cells, and also on B cell dynamics [49,50]. Interaction times are likely to reflect the functionality of the immunological synapse, an essential element in activation of the immune system and establishment of the immunological memory. Experiments have been carried out with model antigens. It is now time to exploit these systems to address how, and through which route, microbial antigens are delivered. These experiments would need to be performed with live pathogens. Furthermore, this will determine whether some pathogens are able to disrupt the interaction dynamics of immunological cells, and will represent a novel view of the pathogenimmune cell interface. Recently, multiphoton intravital microscopy has also been applied to follow the infection of a living organ in real time by bacterial pathogens (Figure 2). This important study used in vivo imaging of a kidney infected with uropathogenic E. coli (UPEC) that were detected on the basis of chromosomal GFP expression [51]. The proliferation of UPEC in the nephronic regions and the tissue responses were tracked in real time [51]. At the early stages of infection, proliferation of the pathogen was detected, and tissue damage led to the constriction of blood flow around the infected nephrons. At later stages, blood leakage occurred into the perivascular regions, and the
Review
TRENDS in Microbiology
Vol.15 No.11
487
Figure 2. Multiphoton imaging of UPEC infection in vivo. (a) Schematics of the experimental set-up. The mouse kidney is removed for microscopy to a dish filled with saline solution (x). Bacteria are either added through injection into the bladder (y) or through direct injection into proximal tubuli (z). (b) One hour after infection, the bacteria (green, see arrows) can be spotted at the epithelial lining of nephrons (dull green). Fluorescent 500 kDa dextran labels blood plasma (red), 10-kDa labeled dextran (purple) outlines the injected tubule, and proximal tubules are identified by dull green autofluorescence. (c) Images collected 4.5 h post-injection of GFP-producing bacteria (green) into the proximal tubule (blue) reveals normal blood flow (red, I), plasma streaming (II) and vessel occlusion (III). Scale bars represent 30 mm. Images were kindly provided by Lisa Ma˚nsson and Agneta Richter-Dahlfors.
tissue was remodeled several hours after infection. This report highlights the potential use of multiphoton microscopy to investigate host–pathogen interactions. Unfortunately, most organs are not easily accessible, and therefore approaches have to be devised to enable ‘microscopic access’ to specific tissues. One way to monitor host–pathogen interactions in vivo is to use the dorsal skinfold chamber [52]. This device, developed more than 25 years ago, renders the subcutaneous dermis and the striated muscles of the dorsal region of various mammals accessible to fluorescence microscopy [52]. The skinfold chamber has been used with hamsters, rats and mice to study events in vascular biology such as ischemia, inflammation and tumor growth biology. Recently, the skinfold chamber has been adapted to investigate the invasion of Staphylococcus aureus, indicating the targeting of the host endothelial structures by this pathogen [53]. Another way to gain access to the abdominal region of the host has been the implantation of a metallic ring into the rodent skin above the abdomen as a portal for multiphoton microscopy [54]. This study investigated fundamental physiological and metabolic functions of the liver; however, it seems possible to adopt this technique for the study of other organs and for following host–pathogen interactions in these organs.
Leave your microscope at home: alternative technologies to track infection Integrating the study of host–pathogen interactions on the level of the whole body requires a change in imaging modalities (Table 1). Three main imaging techniques that can be used to study infectious processes will be presented here. BLI is easy to set up and is used widely [55]. It enables the visualization of either bacteria or host cells that have been modified to express a luciferase reporter gene. BLI is thus useful to visualize growth, dissemination and clearance of bacteria in a whole body and can sometimes reveal unexpected pathogen localizations. For example, this technique demonstrated that L. monocytogenes grows in the gall bladder in a mouse model [56]. Furthermore, BLI can also give a more precise view of pathogenic processes. In the case of Bacillus anthracis, BLI showed that the pathogen establishes itself in the niche of the initial site of infection, and spreads to other organs and to the blood at later time points [57]. Recently, BLI has been improved using multiple luciferases with different emission spectra to visualize simultaneously both the pathogen and the host response [58]. However, BLI does not provide a high resolution of tissues, and the amount of light collected varies with the emission source localization within the body.
Table 1. The advantages and disadvantages of various imaging techniques Imaging modality Light
Light Radioactivity
Magnetic resonance
www.sciencedirect.com
Method Microscopy: high speed videomicroscopy, multiphoton, confocal Bioluminescence PET
Used to detect Organs, cells and molecules
Sensitivity High
Resolution A few mm in vivo; a few nm in vitro
Pros Multiple fluorophors can be used simultaneously; threedimensional imaging
Cons In vivo imaging is often difficult
Organs Organs
High High
mm in vivo mm to cm
Easy to set up and use Low sensitivity
MRI
Organs to cells
Low to high
A few mm
High sensitivity with contrast agents; three dimensional imaging
Poor resolution Use of radioactive molecules; low resolution Cost of the equipment; not easy to use
488
Review
TRENDS in Microbiology Vol.15 No.11
Figure 3. MRI imaging of lung infection. Three dimensional reconstruction of mouse lungs (a) before and (b) 3 days after infection with a virulent strain of Klebsiella pneumoniae. The red volumes indicate the inflammation and oedema within the total lung volume (transparent gray). Images were obtained after processing MRI images acquired on a 7T horizontal magnet.
Obtaining quantitative information requires a precise calibration before analyzing images. The second imaging modality used to visualize infection processes is PET. However, a drawback of PET is that it requires radioactive probes. Nevertheless, imaging glucose metabolism enables the detection of neutrophils recruited to inflammatory sites [59]. As for BLI, PET can be useful to directly follow bacteria or cells that have been modified to express, for instance, herpes simplex virus type-1 thymidine kinase (HSV1-TK) that is detected through radiolabeled ATP analogs [60]. MRI, unlike BLI and PET, provides good anatomical images with a spatial resolution of a few micrometers instead of millimeters. MRI is best known for imaging the amount of water molecules within adjacent spatial volumes called voxels. Therefore, some organs, such as the brain or muscle, are more easily imaged than others, such as lungs. Because inflammation is characterized by an influx of polymorphonuclear cells sometimes accompanied by extravascular leakages, inflammation following an infection can be detected, visualized in three dimensions, and quantified by MRI, even in tissues that are difficult to visualize (Figure 3) [61,62]. In lungs, the development and regression of pneumonia caused by Klebsiella pneumoniae was followed spatially and temporally in individual animals (Figure 3) [61]. This method can be useful for characterizing the effect of pathogens or specific mutants on the inflammatory processes. BLI and PET are both imaging modalities for which sensitivity can be extremely high. However their resolution is fairly poor, in the millimeter range and does not provide good spatial details. Although MRI imaging of water is not sensitive, there are ways to increase this sensitivity by using contrast agents. Using magnetic iron particles and adapted acquisition sequences, it is possible to enhance the contrast of cells by destroying the magnetic resonance signal. In organs, such as the brain or liver, with a high magnetic resonance signal, single labeled cells can thus be detected in vivo [63,64]. In addition, rapidly exchanging amide protons present in lysine-rich reporter proteins provide a MRI www.sciencedirect.com
contrast that can be switched on and off [63]. Altogether, the use of these new technical developments is promising for the study of host–pathogen interactions. Concluding remarks and future perspectives Recent imaging developments have shed light on the interaction between bacterial pathogens and the host. Fluorescence has emerged as an excellent physical parameter that can be exploited to monitor the functional mechanisms of these processes with single-cell resolution and with high temporal dynamics. Furthermore, breaking the resolution limit of light microscopy has been achieved by introducing new microscopy techniques. Host–pathogen interactions are finely organized on the spatial level, and therefore technologies with a 10 to 100 nm resolution will Box 2. Future directions Fluorescent reporter assays First, novel fluorescent probes have to be developed, such as the FlAsH molecule, as alternatives to GFP that enable tracking of biological processes in the context of living cells or pathogens. Second, functional assays must be designed to address the specific physiological aspects of host–pathogen interactions, such as the tracking of particular signaling pathways or gene-expression patterns. New microscope developments New technologies to break the resolution barrier have to be adjusted to investigate host–pathogen interactions. This has been achieved for fixed samples that can be readily investigated with the novel technologies such as 4Pi or STORM. However, these technologies must be improved to enable live cell tracking in the context of the host–pathogen crosstalk. The functional analysis on the cellular level needs to be bridged with whole organ or body investigations In the future it will be important to link cellular studies with intravital microscopy and techniques such as BLI, PET or MRI. This will enable functional investigations of physiological processes in the context of intact organs. The in vivo imaging approach PET can already be combined with computerized tomography to combine the highly sensitive PET signals into three-dimensional high-resolution images. A breakthrough would be the combination of PET and MRI.
Review
TRENDS in Microbiology
give a new picture of the organization of the host–pathogen contact site. Furthermore, adopting these technologies for dynamic imaging will be important for dissecting the sequence of events during the interaction between bacteria and host cells. Additional developments combining multiple imaging approaches needed for the future are outlined in Box 2. Acknowledgements The authors thank Lisa Ma˚nsson and Agneta Richter-Dahlfors for contributing Figure 2, and also for critical comments on the manuscript. All members of the Sansonetti laboratory are acknowledged for their valuable discussions, particularly Allison Marty for critical reading of the manuscript. J.E. was supported by the International Human Frontiers Science Program (H.F.S.P.), and P.S. is an international scholar of the Howard Hughes Medical Institute (H.H.M.I.).
References 1 Sousa, S. et al. (2005) Microbial strategies to target, cross or disrupt epithelia. Curr. Opin. Cell Biol. 17, 489–498 2 Mu¨nter, S. et al. (2006) Signaling during pathogen infection. Sci. STKE 335, re5 3 Sansonetti, P.J. and Di Santo, J.P. (2007) Debugging how bacteria manipulate the immune response. Immunity 26, 149–161 4 Frischknecht, F. et al. (2006) Imaging today’s infectious animalcules. Curr. Opin. Microbiol. 9, 297–306 5 Giepmans, B.N. et al. (2006) The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 6 Heintzmann, R. and Ficz, G. (2006) Breaking the resolution limit in light microscopy. Brief. Funct. Genomic. Proteomic. 5, 289–301 7 Pizarro-Cerda, J. and Cossart, P. (2006) Bacterial adhesion and entry into host cells. Cell 124, 715–727 8 Gruenberg, J. and van der Goot, F.G. (2006) Mechanisms of pathogen entry through the endosomal compartments. Nat. Rev. Mol. Cell Biol. 7, 495–504 9 Valdivia, R.H. and Falkow, S. (1997) Probing bacterial gene expression within host cells. Trends Microbiol. 5, 360–363 10 Bumann, D. and Valdivia, R.H. (2007) Identification of host-induced pathogen genes by differential fluorescence induction reporter systems. Nat. Protoc. 2, 770–777 11 Valdivia, R.H. and Falkow, S. (1997) Fluorescence-based isolation of bacterial genes expressed within host cells. Science 277, 2007–2011 12 Rathman, M. et al. (2000) The development of a FACS-based strategy for the isolation of Shigella flexneri mutants that are deficient in intercellular spread. Mol. Microbiol. 35, 974–990 13 Rietsch, A. and Mekalanos, J.J. (2006) Metabolic regulation of type III secretion gene expression in Pseudomonas aeruginosa. Mol. Microbiol. 59, 807–820 14 Rollenhagen, C. et al. (2004) Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen. Proc. Natl Acad. Sci. U. S. A. 101, 8739–8744 15 Rollenhagen, C. and Bumann, D. (2006) Salmonella enterica highly expressed genes are disease specific. Infect. Immun. 74, 1649–1660 16 Becker, D. et al. (2006) Robust Salmonella metabolism limits possibilities for new antimicrobials. Nature 440, 303–307 17 Cambronne, E.D. and Roy, C.R. (2006) Recognition and delivery of effector proteins into eukaryotic cells by bacterial secretion systems. Traffic 7, 929–939 18 Cornelis, G.R. (2006) The type III secretion injectisome. Nat. Rev. Microbiol. 4, 811–825 19 Galan, J.E. and Wolf-Watz, H. (2006) Protein delivery into eukaryotic cells by type III secretion machines. Nature 444, 567–573 20 Cain, R.J. et al. (2004) The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay. Mol. Microbiol. 54, 887–904 21 Charpentier, X. and Oswald, E. (2004) Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 betalactamase as a new fluorescence-based reporter. J. Bacteriol. 186, 5486–5495 www.sciencedirect.com
Vol.15 No.11
489
22 Wallrabe, H. and Periasamy, A. (2005) Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotechnol. 16, 19–27 23 Jares-Erijman, E.A. and Jovin, T.M. (2006) Imaging molecular interactions in living cells by FRET microscopy. Curr. Opin. Chem. Biol. 10, 409–416 24 Marketon, M.M. et al. (2005) Plague bacteria target immune cells during infection. Science 309, 1739–1741 25 Schlumberger, M.C. et al. (2005) Real-time imaging of type III secretion: Salmonella SipA injection into host cells. Proc. Natl Acad. Sci. U. S. A. 102, 12548–12553 26 Enninga, J. et al. (2005) Secretion of type III effectors into host cells in real time. Nat. Methods 2, 959–965 27 Backert, S. and Meyer, T.F. (2006) Type IV secretion systems and their effectors in bacterial pathogenesis. Curr. Opin. Microbiol. 9, 207–217 28 Vergunst, A.C. et al. (2000) VirB/D4-dependent protein translocation from Agrobacterium into plant cells. Science 290, 979–982 29 Vergunst, A.C. et al. (2005) Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium. Proc. Natl Acad. Sci. U. S. A. 102, 832–837 30 Schulein, R. et al. (2005) A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells. Proc. Natl Acad. Sci. U. S. A. 102, 856–861 31 Hoppe, A.D. and Swanson, J.A. (2004) Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis. Mol. Biol. Cell 15, 3509–3519 32 Violin, J.D. et al. (2003) A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol. 161, 899–909 33 Wang, Y. et al. (2005) Visualizing the mechanical activation of Src. Nature 434, 1040–1045 34 Wong, K.W. and Isberg, R.R. (2005) Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1. PLoS Pathog. 1, e16 35 Seveau, S. et al. (2007) A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway. Cell. Microbiol. 9, 790–803 36 Yudushkin, I.A. et al. (2007) Live-cell imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B. Science 315, 115–119 37 Yates, R.M. et al. (2005) The kinetics of phagosome maturation as a function of phagosome/lysosome fusion and acquisition of hydrolytic activity. Traffic 6, 413–420 38 Yates, R.M. and Russell, D.G. (2005) Phagosome maturation proceeds independently of stimulation of toll-like receptors 2 and 4. Immunity 23, 409–417 39 Hell, S.W. (2007) Far-field optical nanoscopy. Science 316, 1153–1158 40 Egner, A. and Hell, S.W. (2005) Fluorescence microscopy with superresolved optical sections. Trends Cell Biol. 15, 207–215 41 Egner, A. et al. (2002) Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast. Proc. Natl Acad. Sci. U. S. A. 99, 3370–3375 42 Egner, A. et al. (2004) 4Pi-microscopy of the Golgi apparatus in live mammalian cells. J. Struct. Biol. 147, 70–76 43 Betzig, E. et al. (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 44 Rust, M.J. et al. (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793– 795 45 Bates, M. et al. (2007) Multicolor super-resolution imaging with photoswitchable fluorescent probes. Science 317, 1749–1753 46 Willig, K.I. et al. (2006) Nanoscale resolution in GFP-based microscopy. Nat. Methods 3, 721–723 47 Willig, K.I. et al. (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935– 939 48 Sumen, C. et al. (2004) Intravital microscopy: visualizing immunity in context. Immunity 21, 315–329 49 Lindquist, R.L. et al. (2004) Visualizing dendritic cell networks in vivo. Nat. Immunol. 5, 1243–1250 50 Schwickert, T.A. et al. (2007) In vivo imaging of germinal centres reveals a dynamic open structure. Nature 446, 83–87 51 Mansson, L.E. et al. (2007) Real-time studies of the progression of bacterial infections and immediate tissue responses in live animals. Cell. Microbiol. 9, 413–424
Review
490
TRENDS in Microbiology Vol.15 No.11
52 Menger, M.D. et al. (2002) Viewing the microcirculation through the window: some twenty years experience with the hamster dorsal skinfold chamber. Eur. Surg. Res. 34, 83–91 53 Laschke, M.W. et al. (2005) Intravital fluorescence microscopy: a novel tool for the study of the interaction of Staphylococcus aureus with the microvascular endothelium in vivo. J. Infect. Dis. 191, 435–443 54 Liu, Y. et al. (2007) Visualization of hepatobiliary excretory function by intravital multiphoton microscopy. J. Biomed. Opt. 12, 014014 55 Doyle, T.C. et al. (2004) In vivo bioluminescence imaging for integrated studies of infection. Cell. Microbiol. 6, 303–317 56 Hardy, J. et al. (2004) Extracellular replication of Listeria monocytogenes in the murine gall bladder. Science 303, 851–853 57 Glomski, I.J. et al. (2007) Primary involvement of pharynx and peyer’s patch in inhalational and intestinal anthrax. PLoS Pathog. 3, e76 58 Kadurugamuwa, J.L. et al. (2005) Reduction of astrogliosis by early treatment of pneumococcal meningitis measured by simultaneous
59
60
61 62 63 64
imaging, in vivo, of the pathogen and host response. Infect. Immun. 73, 7836–7843 Schuster, D.P. et al. (2003) Imaging lung inflammation in a murine model of Pseudomonas infection: a positron emission tomography study. Exp. Lung Res. 29, 45–57 Bettegowda, C. et al. (2005) Imaging bacterial infections with radiolabeled 1-(20 -deoxy-20 -fluoro-beta-D-arabinofuranosyl)-5iodouracil. Proc. Natl Acad. Sci. U. S. A. 102, 1145–1150 Tournebize, R. et al. (2006) Magnetic resonance imaging of Klebsiella pneumoniae-induced pneumonia in mice. Cell. Microbiol. 8, 33–43 Marzola, P. et al. (2005) (1)H MRI of pneumococcal pneumonia in a murine model. J. Magn. Reson. Imaging 22, 170–174 Shapiro, E.M. et al. (2006) In vivo detection of single cells by MRI. Magn. Reson. Med. 55, 242–249 Heyn, C. et al. (2006) In vivo MRI of cancer cell fate at the single-cell level in a mouse model of breast cancer metastasis to the brain. Magn. Reson. Med. 56, 1001–1010
Elsevier.com – linking scientists to new research and thinking Designed for scientists’ information needs, Elsevier.com is powered by the latest technology with customer-focused navigation and an intuitive architecture for an improved user experience and greater productivity. The easy-to-use navigational tools and structure connect scientists with vital information – all from one entry point. Users can perform rapid and precise searches with our advanced search functionality, using the FAST technology of Scirus.com, the free science search engine. Users can define their searches by any number of criteria to pinpoint information and resources. Search by a specific author or editor, book publication date, subject area – life sciences, health sciences, physical sciences and social sciences – or by product type. Elsevier’s portfolio includes more than 1800 Elsevier journals, 2200 new books every year and a range of innovative electronic products. In addition, tailored content for authors, editors and librarians provides timely news and updates on new products and services. Elsevier is proud to be a partner with the scientific and medical community. Find out more about our mission and values at Elsevier.com. Discover how we support the scientific, technical and medical communities worldwide through partnerships with libraries and other publishers, and grant awards from The Elsevier Foundation. As a world-leading publisher of scientific, technical and health information, Elsevier is dedicated to linking researchers and professionals to the best thinking in their fields. We offer the widest and deepest coverage in a range of media types to enhance cross-pollination of information, breakthroughs in research and discovery, and the sharing and preservation of knowledge.
Elsevier. Building insights. Breaking boundaries. www.elsevier.com www.sciencedirect.com
TRENDS in Microbiology
Vol.15 No.11
Roundtrip explorations of bacterial infection: from single cells to the entire host and back Jost Enninga1,2, Philippe Sansonetti1,2 and Regis Tournebize1,2 1 2
Unite´ de Pathoge´nie Microbienne, Institut Pasteur, 28, rue du Dr Roux, 75724 Paris cedex 15, France Unite´ INSERM 786, France
Host–pathogen interactions are highly regulated, dynamic processes that take place at the molecular, cellular and organ level. Innovative imaging technologies have emerged recently to investigate the underlying mechanisms of host–pathogen interactions. Innovations in fluorescence microscopy enable functional studies on the single-cell level. New light microscopes have been developed that improve the resolution to less than 100 nm. At the other extreme, intravital microscopy enables the correlation of cellular events on the organ level. This is also achieved by alternatives to microscopy such as bioluminescence, positron-emission tomography and magnetic resonance imaging. The methodologies described here will have a tremendous effect on our understanding of host–pathogen interactions. Pathogen invasion of single cells and organs During infection, bacterial pathogens surmount various host epithelial barriers in different tissues and host cell types [1]. The microenvironment of the tissues varies enormously, and different host cells react in multiple ways on contact with pathogens. Thus, pathogens have to adapt their lifestyle to successfully disseminate and propagate inside the host. To achieve this, pathogens have developed molecular mechanisms to subvert the host immune response and major host signaling pathways [2,3]. Emerging technologies using fluorescence imaging have been adapted for the study of host–pathogen interactions to delineate their molecular mechanisms in the cellular context [4]. These technologies enable highly sensitive and dynamic studies owing to the accessibility of state-of-theart microscope hardware and development of fluorescence markers and reporters (Box 1) [5]. This has enabled investigation of the molecular mechanisms on both the host and pathogen with high temporal and/or spatial resolution. Furthermore, physicists are currently developing microscopes that overcome the spatial resolution limit of visible light [6]. Such microscopes have the potential to revolutionize the analysis of host–pathogen interactions at the cellular level. On the organ level, multiphoton microscopy of living tissue enables the tracking of the infection process, whereas bioluminescence (BLI), positron-emission Corresponding authors: Enninga, J. ([email protected]). Available online 5 November 2007. www.sciencedirect.com
([email protected]); Tournebize, R.
tomography (PET) and magnetic resonance imaging (MRI) techniques yield the specific visualization of up to a few cells in the whole body. We will outline below how these approaches have enriched our understanding of the progression on infectious processes inside the host. Innovative fluorescent assays Tracking pathogen invasion Bacterial pathogens invade host cells or attach to their surface [7]. When pathogens are recognized and subsequently ingested by phagocytic cells of the host immune system, they use specific strategies to prevent their destruction and to undermine the host immune response [8]. During all these events specific gene- expression programs are triggered inside the intruding pathogen preparing it for combat with the host [9]. Fluorescence methods have been used for studying gene expression of the single bacterium level and on the protein level for host–pathogen interactions. During the past decade, the study of gene-expression profiles in individual living cells has become possible through the use of genetically encoded fluorescent reporters, such as the green fluorescent protein (GFP). Cells containing GFP-encoding sequences engineered downstream of specific promoters light up after promoter induction. For bacteria, this approach, termed differential fluorescence induction (DFI), has been generally combined with fluorescence-activated cell sorting (FACS). However, DFI is also readily applicable using fluorescence microscopy to track individual bacteria [10]. Initially, DFI was developed to investigate differential pathogen gene expression during macrophage uptake of Salmonella typhimurium [11]. Similar approaches have been adopted to study other host–pathogen interactions, for example, to identify virulence factors involved in cell-to-cell spread of intracellularly growing Shigella flexneri [12]. Furthermore, DFI revealed that the pathogen type III secretion (T3S) machinery of Pseudomonas aeruginosa is expressed in a metabolism-dependent fashion [13]. Importantly, DFI approaches have been performed in the context of the entire host organism and, in the case of Salmonella, have identified pathogen promoters that are highly induced at the site of infection [14,15]. Data from such studies can now be merged with proteomic results from systematic analyses of metabolic Salmonella networks [16].
0966-842X/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2007.10.006
484
Review
TRENDS in Microbiology Vol.15 No.11
Box 1. Accessing state-of-the-art microscopes and interpreting your images correctly Even though microbiologists have been teaming up with cell biologists recently, many microbiology laboratories still lack cutting-edge lightmicroscopy equipment. This does not mean that such laboratories cannot perform projects similar to the ones described here. Three possibilities exist. First, a specific microscope can be purchased by a laboratory. This is often expensive, and the choice of instrument has to be balanced between the various projects that the microscope is supposed to be used for. Furthermore, space has to be considered owing to the size of a microscope. The second option is to work together with light-microscopy core facilities that are now established at a large number of research institutions. Sometimes, core facilities acquire equipment in collaboration with research laboratories to the benefit of both groups. This way, the core facility attracts a more diverse group of users, and the research group is helped with equipment maintenance and upgrading. Third, it is
Gene-expression analyses have indicated that bacterial secretion machineries are expressed by pathogens at increased levels during infections. These machineries translocate a cocktail of toxins, or effector proteins, into host cells [17]. New approaches have been developed to reveal the molecular mechanism of effector-protein secretion through the various secretion devices (Figure 1). For example, the
possible to collaborate with research groups or core facilities working with a particular piece of equipment. Working with core facilities or addressing questions with expert groups (for example, bioinformaticians) helps tremendously with one of the most difficult problems in imaging — the correct, unbiased and quantitative interpretation of the imaging data. During recent years, the amount of imaging data presented in research papers has exploded, and with it the amount of artifacts. Working together with imaging experts who are data-processing specialists is of highest importance to interpret raw imaging data correctly. Such personnel should be present at each imaging core facility. In the coming years, it will be important to increase the stringency of imaging-data interpretation to profit from the strength of imaging as a method, and to keep imaging and the scientific advances it provides credible to the research community.
T3S apparatus that functions as a molecular syringe has been a major research focus [18,19]. However, studies have been hampered because secreted effectors cannot readily be rendered fluorescent through GFP fusion because GFP cannot pass through the T3S syringes. This difficulty has been overcome using various small protein tags in conjunction with indirect immunofluorescence. Such studies
Figure 1. Fluorescent assays to monitor the dynamics of bacterial effector secretion. (a) Translocation of the effectors into host cells is monitored through a FRET assay. Effectors are fused to b-lactamase that cleaves a FRET probe inside the host cells. If FRET takes placde, the cells fluoresce green (no effector injection), if no FRET takes place (effector injection), cells fluoresce blue. This approach is well suited for screenings. Symbols: A, FRET acceptor; D, FRET donor. (b) Arrival of the translocated effector proteins in the host cell is measured through its binding to an effector-specific chaperone (InvB) that has been fluorescently labeled with GFP yielding GFP-InvB (in green). This is the only approach that does not alter the effectors; however, this approach requires their specific localization at the entry site. (c) The effectors are tagged with a small tetracysteine motif that binds with high affinity the small metallo-organic FlAsH compound. Depletion of the effector–4Cys–FlAsH molecules inside the bacterium is directly observed during effector secretion. (d) Effectors are fused to the Cre recombinase. After translocation into cell lines containing a LoxP reporter plasmid, the LoxP sites are cleaved and transcription of the reporter (for example, GFP) is triggered (geen). This approach does not yield real-time resolution owing to GFP maturation. This approach has been used to track type IV secretion. T1 indicates a time point before secretion, and T2 a time point during the secretion process. www.sciencedirect.com
Review
TRENDS in Microbiology
showed localization of secreted Salmonella T3S effectors upon translocation in single cells using microscopy on fixed samples [20]. Novel reporters using fluorescence resonance energy transfer (FRET) carry the potential to track the translocation of T3S effectors into host cells dynamically (Figure 1a) [21]. FRET describes the ability of one fluorophor (the FRET donor, D) to transfer light directly without emission to another nearby fluorophor (the FRET acceptor, A) [22]. FRET from D to A leads to an emission spectrum with the characteristics of A [22]. Often, FRET is used to study protein–protein interactions through live cell imaging or in fixed samples using confocal or epifluorescent set-ups [23]. However, FRET probes can also be used to study various cellular phenomena as discussed below [23]. One FRET-based approach used enteropathogenic Escherichia coli (EPEC) expressing chimeras that consist of a T3S signal sequence fused to b-lactamase (Figure 1a). These bacteria were then used to challenge host cells that had been loaded with a b-lactamase-cleavable FRET substrate. Upon secretion of the T3S effector fusion, the FRET substrate was cleaved, and the fluorescence signal changed to enable the identification of novel T3S signal sequences. This approach has also been adapted to investigate which immune cells are attacked by Yersinia pestis in living rodents [24]. Recently, tracking the type III effector secretion of Salmonella and Shigella has been tackled with different strategies (Figure 1b and c) [25,26]. Translocation of the T3S effectors from both pathogens took place with similar kinetics, emphasizing that this process is functionally conserved. The secretion of the Salmonella T3S effectors SipA took 100 to 600 s [25]. SipA binds within bacteria to the chaperone InvB, and translocation into host cells was measured using host cells expressing GFP–InvB fusions. Upon translocation, the chaperone–GFP chimera accumulated at the bacterial entry site representing the amount of secreted SipA [25]. In the case of Shigella, effectors IpaB and IpaC within bacteria were directly visualized through tetracysteine–FlAsH, which does not impede the secretion of the labelled effectors [5,26]. This indicated their concomitant secretion with half of the effectors secreted 240 s after host cellular contact [26]. The type IV secretion apparatus is another machinery employed by pathogens to secrete various effector substrates from the bacterium into host cells [27]. Various approaches based on fluorescence microscopy have been developed to monitor the translocation of the type IV effectors. For the plant pathogen Agrobacterium, a Cre reporter has been used to track the arrival of the reporter–effector chimera into the host cell containing plasmids with lox sites for recombination [28]. The type IV secretion substrates VirE2 and VirF were fused to the Cre recombinase, and their arrival inside the host cells could be detected through recombination. The sensitivity of this approach was improved through a GFP reporter that was expressed after Cre/Lox recombination as a direct result of translocation (Figure 1d) [29]. Recently, this approach has been adapted for various pathogens, such as Bartonella, to identify novel substrates for the type IV secretion machinery [30]. Furthermore, this reporter enabled the characterization of the type IV secretion signal www.sciencedirect.com
Vol.15 No.11
485
sequence at the C-terminus of the secreted effector proteins. Tracking host responses The contact between pathogenic bacteria and host cells triggers particular morphological changes at the pathogen contact site and leads to a pathogen-specific immune response. Innovative approaches have been used to monitor induced signaling cascades as well as new methods to study how invasive pathogens alter the endocytic pathway. The activation of specific signaling cascades can be measured in single cells using methods that are based on FRET [31]. In recent years, multiple FRET-based assays have been developed to measure the active state and the sites of activation of various signaling molecules in fixed and living cells, such as small GTPases and kinases [31–33]. Increasingly, these approaches are being adopted for the study of host–pathogen interactions. For example, the localization and the active state of the small GTPase Rac1 were investigated during the invasion of Yersinia pseudotuberculosis [34]. Another important signaling pathway during pathogen invasion involves the regulation of phosphatidyl-inositol (PtdIns). During the invasion by Listeria monocytogenes, activation of Rac1 takes place in concert with the PtdIns pathway [34]. Using a PtdIns FRET-based reporter, the PtdIns-3-kinase was activated upstream and independently of Rac1 at the site of bacterial entry [35]. Interpreting FRET data is not simple, and multiple quantification procedures, for example, ratiometric analysis, have been derived to yield reliable data [31]. Another way to overcome the difficulty of FRET data analysis is fluorescence lifetime imaging (FLIM), which measures the lifetime of the donor fluorophor of a FRET pair [22]. When FRET takes place, the electrons of the donor stay in an excited state for a shorter time period than without FRET. Therefore, the fluorescence lifetime of the donor fluorophor is reduced and can be correlated with the proximity of the interacting FRET acceptor. FLIM measurements require either a pulsed excitation light source that is expensive, or they can be performed during phase shifts of the coherent light source during FRET [36]. A great advantage of FLIM is that it relies only on the fluorescence of the FRET donor. Innovative imaging technologies have not only been used to investigate signaling cascades and the orchestration of the activation of host factors at the site of bacterial entry, but also to follow the dynamics of intracellular trafficking events [8]. In this regard, David Russell’s group has implemented interesting approaches to follow the spatiotemporal coordination of enzymatic activities and pH changes in the endosomal pathway [37,38]. Their approaches measure the hydrolytic activity inside the endosomal pathway with specifically designed fluorescent probes linked to phagocytosed beads [37]. One probe profiles the activity of cysteine proteases inside maturing phagosomes, another measures the activity of triglyceride lipases [37]. Such methodologies have the potential to be adopted for the study of invasive bacteria that interfere with membrane trafficking, such as Salmonella or Mycobacterium.
486
Review
TRENDS in Microbiology Vol.15 No.11
Microscope innovations Microscope hardware has been improved tremendously over recent years to study host–pathogen interactions (as reviewed in [4]). In particular, laser scanning confocal microscopy (LSCM) yields images of host–pathogen interactions in three dimensions with a spatial resolution down to a few hundred nanometers. Furthermore, rapid confocal microscopic systems equipped with ‘Nipkow Discs’, so called spinning-disc confocal (SDCM) microscopes, yield threedimensional information and have a temporal resolution to track the dynamics of host–pathogen interactions. New detectors, such as back-illuminated electron multiplying CCD cameras, lower the acquisition times of SDCM, giving up to 102–103 images per second. Recently, improved LSCM technology also enables fast image acquisition. The spatial resolution that can be measured with conventional microscopes is limited by the wavelength of visual light (400 to 800 nm) and by the numerical aperture of the optical lenses used. Resolution (r) is defined by the equation r = l/2*NA, where wavelength is l and the numerical aperture is NA. Under optimal conditions, two points of similar color in the red range of the spectrum that are 250 nm apart can still be resolved with an objective with a NA of 1.3. Physicists have started to tackle this barrier [6,39]. The physics and optics basis of the new approaches have been reviewed elsewhere in detail [39]. One promising way to break the perceived resolution limit of diffraction of a general light microscope is to use the 4Pi method that uses a specialized lens system with two opposing lenses on both sides of the specimen [40]. If light is illuminating the specimen coherently from both lenses, the illuminating light almost perfectly mimics a spherical wavefront that does not result in a distorted focal spot in the z-axis. Such a system yields up to a sevenfold increase in resolution along the z-axis. This technique has already resolved organelles in living cells, such as mitochondria in yeast or the mammalian Golgi apparatus with an equilateral resolution of 100 nm [41,42]. However, this technique has not been adopted for the study of host–pathogen interactions yet. Another way to obtain images with the resolution of individual biological macromolecules is through photoactivated localization microscopy (PALM) or through stochastic optical reconstruction microscopy (STORM) [43,44]. These techniques take into account that fluorescence activation takes place stochastically. Using short laser pulses to activate the fluorescence of some molecules, and then repeating this process up to 10,000 times gives datasets that can be used to calculate the exact location of individual macromolecules. Very recently, STORM has also been adopted for multicolor imaging [45]. Although, these methodologies yield very high resolution below 100 nm, they require repeated imaging of one sample; they can therefore currently be used only on fixed samples. Another pulsed approach that has been used to overcome the perceived resolution limit is stimulated emission depletion (STED). This form of microscopy uses a second red-shifted light beam that impinges on the specimen with a time lag after initial excitation of the fluorophor. The second beam is overlaid over the first beam in a donut shape depleting the fluorophors in the excited state. The www.sciencedirect.com
donut shape of the second beam leads to a specific regional depletion, and increasing its intensity improves the resolution of the objects that can be resolved [46]. STED can be performed with multiple fluorophors and possibly also in living cells. Importantly, STED resolved the clustering of vesicular components during the process of exocytosis with a resolution below 100 nm [47]. Potentially, STED could easily be adopted for studies on host-pathogen interactions. Going beyond single cells: infection in the context of living organs Bacterial pathogens do not encounter isolated cells during their challenge of the host. Depending on the organ of infection and the spread of the bacterial pathogen, the intruding bacteria have to circumvent multiple defense lines to succeed. Therefore, a major aim in the study of host–pathogen interaction has been to follow the dynamics of infection in the context of living organs. Multiphoton microscopy is changing this research area. The main advantage of multiphoton imaging is the depth of tissue that can be imaged (approximately 400 mm), and this is crucial for studies of intact organs [48]. Single photon microscopy does not penetrate deep into the specimen, and is therefore limited to events in cell culture, or in the distal zones of an organ. Nevertheless, there are difficulties that need to be addressed with intravital multiphoton microscopy, such developing fluorescent labeling of specific cellular structures, speeding up the relatively slow pace of image acquisition, accessing the organ of interest, and keeping the organ still throughout image acquisition. During recent years, intravital multiphoton microscopy has been used to study the activation of the immune system. Thus far, studies have been carried out on mouse popliteal lymph node cortex, providing interesting evidence of the dynamics of interactions between dendritic and T cells, and also on B cell dynamics [49,50]. Interaction times are likely to reflect the functionality of the immunological synapse, an essential element in activation of the immune system and establishment of the immunological memory. Experiments have been carried out with model antigens. It is now time to exploit these systems to address how, and through which route, microbial antigens are delivered. These experiments would need to be performed with live pathogens. Furthermore, this will determine whether some pathogens are able to disrupt the interaction dynamics of immunological cells, and will represent a novel view of the pathogenimmune cell interface. Recently, multiphoton intravital microscopy has also been applied to follow the infection of a living organ in real time by bacterial pathogens (Figure 2). This important study used in vivo imaging of a kidney infected with uropathogenic E. coli (UPEC) that were detected on the basis of chromosomal GFP expression [51]. The proliferation of UPEC in the nephronic regions and the tissue responses were tracked in real time [51]. At the early stages of infection, proliferation of the pathogen was detected, and tissue damage led to the constriction of blood flow around the infected nephrons. At later stages, blood leakage occurred into the perivascular regions, and the
Review
TRENDS in Microbiology
Vol.15 No.11
487
Figure 2. Multiphoton imaging of UPEC infection in vivo. (a) Schematics of the experimental set-up. The mouse kidney is removed for microscopy to a dish filled with saline solution (x). Bacteria are either added through injection into the bladder (y) or through direct injection into proximal tubuli (z). (b) One hour after infection, the bacteria (green, see arrows) can be spotted at the epithelial lining of nephrons (dull green). Fluorescent 500 kDa dextran labels blood plasma (red), 10-kDa labeled dextran (purple) outlines the injected tubule, and proximal tubules are identified by dull green autofluorescence. (c) Images collected 4.5 h post-injection of GFP-producing bacteria (green) into the proximal tubule (blue) reveals normal blood flow (red, I), plasma streaming (II) and vessel occlusion (III). Scale bars represent 30 mm. Images were kindly provided by Lisa Ma˚nsson and Agneta Richter-Dahlfors.
tissue was remodeled several hours after infection. This report highlights the potential use of multiphoton microscopy to investigate host–pathogen interactions. Unfortunately, most organs are not easily accessible, and therefore approaches have to be devised to enable ‘microscopic access’ to specific tissues. One way to monitor host–pathogen interactions in vivo is to use the dorsal skinfold chamber [52]. This device, developed more than 25 years ago, renders the subcutaneous dermis and the striated muscles of the dorsal region of various mammals accessible to fluorescence microscopy [52]. The skinfold chamber has been used with hamsters, rats and mice to study events in vascular biology such as ischemia, inflammation and tumor growth biology. Recently, the skinfold chamber has been adapted to investigate the invasion of Staphylococcus aureus, indicating the targeting of the host endothelial structures by this pathogen [53]. Another way to gain access to the abdominal region of the host has been the implantation of a metallic ring into the rodent skin above the abdomen as a portal for multiphoton microscopy [54]. This study investigated fundamental physiological and metabolic functions of the liver; however, it seems possible to adopt this technique for the study of other organs and for following host–pathogen interactions in these organs.
Leave your microscope at home: alternative technologies to track infection Integrating the study of host–pathogen interactions on the level of the whole body requires a change in imaging modalities (Table 1). Three main imaging techniques that can be used to study infectious processes will be presented here. BLI is easy to set up and is used widely [55]. It enables the visualization of either bacteria or host cells that have been modified to express a luciferase reporter gene. BLI is thus useful to visualize growth, dissemination and clearance of bacteria in a whole body and can sometimes reveal unexpected pathogen localizations. For example, this technique demonstrated that L. monocytogenes grows in the gall bladder in a mouse model [56]. Furthermore, BLI can also give a more precise view of pathogenic processes. In the case of Bacillus anthracis, BLI showed that the pathogen establishes itself in the niche of the initial site of infection, and spreads to other organs and to the blood at later time points [57]. Recently, BLI has been improved using multiple luciferases with different emission spectra to visualize simultaneously both the pathogen and the host response [58]. However, BLI does not provide a high resolution of tissues, and the amount of light collected varies with the emission source localization within the body.
Table 1. The advantages and disadvantages of various imaging techniques Imaging modality Light
Light Radioactivity
Magnetic resonance
www.sciencedirect.com
Method Microscopy: high speed videomicroscopy, multiphoton, confocal Bioluminescence PET
Used to detect Organs, cells and molecules
Sensitivity High
Resolution A few mm in vivo; a few nm in vitro
Pros Multiple fluorophors can be used simultaneously; threedimensional imaging
Cons In vivo imaging is often difficult
Organs Organs
High High
mm in vivo mm to cm
Easy to set up and use Low sensitivity
MRI
Organs to cells
Low to high
A few mm
High sensitivity with contrast agents; three dimensional imaging
Poor resolution Use of radioactive molecules; low resolution Cost of the equipment; not easy to use
488
Review
TRENDS in Microbiology Vol.15 No.11
Figure 3. MRI imaging of lung infection. Three dimensional reconstruction of mouse lungs (a) before and (b) 3 days after infection with a virulent strain of Klebsiella pneumoniae. The red volumes indicate the inflammation and oedema within the total lung volume (transparent gray). Images were obtained after processing MRI images acquired on a 7T horizontal magnet.
Obtaining quantitative information requires a precise calibration before analyzing images. The second imaging modality used to visualize infection processes is PET. However, a drawback of PET is that it requires radioactive probes. Nevertheless, imaging glucose metabolism enables the detection of neutrophils recruited to inflammatory sites [59]. As for BLI, PET can be useful to directly follow bacteria or cells that have been modified to express, for instance, herpes simplex virus type-1 thymidine kinase (HSV1-TK) that is detected through radiolabeled ATP analogs [60]. MRI, unlike BLI and PET, provides good anatomical images with a spatial resolution of a few micrometers instead of millimeters. MRI is best known for imaging the amount of water molecules within adjacent spatial volumes called voxels. Therefore, some organs, such as the brain or muscle, are more easily imaged than others, such as lungs. Because inflammation is characterized by an influx of polymorphonuclear cells sometimes accompanied by extravascular leakages, inflammation following an infection can be detected, visualized in three dimensions, and quantified by MRI, even in tissues that are difficult to visualize (Figure 3) [61,62]. In lungs, the development and regression of pneumonia caused by Klebsiella pneumoniae was followed spatially and temporally in individual animals (Figure 3) [61]. This method can be useful for characterizing the effect of pathogens or specific mutants on the inflammatory processes. BLI and PET are both imaging modalities for which sensitivity can be extremely high. However their resolution is fairly poor, in the millimeter range and does not provide good spatial details. Although MRI imaging of water is not sensitive, there are ways to increase this sensitivity by using contrast agents. Using magnetic iron particles and adapted acquisition sequences, it is possible to enhance the contrast of cells by destroying the magnetic resonance signal. In organs, such as the brain or liver, with a high magnetic resonance signal, single labeled cells can thus be detected in vivo [63,64]. In addition, rapidly exchanging amide protons present in lysine-rich reporter proteins provide a MRI www.sciencedirect.com
contrast that can be switched on and off [63]. Altogether, the use of these new technical developments is promising for the study of host–pathogen interactions. Concluding remarks and future perspectives Recent imaging developments have shed light on the interaction between bacterial pathogens and the host. Fluorescence has emerged as an excellent physical parameter that can be exploited to monitor the functional mechanisms of these processes with single-cell resolution and with high temporal dynamics. Furthermore, breaking the resolution limit of light microscopy has been achieved by introducing new microscopy techniques. Host–pathogen interactions are finely organized on the spatial level, and therefore technologies with a 10 to 100 nm resolution will Box 2. Future directions Fluorescent reporter assays First, novel fluorescent probes have to be developed, such as the FlAsH molecule, as alternatives to GFP that enable tracking of biological processes in the context of living cells or pathogens. Second, functional assays must be designed to address the specific physiological aspects of host–pathogen interactions, such as the tracking of particular signaling pathways or gene-expression patterns. New microscope developments New technologies to break the resolution barrier have to be adjusted to investigate host–pathogen interactions. This has been achieved for fixed samples that can be readily investigated with the novel technologies such as 4Pi or STORM. However, these technologies must be improved to enable live cell tracking in the context of the host–pathogen crosstalk. The functional analysis on the cellular level needs to be bridged with whole organ or body investigations In the future it will be important to link cellular studies with intravital microscopy and techniques such as BLI, PET or MRI. This will enable functional investigations of physiological processes in the context of intact organs. The in vivo imaging approach PET can already be combined with computerized tomography to combine the highly sensitive PET signals into three-dimensional high-resolution images. A breakthrough would be the combination of PET and MRI.
Review
TRENDS in Microbiology
give a new picture of the organization of the host–pathogen contact site. Furthermore, adopting these technologies for dynamic imaging will be important for dissecting the sequence of events during the interaction between bacteria and host cells. Additional developments combining multiple imaging approaches needed for the future are outlined in Box 2. Acknowledgements The authors thank Lisa Ma˚nsson and Agneta Richter-Dahlfors for contributing Figure 2, and also for critical comments on the manuscript. All members of the Sansonetti laboratory are acknowledged for their valuable discussions, particularly Allison Marty for critical reading of the manuscript. J.E. was supported by the International Human Frontiers Science Program (H.F.S.P.), and P.S. is an international scholar of the Howard Hughes Medical Institute (H.H.M.I.).
References 1 Sousa, S. et al. (2005) Microbial strategies to target, cross or disrupt epithelia. Curr. Opin. Cell Biol. 17, 489–498 2 Mu¨nter, S. et al. (2006) Signaling during pathogen infection. Sci. STKE 335, re5 3 Sansonetti, P.J. and Di Santo, J.P. (2007) Debugging how bacteria manipulate the immune response. Immunity 26, 149–161 4 Frischknecht, F. et al. (2006) Imaging today’s infectious animalcules. Curr. Opin. Microbiol. 9, 297–306 5 Giepmans, B.N. et al. (2006) The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 6 Heintzmann, R. and Ficz, G. (2006) Breaking the resolution limit in light microscopy. Brief. Funct. Genomic. Proteomic. 5, 289–301 7 Pizarro-Cerda, J. and Cossart, P. (2006) Bacterial adhesion and entry into host cells. Cell 124, 715–727 8 Gruenberg, J. and van der Goot, F.G. (2006) Mechanisms of pathogen entry through the endosomal compartments. Nat. Rev. Mol. Cell Biol. 7, 495–504 9 Valdivia, R.H. and Falkow, S. (1997) Probing bacterial gene expression within host cells. Trends Microbiol. 5, 360–363 10 Bumann, D. and Valdivia, R.H. (2007) Identification of host-induced pathogen genes by differential fluorescence induction reporter systems. Nat. Protoc. 2, 770–777 11 Valdivia, R.H. and Falkow, S. (1997) Fluorescence-based isolation of bacterial genes expressed within host cells. Science 277, 2007–2011 12 Rathman, M. et al. (2000) The development of a FACS-based strategy for the isolation of Shigella flexneri mutants that are deficient in intercellular spread. Mol. Microbiol. 35, 974–990 13 Rietsch, A. and Mekalanos, J.J. (2006) Metabolic regulation of type III secretion gene expression in Pseudomonas aeruginosa. Mol. Microbiol. 59, 807–820 14 Rollenhagen, C. et al. (2004) Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen. Proc. Natl Acad. Sci. U. S. A. 101, 8739–8744 15 Rollenhagen, C. and Bumann, D. (2006) Salmonella enterica highly expressed genes are disease specific. Infect. Immun. 74, 1649–1660 16 Becker, D. et al. (2006) Robust Salmonella metabolism limits possibilities for new antimicrobials. Nature 440, 303–307 17 Cambronne, E.D. and Roy, C.R. (2006) Recognition and delivery of effector proteins into eukaryotic cells by bacterial secretion systems. Traffic 7, 929–939 18 Cornelis, G.R. (2006) The type III secretion injectisome. Nat. Rev. Microbiol. 4, 811–825 19 Galan, J.E. and Wolf-Watz, H. (2006) Protein delivery into eukaryotic cells by type III secretion machines. Nature 444, 567–573 20 Cain, R.J. et al. (2004) The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay. Mol. Microbiol. 54, 887–904 21 Charpentier, X. and Oswald, E. (2004) Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 betalactamase as a new fluorescence-based reporter. J. Bacteriol. 186, 5486–5495 www.sciencedirect.com
Vol.15 No.11
489
22 Wallrabe, H. and Periasamy, A. (2005) Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotechnol. 16, 19–27 23 Jares-Erijman, E.A. and Jovin, T.M. (2006) Imaging molecular interactions in living cells by FRET microscopy. Curr. Opin. Chem. Biol. 10, 409–416 24 Marketon, M.M. et al. (2005) Plague bacteria target immune cells during infection. Science 309, 1739–1741 25 Schlumberger, M.C. et al. (2005) Real-time imaging of type III secretion: Salmonella SipA injection into host cells. Proc. Natl Acad. Sci. U. S. A. 102, 12548–12553 26 Enninga, J. et al. (2005) Secretion of type III effectors into host cells in real time. Nat. Methods 2, 959–965 27 Backert, S. and Meyer, T.F. (2006) Type IV secretion systems and their effectors in bacterial pathogenesis. Curr. Opin. Microbiol. 9, 207–217 28 Vergunst, A.C. et al. (2000) VirB/D4-dependent protein translocation from Agrobacterium into plant cells. Science 290, 979–982 29 Vergunst, A.C. et al. (2005) Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium. Proc. Natl Acad. Sci. U. S. A. 102, 832–837 30 Schulein, R. et al. (2005) A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells. Proc. Natl Acad. Sci. U. S. A. 102, 856–861 31 Hoppe, A.D. and Swanson, J.A. (2004) Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis. Mol. Biol. Cell 15, 3509–3519 32 Violin, J.D. et al. (2003) A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol. 161, 899–909 33 Wang, Y. et al. (2005) Visualizing the mechanical activation of Src. Nature 434, 1040–1045 34 Wong, K.W. and Isberg, R.R. (2005) Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1. PLoS Pathog. 1, e16 35 Seveau, S. et al. (2007) A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway. Cell. Microbiol. 9, 790–803 36 Yudushkin, I.A. et al. (2007) Live-cell imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B. Science 315, 115–119 37 Yates, R.M. et al. (2005) The kinetics of phagosome maturation as a function of phagosome/lysosome fusion and acquisition of hydrolytic activity. Traffic 6, 413–420 38 Yates, R.M. and Russell, D.G. (2005) Phagosome maturation proceeds independently of stimulation of toll-like receptors 2 and 4. Immunity 23, 409–417 39 Hell, S.W. (2007) Far-field optical nanoscopy. Science 316, 1153–1158 40 Egner, A. and Hell, S.W. (2005) Fluorescence microscopy with superresolved optical sections. Trends Cell Biol. 15, 207–215 41 Egner, A. et al. (2002) Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast. Proc. Natl Acad. Sci. U. S. A. 99, 3370–3375 42 Egner, A. et al. (2004) 4Pi-microscopy of the Golgi apparatus in live mammalian cells. J. Struct. Biol. 147, 70–76 43 Betzig, E. et al. (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 44 Rust, M.J. et al. (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793– 795 45 Bates, M. et al. (2007) Multicolor super-resolution imaging with photoswitchable fluorescent probes. Science 317, 1749–1753 46 Willig, K.I. et al. (2006) Nanoscale resolution in GFP-based microscopy. Nat. Methods 3, 721–723 47 Willig, K.I. et al. (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935– 939 48 Sumen, C. et al. (2004) Intravital microscopy: visualizing immunity in context. Immunity 21, 315–329 49 Lindquist, R.L. et al. (2004) Visualizing dendritic cell networks in vivo. Nat. Immunol. 5, 1243–1250 50 Schwickert, T.A. et al. (2007) In vivo imaging of germinal centres reveals a dynamic open structure. Nature 446, 83–87 51 Mansson, L.E. et al. (2007) Real-time studies of the progression of bacterial infections and immediate tissue responses in live animals. Cell. Microbiol. 9, 413–424
Review
490
TRENDS in Microbiology Vol.15 No.11
52 Menger, M.D. et al. (2002) Viewing the microcirculation through the window: some twenty years experience with the hamster dorsal skinfold chamber. Eur. Surg. Res. 34, 83–91 53 Laschke, M.W. et al. (2005) Intravital fluorescence microscopy: a novel tool for the study of the interaction of Staphylococcus aureus with the microvascular endothelium in vivo. J. Infect. Dis. 191, 435–443 54 Liu, Y. et al. (2007) Visualization of hepatobiliary excretory function by intravital multiphoton microscopy. J. Biomed. Opt. 12, 014014 55 Doyle, T.C. et al. (2004) In vivo bioluminescence imaging for integrated studies of infection. Cell. Microbiol. 6, 303–317 56 Hardy, J. et al. (2004) Extracellular replication of Listeria monocytogenes in the murine gall bladder. Science 303, 851–853 57 Glomski, I.J. et al. (2007) Primary involvement of pharynx and peyer’s patch in inhalational and intestinal anthrax. PLoS Pathog. 3, e76 58 Kadurugamuwa, J.L. et al. (2005) Reduction of astrogliosis by early treatment of pneumococcal meningitis measured by simultaneous
59
60
61 62 63 64
imaging, in vivo, of the pathogen and host response. Infect. Immun. 73, 7836–7843 Schuster, D.P. et al. (2003) Imaging lung inflammation in a murine model of Pseudomonas infection: a positron emission tomography study. Exp. Lung Res. 29, 45–57 Bettegowda, C. et al. (2005) Imaging bacterial infections with radiolabeled 1-(20 -deoxy-20 -fluoro-beta-D-arabinofuranosyl)-5iodouracil. Proc. Natl Acad. Sci. U. S. A. 102, 1145–1150 Tournebize, R. et al. (2006) Magnetic resonance imaging of Klebsiella pneumoniae-induced pneumonia in mice. Cell. Microbiol. 8, 33–43 Marzola, P. et al. (2005) (1)H MRI of pneumococcal pneumonia in a murine model. J. Magn. Reson. Imaging 22, 170–174 Shapiro, E.M. et al. (2006) In vivo detection of single cells by MRI. Magn. Reson. Med. 55, 242–249 Heyn, C. et al. (2006) In vivo MRI of cancer cell fate at the single-cell level in a mouse model of breast cancer metastasis to the brain. Magn. Reson. Med. 56, 1001–1010
Elsevier.com – linking scientists to new research and thinking Designed for scientists’ information needs, Elsevier.com is powered by the latest technology with customer-focused navigation and an intuitive architecture for an improved user experience and greater productivity. The easy-to-use navigational tools and structure connect scientists with vital information – all from one entry point. Users can perform rapid and precise searches with our advanced search functionality, using the FAST technology of Scirus.com, the free science search engine. Users can define their searches by any number of criteria to pinpoint information and resources. Search by a specific author or editor, book publication date, subject area – life sciences, health sciences, physical sciences and social sciences – or by product type. Elsevier’s portfolio includes more than 1800 Elsevier journals, 2200 new books every year and a range of innovative electronic products. In addition, tailored content for authors, editors and librarians provides timely news and updates on new products and services. Elsevier is proud to be a partner with the scientific and medical community. Find out more about our mission and values at Elsevier.com. Discover how we support the scientific, technical and medical communities worldwide through partnerships with libraries and other publishers, and grant awards from The Elsevier Foundation. As a world-leading publisher of scientific, technical and health information, Elsevier is dedicated to linking researchers and professionals to the best thinking in their fields. We offer the widest and deepest coverage in a range of media types to enhance cross-pollination of information, breakthroughs in research and discovery, and the sharing and preservation of knowledge.
Elsevier. Building insights. Breaking boundaries. www.elsevier.com www.sciencedirect.com