Huntington's disease: Dancing in a dish

In a recent landmark paper, the Huntington''s disease (HD) iPSC Consortium reports on the establishment and characterization of a panel of iPSC lines from HD patients, and more importantly, the successful modeling of HD in vitro. In the same issue of Cell Stem Cell, An et al. reports on the successful targeted gene correction of HD in human iPSCs. Both advances are exciting, provide new resources for current and future HD research, and uncover new challenges to better understand and, most importantly, treat this devastating disease in the near future.Modeling human diseases using induced pluripotent stem cells (iPSCs) has created novel opportunities for both mechanistic studies as well as for the discovery of new disease therapies. Combined with advanced gene correction technology, human iPSCs hold great promise to provide patient-specific and mutation-free cells for potential cell replacement therapy. Huntington''s disease (HD) is an autosomal dominant neurodegenerative disorder, which causes motor dysfunction, psychiatric disturbances and cognitive impairment¹. HD is caused by an expanded cystosine adenine guanine (CAG) tri-nucleotide repeat encoding polyglutamine in the first exon of the Huntingtin (HTT) gene. To date, there is no effective therapy for preventing the onset or slowdown of this disorder. Preliminary clinical trials using fetal neural grafts had shown long-lasting functional benefits in patients². Though only effective in limited cases, these results suggest that cell-based therapy could be a potential treatment if a reliable and consistent cell source is available. For this purpose, an alternative cell source to overcome the logistical and biological hurdles of this disease had been actively explored in the past decade. With recent advancement in human iPSCs technology, HD patient-specific iPSCs coupled with an efficient directed cell differentiation protocol offers hope for an unlimited supply of autologous cells. Since HD is a monogenic disease, with a very well-established correlation between the number of CAG repeats and the age of disease onset, it provides an ideal target for iPSC-based gene correction that will allow for the production of disease-free cells for potential autologous cell therapy, and at the same time provide a much needed, valuable platform to further study the pathogenesis of the disease^3,4.This is in fact what has been recently accomplished in two reports published in Cell Stem Cell^5,6. The HD iPSC Consortium reports on the generation of HD patient-specific iPSC lines that showed CAG-repeat-expansion-associated phenotypes⁵. The study from An et al.⁶ reports on the successful targeted correction of expanded CAG repeat in HD patient iPSCs and the reversion of disease phenotypes.In the study reported from the HD iPSC Consortium, the authors generated 14 iPSC lines from HD patients and controls (listed in Open in a separate window

A new HCV mouse model on the block

The investigation of virus-induced liver disease and hepatocellular carcinoma needs small animal models modeling hepatitis C virus (HCV) infection and liver disease biology. A recent study published in Cell Research reports a novel mouse model which is permissive for chronic HCV infection and shows chronic liver injury including inflammation, steatosis and fibrosis.Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide. The development of direct-acting antivirals has revolutionized treatment by offering cure¹. However, several hurdles remain. High costs limit treatment access in the majority of patients. Infection is often diagnosed at a late stage when advanced liver disease and cancer are established. Cure in advanced liver disease does not eliminate the risk of hepatocellular carcinoma (HCC). Re-infection remains possible and a vaccine is not available².To better understand the pathogenesis of virus-induced liver disease and HCC, a small animal model permissive for HCV infection and modeling liver disease biology is needed³. HCV infection is limited to humans and chimpanzees, predominantly due to distinct host-dependency factors and innate antiviral immune responses precluding cross-infection of other species⁴. Research efforts have focused on humanizing mice permissive to HCV infection. This has led to the development of conceptually three different types of mouse models.The human liver chimeric mouse is based on immune- and hepato-deficient mice repopulated with human hepatocytes. While the uPA-SCID⁵ and FRG⁶ models are extremely useful to study the viral life cycle and antivirals, the lack of an adaptive immune system and liver disease precludes the use for the study of liver disease biology and vaccine evaluation (7 based on modified Rag-2^−/− mice, activation of the overexpressed FK506-binding protein and caspase-8 fusion protein in the liver induces death of mouse hepatocytes and facilitates engraftment of human hepatocyte progenitor and CD34⁺ haematopoetic stem cells. While infected mice exhibit liver inflammation and fibrosis, this model appears to be limited with detection of virus only in the liver (8. Sustained and robust HCV infection for 90 days was achieved by crossing the 4hEF mice with mice knocked out for STAT1⁹. Furthermore, HCV infection in these mice elicited antiviral cellular and humoral immune responses. Although the animals were not reported to develop liver disease, this robust model represents a major breakthrough since it allows for studying HCV-induced immune responses and the preclinical evaluation of vaccine candidates in a small animal model (

Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages

Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.Classically, transducing phages use the pac site-headful system for DNA packaging. Packaging is initiated on concatemeric post-replicative DNA by terminase cleavage at the sequence-specific pac site, a genome slightly longer than unit length is packaged, and packaging is completed by non-sequence-specific cleavage (reviewed in Rao and Feiss, 2008). Generalized transduction results from the initiation of packaging at pac site homologs in host chromosomal or plasmid DNA, and typically represents ∼1% of the total number of phage particles. In the alternative cos site mechanism packaging is also initiated on concatemeric post-replicative DNA by terminase cleavage at a sequence-specific (cos) site. Here, however, packaging is completed by terminase cleavage at the next cos site, generating a precise monomer with the cohesive termini used for subsequent circularization (Rao and Feiss, 2008). Although cos site homologs may exist in host DNA, it is exceedingly rare that two such sites would be appropriately spaced. Consequently, cos phages, of which lambda is the prototype, do not engage in generalized transduction. For this reason, cos-site phages have been preferred for possible phage therapy, since they would not introduce adventitious host DNA into target organisms.The Staphylococcus aureus pathogenicity islands (SaPIs) are the best-characterized members of the phage-inducible chromosomal island family of mobile genetic elements (MGEs; Novick et al., 2010). SaPIs are ∼15 kb mobile elements that encode virulence factors and are parasitic on specific temperate (helper) phages. Helper phage proteins are required to lift their repression (Tormo-Más et al., 2010, 2013), thereby initiating their excision, circularization and replication. Phage-induced lysis releases vast numbers of infectious SaPI particles, resulting in high frequencies of transfer. Most SaPI helper phages identified to date are pac phages, and many well-studied SaPIs are packaged by the headful mechanism (Ruzin et al., 2001; Ubeda et al., 2007). Recently, we have reported that some SaPIs, of which the prototype is SaPIbov5 (Viana et al., 2010), carry phage cos sequences in their genomes, and can be efficiently packaged and transferred by cos phages to S. aureus strains at high frequencies (Quiles-Puchalt et al., 2014). Here we show that this transfer extends to non-aureus staphylococci and to Listeria monocytogenes.Since the pac phages transfer SaPIs to non-aureus staphylococci and to the Gram-positive pathogen Listeria monocytogenes (Maiques et al., 2007; Chen and Novick, 2009), we reasoned that cos phages might also be capable of intra- and intergeneric transfer. We tested this with SaPIbov5, into which we had previously inserted a tetracycline resistance (tetM) marker to enable selection, and with lysogens of two helper cos phages, φ12 and φSLT, carrying SaPIbov5 (strains JP11010 and JP11194, respectively; Supplementary Table 1). The prophages in these strains were induced with mitomycin C, and the resulting lysates were adjusted to 1 μg ml⁻¹ DNase I and RNase A, filter sterilized (0.2 μm pore), and tested for SaPI transfer with tetracycline selection, as previously described (Ubeda et al., 2008). To test for trans-specific or trans-generic transduction, coagulase-negative staphylococci species and L. monocytogenes strains were used as recipients for SaPIbov5 transfer, respectively, as previously described (Maiques et al., 2007; Chen and Novick, 2009). As shown in Figure 1 and Supplementary Table 2). In contrast, deletion of the SaPIbov5 cos site (strains JP11229 and JP11230) did not affect SaPI replication (Supplementary Figure 1), but completely eliminated SaPIbov5 transfer (Supplementary Table 2). The TerS protein is essential for φ12 and SaPIbov5 DNA packaging, but not for phage-mediated lysis (Quiles-Puchalt et al., 2014). As expected, this mutation abolished SaPIbov5 transfer (Open in a separate windowFigure 1(a) Map of SaPIbov5. Arrows represent the localization and orientation of ORFs greater than 50 amino acids in length. Rectangles represent the position of the ori (in purple) or cos (in red) sites. Positions of different primers described in the text are shown. (b) Amplimers generated for detection of SaPIbov5 in the different recipient strains. Supplementary Table 2 lists the sequence of the different primers used. The element was detected in S. epidermidis JP829 (Se-1), S. epidermidis JP830 (Se-2), L. monocytogenes SK1351 (Lm-1), L. monocytogenes EGDe (Lm-2), S. xylosus C2a (Sx) and S. aureus JP4226 (Sa).

Table 1

Intra- and intergeneric SaPIbov5 transfer^a

Comparative Analysis of Myxococcus Predation on Soil Bacteria

Predator-prey relationships among prokaryotes have received little attention but are likely to be important determinants of the composition, structure, and dynamics of microbial communities. Many species of the soil-dwelling myxobacteria are predators of other microbes, but their predation range is poorly characterized. To better understand the predatory capabilities of myxobacteria in nature, we analyzed the predation performance of numerous Myxococcus isolates across 12 diverse species of bacteria. All predator isolates could utilize most potential prey species to effectively fuel colony expansion, although one species hindered predator swarming relative to a control treatment with no growth substrate. Predator strains varied significantly in their relative performance across prey types, but most variation in predatory performance was determined by prey type, with Gram-negative prey species supporting more Myxococcus growth than Gram-positive species. There was evidence for specialized predator performance in some predator-prey combinations. Such specialization may reduce resource competition among sympatric strains in natural habitats. The broad prey range of the Myxococcus genus coupled with its ubiquity in the soil suggests that myxobacteria are likely to have very important ecological and evolutionary effects on many species of soil prokaryotes.Predation plays a major role in shaping both the ecology and evolution of biological communities. The population and evolutionary dynamics of predators and their prey are often tightly coupled and can greatly influence the dynamics of other organisms as well (1). Predation has been invoked as a major cause of diversity in ecosystems (11, 12). For example, predators may mediate coexistence between superior and inferior competitors (2, 13), and differential trajectories of predator-prey coevolution can lead to divergence between separate populations (70).Predation has been investigated extensively in higher organisms but relatively little among prokaryotes. Predation between prokaryotes is one of the most ancient forms of predation (27), and it has been proposed that this process may have been the origin of eukaryotic cells (16). Prokaryotes are key players in primary biomass production (44) and global nutrient cycling (22), and predation of some prokaryotes by others is likely to significantly affect these processes. Most studies of predatory prokaryotes have focused on Bdellovibrionaceae species (e.g., see references 51, 55, and 67). These small deltaproteobacteria prey on other Gram-negative cells, using flagella to swim rapidly until they collide with a prey cell. After collision, the predator cells then enter the periplasmic space of the prey cell, consume the host cell from within, elongate, and divide into new cells that are released upon host cell lysis (41). Although often described as predatory, the Bdellovibrionaceae may also be considered to be parasitic, as they typically depend (apart from host-independent strains that have been observed [60]) on the infection and death of their host for their reproduction (47).In this study, we examined predation among the myxobacteria, which are also deltaproteobacteria but constitute a monophyletic clade divergent from the Bdellovibrionaceae (17). Myxobacteria are found in most terrestrial soils and in many aquatic environments as well (17, 53, 74). Many myxobacteria, including the model species Myxococcus xanthus, exhibit several complex social traits, including fruiting body formation and spore formation (14, 18, 34, 62, 71), cooperative swarming with two motility systems (64, 87), and group (or “wolf pack”) predation on both bacteria and fungi (4, 5, 8, 9, 15, 50). Using representatives of the genus Myxococcus, we tested for both intra- and interspecific variation in myxobacterial predatory performance across a broad range of prey types. Moreover, we examined whether prey vary substantially in the degree to which they support predatory growth by the myxobacteria and whether patterns of variation in predator performance are constant or variable across prey environments. The latter outcome may reflect adaptive specialization and help to maintain diversity in natural populations (57, 59).Although closely related to the Bdellovibrionaceae (both are deltaproteobacteria), myxobacteria employ a highly divergent mode of predation. Myxobacteria use gliding motility (64) to search the soil matrix for prey and produce a wide range of antibiotics and lytic compounds that kill and decompose prey cells and break down complex polymers, thereby releasing substrates for growth (66). Myxobacterial predation is cooperative both in its “searching” component (6, 31, 82; for details on cooperative swarming, see reference 64) and in its “handling” component (10, 29, 31, 32), in which secreted enzymes turn prey cells into consumable growth substrates (56, 83). There is evidence that M. xanthus employs chemotaxis-like genes in its attack on prey cells (5) and that predation is stimulated by close contact with prey cells (48).Recent studies have revealed great genetic and phenotypic diversity within natural populations of M. xanthus, on both global (79) and local (down to centimeter) scales (78). Phenotypic diversity includes variation in social compatibility (24, 81), the density and nutrient thresholds triggering development (33, 38), developmental timing (38), motility rates and patterns (80), and secondary metabolite production (40). Although natural populations are spatially structured and both genetic diversity and population differentiation decrease with spatial scale (79), substantial genetic diversity is present even among centimeter-scale isolates (78). No study has yet systematically investigated quantitative natural variation in myxobacterial predation phenotypes across a large number of predator genotypes.Given the previous discovery of large variation in all examined phenotypes, even among genetically extremely similar strains, we anticipated extensive predatory variation as well. Using a phylogenetically broad range of prey, we compared and contrasted the predatory performance of 16 natural M. xanthus isolates, sampled from global to local scales, as well as the commonly studied laboratory reference strain DK1622 and representatives of three additional Myxococcus species: M. flavescens (86), M. macrosporus (42), and M. virescens (63) (Table ​(Table1).1). In particular, we measured myxobacterial swarm expansion rates on prey lawns spread on buffered agar (31, 50) and on control plates with no nutrients or with prehydrolyzed growth substrate.

TABLE 1.

List of myxobacteria used, with geographical origin

Evidence for a New Avian Paramyxovirus Serotype 10 Detected in Rockhopper Penguins from the Falkland Islands

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.Viruses from the Paramyxoviridae family have caused disease in humans and animals for centuries. Over the last 40 years, many paramyxoviruses isolated from animals and people have been newly described (16, 17, 22, 29, 31, 32, 36, 42, 44, 46, 49, 58, 59, 62-64). Viruses from this family are pleomorphic, enveloped, single-stranded, nonsegmented, negative-sense RNA viruses that demonstrate serological cross-reactivity with other paramyxoviruses related to them (30, 46). The subfamily Paramyxovirinae is divided into five genera: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, and Avulavirus (30). The Avulavirus genus contains nine distinct avian paramyxovirus (APMV) serotypes (Table ​(Table1),1), and information on the discovery of each has been reported elsewhere (4, 6, 7, 9, 12, 34, 41, 50, 51, 60, 68).

TABLE 1.

Characteristics of prototype viruses APMV1 to APMV9 and the penguin virus

A Targeted Multilocus Genotyping Assay for Lineage,Serogroup, and Epidemic Clone Typing of Listeria monocytogenes

A 30-probe assay was developed for simultaneous classification of Listeria monocytogenes isolates by lineage (I to IV), major serogroup (4b, 1/2b, 1/2a, and 1/2c), and epidemic clone (EC) type (ECI, ECIa, ECII, and ECIII). The assay was designed to facilitate rapid strain characterization and the integration of subtype data into risk-based inspection programs.Listeria monocytogenes is a facultative intracellular pathogen that can cause serious invasive illness (listeriosis) in humans and other animals. L. monocytogenes is responsible for over 25% of food-borne-disease-related deaths attributable to known pathogens and is a leading cause of food recalls due to microbial adulteration (12, 21). However, not all L. monocytogenes subtypes contribute equally to human illness, and substantial differences in the ecologies and virulence attributes of different L. monocytogenes subtypes have been identified (9, 13, 14, 23, 24, 33, 35, 36). Among the four major evolutionary lineages of L. monocytogenes, only lineages I and II are commonly isolated from contaminated food and human listeriosis patients (19, 27, 29, 33). Lineage I strains are overrepresented among human listeriosis isolates, particularly those associated with epidemic outbreaks, whereas lineage II strains are overrepresented in foods and the environment (13, 14, 24). Lineage III strains account for approximately 1% of human listeriosis cases but are common among animal listeriosis isolates and appear to be a host-adapted group that is poorly adapted to food-processing environments (6, 34-36). The ecological and virulence attributes of lineage IV are poorly understood, as this lineage is rare and was only recently described based on a small number of strains (19, 26, 29, 33).L. monocytogenes is differentiated into 13 serotypes; however, four major serogroups (4b, 1/2b, 1/2a, and 1/2c) from within lineages I and II account for more than 98% of human and food isolates (16, 31). Serogroups refer to evolutionary complexes typified by a predominant serotype but which include very rare serotypes that represent minor evolutionary variants (7, 9, 33). Phylogenetic analyses have indicated that rare serotypes may have evolved recently, or even multiple times, from one of the major serotypes (9), and numerous molecular methods fail to discriminate minor serotypes as independent groups (1, 4, 7, 9, 18, 22, 33, 38, 39). Serotyping is one of the most common methods for L. monocytogenes subtyping, and serogroup classifications are a useful component of strain characterization because ecotype divisions appear largely congruent with serogroup distinctions (16, 34). Serogroup 4b strains are of particular public health concern because contamination with these strains appears to increase the probability that a ready-to-eat (RTE) food will be implicated in listeriosis (16, 28). Serogroup 4b strains account for approximately 40% of sporadic listeriosis and also are responsible for the majority of listeriosis outbreaks despite being relatively rare contaminants of food products (9, 13, 17, 30, 34). In addition, serogroup 4b strains are associated with more severe clinical presentations and higher mortality rates than other serogroups (11, 16, 20, 31, 34). Serogroups 1/2a and 1/2b are overrepresented among food isolates but also contribute significantly to human listeriosis, whereas serogroup 1/2c rarely causes human illness and may pose a lower risk of listeriosis for humans (16). Serogroup-specific differences in association with human listeriosis are consistent with the prevalence of virulence-attenuating mutations in inlA within these serogroups (32, 34); however, a number of additional factors likely contribute to these differences.Four previously described epidemic clones (ECs; ECI, ECIa, ECII, and ECIII) of L. monocytogenes have been implicated in numerous listeriosis outbreaks and have contributed significantly to sporadic illness (15, 34). ECI, ECIa, and ECII are distinct groups within serogroup 4b that were each responsible for repeated outbreaks of listeriosis in the United States and Europe. ECIII is a lineage II clone of serotype 1/2a that persisted in the same processing facility for more than a decade prior to causing a multistate outbreak linked to contaminated turkey (15, 25). While there has been speculation that epidemic clones possess unique adaptations that explain their frequent involvement in listeriosis outbreaks (9, 34, 37), it is not clear that epidemic clones are more virulent than other strains with the same serotype. However, contamination of RTE food with EC strains would be cause for increased concern due to the previous involvement of these clones in major outbreaks of listeriosis (16).As a result of the L. monocytogenes subtype-specific differences in ecology, virulence, and association with human illness, molecular subtyping technologies have the potential to inform assessments of relative risk and to improve risk-based inspection programs. The objective of the present study was to develop a single assay for rapid and accurate classification of L. monocytogenes isolates by lineage, major serogroup, and epidemic clone in order to facilitate strain characterization and the integration of subtype data into inspection programs that are based on assessment of relative risk.A database of more than 5.3 Mb of comparative DNA sequences from 238 L. monocytogenes isolates (9, 33-35) was scanned for single nucleotide polymorphisms that could be used to differentiate lineages, major serogroups, and epidemic clones via a targeted multilocus genotyping (TMLGT) approach. The acronym TMLGT is used to distinguish this approach from previously published multilocus genotyping (MLGT) assays that were lineage specific and designed for haplotype discrimination (9, 33). To provide for simultaneous interrogation of the selected polymorphisms via TMLGT, six genomic regions (Table ​(Table1)1) were coamplified in a multiplex PCR. While the previous MLGT assays were based on three lineage-specific multiplexes and required prior identification of lineage identity, TMLGT was designed to target variation across all of the lineages simultaneously and is based on a unique set of amplicons. PCR was performed in 50-μl volumes with 1× High Fidelity PCR buffer (Invitrogen Life Technologies), 2 mM MgSO₄, 100 μM deoxynucleoside triphosphate (dNTP), 300 nM primer, 1.5 U Platinum Taq high-fidelity DNA polymerase (Invitrogen Life Technologies), and 100 ng of genomic DNA. PCR consisted of an initial denaturation of 90 s at 96°C, followed by 40 cycles of 30 s at 94°C, 30 s at 50°C, and 90 s at 68°C. Amplification products were purified using Montage PCR cleanup filter plates (Millipore) and served as a template for allele-specific primer extension (ASPE) reactions utilizing subtype-specific probes.

TABLE 1.

Primers used in multiplex amplification for the TMLGT assay

Linking metabolite production to taxonomic identity in environmental samples by (MA)LDI-FISH

One of the greatest challenges in microbial ecology remains to link the metabolic activity of individual cells to their taxonomic identity and localization within environmental samples. Here we combined mass-spectrometric imaging (MSI) through (matrix-assisted) laser desorption ionization time-of-flight MSI ([MA]LDI-TOF/MSI) with fluorescence in situ hybridization (FISH) to monitor antibiotic production in the defensive symbiosis between beewolf wasps and ‘Streptomyces philanthi'' bacteria. Our results reveal similar distributions of the different symbiont-produced antibiotics across the surface of beewolf cocoons, which colocalize with the producing cell populations. Whereas FISH achieves single-cell resolution, MSI is currently limited to a step size of 20–50 μm in the combined approach because of the destructive effects of high laser intensities that are associated with tighter laser beam focus at higher lateral resolution. However, on the basis of the applicability of (MA)LDI-MSI to a broad range of small molecules, its combination with FISH provides a powerful tool for studying microbial interactions in situ, and further modifications of this technique could allow for linking metabolic profiling to gene expression.Ecological analyses of microbial metabolites have thus far been hampered by the difficulty of localizing and quantifying these compounds in situ and tying their production to subpopulations or even single cells of individual microbial taxa. However, recent advances in mass-spectrometric imaging (MSI) techniques provide excellent tools to monitor metabolic processes and chemical communication in an ecological context (Svatoš, 2010, 2011). For example, matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) has successfully been employed to observe antagonistic interactions between Streptomyces and Bacillus strains in vitro (Yang et al., 2009). The analysis of microbial interactions in situ, however, requires the combination of metabolic profiling with taxonomic identification and localization of the involved microorganisms. Previous studies employing microautoradiography or high-resolution secondary ion mass spectrometry in combination with in situ hybridization have provided insights into the metabolism of individually identified bacterial cells in environmental samples (Orphan et al., 2001; Kindaichi et al., 2004; Behrens et al., 2008; Musat et al., 2008). However, the need for isotopic labeling limits the application of these techniques to a subset of biological questions.Here we combine MSI with fluorescence in situ hybridization (FISH) for simultaneous metabolite profiling and taxonomic identification of bacteria, using the defensive symbiosis between beewolf wasps and Streptomyces bacteria as a model. Beewolves of the genera Philanthus, Trachypus and Philanthinus (Hymenoptera, Crabronidae) cultivate ‘Streptomyces philanthi'' in specialized antennal gland reservoirs (Kaltenpoth et al., 2006; Goettler et al., 2007; Kaltenpoth et al., 2014) and secrete the bacteria into their subterranean brood cells before oviposition (Kaltenpoth et al., 2010a). Later, the larva incorporates the symbionts into the cocoon silk, where the streptomycetes produce a cocktail of at least nine different antibiotics (Kroiss et al., 2010) and thereby protect the larva against pathogenic fungi and bacteria during the long (up to 9 months) and vulnerable phase of hibernation (Kaltenpoth et al., 2005; Koehler et al., 2013). Previous studies using MSI revealed that the antibiotics abound on the outer surface of the cocoon, while they are virtually absent from the inner surface (Kroiss et al., 2010).We used (matrix-assisted) laser desorption ionization time-of-flight MSI ([MA]LDI-TOF/MSI) to visualize the abundance of two different antibiotics (piericidin A1 and B1) and subsequently localized the symbionts producing these compounds on beewolf cocoons using FISH. Pieces of beewolf cocoons were fixed to MALDI target plates without any pre-treatment using double-sided adhesive tape, with the outer cocoon surface facing upward. In order to allow for later alignment of ion-intensity maps and FISH images, the cocoon pieces were surrounded by thin paint markings (Edding751, 1–2 mm tip width, white), applied with the tip of a needle. This marker was chosen because it yielded characteristic signals in (MA)LDI-MS (measured at m/z 322.5±0.5) and also showed fluorescence at 640 nm, the excitation wavelength of the fluorescent dye used for FISH (that is, Cy5). MSI was carried out without any pretreatment of the samples (Hoelscher et al., 2009; Kroiss et al., 2010), or after application of 2,5-dihydroxybenzoic acid matrix by sublimation (Svatoš and Mock, 2013). A MALDI micro MX mass spectrometer (Waters, Milford, MA, USA) equipped with a nitrogen laser (337 nm) was used in the reflectron mode and positive polarity for data acquisition as previously reported (Kroiss et al., 2010). The step size in both x and y directions was set to 50 μm corresponding to 508 dots per inch resolution. Two-dimensional ion-intensity maps were reconstructed using the spectral data for the respective potassium adduct ions of piericidin A1 (PA1, m/z 454±0.5 [M+K]⁺) and piericidin B1 (PB1, m/z 468±0.5 [M+K]⁺) with the BioMAP software (Novartis Institutes for BioMedical Research, Basel, Switzerland). After (MA)LDI imaging, samples were subjected to FISH with the ‘S. philanthi''-specific probe SPT177-Cy5 (Kaltenpoth et al., 2005, 2006) as described previously (Kaltenpoth et al., 2010b). Fluorescence images were recorded on a Zeiss AxioImager Z.1 (Zeiss, Jena, Germany) using both the mosaic and z-stack options for obtaining high-resolution images with increased focusing depth. Overlays of (MA)LDI and FISH images were achieved in Adobe Photoshop CS5 Extended 12.0 by using the pen markings as a guide.(MA)LDI-MSI revealed a patchy distribution of antibiotics across the outer cocoon surface of European beewolves. The two measured antibiotic substances showed very similar distributions (Figures 1a–j), suggesting that both compounds—as well as possibly the other seven antibiotics produced by the symbionts on the beewolf cocoon that could not be measured here because of their low concentrations—are produced by individual bacterial cells or subpopulations of cells. This is supported by MSI with a high-resolution atmospheric pressure scanning microprobe (AP-SMALDI-MSI) of PA1 and PB1 produced by ‘S. philanthi'' on beewolf cocoons and in vitro, which confirmed the colocalization of both antibiotics (Figures 1k–n and Supplementary Figure S1, for experimental procedures see Supplementary Online Material). Thus, different symbiont subpopulations apparently do not specialize in the production of individual compounds, but instead produce a mixture of antibiotics.Open in a separate windowFigure 1(MA)LDI-FISH of antibiotics produced by symbiotic ‘Streptomyces philanthi'' bacteria on a beewolf cocoon (Philanthus triangulum) and in vitro. Ion-intensity maps of (a) the paint marker for alignment of LDI and FISH pictures (m/z 322.5); inset: image of the cocoon piece surrounded by white paint markings on the LDI target plate, (b) piericidin A1 (PA1, m/z 454.5 [M+K]⁺) and (c) piericidin B1 (PB1, m/z 468.5 [M+K]⁺). (d–f) The same maps, overlayed with a FISH micrograph of the cocoon piece. Symbiont cells were labeled with the fluorescent oligonucleotide probe SPT177-Cy5. (g–h) Magnifications of (e, f), respectively, with individual bacterial cells visible. (i–j) MALDI-FISH of (i) PA1 (m/z 454.5 [M+K]⁺) and (j) PB1 (m/z 468.5 [M+K]⁺) on another cocoon piece. (k–n) AP-SMALDI imaging of antibiotics produced by ‘S. philanthi'' in vitro. (k) Light microscopic image of an ‘S. philanthi'' colony, (l) PA1 (m/z 416.27 [M+H]⁺), (m) PB1 (m/z 430.25 [M+H]⁺), (n) overlay of PA1 (green) and PB1 (red).On the cocoon, FISH allowed for the visualization of individual symbiont cells, which were abundant across the entire cocoon surface and often occurred in highest densities along the outer cocoon threads (Supplementary Figure S2). The presence of the matrix had no influence on the efficiency of FISH after MSI (data not shown). The alignment of ion-intensity maps with FISH images revealed high concentrations of antibiotics around some subpopulations of cells, whereas other cell aggregations were surrounded by much lower amounts of antibiotics (Figures 1g–j), highlighting the possibility for cheating in the symbiosis. However, the current limitations in sensitivity and lateral resolution of MALDI-MSI do not permit the visualization of compounds on the single-cell level (~1 μm) and thereby may obscure fine-scale patterns of antibiotic production. This is supported by AP-SMALDI-MSI of beewolf cocoons at two different resolutions (step sizes 20 and 5 μm, Supplementary Figure S1): Whereas the high-resolution measurement revealed high concentrations of antibiotics along the outer cocoon threads, which agrees with the FISH experiments showing a similar pattern of symbiont cell densities (Supplementary Figure S2), this pattern was not as apparent at lower resolutions (Supplementary Figure S1). However, the high laser intensities required for a step size of 5 μm were destructive for the samples; therefore, subsequent FISH experiments could not be performed.The combination of (MA)LDI imaging and FISH provides a powerful tool for tying metabolite profiling to taxonomic identification in environmental samples. However, the laser intensity needs to be carefully adjusted for the desired application to achieve maximum sensitivity and resolution while at the same time conserving the structure of the sample. This problem can be circumvented by using desorption electrospray ionization (DESI) imaging, which we also successfully combined with FISH in preliminary experiments (data not shown). Still, the limitations of both (MA)LDI and DESI in lateral resolution and sensitivity currently prohibit single-cell resolution of metabolic profiling and restrict the technique to mapping the distribution of metabolites on the subpopulation level (Svatoš, 2011). Thus, the exploration of complex environmental samples is at present limited to microbial communities with distinct spatial structure. However, the major strength of MSI-FISH is its broad applicability to a wide range of small molecules as well as proteins (Svatoš, 2010). Therefore, (MA)LDI-FISH and DESI-FISH allow for addressing a multitude of questions in microbial ecology, ranging from interactions in mixed-species biofilms or cross-feeding associations to the chemical basis and dynamics of mutualistic and antagonistic encounters. As several signal enhancement techniques for FISH have been developed (for example, Schönhuber et al., 1997; Zwirglmaier, 2005), modifications of the approach described here could also be employed to tie metabolic profiling by (MA)LDI or DESI imaging to the presence (Moraru et al., 2010) or expression (Pernthaler and Amann, 2004) of particular genes of interest in microbial communities or eukaryotic tissues. Future studies should explore the possibility for using oligonucleotide labels and hybridization protocols that allow for simultaneous MSI of labeled cells and metabolites of interest, which would obviate the necessity for subsequent FISH and thereby circumvent the problems with high laser intensities. Alternatively, new MALDI matrices capable of dissipating the high ultraviolet-laser intensities and thus preventing DNA damage could be developed.

Table 1

Comparison of established methods for linking localization and taxonomic identification of microbes to the production of particular metabolites of interest in environmental samples