source: proto/SymbolTable/test/test_files/wcd_bio_papers.txt @ 1793

Last change on this file since 1793 was 1793, checked in by vla24, 8 years ago

Added some text files for wdc. updated performance test.

File size: 71.1 KB
Line 
1Intracellular invasion of green algae in a salamander host
2Ryan Kerneya,1, Eunsoo Kimb, Roger P. Hangarterc, Aaron A. Heissa, Cory D. Bishopd, and Brian K. Halla
3+ Author Affiliations
4
5aDepartment of Biology, Dalhousie University, Halifax, NS, Canada B3H 4J1;
6bDepartment of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada B3H 1X5;
7cDepartment of Biology, Indiana University, Bloomington, IN 47405; and
8dDepartment of Biology, St. Francis Xavier University, Antigonish, NS, Canada B2G 2W5
9Edited by David B. Wake, University of California, Berkeley, CA, and approved February 18, 2011 (received for review December 6, 2010)
10
11 
12Next Section
13Abstract
14
15The association between embryos of the spotted salamander (Ambystoma maculatum) and green algae (“Oophila amblystomatis” Lamber ex Printz) has been considered an ectosymbiotic mutualism. We show here, however, that this symbiosis is more intimate than previously reported. A combination of imaging and algal 18S rDNA amplification reveals algal invasion of embryonic salamander tissues and cells during development. Algal cells are detectable from embryonic and larval Stages 26–44 through chlorophyll autofluorescence and algal 18S rDNA amplification. Algal cell ultrastructure indicates both degradation and putative encystment during the process of tissue and cellular invasion. Fewer algal cells were detected in later-stage larvae through FISH, suggesting that the decline in autofluorescent cells is primarily due to algal cell death within the host. However, early embryonic egg capsules also contained encysted algal cells on the inner capsule wall, and algal 18S rDNA was amplified from adult reproductive tracts, consistent with oviductal transmission of algae from one salamander generation to the next. The invasion of algae into salamander host tissues and cells represents a unique association between a vertebrate and a eukaryotic alga, with implications for research into cell–cell recognition, possible exchange of metabolites or DNA, and potential congruence between host and symbiont population structures.
16
17photosymbiont endosymbiosis amphibian chlorophyte
18Mutualistic endosymbiosis occurs in many protist and even metazoan groups. One of the most prominent forms of endosymbiosis is between photosynthetic microbes and eukaryotic hosts. Photosynthetic endosymbionts and plastids are hosted by many eukaryote lineages, including several invertebrate animals (1). However, despite the ability of pathogenic organisms to enter vertebrate cells (2), there is an apparent absence of mutualist endosymbionts in vertebrates (3). The inability of symbionts to enter vertebrate host cells may be attributable to the adaptive immune system (3), a gnathostome synapomorphy that recognizes and destroys foreign cells (4). Associations between microbes and vertebrate embryos are possible candidates for intracellular symbioses because such early associations may precede an adaptive immune response.
19
20The symbiosis between algae and embryos of Ambystoma maculatum (spotted salamander) was first reported more than 120 y ago (5). These salamanders spend most of their adult lives underground but emerge for semiannual spring breeding congregations in vernal pools. Eggs are deposited in these pools, where embryos develop through metamorphosis. Once transformed, the young salamanders leave these pools to live underground. Algae live in direct association with embryos inside salamander egg capsules, which are contained in large jelly masses (6). Individual egg capsules appear green due to dense accumulations of algae surrounding the embryo (Fig. 1A). The mutual benefit of this facultative association has been clearly established through exclusion experiments. Clutches raised in the dark do not accrue detectable algae (6–9). The presence of algae in these experiments correlates with earlier hatching (6, 9), decreased embryonic mortality (7, 8), more synchronous hatching (8, 9), and reaching a larger size (8) and later developmental stage (9) at hatching. Additionally, algal growth is minimal in egg capsules after embryos are removed (8), indicating that the embryos, and not the egg capsules, aid algal growth. Algae are thought to benefit from nitrogenous wastes released by the embryos [ref. 6; supported by research in the closely related Ambystoma gracilis (10)], whereas salamander embryos benefit from increased oxygen concentrations associated with the algae [refs. 11–13; however, others (14) found no oxygen benefit using older techniques]. Presence of the algae also correlates with decreased cilium-mediated rotation of early-stage embryos and increased muscular contractions during later embryonic stages (9), both of which may be secondary effects of modulated oxygen levels.
21
22
23View larger version:
24In this page In a new window
25Download as PowerPoint Slide
26Fig. 1. Algal cells from the egg capsule invade salamander tissues. (A) A Stage-44 embryo inside its egg capsule (Ec). (B) Still frame taken from time-lapse recordings of Stage-15 embryos. Concentrations of algae occur synchronously, adjacent to the blastopore (arrows). (C) Black-and-white and fluorescent overlay of a Stage-39 embryo removed from its egg capsule; lateral view of the head. Red spots are autofluorescing cells (A) described in text. (D and E) Scattered algal cells embedded in Stage-37 cranial mesenchyme appear green under light microscopy. Boxed region in D shown magnified in E. (F–H) FISH-stained algal cells embedded in (F) Stage-37 cranial mesenchyme, (G) Stage-46 liver, and (H) Stage-46 ceratohyal chondrocyte. Nt, neural tube; Ph, pharynx. (Scale bars: 1 mm in A–D; 50 ÎŒm in E; 20 ÎŒm in F–H.)
27Despite these physiological studies, structural and developmental aspects of this symbiosis remain unclear. Early attempts to identify or culture the algae from oviducts of gravid female salamanders failed, discouraging further investigations into the anatomical associations between host and symbiont (8, 15). All previous research describes the relationship between the algae and embryo as an ectosymbiotic mutualism, with no intratissue or intracellular stages.
28
29In this study we show that algae invade embryonic salamander tissues (Figs. 1 and 2) and cells (Figs. 3 and 4) during embryonic development. This intracellular invasion resembles the photosynthetic endosymbiosis that has occurred in many protists (16) and metazoan invertebrates (1, 17, 18) but has not been reported in a vertebrate host.
30
31
32View larger version:
33In this page In a new window
34Download as PowerPoint Slide
35Fig. 2. Distribution of algal cells in salamander embryos. (A and B) Coronal and (C–F) transverse vibratome sections imaged for chlorophyll autofluorescence. Boxed region in C shown in higher magnification in D. Arrows indicate autofluorescent algal cells. Individual algae were embedded in (A) the otic capsule (Ot); (B) the pharynx (Ph) and pharyngeal clefts; (C and D) several regions of the head, including the neural tube (Nt); and several regions of the anterior (E) and posterior trunk (F), including the alimentary canal (Al) and notochord (No). Ba, branchial arch; He, heart; Hy, hyoid arch. (Scale bars: 100 ÎŒm in A and D; 1 mm in B, C, E, and F.)
36
37View larger version:
38In this page In a new window
39Download as PowerPoint Slide
40Fig. 3. Intracapsular and intracellular algae. (A) Whole-mount image of egg capsule (Ec) showing clusters of algal cells. (B) TEM image of algal cells encased by an outer envelope (En; boxed region in 3A) on the inner border of the innermost egg capsule. (C and D) TEM images of intracapsular algae revealing starch granules (S), chloroplast grana (G), nuclei (N), and vacuoles (V). (E) Coronal vibratome section through the trunk region of a Stage-37 embryo. White spots are autofluorescing cells embedded in the somites (So). (F–J) Intracellular algae found within somites. (F) Algal cell embedded inside a salamander myocyte (M). (G) Higher magnification of intracellular alga in F. Salamander mitochondria (Mi) and myofibrils (My) occur in the surrounding host cytoplasm. (H) Paired algal cells within a host cell. (I) Algal cell surrounded by a thickened outer envelope. (J) Degraded algal cell without an outer envelope. F, flagella; Nt, neural tube; No, notochord; Yp, yolk platelet. (Scale bars: 100 ÎŒm in A; 2 ÎŒm in B–D; 1 mm in E; 2 ÎŒm in F–J.)
41
42View larger version:
43In this page In a new window
44Download as PowerPoint Slide
45Fig. 4. Algal cells within the alimentary canal during the process of cellular invasion. (A) Fluorescent image of a coronal vibratome section through a Stage-35 embryo showing an aggregation of algae (A) inside the alimentary canal (Al) and invading the surrounding endoderm. (B–F) TEM images from boxed region in A. (B–D) Algal cells in the alimentary canal, adjacent to salamander endoderm. (E) Algal cell inside endodermal cell of the alimentary canal containing many large vacuoles (V) and paired flagella (F). (F) Free thylakoid membranes (T) and starch granules (S) inside cytoplasm of host cell shown in E. H, heart; LD, lipid droplet; N, nucleus; Yp, yolk platelet. (Scale bars: 1 mm in A; 2 ÎŒm in B and C; 5 ÎŒm in D; 2 ÎŒm in E; 1 ÎŒm in F.)
46Previous Section
47Next Section
48Results
49
50Phylogenetic reconstruction, using chloroplast 16S (1,362 bp; GenBank accession no. HM590633) and nuclear 18S (1,714 bp; GenBank accession no. HM590634) rDNA sequences from intracapsular algae, places the algae in the Chlorophyceae, specifically the Chlamydomonadales (Fig. S1). Two algal clones of each PCR amplification had >99% sequence identity to each other, and the 16S and 18S rDNA phylogenies were congruent. These observations suggest that intracapsular algal cells in our samples comprise a single species. The published literature on this association (6, 8, 9) refers to the algae as “Oophila amblystomatis” (Lambert ex Printz) (19) on the basis of an informal species designation by Lambert (6), although—to date—no formal taxonomic description exists. Previous studies reported additional bacteria and protists within the egg capsules (6, 14). Our 18S rDNA amplification clones included sequences from ciliate and cercozoan protists, and our 16S rDNA amplification clones included sequences for the prokaryotes Pedobacter sp., Acidovorax sp., Duganella zoogloeoides, Janthinobacterium sp., Verrucomicrobiales sp., Chitinophaga sp., and Flavobacterium sp. However, no other green algae were identified from the egg capsules aside from the conserved Oophila sequences.
51
52Intratissue algae were first detected through their chlorophyll autofluorescence (480 nm excitation) in a Stage-39 embryo (Fig. 1C). Autofluorescent cells were scattered throughout the embryo, with a high concentration in the anterior end of the ventral abdomen. Algal cell entry into host tissues was verified through PCR amplification of symbiont 18S rDNA (20), time-lapse photography, and fluorescence microscopy.
53
54The timing of algal cell entry was determined through algal 18S rDNA PCR. Embryos were extracted from their egg capsules and rinsed thoroughly before DNA extraction. Oophila-specific 18S rDNA was amplified and sequenced from Harrison (21) Stage-26 (pharyngula), and older, salamander embryos. It was not detected in Stage-17 (neurula), or earlier, embryos (Fig. S2). However, algae were detected microscopically on the innermost egg capsule during these early stages (Fig. 3 A and B).
55
56Gilbert (6) described a concentration of algae around the salamander blastopore (“proctodeum”). Time-lapse photography of A. maculatum embryos revealed this concentration forming in Stage-15 embryos (Fig. 1B and Movie S1). This concentration of algal cells precedes their detection inside salamander tissues through PCR, indicating that the algal bloom outside the blastopore precedes subsequent algal cell invasion into the salamander embryo host.
57
58By imaging chlorophyll autofluorescence, we identified algal cells in embryonic epidermis, optic cup, neural tube, cranial mesenchyme, presumptive lens, somitic myotome, and otic capsule between Stages 35 and 44 (Figs. 2, 3E, and 4A). Autofluorescing cells were more abundant in the yolk and within the alimentary canal (Fig. 4A). Intratissue autofluorescent cells and algae from the intracapsular fluid both fluoresced red under the same excitation wavelength (480 nm). Many of the intratissue autofluorescing cells appeared green under light microscopy in vibratome sections (Fig. 1 D and E). Autofluorescing cells were no longer detectable in feeding larvae (Stage 46, 3 to 4 wk of development). Although many algal cells invaded the alimentary canal and surrounding tissues, most algae persist in the intracapsular fluid and are released into the surrounding water upon hatching.
59
60The genetic identity of intratissue algae was verified through fluorescence in situ hybridization (FISH) using an oligonucleotide probe that targets Oophila 18S rRNA. FISH-positive algal cells were detected in several Stage-37 tissues, including the cranial mesenchyme (Fig. 1F) and endoderm. By Stage 46, FISH-positive cells were found in the larval liver (n = 2; Fig. 1G) and ceratohyal cartilage (n = 1; Fig. 1H) after the cessation of detectable algal autofluorescence. We detected few algal cells in our Stage-46 sample through FISH, in comparison with the widespread distribution of algae during embryonic Stages 35–44.
61
62Transmission electron microscopy (TEM) was used to determine the ultrastructure of algae attached to the egg capsules (Fig. 3B), motile algae within the egg capsule fluid (Fig. 3 C and D), algae within the alimentary canal (Fig. 4 B–D), and algae embedded in salamander tissues (Figs. 3 F–J and 4E). Algal cells were initially present in small clusters on individual egg capsules of early embryos (Fig. 3A). Algae on the egg capsules were encased in an outer envelope (Fig. 3B) and lacked flagella. These cells were recessed from the outer envelope walls.
63
64Vibratome sections containing autofluorescent cells were dissected and prepared for TEM, allowing ultrastructural analysis of 26 algal cells inside salamander tissues during Stages 35, 37, and 42 (e.g., Fig. 3 F–I). Each intratissue alga contained starch granules inside the chloroplast, chloroplast grana, and numerous thylakoid membranes (e.g., Fig. 3G), which were also found in intracapsular algae (Fig. 3 C and D). These conserved ultrastructural features also verify the green algal identity of intratissue autofluorescent cells (22).
65
66Surprisingly, many autofluorescent algal cells were embedded within the cytoplasm of differentiated salamander host cells. Intracellular algae were found in autofluorescent somitic cells (n = 11; Fig. 3 F–I) and embedded within endodermal cells surrounding the alimentary canal (n = 13; Fig. 4E). Intracellular algae often occurred in close proximity to host cell mitochondria (Fig. 3 G–I) and occasionally bordered host cell nuclei. Host cells showed no signs of cellular necrosis or apoptosis and were structurally similar to adjacent, alga-free, cells. Although most intracellular algal cells appeared intact (Fig. 3 G–I), several were degraded (Fig. 3J). All host somite cells were undergoing myocyte differentiation, apparent through myofibril formation within the cytoplasm (Fig. 3 G–I). Most intracellular algae occurred as single cells, although three pairs of intracellular algae were also observed (Fig. 3H). The absence of detectable mitosis or cytokinesis in any of the singular or paired intracellular algae suggests that these paired cells invaded the salamander host cell together.
67
68Several changes to algal cells were associated with host tissue and cellular invasion. Relative area measurements of starch granules and vacuoles were compared between intracapsular (n = 26), intracellular (n = 24), and extracellular algae found within the embryonic alimentary canal (n = 18). There were no differences in starch granule sizes [one-way ANOVA, F(2,56) = 2.89, P > 0.05]; however, starch granules constituted more cross-sectional area of intracapsular algae (10.5% SD 9.2%) than intracellular (5.9% SD 4.2%) or alimentary canal (3.4% SD 3.2%) algae [one-way ANOVA, F(2,56) = 4.70, P < 0.05]. Additionally, vacuoles constituted increasing cross-sectional area of intracapsular (11.0% SD 8.5%), intracellular (48.3% SD 13%), and alimentary canal (56.0% SD 15%) algae [F(2,65) = 135.18, P < 0.05]. Both intracapsular and invaded algae had paired flagella (Fig. 4E), although these were apparent in only a few intracellular algae.
69
70An electron-translucent “perisymbiont zone” (20) surrounded several intracellular algae (Fig. 3 G–J). These perisymbiont zones were not associated with vesicle membranes, suggesting no distinct host symbiosome surrounding each alga (20). Five intracellular algae were encased in an outer envelope (Fig. 3I) similar to the envelope of encysted algae on the innermost egg capsule wall of early-stage embryos (Fig. 3B). Many intracellular algae that did not have an outer envelope appeared degraded (e.g., Fig. 3J), with vacuolar contents recessed and occasionally extruded from the cell.
71
72Salamander endoderm adjacent to algal cells in the alimentary canal had indistinct plasma membranes and dissociated cytoplasm in regions of host–symbiont cellular contact, consistent with an active process of cellular invasion (Fig. 4 B–D). Algal cells within the alimentary canal occurred in loose aggregates (Fig. 4D), often adjacent to the boundary between two endodermal cells. Algae were not found in the alimentary canal of Stage-46 larvae either through autofluorescence, FISH, or TEM imaging.
73
74Nine salamander cells containing algae also contained small collections of acellular thylakoid membranes and starch granules within their cytoplasm (Fig. 4F). These free thylakoids were more common in endodermal cells (n = 7) than in somitic cells (n = 2). They superficially resemble the intracellular kleptoplasts acquired by some sacoglossan mollusks (e.g., Elysia chlorotica) from their algal food (e.g., Vaucheria litorea) (23). However kleptoplasts are often enclosed in distinct vesicular membranes (17, 23), whereas the free thylakoids in salamander cells are not.
75
76We extracted DNA from reproductive tissues of three adult female and three adult male A. maculatum. In female salamanders, Oophila-specific 18S rDNA was amplified from the posterior oviduct (two of three), anterior oviduct (one of three), and posterior ovary (one of three) but was not found in the medial oviduct, anterior or medial ovary. In adult male salamanders, Oophila-specific 18S rDNA was amplified from the Wolffian (one of three) and MÃŒllerian (one of three) ducts but not from the anterior or posterior testes (Fig. S2).
77
78Previous Section
79Next Section
80Discussion
81
82The invasion of green algae into salamander cells reveals that intracellular symbiosis of a phototroph can occur in a vertebrate host. This resembles endosymbioses between cyanobacterial Prochloron sp. and didemnid ascidian hosts (24), dinoflagellates of the genus Symbiodinium and cnidarian, poriferan, mollusk, platyhelminth, and foraminiferan hosts (1, 25), and between the green alga Chlorella and ciliate, poriferan, and cnidarian hosts (26). However, the Ambystoma–alga symbiosis is unique in several features. In the wild, nearly all intratissue algal–animal symbioses are obligatory and consist of photosynthate transfer from symbiont to host (17, 27). However, previous research has shown that the Ambystoma–alga association is facultative (6, 8, 15) and may not result in photosynthate transfer (28). Algal–animal associations are typically limited by the availability of carbon dioxide and photons. The latter are undoubtedly in short supply within the opaque tissues of a primarily fossorial salamander species.
83
84Previous research on invertebrate–alga associations provides ready-made experimental hypotheses for the Ambystoma–alga symbiosis. Two reoccurring features of intracellular photosymbionts include photosynthate transfer and lateral gene transfer. Unlike the case in many invertebrate photosymbionts, photosynthate transfer from algae to A. maculatum has not been detected (27). However, the negative result of this earlier research is not conclusive (10). The methods used to detect carbon fixed by photosynthesis (27) should have revealed algal cells invading host tissues, but did not. Our data on this symbiosis justifies experimentally readdressing the possibility of photosynthate transfer by tracing labeled carbon isotopes. Such experiments will require careful attention to the occurrence of photosynthate transfer vs. algal cell digestion within the host.
85
86Recent research has also revealed lateral gene transfer of algal nuclear genes into the sea slug E. chlorotica (29), which may be associated with maintaining kleptoplast function (however, see ref. 30). Because amphibian nuclei readily acquire foreign DNA from the cytoplasm (31), some scattered intracellular algae found in A. maculatum embryos may have transferred heritable algal DNA. However, unlike E. chlorotica kleptoplasts, there is no obvious role for transferred algal genes in the A. maculatum genome.
87
88Structural Changes to Invading Algae. Algal endosymbionts, such as Chlorella sp., often have a wide range of ultrastructural variation (32). This variation also was apparent in our ultrastructural analyses of Oophila sp. Several structural changes correlate with alimentary canal and cellular invasion. Algae found both within the alimentary canal and inside host cells had a proportional increase in vacuoles and decrease in starch granules compared with intracapsular algae. Vacuoles reached a peak size in algae found within the alimentary canal and often appeared extruded from individual algal cells (Fig. 4D). The vacuoles of intracapsular green algae in Ambystoma gracile hosts have been shown to be sites of ammonia waste storage in the form of proteins (10). This may account for larger vacuole sizes of invading algae, if these algae are following a nitrogenous waste gradient. However, these vacuoles also resemble the accumulation bodies of encysting dinoflagellates, which are a form of autophagic vesicle (22). Therefore, the increase of vacuole size may be associated with alimentary canal and cellular invasion, programmed cell death (33), or even algal cell encystment (22, 26).
89Most intracellular algae were in direct contact with the host cytoplasm and not enclosed in a distinct vesicular membrane. This is similar to the association between Platymonas convolutae (a prasinophycean green alga) and acoel flatworms of the genus Convoluta sp., but unlike the association of Chlorella sp. (a trebouxiophycean green alga) with cnidarian hydras (26), which are contained in a symbiosomal membrane.
90
91Several intracellular algae were enclosed in a thickened envelope. A structurally similar envelope surrounds algae found on the innermost egg capsule of early-stage embryos (before tissue invasion; Fig. 3 A and B). Despite their structural similarity, we do not know whether these envelopes are independently derived or whether they represent a lineage of encysted algal cells. These envelopes are similar to those formed by encysting diatoms (22), indicating that they may be involved in a process of algal cell encystment after invasion.
92
93The process of algal cell integration in the Ambystoma–alga symbiosis differs from that described in other animal–alga symbioses. Invertebrate hosts typically ingest their algal symbionts, and cellular integration is often a result of partial digestive assimilation (17, 18, 27). However, in A. maculatum, algae primarily enter salamander embryos through the blastopore before the formation of a patent stomodeum and therefore before active feeding is possible. Additionally, some algal cells are embedded within epithelial or mesenchymal tissues that are far from the alimentary canal, consistent with algae having entered salamander embryos directly by penetrating their embryonic integument. Algal cells leave direct sunlight by entering opaque salamander tissues. Behavioral stimuli, such as gradients of nitrogenous waste, which may instigate algal proliferation (8, 10), could serve as the behavioral cue for tissue and cellular invasion against this light gradient.
94
95Possibility of Vertical Transmission. Previous research using light microscopy did not find algae in the oviducts of A. maculatum (15) and failed to culture algae from oviduct (6, 15) or oocyte (15) rinses. Additionally, clutches grown in alga-free tap water failed to grow algae in comparison with those raised in pond water (6, 15). This evidence against vertical transmission has led to a general acceptance that the algae are derived from the environment (34). However, neither the process of environmental acquisition (15) nor free-living Oophila sp. from vernal pools (35) has been described. Additionally, previous studies have had difficulty in obtaining alga-free clutches from either the laboratory or the wild (6, 8, 11).
96Although our data are consistent with a process of vertical transmission, this process is unlikely to be the dominant mode of algal acquisition. The amplification of algal 18S rDNA from the oviducts, the encysted algae on the innermost egg capsule (Fig. 3B), and the similar encystment of some intracellular algae (Fig. 3I) indicate possible oviductal transmission. However, we did not amplify 18S rDNA consistently from adult reproductive tracts (Fig. S2), nor did we find algae concentrated in the reproductive tracts of embryos or early-stage larvae. Mixed modes of vertical and horizontal acquisition of symbionts are common. These are often revealed through incongruent phylogenetic topologies between host and symbiont populations (20). Similar phylogenetic comparisons between Ambystoma-Oophila populations could reveal the extent of vertical or horizontal acquisition in different salamander populations and test the controversial (6) species designation “Oophila amblystomatis” (Lambert ex. Printz) (19).
97
98Endosymbiosis. The reciprocal benefits of hatchling survival and growth to the embryo, and population growth to the algae, reveal that this symbiosis is a true mutualism (6, 8, 9). However, the material benefit of algal symbionts has been controversial. Hutchison and Hammen (14) found no net gain of oxygen from the algae and instead attributed the algal contribution to unknown “growth factors” supplied to the embryo. More refined microelectrode studies have since shown that intracapsular algae do produce a net increase of oxygen (11, 12), which confers a meaningful physiological benefit (13). The unexpected intracellular association shown in our study may reveal a benefit to the embryo other than oxygen production. Most intracellular algae, and algae of the alimentary canal, disappear by early larval stages. Their possible absorption may confer a metabolic benefit to their host.
99Intracellular symbionts have been reported for many metazoan taxa, but we know of no other observation for vertebrates (3). As suggested in the Introduction, the embryonic association of the Ambystoma–alga symbiosis may preclude an adaptive immune response that would otherwise remove invading algal cells. V(D)J recombination in B and T cells, as indicated by RAG-1 protein in the thymus, does not occur until 6–8 wk after fertilization in the axolotl Ambystoma mexicanum (4), well after the initial invasion of algae in A. maculatum. The number of potential antibodies used in adult salamander antigen response is restricted (36), which may account for their remarkable regenerative abilities and acceptance of allo- and xenografts (37). The combination of an embryonic symbiosis and an inefficient immune system may, in part, account for the acceptance of an intracellular symbiont in A. maculatum. However, the lack of other intracellular symbiont examples in vertebrates may simply be due to a lack of investigation. The intracellular symbiosis described here reveals unanticipated complexity in the Ambystoma–alga system with implications for the ecology, evolution, and development of both host and symbiont.
100
101Previous Section
102Next Section
103Materials and Methods
104
105Animal Care. Animal procedures followed Dalhousie University Committee on Laboratory Animals protocol (09-029). Embryo clutches were collected from the Halifax Regional Municipality, Nova Scotia, Canada under permits from The Wildlife Division of the Nova Scotia Department of Natural Resources.
106Fluorescence and TEM. Individual embryos were anesthetized in tricaine methanesulfonate (pH 7.4), fixed overnight at 4 °C in Karnovsky's solution (2% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4), cut in 100-Όm sections in 1% agarose (wt/vol) on a Vibratome 1000 (Automatic Tissue Sectioning Systems), and imaged for fluorescence on a Zeiss Axio Observer Z1 compound microscope. Color images were acquired on a Nikon AZ0 stereo dissecting microscope. Tissue regions containing bright autofluorescing cells were manually dissected from vibratome sections, postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer (30 min), dehydrated in an ethanol series, and embedded in Spurr's resin (Structure Probe). Polymerized resin blocks were trimmed and sectioned to a thickness of 70 nm, stained with saturated (2%) uranyl acetate in 50% (vol/vol) ethanol, counterstained with Reynold's lead citrate, and imaged on an FEI Tecnai-12 transmission electron microscope. Additional algae from the intracapsular fluid (Fig. 1A) were aspirated with a 32-gauge needle and processed in parallel to the salamander tissues. All image area measurements were made with Image J (http://rsbweb.nih.gov/ij/). One-way ANOVAs of area measurements with Games-Howell post hoc tests were run on SPSS (version 18).
107Fluorescent in Situ Hybridization. We designed an oligonucleotide probe specific for Oophila 18S rRNA (5′-TCTCTCAAGGTGCTGGCGA-3′) based on the regions of low RNA folding complexity in eukaryotes (38). The horse radish peroxidase-conjugated Oophila-specific probe, along with a positive control bacterial 16S rRNA-targeted probe (EUB338) (39) and negative control bacterial sense probe (NON338) (40), were purchased from biomers.net.
108Salamander tissues were fixed in 4% paraformaldehyde (PFA) overnight, rinsed in sterile phosphate buffered saline (PBS), and stored in 100% methanol (−20 °C). Prehybridization steps followed Zelzer et al. (41) for mRNA in situ hybridizations and included rehydration into PTw (PBS with 0.1% Tween-20), 15 min in 0.02 M HCl, 10 min in 3% H2O2, 10 min in 5 ÎŒg/mL proteinase-K in PBS (37 °C), 2 × 5 min in 0.1 M triethanolamine (pH 7.0–8.0; Sigma), then 2 × 5 min in 0.1 M triethanolamine solution containing 2.5 ÎŒL/mL acetic anhydride. Slides were washed 2 × 5 min in PBS in between each step up to the triethanolamine treatment. Slides were then refixed in 4% PFA for 5 min, rinsed 5 × 1 min in PBS, dehydrated in a graded ethanol series, and air-dried for 30 min.
109
110Each slide was covered with 150 ÎŒL hybridization buffer [900 mM NaCl, 20 mM Tris (pH 7.5), 0.02% SDS, 25% (vol/vol) formamide, 1% blocking reagent, and 10% (wt/vol) dextran sulfate] and included an unlabeled helper oligonucleotide (5′-GTCATCAAAAGAACGCTCGCC-3′) (42) and HRP-conjugated probe (0.15 ng/mL final concentration each). Slides were covered with parafilm strips during the hybridization and tyramide signal amplification (TSA) steps. Slides were hybridized for 3 h in a humidity chamber (46 °C), followed by a brief rinse in prewarmed wash buffer [20 mM Tris (pH 7.5), 159 mM NaCl, 0.01% SDS, and 5 mM EDTA; 46 °C] and an additional 15 min in wash buffer (46 °C). Slides were soaked in TNT buffer [0.1 M Tris (pH 7.5), 150 mM NaCl, and 0.074% Tween 20] for 15 min (25 °C). A TSA plus fluorescence kit (PerkinElmer) was used for signal amplification, following the manufacturer's instructions for tetramethylrhodamine or cyanine-3 tyramide in 40% (wt/vol) dextran sulfate. Slides were covered with 150 ÎŒL of amplification reagent and incubated for 45 min in the dark (25 °C) before sequential washes (20 min and 15 min) with TNT buffer (46 °C) and 1.5 mg/mL DAPI counterstaining (5 min; 25 °C). Slides were cover-slipped with 150 ÎŒL of AF1 (Citifluor) and sealed with nail polish.
111
112Supporting Methods. SI Materials and Methods provides additional methods pertaining to the online supporting material. These methods include amplification and sequencing of nuclear 18S and chloroplast 16S rDNA of Oophila, Oophila-specific 18S rDNA amplification in salamander tissues, phylogenetic reconstructions, and in vivo time-lapse microscopy.
113Previous Section
114Next Section
115Acknowledgments
116
117We thank Joe Martinez, John Gilhen, David Hewitt, and Robin Kodner. This work was partially completed in the laboratories of James Hanken, Alastair Simpson, and John Archibald. Michelle Leger and Yana Eglit provided useful comments on an earlier draft. R.K. is an American Association of Anatomists Scholar. E.K. is supported by the Tula Foundation. A.A.H is supported in part by Natural Sciences and Engineering Research Council (NSERC) Grant 298366-2009 to Alastair Simpson. This research was funded by the American Association of Anatomists (R.K.), an NSERC grant (to B.K.H.), and National Science Foundation Grant MCB-0848083 (to R.P.H.).
118
119Previous Section
120Next Section
121Footnotes
122
123↵1To whom correspondence should be addressed. E-mail: ryankerney@gmail.com.
124Author contributions: R.K. designed research; R.K., E.K., R.P.H., A.A.H., and C.D.B. performed research; E.K., R.P.H., A.A.H., and C.D.B. contributed new reagents/analytic tools; R.K. and B.K.H. analyzed data; and R.K., E.K., R.P.H., A.A.H., C.D.B., and B.K.H. wrote the paper.
125The authors declare no conflict of interest.
126This article is a PNAS Direct Submission.
127Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. HM590633 (Oophila sp. 16S) and HM590634 (Oophila sp. 18S)].
128This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1018259108/-/DCSupplemental.
129Previous Section
130 
131References
132
133↵ Venn AA, Loram JE, Douglas AE (2008) Photosynthetic symbioses in animals. J Exp Bot 59:1069–1080. Abstract/FREE Full Text
134↵ Bhavsar AP, Guttman JA, Finlay BB (2007) Manipulation of host-cell pathways by bacterial pathogens. Nature 449:827–834. CrossRefMedlineWeb of Science
135↵ Douglas A (2010) The Symbiotic Habit (Princeton Univ Press, Princeton).
136↵ Flajnik M, Du Pasquier L (2009) in Fundamental Immunology, Evolution of the immune system, ed Paul W (Wolters Kluwer, Lippincott Williams & Wilkins, Philadelphia), 6th Ed, pp 56–124.
137↵ Orr H (1888) Note on the development of amphibians, chiefly concerning the central nervous system; with additional observations on the hypophysis, mouth, and the appendages and skeleton of the head. Q J Micro Sci N S 115:483–489.
138↵ Gilbert PW (1942) Observations on the eggs of Ambystoma maculatum with especial reference to the green algae found within the egg envelopes. Ecology 23:215–227. CrossRefWeb of Science
139↵ Breder R (1927) The courtship of the spotted salamander. Bull N Y Zool Soc 30:51–56.
140↵ Gilbert PW (1944) The alga-egg relationship in Ambystoma maculatum. A case of symbiosis. Ecology 25:366–369. CrossRefWeb of Science
141↵ Tattersall G, Spiegelaar N (2008) Embryonic motility and hatching success of Ambystoma maculatum are influenced by a symbiotic alga. Can J Zool 86:1289–1298. CrossRef
142↵ Goff LJ, Stein JR (1978) Ammonia: Basis for algal symbiosis in salamander egg masses. Life Sci 22:1463–1468. CrossRefMedlineWeb of Science
143↵ Bachmann M, Carlton R, Burkholder J, Wetzel R (1985) Symbiosis between salamander eggs and green algae: Microelectrode measurements inside eggs demonstrate effect of photosynthesis on oxygen concentration. Can J Zool 64:1586–1588.
144↵ Pinder A, Friet S (1994) Oxygen transport in egg masses of the amphibians Rana sylvatica and Ambystoma maculatum: Convection, diffusion and oxygen production by algae. J Exp Biol 197:17–30. Abstract
145↵ Valls JH, Mills NE (2007) Intermittent hypoxia in eggs of Ambystoma maculatum: Embryonic development and egg capsule conductance. J Exp Biol 210:2430–2435. Abstract/FREE Full Text
146↵ Hutchison V, Hammen C (1958) Oxygen utilization in the symbiosis of embryos of the salamander, Ambystoma maculatum and the alga, Oophila amblystomatis. Biol Bull Mar Biol Lab Woods Hole 115:483–489. Abstract/FREE Full Text
147↵ Gatz J (1973) Algal entry into the eggs of Ambystoma maculatum. J Herpetol 7:137–138. CrossRef
148↵ Lane CE, Archibald JM (2008) The eukaryotic tree of life: Endosymbiosis takes its TOL. Trends Ecol Evol 23:268–275. CrossRefMedline
149↵ Trench R (1979) The cell biology of plant-animal symbiosis. Annu Rev Plant Physiol 30:485–531. Web of Science
150↵ Buchner P (1965) Endosymbiosis of Animals with Plant Microorganisms (John Wiley, New York).
151↵ Printz H (1928) in Die natÃŒrlichen Pflanzenfamilien [The Natural Plant Families] Chlorophyceae, eds Engler A, Prantl K (W. Engelmann, Leipzig, Germany), 3, pp 1–463.
152↵ Bright M, Bulgheresi S (2010) A complex journey: Transmission of microbial symbionts. Nat Rev Microbiol 8:218–230. CrossRefMedlineWeb of Science
153↵ Harrison R (1969) in Organization and Development of the Embryo, Harrison stages and description of normal development of the spotted salamander, Ambystoma punctatum (Linn) ed Wilens S (Yale Univ Press, New Haven, CT), pp 44–66.
154↵ Dodge J (1973) The Fine Structure of Algal Cells (Academic Press, New York).
155↵ Rumpho M, Dastoor FP, Manhart JR, Lee J (2006) in The Structure and Function of Plastids (Advances in Photosynthesis and Respiration) The kleptoplast, eds Wise R, Hoober J (Springer, Dordrecht, The Netherlands), 23, pp 451–473.
156↵ Hirose E, Maruyama T, Cheng L, Lewin R (1996) Intracellular symbiosis of a photosynthetic prokaryote, Prochloron sp., in a colonial ascidian. Invertebr Biol 115:343–348. CrossRefWeb of Science
157↵ Stat M, Carter D, Hoegh-Guldberg O (2006) The evolutionary history of Symbiodinium and scleractinium hosts—symbiosis, diversity, and the effect of climate change. Perspect Plant Ecol Evol Syst 8:23–43. CrossRef
158↵ Muscatine L, Pool RR, Trench RK (1975) Symbiosis of algae and invertebrates: Aspects of the symbiont surface and the host-symbiont interface. Trans Am Microsc Soc 94:450–469. Medline
159↵ Pardy R (1983) in Phycozoans, Phycozoology, Phycozoologists? Algal Symbiosis: A Continuum of Interaction Strategies, ed Goff L (Cambridge Univ Press, Cambridge, UK), pp 5–18.
160↵ Hammen C, Hutchison V (1962) Carbon dioxide assimilation in the symbiosis of the salamander Ambystoma maculatum and the algae Oophila amblystomatis. Life Sci 1:527–532. CrossRef
161↵ Rumpho ME, et al. (2008) Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica. Proc Natl Acad Sci USA 105:17867–17871. Abstract/FREE Full Text
162↵ WÀgele H, et al. (2011) Transcriptomic evidence that longevity of acquired plastids in the photosynthetic slugs Elysia timida and Plakobranchus ocellatus does not entail lateral transfer of algal nuclear genes. Mol Biol Evol 28:699–706. Abstract/FREE Full Text
163↵ Chesneau A, et al. (2008) Transgenesis procedures in Xenopus. Biol Cell 100:503–521. CrossRefMedlineWeb of Science
164↵ Karakashian S (1970) Morphological plasticity and the evolution of algal symbionts. Ann N Y Acad Sci 175:474–487. CrossRefWeb of Science
165↵ van Doorn WG, Yoshimoto K (2010) Role of chloroplasts and other plastids in ageing and death of plants and animals: A tale of Vishnu and Shiva. Ageing Res Rev 9:117–130. CrossRefMedlineWeb of Science
166↵ Epel D, Gilbert SF (2008) Ecological Developmental Biology: Integrating Epigenetics, Medicine, and Evolution (Sinauer Associates, Sunderland, MA).
167↵ Shudert E (2003) in Freshwater Algae of North America, Nonmotile coccoid and colonial green algae, eds Wehr TD, Sheath RG (Academic Press, New York), pp 253–307.
168↵ Charlemagne J (1987) Antibody diversity in amphibians. Noninbred axolotls used the same unique heavy chain and a limited number of light chains for their anti-2,4-dinitrophenyl antibody responses. Eur J Immunol 17:421–424. CrossRefMedlineWeb of Science
169↵ Mescher AL, Neff AW (2006) Limb regeneration in amphibians: Immunological considerations. Sci World J 6(Suppl 1):1–11.
170↵ Behrens S, et al. (2003) In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes. Appl Environ Microbiol 69:1748–1758. Abstract/FREE Full Text
171↵ Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770. Abstract/FREE Full Text
172↵ Worden AZ, Chisholm SW, Binder BJ (2000) In situ hybridization of Prochlorococcus and Synechococcus (marine cyanobacteria) spp. with rRNA-targeted peptide nucleic acid probes. Appl Environ Microbiol 66:284–289. Abstract/FREE Full Text
173↵ Zelzer E, et al. (2001) Tissue specific regulation of VEGF expression during bone development requires Cbfa1/Runx2. Mech Dev 106:97–106. CrossRefMedlineWeb of Science
174↵ Fuchs BM, Glöckner FO, Wulf J, Amann R (2000) Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 66:3603–3607. Abstract/FREE Full Text
175
176Infect Immun. 2002 December; 70(12): 6518–6523.
177doi:  10.1128/IAI.70.12.6518-6523.2002
178PMCID: PMC132940
179Copyright © 2002, American Society for Microbiology
180In Vivo Expression Technology
181Michael J. Angelichio and Andrew Camilli*
182Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
183*Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-2144. Fax: (617) 636-0337. E-mail: Andrew.Camilli@Tufts.edu.
184 This article has been cited by other articles in PMC.
185 Other Sectionsâ–Œ
186 
187The capacity to quickly and efficiently regulate gene expression has helped bacteria to colonize virtually every available niche in the biosphere, including dynamic and extreme ones. In accordance with this, bacterial populations must be ever ready to either take advantage of a favorable change or hunker down when the going gets tough. This proficiency is nicely demonstrated by infection of humans by facultative pathogens, for which up-regulation of genes necessary for survival and growth and down-regulation of genes deleterious to infectivity must occur on cue. To better understand this transition from ex vivo to in vivo conditions and to further our understanding of pathogenesis, it is necessary to identify genes that are specific to infection. Toward this end, in vivo expression technology (IVET) was developed (27). The purpose of this short review is to update the reader on the many variations of IVET that have been developed, to discuss nuances of each method that may be helpful to investigators embarking on studies using this technology, and to discuss offshoot technologies of IVET as tools for studying regulation of virulence genes. The many individual microbial virulence factors that have been identified using IVET are not reviewed here but have been recently reviewed by Mahan et al. (26). Because IVET is but one of several methods that can be used to screen for virulence genes induced during infection of cultured cells but is the only established method for accomplishing the same feat within infected animals, discussion herein is limited to reported uses of IVET in live animal hosts.
188 Other Sectionsâ–Œ
189GETTING CLOSER TO THE ACTION
190IVET was originally conceived upon the premise (now considered fact) that most virulence genes are transcriptionally induced at one or more times during infection (27). Although certain host environmental parameters can be mimicked in vitro to induce a subset of virulence genes, the full repertoire is only expressed in vivo. The beauty of IVET is that a live host, with tissue barriers and immune system intact, is used to signal induction of virulence genes. Genetic trickery, the modus operandi of IVET, is then used to identify the in vivo-induced (ivi) genes. As is true of all genetic screens and selections, IVET does have its limitations. The most significant of these is that the relative level and timing of transcription of an ivi gene largely dictates whether the gene will be identified in a particular IVET selection or screen. To date, there are four variations of IVET, and each relies on the generation of transcriptional fusions of genomic sequences to a reporter gene encoding an enzymatic activity. The variation in the four methods lies in the particular reporter gene utilized.
191In the original utilization of IVET (27), advantage was taken of the fact that purine auxotrophs (in this case ΔpurA) of Salmonella enterica serovar Typhimurium (referred to hereafter as Salmonella) are rapidly eliminated from the mouse unless they are complemented. To identify ivi genes, we cloned random genomic fragments directly upstream of a promoterless purA-lacZY synthetic operon present on a suicide vector (Fig. ​(Fig.1).1). This library was transferred by conjugation into ΔpurA Salmonella and was integrated by homologous recombination to form merodiploids. A particular advantage of generating merodiploids as opposed to clean insertions (by double-crossover homologous recombination) is that ivi genes that are essential for survival and growth in the host have a greater likelihood of being identified. The Salmonella library was injected into the peritoneal cavity of mice, and systemic spread and growth in the mouse provided positive selection for strains in which an ivi gene was driving expression of purA-lacZY. Why? Because strains expressing the gene fusion in vivo became prototrophic and thrived, while strains not expressing the fusion remained auxotrophic and died. To avoid the subsequent study of strains containing constitutively active fusions, output bacteria were screened for lacZY expression on lactose-MacConkey indicator medium. This seminal study identified five ivi genes, of which three were shown to play essential roles in virulence (Table ​(Table1).1). Similar IVET selections, incorporating purA or other selectable complementing genes, have subsequently been used to identify ivi genes in a large number of gram-negative and gram-positive pathogens, as well as in a fungal pathogen (Table ​(Table11).
192
193FIG. 1.
194Graphic depiction of four variations of IVET. Auxotrophy complementation-based selections are conducted using fusions to a promoterless purA or other such gene (plasmid 1), antibiotic selections are conducted using fusions to a promoterless antibiotic (more ...)
195
196TABLE 1.
197IVET selections and screens used to identify pathogen genes induced during infectiona
198The primary advantage of the IVET selection, in comparison with other methods for identifying virulence genes en masse, lies in its simplicity: one need only generate a gene fusion library in an auxotrophic strain background and then infect a suitable host. Using positive selection in order to identify ivi genes makes this technique even more appealing. There are two limitations of IVET selections as follows: (i) ivi genes that are transiently expressed or expressed at a low level in vivo are difficult or impossible to detect because they either do not produce PurA long enough or do not produce enough of it to allow survival and growth of the strain and (ii) not all ivi genes are essential for infectivity. The first of these limitations is of unknown magnitude; however, it is reasonable to predict that only a subset of virulence genes that are transcriptionally silent in vitro will be expressed at levels sufficient for survival throughout the course of an infection. The second limitation, applicable to all IVET selections and screens, is often misconstrued, particularly since the advent of signature-tagged mutagenesis, which is a genetic screen used to identify genes that are essential for infectivity (16). While it is true that many ivi genes, when mutated singly, do not reduce infectivity in animal models, it is incorrect to conclude that such genes therefore play no role in virulence. It is becoming appreciated more and more that many virulence factors act in a partially or fully redundant manner (e.g., see references 3, 4, 15, 29, and 38) and thus mutation of one such gene is unlikely to attenuate virulence. Indeed, a fuller understanding of redundant genes involved in survival and growth in vivo can be obtained by observing those occasions when the suicide vector contains, not a promoter region, but instead an internal segment of an ivi gene (or operon) which upon insertion inactivates the gene (or downstream genes in an operon) in which it resides. By identifying such genes, the experimenter has not only identified an in vivo-induced gene but has learned that the ivi gene is not coding for an essential function by itself, and a search for possible redundant factors can then be pursued.
199With IVET, as with other techniques that are used to search for virulence genes, the animal model being used often limits the “search light” to particular stages of the infectious life cycle of a pathogen and therefore a particular ivi gene may serve a role in a stage not being examined. For example, one stage that is rarely investigated is transmission of an infectious agent, which, along with multiplication in the host, is of supreme importance to any professional pathogen. To illustrate this point, cholera toxin (an Ivi protein) is not necessary for Vibrio cholerae to adhere to and multiply within the infant mouse small intestine (a widely used animal model to study this pathogen), and yet this potent toxin is clearly a major pathogenicity factor in human disease and in transmission of this waterborne agent back into aqueous environments (17, 33). Perhaps, where possible, we should begin to look at pathogens in their naturally occurring environment to better understand the entire pathogenic lifestyle (31).
200A second variation of IVET involves the use of antibiotic resistance genes as selectable reporters (28) (Fig. ​(Fig.1).1). By this strategy, treatment of the infected host with the appropriate antibiotic selects for bacterial strains harboring active gene fusions. This technique was used by Heithoff et al. (12) to identify more than 100 ivi genes in Salmonella, though the identities of most of these genes have not yet been reported. This type of IVET approach was actually used prior to the inception of the term “in vivo expression technology” to select Xanthomonas campestris pv. campestris genes induced during infection of a plant host (32). This antibiotic-based method has subsequently been applied to a number of other pathogens (Table ​(Table1).1). The primary advantage of this IVET strategy lies in the fact that complementable auxotrophy in the strain of study is not needed. However, this strategy still requires that the pathogen be transformable and exhibit either (i) homologous recombination for generating chromosomal fusions to IVET reporter genes or (ii) plasmid maintenance for generating promoter fusions to IVET reporters on an episome. A disadvantage of this approach is that the antibiotic must be administered to the host animal and must penetrate to the site of infection. This requirement, in turn, provides some flexibility to the IVET selection in that the antibiotic can be given at lower doses or at specific times of infection in order to increase the breadth of ivi genes identified. The latter was nicely demonstrated by the differential selection for populations of ivi genes that were induced during different stages of Yersinia enterocolitica infection (10, 42) (Table ​(Table1).1). Administration of the antibiotic at an early stage of infection, soon after intragastric inoculation, allowed the investigators to identify Y. enterocolitica ivi genes induced during colonization of the Peyer's patches (42), and in a separate study, the antibiotic was administered at a later stage to intraperitoneally infected mice to identify ivi genes induced during systemic infection in the liver and spleen (10). Of the genes identified, only the siderophore receptor fyuA was found in both screens. These two implementations of IVET exemplify the ability to inventory genes necessary for different sites and stages of colonization and the ability of IVET to tease out tissue-specific virulence factors.
201A third type of IVET selection uses a single gene as a dual reporter, providing for both in vivo selection of active gene fusions and later screening of fusions that are transcriptionally silent during in vitro growth. The first such dual reporter used was hly, encoding the pore-forming hemolysin listeriolysin O (LLO) of Listeria monocytogenes (8). LLO mediates lysis of the phagosomal membrane in macrophages and in other cell types that have engulfed L. monocytogenes (39). This reporter provides an in vivo selection for active fusions that allow for escape from the phagosomal compartment and subsequent multiplication of L. monocytogenes in the cytoplasm as well as a convenient screen for inactive fusions on blood agar plates in vitro (such colonies show no hemolysis). Because expression of the reporter is required at the stage of phagosomal containment, ivi genes that are expressed in the phagosomal environment are identified. Another dual reporter is galU from Klebsiella pneumoniae (21). GalU is required for lipopolysaccharide and capsule synthesis, which, in turn, is required for survival in vivo (6). GalU also allows for a convenient plate screen of failure to ferment galactose on MacConkey agar to identify fusions that are transcriptionally silent in vitro (6). The use of a dual reporter gene simplifies the design of an IVET vector and, in the case of hly, provides unique specificity to the class of ivi genes identified.
202Recombination-based IVET (RIVET) is the fourth IVET strategy for identifying ivi genes and the only one developed so far that functions as a genetic screen. In this case, fusions are made to a promoterless resolvase gene such as tnpR from TnγΎ (35). Prior to this step, a gene cassette that serves as the substrate for resolvase is placed at a neutral site in the bacterial genome. Typically, the substrate is an antibiotic resistance gene flanked by resolvase recognition sequences. An ivi gene fused to tnpR results in resolvase production, whose action results in the permanent excision of the antibiotic marker (a reaction termed resolution). This event marks the bacterium by endowing it with an inheritable antibiotic-sensitive phenotype. Resolved strains are then screened for (by replica plating of colonies) after recovery of the bacteria from infected tissues.
203The RIVET method has distinct advantages and disadvantages relative to the other IVET approaches. Because only a small pulse of resolvase expression is needed to mediate resolution, the method is exquisitely sensitive to low or transient expression of the ivi gene during infection and is therefore capable of identifying these potentially interesting classes of genes. This sensitivity is a double-edged sword, though, in that ivi genes with low to moderate basal levels of expression in vitro cannot be identified because such genes result in immediate resolution during strain construction. In the absence of taking steps to reduce the sensitivity of the system (23), the number of ivi genes that can be identified in any particular pathogen is likely to be restricted. A second advantage of RIVET is that no selective pressure is placed upon the bacteria during infection, which is not true for IVET selections, and thus the infection is guaranteed to proceed on a natural course. Finally, use of RIVET to study induction of virulence genes in vivo is limited in two other ways: first, only the initial induction of an ivi gene can be assayed, since resolution is irreversible, and thus expression at later times or within downstream host tissues cannot be detected; and second, no quantitative information concerning gene expression levels is provided. Because of these and other unique features (described below), the acronym RIVET is often used to distinguish it from the more commonly used IVET selection methods. RIVET has been used to identify ivi genes in V. cholerae and with greater success in Staphylococcus aureus (5, 25).
204 Other Sectionsâ–Œ
205OFFSHOOT TECHNOLOGIES TO STUDY THE REGULATION OF IVI GENES
206Because ivi genes, by definition, are transcriptionally silent during in vitro growth and induced during infection, it is difficult to study their regulation using standard methods. Several approaches have been developed, however, which circumvent or even take advantage of this limitation in order to study the regulation of ivi genes.
207Examining the spatial and temporal patterns of induction of ivi genes.
208An interesting alternative use of RIVET is the monitoring of induction of transcription of ivi genes as a function of time and location in the host. To do this, a pathogen (containing an ivi-tnpR fusion) is isolated at different times during infection or from specific tissues, and then the recovered bacteria are assayed to determine the percentage that have resolved. This technique was utilized by Lee et al. (23) to study the induction patterns of several virulence genes within wild-type and mutant strains of V. cholerae during infection. Induction of two major virulence genes that were thought to be coinduced based on in vitro studies were found to be induced in a sequential manner in the infant mouse small intestine. Moreover, it was shown that induction of the first of these was needed in order for the second to be expressed. These results provide tantalizing clues to what appears to be a highly coordinated and host-pathogen interaction-dependent program of virulence gene expression by V. cholerae.
209In addition to these findings, Lee et al. (22) used RIVET to assess the roles of upstream regulatory factors for induction of these virulence genes. To accomplish this, individual genes encoding known regulators were mutated in an ivi-tnpR fusion strain background. Next, the mutant strains were inoculated into animals and the temporal patterns of induction were determined. If the pattern of induction is unchanged from the parent strain, then the regulator tested plays no essential role in regulating the ivi gene in vivo. On the other hand, any change in the temporal pattern of resolution, such as loss of induction altogether, reflects a role for the regulator in regulating the virulence gene during infection. Once again, it was shown that the insights gained from in vitro studies were not borne out in vivo; Lee et al. found that there were differences in the requirements for particular virulence regulators in vivo versus in vitro. For example, it was found that the transcriptional regulator TcpP and its accessory protein TcpH, although required for cholera toxin gene expression in vitro, are not required for expression during infection in the infant mouse model of cholera.
210As mentioned earlier, because resolution is irreversible, spatio-temporal experiments using RIVET are limited to assaying the initial induction patterns. For these reasons, quantitative reverse transcriptase PCR is currently the method of choice for quantitative spatio-temporal studies of in vivo gene expression, at least for experimental systems in which sufficient numbers of bacterial cells for mRNA isolation are present in host tissues (9, 37). An alternative method for spatio-temporal studies is the use of a light-emitting reporter, such as Gfp, to detect gene expression (1). Use of Gfp is limited in some cases by background fluorescing particles which interfere with readings from bacteria recovered from infected host tissues (1).
211Identifying transcriptional regulators of ivi genes.
212Because ivi genes are transcriptionally silent during in vitro growth, it is possible, in some cases, to screen or select for mutations in loci encoding regulators of the ivi genes. For example, consider the generic case of a strain containing a fusion of an ivi gene to purA-lacZY: selection for strains with mutations in a repressor of ivi can be carried out simply by demanding growth on media lacking purines. Alternatively, one could screen for mutant strains that form blue colonies on agar plates containing X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). Heithoff et al. (14) used the latter method to identify a semiglobal repressor of Salmonella ivi genes. The repressor turned out to be Dam (DNA adenine methylase). A null mutation in dam resulted in derepression of ~20% of the previously identified ivi genes in Salmonella and also attenuated virulence substantially. Because a dam strain both expresses ivi genes inappropriately and is avirulent, such strains have potential as live or killed vaccine strains (13, 14).
213Recently, Lee et al. (22) developed a genetic selection to identify positive regulators of ivi genes. This method takes advantage of a property of RIVET, excision of the antibiotic resistance marker in vivo, to select for mutant strains that instead retain the antibiotic marker after animal passage. Such strains contain mutations in positive regulators of the particular ivi gene. A unique aspect of this selection method is that the regulators identified must function during infection. This is likely to be a useful genetic tool for probing regulatory networks that are active during infections but which may not be active during growth in vitro. In the above study, a number of positive regulators of the cholera toxin genes (ctxAB) were identified, and these included genes involved in chemotaxis and other signal transduction pathways. These signaling pathways were not required for ctxAB induction in vitro during growth under specialized conditions that induce ctxAB (22).
214 Other Sectionsâ–Œ
215CONCLUDING REMARKS
216The coordinated regulation of bacterial virulence factors is critical to successful infection. This requires that a set of genes be up-regulated during infection while, concurrently, another set is down-regulated. If a pathogen has in effect been keeping a particular set of genes in reserve for the appropriate moment in host tissues, then it is likely that these genes play some role in virulence: it is up to us to figure out what these specific roles are. Understanding the timing, tissue specificity, and regulation of an ivi gene can set the stage for additional studies directed at deciphering the precise role of the encoded protein.
217Here we have given a description of the various forms of IVET and the major advantages and disadvantages of each. While each IVET approach has been shown to be limited in one way or another, the importance of this technology is indisputable. Many reports listed in Table ​Table11 have shown the requirement of one or more ivi genes for infection of a host, and many more bona fide virulence genes have been identified through IVET screens and selections done using cultured cells or using other in vitro models of infections that were not discussed here. Indeed, 9 years after the advent of IVET, the technology has become more than a bellwether (2)—it has become a utility for virulence gene discovery in many pathogens and a stimulus for the creation of new tools to investigate pathogenicity.
218NOTES
219Editor: D. A. Portnoy
220 Other Sectionsâ–Œ
221REFERENCES
2221. Allaway, D., N. A. Schofield, M. E. Leonard, L. Gilardoni, T. M. Finan, and P. S. Poole. 2001. Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes. Environ. Microbiol. 3:397-406. [PubMed]
2232. Barinaga, M. 1993. New technique offers a window on bacteria's secret weapons. Science 259:595. [PubMed]
2243. Berry, A. M., and J. C. Paton. 2000. Additive attenuation of virulence of Streptococcus pneumoniae by mutation of the genes encoding pneumolysin and other putative pneumococcal virulence proteins. Infect. Immun. 68:133-140. [PMC free article] [PubMed]
2254. Brown, J. S., S. M. Gilliland, and D. W. Holden. 2001. A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence. Mol. Microbiol. 40:572-585. [PubMed]
2265. Camilli, A., and J. J. Mekalanos. 1995. Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection. Mol. Microbiol. 18:671-683. [PubMed]
2276. Chang, H. Y., J. H. Lee, W. L. Deng, T. F. Fu, and H. L. Peng. 1996. Virulence and outer membrane properties of a galU mutant of Klebsiella pneumoniae CG43. Microb. Pathog. 20:255-261. [PubMed]
2287. Fuller, T. E., R. J. Shea, B. J. Thacker, and M. H. Mulks. 1999. Identification of in vivo induced genes in Actinobacillus pleuropneumoniae. Microb. Pathog. 27:311-327. [PubMed]
2298. Gahan, C. G., and C. Hill. 2000. The use of listeriolysin to identify in vivo induced genes in the gram-positive intracellular pathogen Listeria monocytogenes. Mol. Microbiol. 36:498-507. [PubMed]
2309. Goerke, C., U. Fluckiger, A. Steinhuber, W. Zimmerli, and C. Wolz. 2001. Impact of the regulatory loci agr, sarA and sae of Staphylococcus aureus on the induction of alpha-toxin during device-related infection resolved by direct quantitative transcript analysis. Mol. Microbiol. 40:1439-1447. [PubMed]
23110. Gort, A. S., and V. L. Miller. 2000. Identification and characterization of Yersinia enterocolitica genes induced during systemic infection. Infect. Immun. 68:6633-6642. [PMC free article] [PubMed]
23211. Handfield, M., D. E. Lehoux, F. Sanschagrin, M. J. Mahan, D. E. Woods, and R. C. Levesque. 2000. In vivo-induced genes in Pseudomonas aeruginosa. Infect. Immun. 68:2359-2362. [PMC free article] [PubMed]
23312. Heithoff, D. M., C. P. Conner, P. C. Hanna, S. M. Julio, U. Hentschel, and M. J. Mahan. 1997. Bacterial infection as assessed by in vivo gene expression. Proc. Natl. Acad. Sci. USA 94:934-939. [PMC free article] [PubMed]
23413. Heithoff, D. M., E. Y. Enioutina, R. A. Daynes, R. L. Sinsheimer, D. A. Low, and M. J. Mahan. 2001. Salmonella DNA adenine methylase mutants confer cross-protective immunity. Infect. Immun. 69:6725-6730. [PMC free article] [PubMed]
23514. Heithoff, D. M., R. L. Sinsheimer, D. A. Low, and M. J. Mahan. 1999. An essential role for DNA adenine methylation in bacterial virulence. Science 284:967-970. [PubMed]
23615. Henderson, D. P., and S. M. Payne. 1994. Vibrio cholerae iron transport systems: roles of heme and siderophore iron transport in virulence and identification of a gene associated with multiple iron transport systems. Infect. Immun. 62:5120-5125. [PMC free article] [PubMed]
23716. Hensel, M., J. E. Shea, C. Gleeson, M. D. Jones, E. Dalton, and D. W. Holden. 1995. Simultaneous identification of bacterial virulence genes by negative selection. Science 269:400-403. [PubMed]
23817. Herrington, D. A., R. H. Hall, G. Losonsky, J. J. Mekalanos, R. K. Taylor, and M. M. Levine. 1988. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. Exp. Med. 168:1487-1492. [PMC free article] [PubMed]
23918. Hunt, M. L., D. J. Boucher, J. D. Boyce, and B. Adler. 2001. In vivo-expressed genes of Pasteurella multocida. Infect. Immun. 69:3004-3012. [PMC free article] [PubMed]
24019. Khan, M. A., and R. E. Isaacson. 2002. Identification of Escherichia coli genes that are specifically expressed in a murine model of septicemic infection. Infect. Immun. 70:3404-3412. [PMC free article] [PubMed]
24120. Kilic, A. O., M. C. Herzberg, M. W. Meyer, X. Zhao, and L. Tao. 1999. Streptococcal reporter gene-fusion vector for identification of in vivo expressed genes. Plasmid 42:67-72. [PubMed]
24221. Lai, Y. C., H. L. Peng, and H. Y. Chang. 2001. Identification of genes induced in vivo during Klebsiella pneumoniae CG43 infection. Infect. Immun. 69:7140-7145. [PMC free article] [PubMed]
24322. Lee, S. H., S. M. Butler, and A. Camilli. 2001. Selection for in vivo regulators of bacterial virulence. Proc. Natl. Acad. Sci. USA 98:6889-6894. [PMC free article] [PubMed]
24423. Lee, S. H., D. L. Hava, M. K. Waldor, and A. Camilli. 1999. Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell 99:625-634. [PubMed]
24524. Lee, S. W., and D. A. Cooksey. 2000. Genes expressed in Pseudomonas putida during colonization of a plant-pathogenic fungus. Appl. Environ. Microbiol. 66:2764-2772. [PMC free article] [PubMed]
24625. Lowe, A. M., D. T. Beattie, and R. L. Deresiewicz. 1998. Identification of novel staphylococcal virulence genes by in vivo expression technology. Mol. Microbiol. 27:967-976. [PubMed]
24726. Mahan, M. J., D. M. Heithoff, R. L. Sinsheimer, and D. A. Low. 2000. Assessment of bacterial pathogenesis by analysis of gene expression in the host. Annu. Rev. Genet. 34:139-164. [PubMed]
24827. Mahan, M. J., J. M. Slauch, and J. J. Mekalanos. 1993. Selection of bacterial virulence genes that are specifically induced in host tissues. Science 259:686-688. [PubMed]
24928. Mahan, M. J., J. W. Tobias, J. M. Slauch, P. C. Hanna, R. J. Collier, and J. J. Mekalanos. 1995. Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host. Proc. Natl. Acad. Sci. USA 92:669-673. [PMC free article] [PubMed]
25029. Mellado, E., A. Aufauvre-Brown, N. A. Gow, and D. W. Holden. 1996. The Aspergillus fumigatus chsC and chsG genes encode class III chitin synthases with different functions. Mol. Microbiol. 20:667-679. [PubMed]
25130. Merrell, D. S., and A. Camilli. 1999. The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance. Mol. Microbiol. 34:836-849. [PubMed]
25231. Oke, V., and S. R. Long. 1999. Bacterial genes induced within the nodule during the Rhizobium-legume symbiosis. Mol. Microbiol. 32:837-849. [PubMed]
25332. Osbourn, A. E., C. E. Barber, and M. J. Daniels. 1987. Identification of plant-induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoter-probe plasmid. EMBO J. 6:23-28. [PMC free article] [PubMed]
25433. Pierce, N. F., J. B. Kaper, J. J. Mekalanos, and W. J. Cray. 1985. Role of cholera toxin in enteric colonization by Vibrio cholerae O1 in rabbits. Infect. Immun. 50:813-816. [PMC free article] [PubMed]
25534. Rainey, P. B. 1999. Adaptation of Pseudomonas fluorescens to the plant rhizosphere. Environ. Microbiol. 1:243-257. [PubMed]
25635. Reed, R. R. 1981. Transposon-mediated site-specific recombination: a defined in vitro system. Cell 25:713-719. [PubMed]
25736. Retallack, D. M., G. S. Deepe, Jr., and J. P. Woods. 2000. Applying in vivo expression technology (IVET) to the fungal pathogen Histoplasma capsulatum. Microb. Pathog. 28:169-182. [PubMed]
25837. Rokbi, B., D. Seguin, B. Guy, V. Mazarin, E. Vidor, F. Mion, M. Cadoz, and M. J. Quentin-Millet. 2001. Assessment of Helicobacter pylori gene expression within mouse and human gastric mucosae by real-time reverse transcriptase PCR. Infect. Immun. 69:4759-4766. [PMC free article] [PubMed]
25938. Smith, G. A., H. Marquis, S. Jones, N. C. Johnston, D. A. Portnoy, and H. Goldfine. 1995. The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect. Immun. 63:4231-4237. [PMC free article] [PubMed]
26039. Tilney, L. G., and D. A. Portnoy. 1989. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J. Cell Biol. 109:1597-1608. [PMC free article] [PubMed]
26140. Wang, J., A. Mushegian, S. Lory, and S. Jin. 1996. Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection. Proc. Natl. Acad. Sci. USA 93:10434-10439. [PMC free article] [PubMed]
26241. Wu, Y., S. W. Lee, J. D. Hillman, and A. Progulske-Fox. 2002. Identification and testing of Porphyromonas gingivalis virulence genes with a pPGIVET system. Infect. Immun. 70:928-937. [PMC free article] [PubMed]
26342. Young, G. M., and V. L. Miller. 1997. Identification of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis. Mol. Microbiol. 25:319-328. [PubMed]
264Articles from Infection and Immunity are provided here courtesy of
265American Society for Microbiology (ASM)
266PubMed articles by these authors
Note: See TracBrowser for help on using the repository browser.