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Department of Biological Sciences, 500 Glenridge Avenue, Brock University, St Catharines, Ontario, Canada L2S 3A1
Correspondence
Michael J. Bidochka
bidochka{at}brocku.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the ITS region sequence reported in this paper is AY521473.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Opportunistic pathogens persist on a wide range of organic substrates, but exhibit low virulence towards a broad array of living hosts, particularly if the host is injured or immunocompromised. Facultative pathogens efficiently colonize and produce disease in a narrow range of healthy hosts, although they are capable of surviving outside the host. Micro-organisms that require a living host for survival and replication constitute the obligate pathogens and generally have narrow host ranges. Specialized pathogens, such as the obligate and facultative pathogens, probably evolved from less specialized opportunistic pathogens, representing a theoretical framework for the emergence of infectious diseases and host specialization (Scheffer, 1991).
To study the evolution of host specificity and the development of infectious diseases, we utilized an insect–fungal model of infection involving the fungus Aspergillus flavus as the pathogen and wax moth larvae [Galleria mellonella (Lepidoptera)], as the insect host. A. flavus is an opportunistic fungal pathogen capable of thriving saprobically as well as infecting a wide variety of living hosts, including plants, insects and mammals, albeit with low virulence. A. flavus is an ideal model pathogen for the study of adaptation and host restriction, since it possesses a broad spectrum of protein- and polysaccharide-hydrolysing enzymes for exploiting available living and non-living organic resources (St Leger et al., 2000). With respect to insect pathogenicity, A. flavus is an opportunistic pathogen of silkworms, grasshoppers, houseflies and mealy bugs among others (Gupta & Gopal, 2002). A. flavus is the causative agent of aspergillosis documented in the silkworm Bombyx mori (Kumar et al., 2004). A. flavus infection of insects involves conidium germination on the insect surface, followed by a breech of the cuticle and access to the nutrient-rich haemocoel. Inside the infected insect, the hyphae multiply extensively, causing death and subsequent mycelial emergence from and mummification of the cadaver. The emergent mycelia conidiate on the surface of the cadaver, providing the transmissible propagules dispersed to other hosts for the continuation of the pathogenic cycle (Kumar et al., 2004). In the event that a suitable host is not available, the conidia germinate and grow saprobically.
In this study, we employed a serial propagation scheme involving the continuous passage of A. flavus through larvae of G. mellonella, mimicking the repeated infection and transmission of a disease-causing, opportunistic pathogen through a single species of a susceptible host. The result was a strain of A. flavus that exhibited a severe diminution in conidial production on standard artificial media, while retaining pathogenicity towards the insect host, including conidial production on the dead host during the final stage of pathogenesis. In addition, it demonstrated a reduction in host range as a plant pathogen compared to the original strain as well as an auxotrophic requirement for cysteine/methionine due to an inability to reduce sulfate to sulfite. In essence, this strain represents the progression by an opportunistic fungus towards obligate insect pathogenesis in that it can only efficiently complete its life cycle (i.e. mycelial growth followed by conidial production) as a consequence of infection of an insect host. In the opportunistic pathogen A. flavus, we show an association between auxotrophy and severe host restriction constituting a shift towards obligate pathogenesis that emphasizes the role of nutrition in evolving host–pathogen relationships.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), Aspergillus fumigatus FR 2923-97 (Rosehart et al., 2002) and Emericella nidulans 7677 (University of Alberta Microfungus Collection and Herbarium). All strains were maintained on Petri dishes containing 10 ml volumes of Potato Dextrose Agar (PDA) incubated at 30 °C for 7 days. The following insect and plant species were used in this study: mealworms [Tenebrio molitor (Coleoptera)], blowfly larvae [Sarcophaga bullata (Diptera)] and crickets [Acheta domestica (Orthoptera)] from Ward's Scientific, wax moth larvae [Galleria mellonella (Lepidoptera)] from Peterborough Live Bait (Peterborough, ON, Canada) and milkweed bugs [Oncopeltus fasciatus (Hemiptera)] from Boreal, tomato (Lycopersicon sp.), corn (Zea sp.), green bean (Phaseolus sp.), pea (Pisum sp.) and cucumber (Cucumis sp.) from Zehrs (St Catharines, ON, Canada) and alfalfa [Medicago sp. (Boreal)].
Serial propagation of A. flavus through G. mellonella larvae.
To propagate A. flavus 6982 through G. mellonella, 25 larvae were topically inoculated on the dorsal surface with 5 µl 1x105 conidia ml–1 suspension, placed separately into clean plastic vials and incubated at 30 °C. At death, insects were washed for 1 min in 1 % (v/v) NaOCl followed by two washings of 2 min each in sterile dH2O. This procedure effectively removed surface conidia from insect cadavers, allowing only cuticle-breeching, pathogenic conidia to survive (results not shown). Emergent conidia from the first insect to die were harvested in 3 ml 0·01 % (v/v) Triton X-100, 0·02 % (w/v) chloramphenicol solution. After quantifying the suspension microscopically with a haemocytometer, 25 larvae were topically inoculated to obtain the next generation of fungal passage through the insect host. Five replicate lineages of this scheme were performed, each consisting of six generations of fungal passage through the host. Fungal samples from each generation of each lineage were stored at –80 °C in 25 % glycerol and subcultured onto PDA. The initial conidial suspension used in this scheme was mutagenized by exposure to UV-A light (400 µW cm–2) for 3 min, resulting in 80 % mortality of conidia.
From this artificial selection scheme, a single strain of A. flavus was derived that exhibited a drastic reduction in conidial production on PDA, while producing conidia on the infected insect cadaver as the culmination of the pathogenic cycle. This strain was designated Af6982conins because it demonstrated efficient conidial production on the infected insect cadaver, but not on PDA. Af6982conins demonstrated a similar colony phenotype when subcultured onto nutrient agar (NA; Difco) and yeast peptone dextrose agar (YPD; 0·2 % yeast extract, 1 % peptone, 2 % glucose, 1·5 % agar), but demonstrated no growth on Czapek solution agar (Cz; Difco).
DNA extraction, ITS amplification and amplified fragment length polymorphism (AFLP) analysis.
To confirm that the derived strain was not a contaminant, the DNA was extracted using the DNeasy tissue extraction kit (Qiagen), and the ITS region was amplified and sequenced using standard protocols and ITS primers (White et al., 1990).
To further confirm the relationship of the derived strain to the parental strain as well as to other strains and species of Aspergillus, we used AFLP analysis. Genomic DNA of A. flavus, A. fumigatus and E. nidulans isolates was prepared using the DNeasy tissue extraction kit (Qiagen). Five hundred nanograms of fungal DNA was digested with 1 U EcoRI and 1 U MseI in a total volume of 10 µl, following the manufacturer's instructions (New England Biolabs). Following digestion, reactions were incubated at 70 °C for 15 min. Ligation was performed in a total volume of 20 µl by adding 2 pmol E adapter, 20 pmol M adapter (Table 1) and 0·4 U T4 DNA ligase (New England Biolabs) to the total volume of the digestion reaction. The ligation reactions were incubated at 20 °C for 2 h. The digestion–ligation products were diluted 1 : 10 with TE (10 mM Tris/HCl, 1 mM EDTA, pH 8·0).
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) and 15 µl Taq PCR Master Mix (Qiagen). The PCR conditions were: 20 cycles of 94 °C for 30 s, 56 °C for 60 s and 72 °C for 60 s. The preamplification products were diluted 1 : 10 with TE.
Eleven selective amplification reactions were performed using the following primer combinations: EO/M+CAG, E+A/M+A, E+A/M+CAG, E+GC/MO, E+GC/M+T, E+GC/M+A, E+GC/M+CAG, E+AG/MO, E+AG/M+T, E+AG/M+A and E+AG/M+CAG (Table 1). The selective amplification reactions contained 5 µl diluted preamplification product, 0·5 µM each primer and 11 µl Taq PCR Master Mix (Qiagen) in a total volume of 22 µl. The PCR conditions were: 6 cycles of 94 °C for 30 s, 65 °C for 30 s, 72 °C for 60 s; 6 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s; 23 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 60 s; followed by a final extension at 72 °C for 5 min.
The selective amplification products were analysed on 10 % (19 : 1) denaturing polyacrylamide gels (Sequi-Gen GT Nucleic Acid Electrophoresis Cell; Bio-Rad), which were subsequently silver-stained (Blum et al., 1987).
A similarity matrix using simple matching coefficients was constructed using NYSTS 2.1 after scoring the bands ‘1’ for presence and ‘0’ for absence. Cluster analysis was performed using the unweighted pair-group method using arithmetic means (UPGMA).
Insect and plant bioassay.
Virulence towards G. mellonella larvae was determined by topical application of 25 larvae with 5 µl 1x108 conidia ml–1 and recording the percentage mortality at day 10. Fisher's exact tests or 
2 tests were used to compare the percentage mortality of A. flavus strains to each other and to the control bioassay, which was topical application of 5 µl of 0·01 % (v/v) Triton X-100. Pathogenicity tests against all insect species were performed by injecting conidial suspensions (3 µl 1x106 conidia ml–1) into the haemocoel using a clean syringe (gauge=22). All insects were placed individually in clean plastic vials, incubated at 30 °C and monitored daily for mortality and subsequent conidiation on the cadaver. Inoculation via injection was utilized to ensure infection.
For plant pathogenicity tests, various plant species were washed in 1 % NaOCl for 1 min followed by three 1 min washings in sterile dH2O. Plants were inoculated by piercing the tissue with a sterilized needle, followed by inoculation of the wounded area with 2 µl of a 1x106 conidia ml–1 suspension. Some suspensions were supplemented with a 10 mM final concentration of either cysteine or methionine. The plants were incubated on 1 % water agar at 30 °C for 7 days before assessing mycelial growth and conidiation.
For all plant and insect bioassays utilizing auxotrophic A. flavus strains, emergent conidia were tested to determine if they retained the auxotrophic phenotype. The conidia were subcultured onto Cz and Cz supplemented with 10 mM of the required amino acid. Lack of growth in the absence of supplementation confirmed the auxotrophic phenotype. Any plants or insects with conidia that failed to display an auxotrophic phenotype were omitted from the data since this indicated reversion.
Conidial counts.
To quantify conidial production of a subculture grown on PDA with or without 10 mM cysteine or methionine, a 2·54 cm diameter agar plug was removed from halfway between the centre of the Petri dish and the perimeter. The plug was homogenized in 5 ml 0·01 % Triton X-100 for 1 min in a 50 ml polystyrene tube with a motorized homogenizer (Greiner Scientific). Conidia were counted using a haemocytometer.
Quantification of conidia produced on G. mellonella and Zea sp. was performed by vortexing a conidiated insect cadaver or corn kernel in 3 ml 0·01 % Triton X-100, 0·02 % chloramphenicol. The resulting suspension was counted using a haemocytometer.
Microscopy.
Samples of fungal material from G. mellonella larvae and PDA plates were stained with lactophenol cotton blue and examined under a bright-field microscope.
Detection of auxotroph phenotype and reversion.
Wells of 96-well plates were filled with 200 µl Cz and individually supplemented with 1 µmol of one of the following amino acids: L-
alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. Wells were inoculated with 5 µl of a 1x106 conidia ml–1 suspension.
Screening for auxotrophic reversion was performed by plating 50 µl 1x106 conidia ml–1 of Af6982conins suspension onto each of 40 Cz Petri dishes and observing fungal growth.
Sulfur assimilation enzyme assays.
Flasks containing 100 ml YPD broth (0·2 % yeast extract, 1 % peptone, 2 % glucose) were inoculated with 50 µl 1x106 conidia ml–1 and incubated at 27 °C shaking at 250 r.p.m. for 3 days. Fungi were then filtered from the broth, washed with Czapek–Dox broth (Difco) and transferred to flasks containing 100 ml Czapek–Dox broth incubated for 2 days at 27 °C and 250 r.p.m. After filtering from the medium, the fungi were crushed in liquid nitrogen and the resulting material was used to assay the activity of ATP sulfurylase as described by Segel et al. (1987), the conversion of sulfate to sulfite (Breton & Surdin-Kerjan, 1977) and sulfite reductase (De Vito & Dreyfuss, 1964). Sulfate permease function was assessed based on the inhibition of growth by the toxic sulfate analogue chromate on Cz agar containing 10 mM methionine and 10 mM potassium chromate as described by Arst (1968).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A similar reduction was observed on a variety of other agar media, namely YPD and NA (data not shown), and unlike the original strain, no growth occurred on Cz. Conidial production exhibited by this strain when grown on PDA was 75-fold less than that of the original strain previous to passage through the insect host (Fig. 2). Conversely, the amount of conidia produced by this strain on the insect cadaver at the completion of the pathogenic cycle was much higher, exhibiting only a 10-fold reduction compared to the parental strain by either topical application or injection (Fig. 2). These characteristics were displayed by this strain at each of generations 2–6 of passage through the insect (data not shown).
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Virulence and host range of Af6982conins
The virulence of Af6982conins towards G. mellonella larvae (64 % mortality at day 10) remained comparable to that of Af6982 (48 % mortality at day 10, P=0·39; Fisher's exact test) and statistically significant compared to a control inoculation (P=0·001 and 0·03, respectively; Fisher's exact test) during topical application bioassays (5 µl 1x108 conidia ml–1).
Insects of various orders as well as a range of plant species were inoculated with Af6982 and Af6982conins to determine host ranges established by the essential pathogenic property of conidiation on the infected host, indicative of successful colonization. The results indicated that Af6982conins and its parental strain Af6982 both possessed the ability to conidiate on a variety of infected insects (injected with 3 µl 1x106 conidia ml–1). However, unlike strain Af6982, Af6982conins failed to produce lesions or exhibit any growth on the infected plant species except for the corn kernels on which it produced some mycelia and exceptionally few conidia (Table 2, Fig. 2).
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Although supplementation of Af6982conins with either cysteine or methionine failed to restore pathogenicity towards most plant hosts, on PDA and corn conidial production relative to Af6982 was brought to a level similar to that generated on the insect host (Fig. 2).
Sulfur assimilation enzyme activity of Af6982conins
The ability of Af6982conins to import sulfate and convert it to cysteine or methionine was assessed by analysing the functionality of the enzymes of the sulfur assimilation pathway (Fig. 3a). Both Af6982 and Af6982conins failed to grow in the presence of chromate, a toxic sulfate analogue, indicating a functional sulfate permease (Fig. 3b). While both Af6982 and Af6982conins demonstrated comparable levels of ATP sulfurylase (Fig. 3c, t=0·74, P=0·26) and sulfite reductase (Fig. 3e, t=0·13, P=0·46) activity, Af6982conins failed to show any sulfate to sulfite reduction activity (Fig. 3d).
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). In addition, the revertant retained a level of virulence towards G. mellonella larvae that was statistically different from a control inoculation (32 % mortality by day 10, P=0·02; Fisher's exact test) and statistically equivalent to that of the original strain Af6982 and Af6982conins (P=0·08; 2 test).
AFLP analysis
Cluster analysis based on the presence or absence of AFLP bands (Fig. 4) indicated that isolates of the original strain Af6982, the strain demonstrating insect-dependent conidiation Af6982conins and the revertant possessed 99 % similarity by simple matching coefficients. The similarity of these isolates to other A. flavus strains ranged from 57 to 94 %, while their similarity to A. fumigatus and E. nidulans was 49 and 47 %, respectively. Although differences were observed between the isolates of Af6982, Af6982conins and the revertant, there were no diagnostic polymorphisms that distinguished the isolates of the original strain from those of the strain demonstrating insect-dependent conidiation.
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; and confirmed in this study) all exhibited minimal to no growth on Cz. All five auxotrophs exhibited infection and substantial conidiation on G. mellonella cadavers when injected or applied topically to the cuticle. Three of the five auxotrophic strains demonstrated a statistically significant percentage mortality (Table 3) compared to a control inoculation during topical application bioassays (5 µl 1x108 conidia ml–1). However, unlike Af6982conins, most of these auxotrophs retained the ability to infect alfalfa (Table 3). Further investigation of A. flavus 46114, which demonstrated an auxotrophic requirement for methionine similar to that of Af6982conins, showed an ability to infect a variety of both plants and insects (Table 2) and exhibited no diminution in conidial production on PDA, corn or G. mellonella larvae relative to Af6982 (Fig. 2). As with Af6982, Af46114 failed to grow in the presence of chromate (Fig. 3b), demonstrating a functional sulfate permease, and exhibited comparable levels of ATP sulfurylase (Fig. 3c, t=0·01, P=0·50), sulfate to sulfite reduction (Fig. 3d, t=0·66, P=0·28) and sulfite reductase activity (Fig. 3e, t=0·56, P=0·30).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementation of Cz with various amino acids revealed that Af6982conins was a cysteine/methionine auxotroph. This nutrient deficiency provides a plausible explanation for its dependency on the insect host for reproduction. Such nutrient requirements suggest that infection and propagation through the larval host allowed this auxotroph to grow and conidiate as it gained the required amino acids from the host, most likely from the protein- and amino-acid-rich insect cuticle or haemolymph (Hackman & Goldberg, 1976; Paterson et al., 1994; Hanzal & Jegorov, 1991). However, neither standard artificial media nor plant species were able to provide Af6982conins with the nutrients required for adequate conidiation. Accordingly, Af6982conins exhibited properties similar to those of obligate pathogens because it was severely impeded in its ability to complete its lifecycle via the production of conidia unless it was allowed to infect, colonize, gain nutrients from and finally re-emerge from the insect to conidiate on the cadaver surface.
Many pathogens designated obligate can proliferate on artificial media when they are provided with the appropriate nutrients. For example, the obligate mammalian pathogens Mycobacterium tuberculosis and Yersinia pestis can be cultured on Lowenstein–Jensen medium and TMH medium, respectively (Tanoue et al., 2002; Qiu et al., 2005). Cronartium quercuum, the obligate fungal pathogen of oak tree leaves, can be cultivated on PGY medium (Warren & Covert, 2004). In principle, the cysteine/methionine auxotroph described in this study behaves no differently. Besides demonstrating a restricted host range, Af6982conins requires enhanced artificial media for culture growth. These characteristics show similarity with other obligate pathogens and demonstrate an evolution towards obligate pathogenesis.
Unlike Af6982conins, a variety of other A. flavus auxotrophs demonstrating nutrient deficiencies did not show a diminished host range. Four of the five auxotrophs demonstrated mycelial growth and conidiation on the insect host G. mellonella larvae as well as the ability to infect and conidiate on alfalfa leaves. Specifically, A. flavus strain 46114, which displayed an auxotrophic requirement for methionine similar to that of Af6982conins, demonstrated that its nutrient deficiency neither reduced the host range of Af46114 nor hindered its ability to produce conidia on a variety of substrates. Enzyme assays revealed that unlike Af46114, Af6982conins is deficient in the ability to reduce sulfate to sulfite, indicating that the metabolic cause of auxotrophy in these two strains is different. Such metabolic differences are a probable explanation for the observed differences in host range.
Of the 20 essential amino acids, cysteine and methionine are the only two that contain sulfur. Not surprisingly, their biosynthetic and metabolic pathways are highly interconnected (Ono et al., 1999), leading to the hypothesis that the cysteine/methionine auxotrophy of Af6982conins was a result of a single-gene mutation in a common biosynthetic pathway. We obtained a spontaneous revertant of Af6982conins that regained the characteristics of the original opportunistic pathogen Af6982, namely conidiation on artificial media and plant species while preserving insect pathogenicity and a similar level of virulence towards G. mellonella larvae. The recovery of a spontaneous revertant implied that the obligate pathogen characteristics of Af6982conins were due to a single gene mutation, since the probability of obtaining a spontaneous revertant after multiple mutations was unlikely.
Because fungi are able to interconvert cysteine and methionine (Ono et al., 1999), a single gene mutation resulting in auxotrophy that is recovered by either cysteine or methionine implies a mutation in the sulfur assimilation pathway essential for the synthesis of either amino acid (Marzluf, 1997). Indeed, we showed that Af6982conins was unable to reduce sulfate to sulfite. We hypothesize that the auxotrophy of Af6982conins is due to an incapacitating mutation in one of the enzymes essential for the reduction of sulfate to sulfite, presumably either adenosine 5'-phosphosulfate (APS) kinase or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase, since ATP sulfurylase activity is detected in Af6982conins. Further experiments are under way to genetically characterize this mutation.
The high level of similarity between the original opportunistic strain Af6982, the strain demonstrating insect-dependent conidiation Af6982conins and the revertant indicated by the AFLP cluster analysis suggested that they are derived from the same strain (Savelkoul et al., 1999; Rademaker et al., 2000; Janssen et al., 1997). In all likelihood, a mutant produced during the UV mutagenesis of the initial conidial suspension was selected during passage through the insect that resulted in the derivation of a strain, which displayed insect-dependent conidiation. Subsequent reversion of the mutation allowed for recovery of the revertant. Although a small number of differences was observed among the isolates of the Af6982, Af6982conins and the revertant, this degree of dissimilarity has been observed between multiple isolates of a single strain of other asexually reproducing microbes (Savelkoul et al., 1999; Rademaker et al., 2000; Janssen et al., 1997). These differences may be due to noise or hysteresis in this PCR-based technique. Alternatively, they may be legitimately due to mutations as asexually reproducing microbes are not genetically static during subculturing.
Our results indicated an association between a single gene mutation resulting in auxotrophy and host restriction, in turn resulting in evolution towards obligate insect pathogenesis by an opportunistic pathogen. As shown in Fig. 5, such a mutation may represent a decisive step in the emergence of a highly virulent obligate pathogen as it may provide selection pressure for further adaptation towards increased virulence. This hypothesis recognizes that an essential prerequisite to pathogenicity is the capacity of the host to provide suitable nutrients for the pathogen. The host must satisfy the nutritional requirements of the pathogen for the latter to proliferate and cause disease (Garber, 1956). Recent reviews highlight the importance of host–microbe interactions in the production of tissue damage and disease, recognizing that host nutritional status and the ability of a microbe to survive and replicate in a given host are important aspects of pathogenicity (Casadevall & Pirofski, 1999, 2001).
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). The facultative bacterial pathogen Listeria monocytogenes auxotrophic for uracil, phenylalanine, glycine or proline displayed virulence and growth rates similar to the parental strain when bioassayed against mice (Marquis et al., 1993). Similarly, cysteine or methionine auxotrophs of a variety of pathogens retain pathogenicity towards their hosts. Methionine auxotrophs of Pseudomonas syringae pv. glycinea retain pathogenicity towards soybeans (Thomas & Leary, 1980), and methionine or cysteine-methionine auxotrophs of Candida albicans show no decrease in virulence towards mice (Manning et al., 1984). With respect to survival in mice, methionine auxotrophs of Mycobacterium bovis show no attenuation compared to the wild-type (Wooff et al., 2002).
It follows from this explanation of the role of nutrition in pathogenicity that host specificity of obligate pathogens may exist in part because the host is a source of adequate nutritional supplementation (Garber, 1960). Indeed, many obligate pathogens exhibit very specific nutrient requirements that must be supplied by the host for pathogenicity to occur, including the completion of the pathogen life cycle. Rickettsia prowazekii, the causative agent of louse-borne typhus, requires serine, glycine and proline from the host (Austin et al., 1987; Austin & Winkler, 1988), while Eubacterium suis, an obligate bacterial pig pathogen causing pyelonephritis and cystitis, utilizes a multitude of carbohydrates, peptides and vitamins from its host (Wegienek & Reddy, 1982). Yersinia pestis, the causative agent of plague, possesses a phenotype devoid of the ability to synthesize many vitamins, amino acids and enzymes. As a consequence, Y. pestis cannot survive outside its mammalian or flea hosts. Comparison of the Y. pestis genome with that of a closely related, free-living, facultative pathogen, Yersinia pseudotuberculosis, implies that their divergence involved host-range restriction accompanied by metabolic gene inactivation in Y. pestis (Hinnebusch, 1997).
These studies indicate that at least part of the adaptation and restriction to a specific host of an obligate pathogen is correlated with loss of a biosynthetic component concomitantly supplemented by the host. The derivation of the auxotrophic strain Af6982conins in this study also implies a direct association between loss of a biosynthetic component and host restriction. Furthermore, this may be the first step in the emergence of infectious diseases because restriction of the pathogen to the host provides the opportunity for further adaptation via selection, involving the acquisition of virulence factors by spontaneous mutation, recombination or horizontal transmission.
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ACKNOWLEDGEMENTS |
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Received 17 August 2005;
revised 21 October 2005;
accepted 24 October 2005.
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