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Chapter 4 : Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia

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Abstract:

The recognition of microsporidia as opportunistic pathogens in humans has led to increased interest in their molecular biology. Much of the recent work has focused on determination of the nucleotide sequences for rRNA genes. These sequences have been used in the development of diagnostic tools for species identification. In addition, such rRNA gene sequences have facilitated the development of a molecular phylogeny of these organisms. Given the increasing evidence of the importance of the microsporidia as both human and agricultural pathogens, recent work has focused on the identification of microsporidial genes that could serve as potential therapeutic targets. Prior to the advent of comparative analysis of molecular data, the classification of eukaryotes was based on morphological, ecological, and physiological characteristics. Speculation based on this information is problematic because the homology among such characters is often not discernible and characters that could be considered were thought to be either shared or derived depending on the underlying hypothesis used in construction of the phylogeny. A much different view of the phylogenetic placement of the Microspora has been suggested based on analysis of β-tubulin genes. The molecular data present an excellent means of identifying a species and provide an excellent data set for proposing evolutionary relatedness through phylogenetic analysis. Molecular techniques for the diagnosis of microsporidiosis appear to have high sensitivity and specificity. These methods have proven extremely useful both in the identification of animal models as well as in investigations of the epidemiology of microsporidia pathogenic to humans.

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4

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Image of FIGURE 1
FIGURE 1

PCR of with the microsporidian rRNA primers described in Table 2 . Lane S, standard (50, 100, 200, 300, 400, 500, 700, 1,000, 1,500, and 2,000 bp); lane 1, 18f::350r; lane 2, 18f::530r; lane 3, 18f:;1047r; lane 4, 18f::1492r;lane 5 , 18f-212rl; lane 6, 350f-1492r; lane 7, 530f::1492r; lane 8, 1061f-1492r; lane 9, 530f- 212rl . Products of the predicted sizes are present w i t h all t h e primer sets. In cases in which more than one band is present, cloning of the amplicon of the correct size usually yields the rRNA fragment of interest. Additional bands are due to hybridization to unrelated genes in the samples.

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 2
FIGURE 2

Multikingdom phylogeny inferred from 16S rRNA sequences.The phylogenetic tree was inferred by using unambiguously aligned sequences with the neighbor joining method in the PHYLIP program. Bootstrap support for topological elements in the tree is based on 200 resamplings. Horizontal distances between nodes of the tree represent relative evolutionary distances. The bar scale corresponds to 10 changes per 100 positions. (Reprinted with permission from )

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 3
FIGURE 3

EF-lα tree of e u karyotes with archaebacteria as an outgroup. The phylogenetic tree was constructed by the maximun likelihood method based on the JTT model. The horizontal length of each branch is proportional to the estimated number of substitutions. Bootstrap probabilities are shown in parentheses. (Reprinted with permission from )

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 4
FIGURE 4

Phylogenetic tree of representative partial β-tubulin sequences (residues 108 to 259). Selected α-tubulin and γ-tubulin sequences are included, with α-tubulin as the designated outgroup. Parsimony analysis (100 bootstrap resamplings) was used for the consensus tree. Numbers are percentages of trees in which the group of species to the right of that branch was found. A nearly identical tree, except that and branches were reversed, was obtained with a distance matrix method (500 resamplings). (Reprinted with permission from )

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 5
FIGURE 5

phylogenetic analysis, (a) Maximum parsimony (MP) bootstrap (500 replicates) consensus tree for 41 sequences; (b) maximum likelihood (ML) consensus tree (using the PUZZLE 3.0 program with 1,000 puzzling steps) for 37 sequences. B o t h trees demonstrate that the sequence is within the mitochondrial clade. Support values above branches correspond to the analysis shown. Support values below relevant branches are from analyses of t h e same data set by different methods: (a) an M L and a least squares (LS) distance (100 bootstrap replicates); (b), an M P and an LS distance analyses. Boxed values in panel b correspond to support values with the sequence removed from the analysis. (Reprinted with permission from )

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 6
FIGURE 6

Hypothetical phylogenetic tree.

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 7
FIGURE 7

Microsporidian phylogeny: molecular and morphologic characteristics. Bootstrap analysis (400 replicates) of t h e most parsimonious tree. Numbers represent percentages of bootstrap replicates. Characters represented are as follows. (1) Nuclear condition of the spore: D, diplokaryotic; U, uninucleate. (2) Host: C, Crustacea; I, Insecta; M, Mammalia (h, human); P, Pisces. (3) Membrane surrounding spores: N, none; P, parasitophorous vacuole; S, sporophorous vesicle. (4) Sporogony: D, disporous; O, octosporous; P, polysporous;T, tetrasporous. (5) Chromosome cycle: D l , dihaplophasic/haplophasic with meiosis; D2, dihaplophasic/haplophasic with nuclear dissociation; H, haplophasic only. sp. characters are based o n genomic placement. (Reprinted with permission from )

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 8
FIGURE 8

Microsporidian smallsubunit rRNA phylogeny. This is a bootstrap analysis of the most parsimonious tree containing rRNA sequences from GenBank ( Table 1 ).

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 9
FIGURE 9

phylogeny. This is an analysis of and rRNA sequences in GenBank ( Table 1 ). As can be seen, despite the difference in nuclear number, there is overlap in these two genera at the molecular level of analysis.

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 10
FIGURE 10

ITS o f isolates. This analysis demonstrates the variation in the number of G T TT repeats in the I TS region of t he various isolates and t h e relationship to host species.

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 11
FIGURE 11

PCR for in stool specimens. Amplification of a band of the correct size (375 bp) is demonstrated by P C R in stool specimens from two patients with primer s e t V l : : S I 5 0 0 to the smallsubunit r R N A gene. Lanes 1 and 2, stool specimens from two patients with infection; lane 3, stool from a patient with infection; lane 4, negative control (no DNA).

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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Image of FIGURE 12
FIGURE 12

In situ hybridization for , demonstrating the detection of in an intestinal biopsy specimen using an rRNA hybridization probe. (Reprinted with permission from )

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
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References

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190. Weber, R.,, B. Sauer,, M. A. Spycher,, R. Deplazes,, R. Keller,, R. Ammann,, J. Briner,, and R. Luthy. 1994b. Detection of Septata intestinalis in stool specimens and coprodiagnostic monitoring of successful treatment with albendazole. Clin. Infect. Dis. 19: 342 345.
191. Weiser, J. 1959. Nosema laphygmae n. sp., and the internal structure of the microsporidian spore (not). J. Insect Pathol. 1: 52 59.
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197. Weiss, L. M.,, X. Zhu,, A. Cali,, H. B. Tanowitz,, and M. Wittner. 1994. Utility of microsporidian rRNA in diagnosis and phylogeny: a review. Folia Parasitol. (Prague) 41: 81 90.
198. Wittner, M.,, H. B. Tanowitz,, and L. M. Weiss. 1993. Parasitic infection in AIDS patients: cryp-tosporidiosis, isosporiasis, microsporidiosis, cyclosporiasis. Infect. Dis. Clin. N.Am. 7: 569 586.
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201. Zhu, X.,, M. Wittner,, H. B. Tanowitz,, A. Cali,, and L. M. Weiss. 1993b. Nucleotide sequence of the small ribosomal RNA of Encephalitozoon cuniculi. Nucleic Acids Res. 21: 1315.
202. Zhu, X.,, M. Wittner,, H. B. Tanowitz,, A. Cali,, and L. M. Weiss. 1994. Ribosomal RNA sequences of Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis: phylogenetic construction and structural correspondence. J. Eukaryot. Microbiol. 41: 204 209.
203. Zhu, X.,, M. Wittner,, H. B. Tanowitz,, D. Kotler,, A. Cali,, and L. M. Weiss. 1993c. Small subunit rRNA sequence of Enterocytozoon bieneusi and its potential diagnostic role with use of the polymerase chain reaction. J. Infect. Dis. 168: 1570 1575.

Tables

Generic image for table
TABLE 1

Microsporidian genes in GenBank

Adapted from with permission.

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
Generic image for table
TABLE 2

Primers for the identification and sequencing of microsporidian rDNA1

Primers 18f and 1492r amplify most of the small-subunit rRNA of the microsporidia. Primers 530f and 212rl or 212r2 are used to amplify the small-subunit rRNA and the ITS region.The remaining primers are used to sequence, with overlap, the forward and reverse strands of the entire small-subunit rRNA and ITS region. Is580r amplifies a variable region of the 5' end of the large subunit rRNA gene of many microsporidia (e.g., and ),but it does not work on all microsporidia. ssl537 allows sequencing closer to the 3' end of the small-subunit rRNA of many but not all microsporidia. ss350f and ss350r may not be needed for sequencing reactions if 18f and 530r provide sufficient overlap to obtain clear sequence data. The table is adapted from with permission.

ss, primers in the small-subunit rRNA gene; Is, primers in the large subunit rRNA gene; f, forward primer (positive strand); r, reverse primer (negative strand).

Similar toV1 primer ( ).

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
Generic image for table

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
Generic image for table

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
Generic image for table

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4
Generic image for table
TABLE 3

Diagnostic P C R primers for microsporidia pathogenic in humans

Adapted from with permission.

Designation of primers in references.

Annealing temperature in PCR.

Size of amplified fragment in base pairs. Eb, Ec, Ei, Eh,

and IIIrestriction analysis differentiates these amplicons.

and fl restriction analysis differentiates these amplicons.

Citation: Weiss L, Vossbrinck C. 1999. Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia, p 129-171. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch4

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