Genetic diversity of the dnaJ gene in the Mycobacterium avium complex

The Mycobacterium avium complex (MAC) can infect various animal hosts, including humans, swine and fowl (Ashford et al., 2001; Pavlik et al., 2000; Thorel et al., 2001). MAC infections are especially likely to be lethal in fowl (Mijs et al., 2002). M. avium consists of three subspecies: M. avium subsp. avium, M. avium subsp. paratuberculosis and M. avium subsp. silvaticum (Anz et al., 1970; Thorel et al., 1990). In humans, MAC can infect various tissues including lung, bone marrow and lymph node (Jagadha et al., 1985). Most MAC strains reportedly show multiple drug resistance, impeding treatment of MAC infections (Reddy et al., 1999). Importantly, MAC can cause systemic infections in immunocompromised hosts, including patients with AIDS (Aily et al., 1999; Jagadha et al., 1985; Pozniak, 2002). Recent reports have demonstrated a relatively increased prevalence of MAC infections in AIDS patients (Pozniak, 2002).

MAC is composed of 28 Schaefer's serotypes, with serotypes 13 representing classical M. avium and serotypes 428 representing classical Mycobacterium intracellulare (Wayne & Kubica, 1986). An international working group concerned with mycobacterial taxonomy has classified these into four MAC groups, with serotypes 16 and 811 belonging to M. avium, serotypes 7, 1220, 23 and 25 belonging to M. intracellulare, serotype 27 resembling Mycobacterium scrofulaceum, and serotypes 21, 24, 26 and 28 remaining unclassified (Wayne et al., 1993). Serologic determination in MAC is of practical value in epidemiological studies of infections in humans and animals (Schaefer, 1965, 1968; Wayne & Kubica, 1986). Genetic determinations in MAC also may be performed, using species-specific PCR analysis (Comincini et al., 1998; Sola et al., 1996; Thierry et al., 1993; Valente et al., 1997). However, relationships between MAC serotypes and genotypes are poorly understood.

The dnaJ gene, which encodes a stress protein, is highly conserved among bacterial genera (Bardwell et al., 1986; Ohki et al., 1986). Members of the Mycobacteriaceae possess the dnaJ gene, and its sequence in these organisms has proved useful in identification of species (Lathigra et al., 1988; Takewaki et al., 1993, 1994). Accumulating evidence suggests that phylogenetic analyses including the neighbour-joining method or/and the unweighted pair group method are useful for molecular epidemiological analysis in various micro-organisms. However, such methods have seen little application in homologue and phylogenetic analyses of the dnaJ gene in MAC. We performed partial sequencing of the dnaJ gene as a basis for phylogenetic analysis in various MAC strains.

Isolates.
Schaefer's 28 reference strains of MAC are listed in Table 1. These reference strains were kindly donated by J. K. McClatchy and A. Y. Tsang of the National Jewish Hospital, Denver, CO, USA. Fourteen strains representing human clinical isolates were kindly donated by C. Abe (Research Institute of Tuberculosis of the Japan Anti-tuberculosis Association). Twenty-two isolates from swine, birds and cats also were studied and are listed in Table 2.

Table 1. Reference strains examined in this study Accession numbers refer to dnaJ sequences.

Table 2. Human clinical and veterinary isolates examined in this study

DNA extraction, PCR assay and DNA sequence of the dnaJ amplicons.
A loopful of MAC grown on 1 % Ogawa egg medium (Nissui) was suspended in 1 ml double-distilled water for washing. To extract DNA, the pellet remaining after centrifugation was resuspended in 200 µl InstaGene Matrix (Bio-Rad) and incubated at 56 °C for 30 min. The mixture was heated at 100 °C for 8 min, agitated vigorously and centrifuged at 10 000 g for 2 min. The supernatant was used as a template for PCR. We used a set of previously reported modified primers (Takewaki et al., 1993) for amplification of the dnaJ gene. Their sequences were 5'-GGGTGACGCG(G/A)CATGGCCCA-3' and 5'-CGGGTTTCGTCGTACTCCTT-3', producing a theoretical amplicon of 236 nucleotides. The PCR contained 1 µl template DNA and 1 µl of the set of primers (20 pmol each), 12.5 µl PCR Master Mix (Promega) and 9.5 µl of DNase- and RNase-free double-distilled water (total volume 25 µl). The PCR protocol was as follows: incubation for 2 min at 94 °C; 35 cycles of 94 °C for 1 min, 65 °C for 1 min and 72 °C for 2 min; and an additional 5 min final elongation step at 72 °C. The size of the amplified DNA fragment was confirmed by electrophoresis on a 1.5 % agarose gel. After purification of DNA fragments using a QIAquick PCR purification kit (Qiagen), the nucleotide sequence was determined with an ABI310 automated DNA sequencer (Applied Biosystems) using a dye terminator cycle-sequencing ready reaction kit (Applied Biosystems). Nucleotide sequences of 236 bp were analysed phylogenetically using the CLUSTAL W program on the DNA database of Japan (DDBJ) home page (http://hypernig.nig.ac.jp/homology/clustalw-e.shtml). Evolutionary distances were estimated using Kimura's two-parameter method (Kimura, 1980) and phylogenetic trees were constructed using the neighbour-joining method (Saitou & Nei, 1987). The reliability of the tree was estimated using 1000 bootstrap replications.

The dnaJ gene encodes a heat-shock protein and is located in a single operon in Escherichia coli (Lathigra et al., 1988). Genetic diversity of the dnaJ gene is seen in various bacteria and is used as a tool for molecular epidemiological analysis (Liu et al., 2003). In addition, this gene is used as an alternative method for identification of some mycobacteria (Takewaki et al., 1993, 1994). Thus, we examined the diversity of the dnaJ gene in MAC. In this study, the dnaJ gene was successfully amplified in all strains. To detect the amplicons of 236 bp, we performed electrophoresis on 1.5 % agarose gels. The G+C content of the dnaJ gene ranged from 62.3 to 64.8 mol%, which is representative of the G+C content of the DNA in Mycobacterium species (6270 mol%) (Wayne & Kubica, 1986). No insertions or deletions were observed in these amplicons. From the dnaJ gene nucleotide sequences (nt 14061627) (Lathigra et al., 1988), all MAC strains could be classified into 14 groups (Fig. 1); 2132 nucleotide substitutions were found in the dnaJ gene depending on the strain. Among the reference strains, identical nucleotide sequences were seen in serotypes 1 and 11, serotypes 2, 3, 4 and 9, serotypes 5, 6, 8, 10 and 21, serotypes 14, 16, 17 and 20, serotypes 15, 23 and 24, serotypes 18 and 28, and serotypes 25 and 27. The human clinical and veterinary isolates could be classified into four groups: three groups belonged to serotypes 1, 2 and 5, respectively, while the remaining group consisting of five strains from humans (H1, H2, H9, H10, H11) and one strain from fowl (B5) represented an independent cluster. Serotypes of human clinical and veterinary MAC isolates were not completely consistent with their phylogenetic clusters. When we deduced and compared 74 amino acid residues of the dnaJ gene in all reference strains, only two amino acid substitutions (codon 13, Q→K; codon 44, G→D) were observed in comparison with the Mycobacterium tuberculosis prototype (GenBank accession no. X06422). The results suggested that most nucleotide substitutions found in our study were synonymous and that the amino acid sequence in dnaJ gene is highly conserved.

(45K): Fig. 1. Nucleotide and amino acid sequences of the dnaJ gene segments in prototype strains, human clinical strains and veterinary strains of MAC. Numbers of nucleotides are given at the beginning of the sequence and the deduced amino acid sequence is shown in single-letter code. Nucleotides and amino acids are noted only where different from those of M. tuberculosis (GenBank accession no. X06422); where identical, they are shown by dashes. Serotypes of the human clinical and veterinary isolates in this study are indicated in bold in parentheses. In codon 13, Gln was replaced by Lys (Q13→K13). In codon 44, Gly was replaced by Asp (G44→D44). Notes: a, strains H3(1), H4(1), H5(1), H6(2), H7(2), H8(2) and H12(3) had sequences identical to those of the reference strains of serotypes 1 and 11; b, strains S2(2), S3(3), S4(4), S5(4), S6(4), B1(1), B2(1), B3(2), B4(2) and B6(9) had sequences identical to those of the reference strains of serotypes 2, 3, 4 and 9; c, strains H13(8), H14(8), S1(1), S7(6), S8(6), S9(6), S10(8), S11(8), S12(8), S13(10), S14(10), S15(10) and C1(6) had sequences identical to those of the reference strains of serotypes 5, 6, 8 10 and 21; d, strains H2(2), H9(2), H10(2), H11(3) and B5(3) had sequences identical to that of strain H1(1).

Phylogenetic trees of the dnaJ gene constructed by the neighbour-joining method are shown in Fig. 2. In the rooted tree, about 8 % genetic diversity was seen in the dnaJ gene among all strains, and our strains divided into two clusters, I and II. M. avium strains including serotypes 16, 811 and 21 belonged to cluster I, while the remaining strains (serotypes 7, 1220 and 2228) belonged to cluster II. Only 0.8 % genetic diversity was seen within cluster I; in contrast, cluster II showed 7 % genetic diversity. Most serotypes of MAC cluster I were located very close to one another, suggesting that in cluster I the dnaJ gene is highly conserved, although only partial sequencing was carried out. No significant genetic diversity was observed among the clinical and veterinary strains included in cluster I, and their serotypes included 14, 6, 8 and 10, indicating M. avium. On the other hand, in cluster II, wide diversity of the dnaJ gene was seen between strains in serotypes 25 and 27 and those in serotype 26.

(33K): Fig. 2. Phylogenetic rooted tree of the dnaJ gene constructed by the neighbour-joining method. The methods of phylogenetic analysis are described in detail in the text. The numbers at each branch indicate bootstrap values for the clusters supported by that branch. Sequences of the strains are indicated in Tables 1 and 2. The serotypes of the human clinical and veterinary isolates in this study are indicated in bold in parentheses.

M. avium and M. intracellulare have been identified genetically using various sequences, such as the 16S rRNA gene, the 16S23S rRNA internal transcribed spacer and the DT1/DT6 sequence (Boddinghaus et al., 1990; Frothingham & Wilson, 1993; Thierry et al., 1993). All these studies indicated that serotypes 16, 811 and 21 were M. avium. Our study using the dnaJ gene showed the same result, with serotypes 16, 811 and 21 belonging to cluster I. The results indicated that several genes in various strains of M. avium are related and can be defined genetically. However, the dnaJ gene displayed no independent or clear clusters within M. intracellulare. Previous reports using molecular analyses have shown disagreement regarding serotypes in M. intracellulare (Boddinghaus et al., 1990; Comincini et al., 1998; Frothingham & Wilson, 1993; Sola et al., 1996; Thierry et al., 1993; Valente et al., 1997; van der Giessen et al., 1993). Since MAC strains in cluster II had wide genetic diversity, differing results with respect to the serotype of M. intracellulare could have been reached depending on the gene examined. Based on these results, these various genes, including those encoding rRNA and the dnaJ gene, are useful target genes for molecular epidemiological analysis of MAC.

MAC is one of the most common bacteria isolated from patients with atypical mycobacterial infections (Aily et al., 1999; Jagadha et al., 1985; Pozniak, 2002; Thorel et al., 2001). We have previously reported that comparative sequence analysis of the amplified dnaJ gene has been used to detect and identify these organisms derived from clinical materials and/or from environmental sources in infant formula (Morita et al., 2002). In conclusion, sequence and phylogenetic analysis of MAC may contribute towards an understanding of the epidemiology of MAC infections.