EM organisms have a significant ability to cause disease in humans and animals. Very little knowledge exists, however, about the mechanisms of disease for this group of pathogens, including those important to fish mycobacteriosis. To learn more about the virulence and genomics of some of these species with zoonotic potential, we used macrophage assays and a custom microarray analysis to compare strains of different EM species to that of M. avium.
Many of the EM strains of M. peregrinum
and M. chelonae
were isolated from zebrafish and have been characterized previously (27
). The strains isolated from salmonid fishes were first classified as M. salmoniphilum
, but the species was eliminated and its members were assigned to M. chelonae
). However, the species has now been resurrected based on molecular analyses (51
). Because all the strains of M. chelonae
and M. salmoniphilum
and the MP102 strain of M. peregrinum
were unable to grow in culture at 37°C, it was notable that many of these isolates were able to grow in human and mouse macrophages at the same temperature. Previous work with M. marinum
, another fish pathogen with zoonotic potential, indicates that strains that are not able to grow in culture at 37°C are capable of growing in macrophages at that temperature (26
). All strains of mycobacteria isolated from fish replicated in the carp cells, and many of those strains also grew in the human and mouse cells. In contrast, neither the MAC101 nor the MAC104 strain (from humans) grew well in carp cells. This result does not seem to be a product of the optimum temperature for the cell type or bacterial strain, as the MAC101 strain was able to grow in human and mouse cells at 28.5°C.
The percentages of growth over 2 and 4 days for the three types of macrophages were not similar in mycobacteria isolated from the same hosts or even in the same species, although there were some trends. The M. salmoniphilum strains, isolated from salmonids, and the MCJA and MCJ78 strains, isolated from zebrafish, showed growth over 2 and 4 days in all three macrophages lines. The MCH strain of M. chelonae, isolated from zebrafish, however, was not able to grow in either mouse or human macrophages or in carp cells incubated at 37°C. When the human and mouse macrophages were incubated at a lower temperature, the MCH strain was able to grow. The MP101 strain of M. peregrinum, isolated from zebrafish, was able to grow in all cell types at either temperature, but the MP102 strain, isolated from tilapia, was not able to grow in mouse macrophages, unless they were incubated at 28.5°C. This strain was also unable to grow in carp cells incubated at 37°C. As indicated previously, the M. avium strains tested were unable to multiply well in carp cells at either temperature.
The macrophage growth profile correlates well with other virulence data. Watral and Kent (50
), using intraperitoneal injection, showed that the MCH strain of M. chelonae
did not cause any significant disease or mortality in zebrafish, nor could it be cultured from the zebrafish weeks after exposure. Moreover, this isolate was obtained from a few fish as an incidental infection in a large research colony (27
). In a different study, in which zebrafish were exposed to mycobacteria by bath immersion or intubation, we showed that the MP101 strain of M. peregrinum
(which did grow well in mammalian macrophages) was consistently able to infect and grow in zebrafish tissues, although signs of disease and mortality were rare (24
). In data not included in that study, the MP102 strain of M. peregrinum
could not be cultured from the zebrafish, nor did it cause any signs of disease. Similarly, we could not culture the M. avium
MAC104 strain from zebrafish infected by bath immersion or by intubation, and the infected fish showed no signs of disease (M. J. Harriff, unpublished observations).
Genomic differences based on microarray analyses also correlated with phenotypic virulence data. A survey of the literature revealed over 120 genes predicted to play a role in the virulence of different species of mycobacteria. Many of the genes selected for the microarray were identified by a number of genome- or proteome-wide screens designed to detect M. avium
or M. tuberculosis
genes or proteins upregulated in human macrophages (7
). Over 50 of these gene products have no known or predicted function, including the ORFs in a 3-kb PI identified in our laboratory, that is important in virulence (14
). Little is known about how the genes contained in the open reading frames in the PI may function in virulence, but it has been shown to allow for the infection of amoeba. This region is not present in M. tuberculosis
but is present in other members of the MAC. As shown by microarray analysis, the island was completely absent in the MCH strain of M. chelonae
, while portions of the island were also predicted to be absent in the ATCC and MP101 strains of M. peregrinum
. Interestingly, the island is putatively duplicated in the M. salmoniphilum
strains. This bacterium is a recognized pathogen of salmonids, based on both laboratory transmission studies (1
) and field observations (8
). The in vivo and in vitro growth of these strains of mycobacteria suggest that the genes making up this PI should be studied further for their role in the virulence of EM.
In addition to genes predicted to play a role in virulence, sequences representing selected genes of many of the metabolic pathways in mycobacteria were included. It is not known if differences or absence of metabolic pathways can play a role in host specificity or levels of pathogenicity. Differences in metabolic pathways are also important to differences in the basic phenotypic characteristics of species, which may play roles in environmental survival, host specificity, and pathogenicity. For example, M. avium
is very closely related to M. avium
at the genomic level (4
); however, multiple studies have revealed that each subspecies has regions and genes that are not present in the other (34
). Among some of the important findings, microarray analysis revealed that M. avium
has a truncation of an early gene in the mycobactin synthesis operon (47
). Further study of this truncation could reveal the reasons for the slow growth of M. avium
as well as for differences in hosts and pathogenicity levels. Some of the species of EM tested in this study were putatively missing genes from metabolic pathways that may play a role in the ability to persist in certain environments.
Although many virulence genes identified in screens and included on this array have no putative function, others have been studied for their role in virulence. The mtr
A gene was scored as absent in the MCH strain of M. chelonae
and three of the four strains of M. salmoniphilum
(with the fourth strain score being very near to that of the cutoff). This gene encodes the response regulator protein of a two-component regulatory system that has been shown by Zahrt and Deretic to be essential to growth in broth and differentially expressed by virulent and avirulent mycobacteria (54
). While the expression of this gene is constitutive and high in M. tuberculosis
, it is induced to very high levels in the BCG vaccine strain upon entry into macrophages. A more recent study, by Fol et al. (19
), showed that the regulation of the ratio of this protein product and its phosphorylated version are important to proliferation in macrophages. The protein product encoded by mtrB
that phosphorylates MtrA is not essential (54
), suggesting that there are other kinases that play a role in the modulation of this ratio. It is possible that in strains of EM without the MtrA protein, other response regulators have evolved to serve in the essential role for growth in the environment. When a host is encountered, however, the lack of this protein leads to an inability to replicate in the host cells. It is possible that the lack of the mtrA
gene is important to these differences in both in vitro and in vivo growth. Although the strains of M. salmoniphilum
were able to grow in the human and mouse macrophages, the change in growth from 2 to 4 days was much reduced from the initial growth over 2 days (with the exception of one strain in human cells), suggesting that these strains are not able to persist as well in macrophages as strains with the mtrA
system was not the only two-component regulatory system to be identified as being absent from certain strains of EM. The kdpE
(MCJA and MCJ78) gene scored as absent in some of the strains, while the phoP
genes also scored as absent (or nearly absent) in one to three of the M. chelonae
or M. salmoniphilum
strains. These response regulatory genes have been shown by a number of groups to be important to the growth and virulence of M. tuberculosis
in animal models (21
), but there is still very little known about the mechanisms by which they do so.
Although microarrays have been a useful tool for comparing the genomes of different strains of sequenced mycobacteria, we determined that there are potential limitations to the use of this tool to compare unsequenced strains of EM. The cutoff score for absent genes was determined by hybridizing the DNA of M. avium and M. tuberculosis to oligonucleotides representing genes known to be present and absent in both strains. Even with a cutoff score of two standard deviations lower than the median absent score for M. tuberculosis, we were nevertheless able to amplify some genes by PCR that were identified as absent by the microarray. Other than 16S rRNA genes, ITS, and hsp60 DNA, there are very few sequence data in the database for M. peregrinum and M. chelonae. This lack of sequence data makes it difficult to know what the cause of the discrepancy is between the microarray and the PCR results. It is possible that, although they are present, the sequences of these genes are divergent enough from the MAC104 gene sequences that they do not hybridize to the oligonucleotide on the array. It is also possible that the oligonucleotide was selected from a region of the gene that is in fact absent, but the primers used for PCR are similar to regions of the genomic DNA that are still present. If this were the case, we would expect the amplified PCR fragments to have different sizes. As this was not the case, it is more likely that the former explanation accounts for the discrepancy. Further evidence for this explanation is that primers designed from M. smegmatis were required to amplify genes expected to be present from some strains. The use of PCR as a technique for verification has limitations as well, as even a single base difference could lead to a false-negative PCR result. The use of additional techniques, such as Southern blotting or reverse transcription-PCR, could be used to verify the presence or absence as well as the expression of specific genes or regions identified in this study. Overall, however, there was good correlation between the microarray and the PCR data, and there were many genes for which the test strain DNA so strongly hybridized to the array spot, compared to that of the MAC104 reference, to be considered putatively duplicated, indicating that these techniques together provide reliable results. Further optimization may improve this custom microarray as a tool for comparing the genomes and gene expression in unsequenced species of mycobacteria.
Given how little we know about the mechanisms of virulence for many of these species, this study has provided a useful set of data from which to base further investigation. Future study may involve an examination of the expression of these genes when the various strains of mycobacteria are exposed to cell types or an animal model, such as zebrafish or mice. Additionally, constructing recombinant strains that express genes or regions that are putatively absent and looking for differences in the ability to grow in cell types or cause disease could provide insight into the relative importance of those genes to virulence. As we isolate more species and strains of EM from hosts with which humans frequently come into contact, such as cattle, zebrafish, and other fishes, further understanding of how virulence is tied to the genomes of these organisms may provide a means by which we are able to determine the isolates with zoonotic potential. Finally, more knowledge about the mechanisms of virulence and pathogenic potential of these organisms may lead to advances in controlling disease in both humans and animals of economic and research importance, such as zebrafish.