Natural mycoplasmal infections in isolator-maintained LEW/Tru rats

For 4 years a colony of cesarean-derived, isolator-maintained LEW/Tru rats was evaluated for mycoplasmal infection by serology, culture and histopathology. Anti-mycoplasmal antibodies were detected by enzyme-linked immunosorbent assay (ELISA), and the colony eventually was found to have inapparent infections of Mycoplasma pulmonis and Mycoplasma arthritidis. Rats, naturally infected with M. pulmonis, remained consistently positive in the M. pulmonis ELISA after their initial seroconversion, and eventually developed clinical signs and lesions of respiratory and genital mycoplasmosis. M. pulmonis was apparently eliminated by serological testing and removal of infected rats. Rats naturally infected with M. arthritidis did not develop clinical or histologic evidence of mycoplasmal disease and their sera gave inconsistent results in the M. pulmonis ELISA, but eventually developed positive M. arthritidis ELISA responses. M. arthritidis was isolated from the genital tract, the intestinal tract, and Harderian gland. In contrast to M. pulmonis, removal of serologically positive animals was not sufficient for elimination of M. arthritidis from the colony.     

Membrane-associated nuclease activities in mycoplasmas.

Membrane-associated nucleases of various mycoplasmal species were investigated by using two nuclease assays. A lambda DNA assay was developed to measure nuclease activity associated with whole-cell suspensions, activity released from intact cells, and activity associated with detergent-disrupted cells. In most species, nuclease activities were entirely membrane associated, and disruption by a detergent had a stimulatory effect on these activities. All mycoplasmal species contained nuclease activity, but Mycoplasma capricolum was unusual because its activity was dependent upon magnesium and was inhibited by calcium. We developed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system that produced reproducible nuclease patterns, and this system was used to determine the apparent molecular weights of the nuclease proteins. An examination of 20 mycoplasmal species failed to identify common bands in their nuclease patterns. An examination of 11 Mycoplasma pulmonis strains, however, indicated that nuclease patterns on polyacrylamide gels may provide a means for categorizing strains within a species. Our results suggest that nucleases are important constituents of mycoplasmal membranes and may be involved in the acquisition of host nucleic acids required for growth.     

The effect of thiol-active compounds and sterols on the membrane-associated hemolysin of Mycoplasma pulmonis

Abstract Previous studies had shown that Mycoplasma pulmonis contained a bovine serum albumin-dependent, membrane-associated hemolysin. Biochemical analyses were performed to further characterize this activity. The membrane-associated hemolytic activity could be activated by dithiothreitol and β-mercaptoethanol, and inactivated by oxidizing compounds, a sulfhydryl inhibitor and heat treatment. Cholesterol and other sterols were inhibitory in a stereo-specific manner, but they did not interfere with adherence of M. pulmonis to red blood cells. These results indicated that once attached, the M. pulmonis hemolysin recognized cholesterol in the opposing membrane leading to red cell lysis. Because of the unique location of this toxin and its sensitivity to cholesterol, the mycoplasma membrane hemolysins may belong to a unique class of bacterial toxins.     

High-frequency rearrangements in the chromosome of Mycoplasma pulmonis correlate with phenotypic switching

Mycoplasma pulmonis is a murine pathogen that causes chronic respiratory disease in laboratory rats and mice. Several examples of high-frequency phenotypic switching have been reported for M. pulmonis, the molecular basis of which is unknown. We report here that during growth the M. pulmonis chromosome undergoes DNA rearrangements at a high frequency. Some of the rearrangements we examined correlated with changes in the susceptibility of the cells to mycoplasma virus P1, an example of phenotypic switching involving changes in surface antigen structure. Other rearrangements, unrelated to phenotypic switching, involved a DNA element present in the chromosome in multiple copies. The high level of DNA recombination that occurred in M. pulmonis indicates that this may be one of the most variable genomes studied to date. High levels of DNA recombination may contribute to the unusually high rate of evolution that mycoplasmas are thought to be undergoing. Understanding the molecular basis for this phenomenon may provide an insight into the chronic nature of many mycoplasmal infections.     

Cross-reactive antibodies to mycoplasmas found in human sera by the enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycoplasma pneumoniae in patients' sera with M. pneumoniae infection were measured by the complement fixation (CF) test and enzyme-linked immunosorbent assay (ELISA). Many patients' sera cross-reacted with heterologous mycoplasmal ELISA antigens such as M. hominis, M. hyorhinis, M. orale, M. pulmonis and M. salivarium. The sera with high CF (CF greater than or equal to 40) titers gave significantly higher ELISA values to M. hyorhinis (P less than 0.001) and M. pulmonis (P less than 0.001), which are not parasitic for humans, than those with low CF (CF less than 20) titer. Human normal immunoglobulin G (human normal IgG) containing 98% or more IgG, prepared from pooled plasma of at least 500 normal human donors, showed ELISA reactions with all mycoplasmal strains used. The nonspecific adsorption of human normal IgG on the surface of plate wells and on medium components which might contaminate mycoplasmal ELISA antigens could be disregarded. These results suggest that cross-reactive antibodies to mycoplasmas exist in human sera, and they affect the results of ELISA for serodiagnosis of M. pneumoniae infection.     

PCR-based detection of Mycoplasma species

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the genomic DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the diagnosis of mycoplasmal contamination in cell culture systems.     

Variations in the surface proteins and restriction enzyme systems of Mycoplasma pulmonis in the respiratory tract of infected rats

Restriction and modification (R-M) systems are generally thought to protect bacteria from invasion by foreign DNA. This paper proposes the existence of an alternative role for the phase-variable R-M systems encoded by the hsd loci of Mycoplasma pulmonis. Populations of M. pulmonis cells that arose during growth in different environments were compared with respect to R-M activity and surface antigen production. When M. pulmonis strain X1048 was propagated in laboratory culture medium, > 95% of colony-forming units (cfu) lacked R-M activity and produced the variable surface protein VsaA. Mycoplasmas isolated from the nose of experimentally infected rats also lacked R-M activity and produced VsaA. In contrast, the cell population of mycoplasmas isolated from the lower respiratory tract of the infected rats was more complex. The most dramatic results were obtained for mycoplasmas isolated from the trachea. At 14 days postinfection, 38% of mycoplasma isolates produced a Vsa protein other than VsaA, and 34% of isolates had active restriction systems. These data suggest that differences in selection pressures in animal tissues affect the surface proteins and the R-M activity of the mycoplasmal cell population. We propose that variations in the production of R-M activity and cell surface proteins are important for the survival of the mycoplasma within the host.     

Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification.

Systematic computer alignment of mycoplasmal 16S rRNA sequences allowed the identification of variable regions with both genus- and species-specific sequences. Species-specific sequences of Mycoplasma collis were elucidated by asymmetric amplification and dideoxynucleotide sequencing of variable regions, using primers complementary to conserved regions of 16S rRNA. Primers selected for Mycoplasma pneumoniae, M. hominis, M. fermentans, Ureaplasma urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis proved to be species specific in the polymerase chain reaction. The genus-specific primers reacted with all mycoplasmal species investigated as well as with members of the genera Ureaplasma, Spiroplasma, and Acholeplasma. No cross-reaction was observed with members of the closely related genera Streptococcus, Lactobacillus, Bacillus, and Clostridium or with any other microorganism tested. On the basis of the high copy number of rRNA, a highly sensitive polymerase chain reaction assay was developed in which the nucleic acid content equivalent to a single organism could be detected.     

Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification.

Systematic computer alignment of mycoplasmal 16S rRNA sequences allowed the identification of variable regions with both genus- and species-specific sequences. Species-specific sequences of Mycoplasma collis were elucidated by asymmetric amplification and dideoxynucleotide sequencing of variable regions, using primers complementary to conserved regions of 16S rRNA. Primers selected for Mycoplasma pneumoniae, M. hominis, M. fermentans, Ureaplasma urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis proved to be species specific in the polymerase chain reaction. The genus-specific primers reacted with all mycoplasmal species investigated as well as with members of the genera Ureaplasma, Spiroplasma, and Acholeplasma. No cross-reaction was observed with members of the closely related genera Streptococcus, Lactobacillus, Bacillus, and Clostridium or with any other microorganism tested. On the basis of the high copy number of rRNA, a highly sensitive polymerase chain reaction assay was developed in which the nucleic acid content equivalent to a single organism could be detected.     

Characterization of Mycoplasma pulmonis Variants Isolated from Rabbits I. Identification and Properties of Isolates

Mycoplasma showing at least two colony types were isolated from the nares and oropharynx of New Zealand white rabbits. Two strains were purified by single-colony passages and characterized. Morphology by phase-contrast and electron microscopy was typical of Mycoplasmataceae. Both grew anaerobically as well as aerobically, caused hemolysis of guinea pig, sheep, and horse red blood cells, and fermented glucose. These characteristics are shared by members of the species M. pulmonis, commonly isolated from the respiratory tracts of laboratory rats and mice. By use of the growth-inhibition test and agar-gel double-diffusion tests, the two strains were found to be serologically related to each other and to M. pulmonis ATCC 14267 but not to other representative Mycoplasma species from man and animals.     

Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC replicative plasmids

Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratory mycoplasmosis. The complete genome of this bacterium, which belongs to the class Mollicutes, has recently been sequenced, but studying the role of specific genes requires improved genetic tools. In silico comparative analysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the region containing the predicted origin of replication (oriC) and the existence, in most of the mollicute genomes examined, of putative DnaA boxes lying upstream and downstream from the dnaA gene. The predicted M. pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transformation of the mycoplasma, which was obtained with a frequency of 3 x 10(-6) transformants/CFU/ micro g of plasmid DNA. However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region. Reduction of this oriC region by subcloning experiments to the region either upstream or downstream from dnaA resulted in plasmids that failed to replicate in M. pulmonis, except when these two intergenic regions were cloned with the tetM determinant as a spacer in between them. An internal fragment of the M. pulmonis hemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transform M. pulmonis. Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over, which results in the disruption of the gene, could be documented. These mycoplasmal oriC plasmids may therefore become valuable tools for investigating the roles of specific genes, including those potentially implicated in pathogenesis.     

Transformation of Mycoplasma pulmonis: demonstration of homologous recombination, introduction of cloned genes, and preliminary description of an integrating shuttle system

The transposons Tn916 and Tn4001 and a series of integrating plasmids derived from their antibiotic resistance genes were used to examine polyethylene glycol-mediated transformation of Mycoplasma pulmonis. Under optimal conditions, Tn916 and Tn4001 could be introduced into M. pulmonis at frequencies of 1 x 10(-6) and 5 x 10(-5) per CFU, respectively. Integrating plasmids were constructed with the cloned antibiotic resistance determinants of Tn916 and Tn4001, a pMB1-derived plasmid replicon, and mycoplasmal chromosomal DNA and were used to examine recombinational events after transformation into M. pulmonis. Under optimal conditions, chromosomal integrations could be recovered at a frequency of 1 x 10(-4) to 1 x 10(-6) per CFU, depending on the size and nature of the chromosomal insert and the parental plasmid. Integrated plasmids were stable in the absence of selection and could be rescued in Escherichia coli along with adjacent mycoplasma DNA. These studies provide the first direct evidence of a recombination system in the Mollicutes and describe the first E. coli-M. pulmonis shuttle vectors.     

Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis

Previous attempts to introduce transposon Tn4001 into Mycoplasma pulmonis and Mycoplasma arthritidis have not been successful, possibly due to functional failure of the transposon's gentamicin resistance determinant. Tn4001C and Tn4001T were constructed, respectively, by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant into Tn4001. Both Tn4001C and Tn4001T transposed in M. pulmonis, and Tn4001T transposed in M. arthritidis. The incorporation of a Tn4001T derivative that contained lacZ into either Mycoplasma species resulted in transformants with readily detectable LacZ activity. Tn4001T may be of general utility for use as a mycoplasma cloning vehicle because tetM functions in all species of Mycoplasma examined thus far.     

Characterization of the ftsZ gene from Mycoplasma pulmonis, an organism lacking a cell wall.

The ftsZ gene is required for cell division in Escherichia coli and Bacillus subtilis. In these organisms, FtsZ is located in a ring at the leading edge of the septum. This ring is thought to be responsible for invagination of the septum, either causing invagination of the cytoplasmic membrane or activating septum-specific peptidoglycan biosynthesis. In this paper, we report that the cell division gene ftsZ is present in two mycoplasma species, Mycoplasma pulmonis and Acholeplasma laidlawii, which are eubacterial organisms lacking a cell wall. Sequencing of the ftsZ homolog from M. pulmonis revealed that it was highly homologous to other known FtsZ proteins. The M. pulmonis ftsZ gene was overexpressed, and the purified M. pulmonis FtsZ bound GTP. Using antisera raised against this purified protein, we could demonstrate that it was expressed in M. pulmonis. Expression of the M. pulmonis ftsZ gene in E. coli inhibited cell division, leading to filamentation, which could be suppressed by increasing expression of the E. coli ftsZ gene. The implications of these results for the role of ftsZ in cell division are discussed.     

Detection of Mycoplasma pulmonis by fluorogenic nuclease polymerase chain reaction analysis

Mycoplasma pulmonis induces persistent infections in laboratory mice and rats and can contaminate biological materials. We developed a fluorogenic nuclease polymerase chain reaction (fnPCR) assay to detect M. pulmonis specifically. Primer and probe sequences for the assay were targeted to 16S rRNA sequences specific to M. pulmonis. The assay consistently detected the equivalent of fewer than 10 copies of template DNA. When evaluated against a panel of 24 species of bacteria, the M. pulmonis assay detected only M. pulmonis isolates. Evaluation of 10-fold serial dilutions of cultured M. pulmonis showed that the M. pulmonis fnPCR assay and culture on Dutch agar had comparable sensitivity in detecting viable M. pulmonis organisms, whereas the mouse antibody production test displayed positive serologic results at dilutions higher than those in which viable organisms could be detected. Finally, the M. pulmonis fnPCR assay was able to detect M. pulmonis DNA in nasopharyngeal wash fluid and trachea, lung, and uterus tissue collected from mice naturally infected with M. pulmonis but did not detect the organism in similar samples collected from uninfected, negative control mice. The M. pulmonis fnPCR assay provides a high-throughput, PCR-based method to detect M. pulmonis in infected rodents and contaminated biological materials.     

Effects of nitrogen dioxide on Mycoplasma pulmonis infection and humoral immune responses in mice

The effects of continuous exposure to nitrogen dioxide (NO2) on the pathologic and immunologic responses of ddY mice to the infection with Mycoplasma pulmonis were investigated. The organisms grew well in the trachea as early as 7 days after infection but barely grew in the lung even after 28 days, causing slight pneumonic lesions in only a few of the infected mice exposed to 1 and 5 ppm NO2. When mice were exposed to 10 ppm NO2 at or after the infection, however, mycoplasmal growth in the lung, but not in the trachea, was greatly enhanced, and pneumonic lesions were evident in the lung of almost all the mice examined. The serum antibody titers to M. pulmonis increased with time after infection regardless of the concentration of NO2 exposed or the mycoplasmal number in the respiratory tract in the infected mice. The in vitro immune responses of the spleen cells of the infected mice were significantly depressed by exposure to 10 ppm NO2 in not only mitogenic response to LPS and ConA but also antibody production to SRBC, whereas uninfected healthy mice were apparently not modulated except for a slight decrease in Con A response.     

Development of a cloning system in Mycoplasma pulmonis

A system suitable for recombinant DNA manipulation in mycoplasmas was developed using the cloned antibiotic-resistance genes of Tn4001 and Tn916. An integrative plasmid containing one of the resistance markers was inserted into the genome of Mycoplasma pulmonis to form a recipient strain. This was accomplished by transformation and homologous recombination between chromosomal DNA sequences cloned onto the integrative plasmid. A second vector, the cloning vector, containing the same plasmid replicon and alternate resistance marker, carried cloned foreign DNA. When transformed into mycoplasmal recipients, homologous recombination between plasmid sequences resulted in integration of the cloning vector and foreign DNA. A Brucella abortus gene coding for a 31-kDa protein and the P1 structural gene and operon from Mycoplasma pneumoniae were introduced to examine the feasibility of developing mycoplasma as cloning hosts. Recombinant plasmids as large as 20 kb were inserted into M. pulmonis, and the integrated foreign DNA was stably maintained. The maximum size of clonable DNA was not determined, but plasmids larger than 22 kb have not been transformed into mycoplasmas using polyethylene glycol. Also the size of genome (800-1200 kb) may affect the stability of larger inserts of foreign DNA. This system is applicable to any mycoplasma capable of transformation, homologous recombination and expression of these resistance markers. Because of their lack of a cell wall, mycoplasmas may be useful cloning hosts for membrane or excreted protein genes from other sources.     

Male germ cell specific sulfogalactoglycerolipid is recognized and degraded by mycoplasmas associated with male infertility

Sulfogalactosylglycerolipid (SGG) is the major mammalian male germ cell glycolipid and has been implicated in sperm/egg binding. Mycoplasma pulmonis, a species of Mollicutes, is associated with male infertility in rodents. Purified SGG incubated in the presence of M. pulmonis was enzymatically degraded by both desulfation and deacylation. Desulfation occurred primarily at alkaline pH, and deacylation also increased with increased pH, indicating that these represent novel enzymatic activities. Digestion was facilitated, but not dependent on, the presence of detergent. Rat spermatozoa exposed to M. pulmonis showed a reduction in SGG content which was particularly marked for cauda (mature) spermatozoa. With the aid of tlc overlay binding procedure, intact M. pulmonis were found to bind specifically to sulfated glycolipids and thus SGG may provide the cell membrane receptor for this organism. The topology of mycoplasma binding to rat sperm was consistent with the known topology of sperm SGG. The reduced binding (and subsequent digestion) of caput spermatozoan SGG correlates with the membrane colocalization of SGG and its endogenous binding protein at this stage. Separation of SGG and its binding protein during epididymal sperm maturation appears to facilitate M. pulmonis binding to and digestion of cauda sperm SGG. The binding and degradation of the sperm SGG by M. pulmonis may play a role in the induction of infertility which follows infection with these organisms by interfering in sperm/egg receptor recognition.     

Gliding mycoplasmas are inhibited by cytochalasin B and contain a polymerizable protein fraction

Studies are presented on the effect of cytochalasin B (CB) on the growth of five Mycoplasma species, three Acholeplasma species, and one Spiroplasma species. The three gliding mycoplasma species (M) gallisepticum, M pneumoniae and M pulmonis are the only mycoplasmas inhibited by CB. These are the only prolaryotes reported to be inhibited by CB. This suggested that these three mycoplasmas might have some sort of cytoskeletal structure. A protein fraction has been isolated from M gallisepticum which polymerizes in 0.6 M KC1 and depolymerizes when KC1 is removed. This fraction contains a major 58,000-dalton protein, a 46,000-dalton protein, and a minor 87,000-dalton protein.     

DNA methylation and Mycoplasma genomes

DNA methylation is one of the many hypotheses proposed to explain the observed deficiency in CpG dinucleotides in a variety of genomes covering a wide taxonomic distribution. Recent studies challenged the methylation hypothesis on empirical grounds. First, it cannot explain why the Mycoplasma genitalium genome exhibits strong CpG deficiency without DNA methylation. Second, it cannot explain the great variation in CpG deficiency between M. genitalium and M. pneumoniae that also does not have CpG-specific methyltransferase genes. I analyzed the genomic sequences of these Mycoplasma species together with the recently sequenced genomes of M. pulmonis, Ureaplasma urealyticum, and Staphylococcus aureus, and found the results fully compatible with the methylation hypothesis. In particular, I present compelling empirical evidence to support the following scenario. The common ancestor of the three Mycoplasma species has CpG-specific methyltransferases, and has evolved strong CpG deficiency as a result of the specific DNA methylation. Subsequently, this ancestral genome diverged into M. pulmonis and the common ancestor of M. pneumoniae and M. genitalium. M. pulmonis has retained methyltransferases and exhibits the strongest CpG deficiency. The common ancestor lost the methyltransferase gene and then diverged into M. genitalium and M. pneumoniae. M. genitalium and M. pneumoniae, after losing methylation activities, began to regain CpG dinucleotides through random mutation. M. genitalium evolved more slowly than M. pneumoniae, gained relatively fewer CpG dinucleotides, and is more CpG-deficient.