Spreading factors of Mycoplasma alligatoris, a flesh-eating mycoplasma

Abstract

Mycoplasma alligatoris causes lethal invasive disease of alligators and caimans. A homolog of the nagH gene, encoding a hyaluronidase secreted by Clostridium perfringens, and a C. perfringens hyaluronidase nagI or nagK pseudogene were discovered in the M. alligatoris genome. The nagH gene was detected by PCR in the closest relative of M. alligatoris, Mycoplasma crocodyli, but not in 40 other species representing the Mycoplasma hominis, Mycoplasma pneumoniae, and Spiroplasma phylogenetic clusters. The hyaluronidase activity in the cellular fraction of M. alligatoris and M. crocodyli SP4 broth cultures was equivalent to 10(-16) U of Streptomyces hyalurolyticus hyaluronidase CFU(-1). Negligible activity was present in the cell-free supernatant fraction. No chondroitinase activity was detected. There is also a novel homolog of the nanI gene, which encodes a sialidase secreted by C. perfringens, in the M. alligatoris genome. The signature YRIP and SXDXGXTW motifs and catalytic residues of the clostridial sialidase are conserved in the mycoplasmal gene, but the leader sequence necessary for its secretion by C. perfringens is absent. The gene was not detected by PCR in any other mycoplasma. Potent cell-associated sialidase activity was present in M. alligatoris colonies on agar but not in the cell-free supernatants of broth cultures or in M. crocodyli. The presence of hyaluronidase and sialidase in M. alligatoris is consistent with the rapid invasiveness and necrotizing effects of this organism, and the lack of sialidase in M. crocodyli is consistent with its comparatively attenuated virulence. This genetic and biochemical evidence suggests that the spreading factors hyaluronidase and sialidase, a combination unprecedented in mycoplasmas, are the basis of the virulence of M. alligatoris.

FIG. 1.

Agarose gel electrophoresis of PCR and RT-PCR amplification products of the M. alligatoris nagH gene and mRNA, showing that the gene is expressed during culture in SP4 complex medium and after 48 h of starvation in CMRL 1066 minimal medium. Lane M, marker; lane 1, PCR with DNA template; lane 2, RT-PCR with no template (negative control); lane 3, RT-PCR of mRNA from SP4 culture; lane 4, RT-PCR of mRNA from SP4 culture with no AMV RT (negative control); lane 5, RT-PCR of mRNA from CMRL 1066 culture.

FIG. 2.

Hyaluronidase standard curve and estimation of M. alligatoris hyaluronidase activity. S. hyalurolyticus hyaluronate lyase (Sigma) was serially diluted in PBS (pH 7.5) to obtain concentrations ranging from 1 × 10⁻² to 2 × 10⁻⁵ U 100 μl⁻¹ (open circles). The representative activity of a 100-μl aliquot of M. alligatoris in the early log phase in SP4 broth was compared to the activities of PBS and sterile SP4 negative controls (arrows).

FIG. 3.

M. alligatoris cell-associated sialidase activity. Colonies (upper center plate) and aliquots of cell-free culture supernatant (lower center plate) were overlaid with top agar containing the fluorogenic sialidase substrate MUAN (Sigma) and illuminated with short-wave UV light for comparison to M. crocodyli (right plates) and the negative control M. pulmonis (left plates). The plates in the foreground were positive (type VI C. perfringens neuraminidase [Sigma]) (left plate) and negative (PBS) (right plate) sialidase controls.