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.Mycoplasma alligatoris causes a lethal invasive disease in adult alligators (Alligator mississippiensis) and closely related caimans (Caiman latirostris). The pathology observed as early as 1 week after infection by instillation via the glottis includes necrotizing pneumonia, severe pericarditis, necrotizing myocarditis, lymphocytic interstitial nephritis, lymphocytic periportal hepatitis, splenic hyperplasia, pyogranulomatous meningitis, and necrotizing synovitis (9,10,11,13,18,33). Hatchling alligators also rapidly developed disseminated M. alligatoris mycoplasmosis after intratracheal instillation, and the lesions are similar to those of adults and include acute multifocal brainstem hemorrhage (35). Mycoplasma crocodyli (23), the closest known relative of M. alligatoris (98% 16S rRNA gene similarity [9]), causes a similar necrotizing synovitis and occasionally subacute pneumonia in Nile crocodiles (Crocodylus niloticus) (27,28). The severity of the lesions correlates with the numbers of M. alligatoris cells in affected tissues, suggesting that a spreading factor(s) contributes to the virulence. The extracellular matrix (ECM)-degrading enzymes that act as bacterial spreading factors include hyaluronidases, sialidases, and mucinases (26...

Genetic variation in sialidase and linkage to N-acetylneuraminate catabolism in Mycoplasma synoviae

“…ABS50356), N-acetylneuraminate lyase nanA, N-acetylmannosamine kinase nagC, Nacetylmannosamine-6-phosphate epimerase nanE, N-acetylglucosamine-6-phosphate deacetylase nagA, and glucosamine-6-phosphate deaminase nagB genes in a locus comprising a canonical sialic acid scavenging and degradation pathway ( Figure 1A) [11]. This was unexpected, although sialidase activity is common in other pathogenic bacteria [12], because it is very rare in mycoplasmas, having been described previously only in the lethal pathogen of alligators Mycoplasma alligatoris [13] and an extinct strain of the avian pathogen Mycoplasma gallisepticum [14,15,16]. The term sialic acid is the family name covering all derivatives of neuraminic acid [17], the aldol condensation product of D-mannosamine and pyruvic acid, which are potential bacterial nutrients.…”

Role of sialidase in Mycoplasma alligatoris-induced pulmonary fibroblast apoptosis

“…Interaction of sialidase with another virulence factor(s) seems necessary to cause the pro-apoptotic effects of M. alligatoris infection. A prominent candidate that emerged from our M. alligatoris genome survey is hyaluronidase (Brown et al, 2004), because in other diseases the host cell HA receptor CD44 is modulated by the specific combination of sialidase and hyaluronidase to transduce signals leading to inflammation, CD95 upregulation, and excessive cell death (Bartolazzi et al, 1996;Fujii et al, 2001;Gee et al, 2004;Hauptschein et al, 2005;Katoh et al, 1995;Lesley et al, 1997;Pure and Cuff, 2001). Although there is no consensus on the molecular mechanisms by which CD44 is linked to apoptosis (Hauptschein et al, 2005), HA fragments generated from the ECM by hyaluronidases stimulate HER2 and c-Src tyrosine kinase phosphorylation of cytoskeletal proteins, and Rho GTPase signaling, through desialylated CD44 receptor-specific networks (Ponta et al, 2003;Turley et al, 2002).…”

“…In addition, even potent competitive inhibitors like DANA probably cannot completely extinguish bacterial surfaceassociated sialidase activity directly at the intimate host-pathogen interface during infection. Because its small genome includes only single copies of sialidase and hyaluronidase genes (Brown et al, 2004), M. alligatoris should be readily amenable to gene inactivation and complementation approaches to model the effects of bacterial sialidase and hyaluronidase on host CD44 receptor and apoptosis signal transduction pathways potentially modulated by many important pathogens. Mycoplasma alligatoris infection promotes surface CD95 expression and apoptosis of primary pulmonary fibroblasts.…”

“…For example, intrapleural inoculation with a dose of M. crocodyli strain MP145 T which was 3 log 10 greater than the LD 50 of M. alligatoris strain A21JP2 T caused only mild respiratory disease (Mohan et al, 2001;Mohan et al, 1995). However, M. crocodyli strain MP145 T does not possess sialidase (Brown et al, 2004).…”

“…M. alligatoris can be cultured in high numbers from the lower respiratory tract of infected individuals, where pathologic changes include fibrinous pleuritis, pulmonary edema and congestion, and diffuse interstitial to fibronecrotic pneumonia as early as 1 wk after exposure (Brown et al, 2001b;Brown et al, 2001c;Pye et al, 2001). A genome survey revealed that M. alligatoris strain A21JP2 T notably possesses the "spreading factors" sialidase and hyaluronidase, a combination unprecedented among mycoplasmas but common among other invasive pathogens, which could degrade host extracellular matrix (ECM) glycans during nutrient scavenging to contribute to disease (Brown et al, 2004). Its comparatively attenuated sibling species Mycoplasma crocodyli also expresses hyaluronidase, so mechanical ECM damage alone seems insufficient to explain the particular severity of M. alligatoris infection.…”