Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion

Abstract

BackgroundArcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD), which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition.ResultsRecombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as a pld mutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, the pld mutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type or pld mutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated with A. haemolyticum strains revealed that the pld mutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities.ConclusionsThese data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis of A. haemolyticum infections.

Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae

“…The effect of hemolysins on the association of bacteria with cells was unexpected, but a directed search of the literature showed that the phenomenon is not unprecedented (62)(63)(64)(65). Our findings add important new information by showing (i) that hemolysins increase the association of Gram-positive or Gram-negative bacteria with mammalian cells, (ii) that the effect does not depend on chemotaxis, (iii) that it is independent of pili, and (iv) that it depends on an active response of target cells, which is apparently under the control of class III PI3Ks.…”

Bacteriological characteristics of Arcanobacterium haemolyticum isolated from seven patients with skin and soft-tissue infections

“…Lucas et al (2010) showed that phospholipase D reorganized lipid rafts, resulting in the clustering of adhesion receptors on host cells; thus, it could play a critical role in bacterial adhesion. Furthermore, phospholipase D-expressing A. haemolyticum was able to escape from vacuoles, and phospholipase D caused cellular necrosis.…”

“…upon Gram staining and weak haemolytic activity on conventional media (García-de-la-Fuente et al, 2008). It is therefore important to assess the bacteriological characteristics and presence of virulence factors, such as phospholipase, to identify A. haemolyticum (Lucas et al, 2010).…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…4) (55). This type of SMase D lacks the HKD motif seen in phospholipase Ds (PLDs), explaining why they do not act on phosphatidylcholine (PC) and other glycerophospholipids, although sometimes they are mistakenly referred to as PLDs (56)(57)(58). Their catalytic site, identifiable by the position of the Mg 2ϩ ion, which is essential for activity and octahedrally coordinated by strictly conserved residues, is made up of hydrophobic loops and a negatively charged surface (55).…”

“…haemolyticum, which causes pharyngitis and several invasive diseases, including septic arthritis, osteomyelitis, and meningitis, produces a SMase D which likely plays a role in pathogenicity (57). A wild-type A. haemolyticum strain escapes the phagosome and causes host cell death, apparently by necrosis, whereas a mutant strain lacking the SMase D-encoding gene has a defective phagosomal escape capacity, indicating that SMase D is involved in phagosomal membrane disruption (57). The chromosomal genome of the emergent pathogen C. ulcerans, which causes among other diseases pharyngeal and pulmonary infections in humans, includes a homologous SMase D among its repertoire of virulence factors (63).…”