Hemolytic anemia as a result of acute M. suis infection is accompanied by coagulopathy, vascular thrombosis, and hemorrhaging of the skin and organs; symptoms that are indicative of the involvement of vascular ECs in the pathogenesis of acute M. suis infection[9, 11, 12]. However, the involvement of ECs in M. suis pathogenesis has received little attention to date because consumption coagulopathy is considered to be the primary cause of hemorrhagic diathesis. Our own recent clinical observations of acute IAP with a fatal outcome led to the hypothesis that interactions with ECs contribute to IAP pathogenesis. Therefore, the aim of the present study was to investigate the interaction between M. suis and ECs, and the resulting endothelial disruption and damage to the vasculature.
SEM and TEM analyses clearly demonstrated marked abnormalities in the endothelium of blood vessels in M. suis-infected pigs. Blood vessels exhibited detached ECs over a considerable portion of the endothelium. In some ultrathin sections, EC were completely missing and the subcellular matrix was exposed. This likely represents the mechanism by which M. suis gained access to the sub-endothelial matrix, where it was detected by TEM. However, the extent of endothelial denudation made the microscopic detection of intracellular M. suis in ECs unfeasible. An analogy can be made with certain Bartonella species, which are also undetectable intracellularly in ECs, even though pathogenesis induced by these species was shown to involve ECs. Additionally, the abnormalities reported herein are typical signs of activated ECs seen in several other diseases, i.e. sickle cell anemia[38, 39], and are highly indicative of dysfunctional activation of the endothelium in IAP.
The formation of microvilli-like membranes on ECs was observed in M. suis-infected animals, which is also characteristic of an activated phenotype, and could serve to increase the frequency of adhesion of M. suis-infected RBCs, as observed in micro vessels from patients infected with P. falciparum. The increased adhesion of M. suis-infected RBCs to the endothelium might be further enhanced by M. suis-induced RBC changes, some of which could trigger a variety of pathologies such as vasoocclusion, endothelial activation, and initiation of the coagulation cascade. The extent of RBC adhesion seen in the liver sinusoids, as well as in heart capillaries, might be a result of a combination of activated ECs with microvilli, altered RBC membranes, and an increased adhesiveness caused by pseudopodia. Similar pathologic patterns are observed in P. falciparum infections.
Numerous arterial thrombi, composed of leukocytes, RBCs, platelets and a network of fibrin fibers, were attached to ECs next to sites of M. suis-induced endothelial injury. This fibrin network acts as a provisional matrix during inflammation and wound healing. Fibrin and fibrinogen metabolites bind to activated platelets and also to vascular EC, thereby activating both cell types[43, 44]. Activated ECs facilitate the translocation of phagocytes into the injured tissue to eliminate bacteria via phagocytosis. Furthermore, in diabetes mellitus and sickle cell anemia, fibrinogen enhances adhesion of pathological RBCs to ECs, and this is associated with a high incidence of thrombosis[46, 47].
The detection of M. suis in direct contact with ECs and attached to the endothelium is an entirely new finding in HM research. To date, HM has been reported to attach and invade a unique host cell, the RBC[1–3]. In the current study, M. suis was attached to ECs either as a single cell or as microcolonies ranging in diameter from 30–50 μm. This direct interaction of M. suis with ECs could trigger the EC destruction and exposure of the subendothelial matrix observed in the M. suis-infected pigs. This would be consistent with other pathogenic Mycoplasma species, such as the large colony strains of M. mycoides subsp. mycoides, which are able to adhere to and destroy caprine ECs, thereby exposing subendothelial collagen and causing vasculitis and coagulation disturbances[18, 20].
The M. suis biofilm-like structures we observed were homogenous aggregates of cross-linked 200–400 nm M. suis cells that formed a three-dimensional network. Occasionally, microcolonies appeared more compact and the bacteria were embedded in a granular matrix. M. suis microcolonies attached to the vascular wall and resembling biofilms have been described for other mycoplasmas. Within these microcolonies, smaller coccoid cells less than 100 nm in diameter were observed on the surface of the larger cells. These M. suis cells probably originated from a budding-like replication mechanism, similar to that proposed for HM, as well as for other mycoplasmas, and they resembled propagating M. suis cells on RBC[1, 49, 50]. These results strongly suggested that M. suis is capable of propagating on ECs.
One of the more novel and more interesting findings of this study was the formation of small fibrils interconnecting several M. suis cells on the surface of RBCs and attached to the vascular wall. M. suis adhere to host RBCs via fibrils[50, 51]. In the current study, the interconnected M. suis cells were observed only on the surface of RBCs attached to the endothelium Further studies are needed to better characterize the composition and function of M. suis fibrils.
In vitro analysis using porcine endothelial cells (PAEC and PEDSV.15 cells) provided further insight into the mechanism of interaction between M. suis and ECs. A considerable number of M. suis cells were attached to the PAEC surface, and actin condensation was evident at the attachment sites. Indications for an interaction between M. suis and porcine actin have been found in a previous study since M. suis-induced autoreactive IgG antibodies also bind to actin[8, 53]. In the present study, we could document by microscopic analysis that M. suis interacts with cytoskeletal actin of endothelial cells leading to cytoskeletal rearrangements in the EC. Various invasive bacteria, such as Yersinia enterocolitica and Listeria monocytogenes, trigger rearrangement of the actin cytoskeleton to facilitate their uptake into eukaryotic cells[54, 55]. Whether M. suis is internalized by a similar mechanism remains to be clarified in further studies. Analysis of the M. suis genome revealed that, similar to other Mycoplasma species, M. suis lack any of the genes required for de novo nucleotide biosynthesis. Thus, establishing itself in a new niche in a nucleated cell would be of critical importance for M. suis in terms of acquiring a source of nucleotides for proliferation. Despite demonstrating a clear and close interaction between M. suis and ECs, definitive proof of the intracellular localization of M. suis in ECs in vivo could not be demonstrated due to the extensive cytopathological effects of M. suis (data not shown). Damaged ECs most likely detached from the endothelium and, thus, were not available for microscopic analysis of intracellular M. suis. Additional studies of sections from infected blood vessels at an early phase of infection may provide the necessary evidence to support this hypothesis.
The activation of ECs by M. suis was demonstrated in vitro by the significant increase of ICAM-1, PECAM-1, E-selectin and P-selectin expressing cells, all of which are important markers of EC activation. Endothelial E-selectin and P-selectin are up-regulated by inflammation and mediate leukocyte capture and rolling on the endothelium. The up-regulation of E-selectin is associated with organ dysfunction and septic shock, both of which are seen in acute IAP. The time point of a maximum upregulation of activation markers by LPS and TNF-alpha might not represent the time point of a maximum upregulation by M. suis. This could explain the relatively smaller but significant difference between M. suis and negative control in case of E-selectin and PECAM-1. ICAM-1 is involved in leukocyte rolling and arrest on endothelial cells, as well as the movement of neutrophils and monocytes on the endothelium. Recently, ICAM-1 was identified as the receptor for rhinovirus. PECAM-1 is involved in the removal of apoptotic neutrophils from the body and makes up a large proportion of the endothelial cell intercellular junction[61, 62], where it mediates transendothelial migration via homotypic binding to PECAM-1 on leukocytes. In summary, these in vitro findings, including the up-regulation of endothelial adhesion receptors, together with the observed structural changes of the endothelial layer in vivo, i.e. microvilli- and gap-formation, demonstrate that M. suis infection results in activation of EC.
Another of the more intriguing findings of the current study was the observation of cardiac muscle damage with disorganized and damaged and/or destroyed cardiac muscles cells. These effects could be explained by RBC aggregation in the blood vessels of M. suis-infected pigs with subsequent occlusion of capillaries leading to ischemia (interruption of the blood supply). This would be consistent with the fact that some cases of acute M. suis infection in pigs result in death within a few days.
In conclusion, we report several novel findings of infection with HM leading to widespread endothelial damage, RBC adhesion to the endothelium, and vascular occlusion. These vascular alterations lead to the development of hemorrhage and organ failure. To our knowledge, this is the first demonstration that HM interacts with host cells other than RBCs. In addition, the ability of M. suis to form biofilm-like microcolonies on the endothelium, which may protect the organism from antimicrobial agents and host immune factors, may contribute to the persistence of HM infections.