Karsten Hueffer

Chondrocyte necrosis and apoptosis in impact damaged articular cartilage

A decrease in chondrocyte numbers is one characteristic of osteoarthritic cartilage. This decrease may be the result of apoptosis or other forms of cell death induced by mechanical damage. Furthermore, cell death may contribute to the structural and metabolic changes found in osteoarthritic cartilage. Therefore, we investigated cell viability and the mode of cell death in cartilage subjected to an increasing severity of impact loads expected to cause compositional damage and osteoarthritic‐like metabolic alterations. Canine cartilage explants were subjected to cyclic indentation impacts of 5 megapascals at 0.3 Hz for 0, 2, 20, and 120 min and then kept in culture for 2, 4, 48, and 144 h. Cell death was assessed by the TUNEL assay and by uptake of propidium iodide. Viable cells were detected by the ability to metabolize fluorescein diacetate. Nuclear morphology and ultrastructure of the cell were examined using Hoechst 33342 fluorescent staining and transmission electron microscopy (TEM). As controls for necrosis and apoptosis, cartilage was, respectively, frozen and thawed or incubated with mitomycin‐C, an apoptosis inducer. In cartilage that had been loaded for 2 h, 32% of the chondrocytes in the loaded core took up propidium iodide within 2 h after loading. Most of these were in the middle to superficial zones and reflected leaky cell membranes usually characteristic of necrosis. Less than 1% of these chondrocytes were positive in the TUNEL assay after 4 h. After additional culture for 2 days, however, the proportion of chondrocytes which were positive in the TUNEL assay reached 73%. A dose dependent response to duration of loading was detected with the TUNEL assay at this time. The TUNEL assay was not specific for apoptosis since 92% of chondrocytes in freeze/thawed cartilage were TUNEL positive. However, some cells with apoptotic bodies and chromatin condensation characteristic of apoptosis were found in the transition zone between necrotic and normal chondrocytes, but not in the superficial and upper zones, in impact damaged cartilage. We concluded that in this study, necrosis occurred first, followed by apoptosis.

The Natural Host Range Shift and Subsequent Evolution of Canine Parvovirus Resulted from Virus-Specific Binding to the Canine Transferrin Receptor

ABSTRACT Canine parvovirus (CPV) is a host range variant of a feline virus that acquired the ability to infect dogs through changes in its capsid protein. Canine and feline viruses both use the feline transferrin receptor (TfR) to infect feline cells, and here we show that CPV infects canine cells through its ability to specifically bind the canine TfR. Receptor binding on host cells at 37°C only partially correlated with the host ranges of the viruses, and an intermediate virus strain (CPV type 2) bound to higher levels on cells than did either the feline panleukopenia virus or a later strain of CPV. During the process of adaptation to dogs the later variant strain of CPV gained the ability to more efficiently use the canine TfR for infection and also showed reduced binding to feline and canine cells compared to CPV type 2. Differences on the top and the side of the threefold spike of the capsid surface controlled specific TfR binding and the efficiency of binding to feline and canine cells, and these differences also determined the cell infection properties of the viruses.

Parvovirus host range, cell tropism and evolution

The past few years have seen major advances in our understanding of the controls of evolution, host range and cell tropism of parvoviruses. Notable findings have included the identification of the transferrin receptor TfR as the cell surface receptor for canine parvovirus and feline panleukopenia virus, and also the finding that specific binding to the canine TfR led to the emergence of canine parvovirus as a new pathogen in dogs. The structures of the adeno-associated virus-2 and porcine parvovirus capsids, along with those of the minute virus of mice, have also advanced our understanding of parvovirus biology. Structure-function studies have shown that in several different parvoviruses the threefold spikes or peaks of the capsid control several aspects of cell tropism and host range, and that those are subject to selective pressures leading to viral evolution. The cell and tissue tropisms of different adeno-associated virus serotypes were demonstrated to be due, in part, to specific receptor binding.

Diversification of a Salmonella Virulence Protein Function by Ubiquitin-Dependent Differential Localization

Many bacterial pathogens and symbionts utilize type III secretion systems to deliver bacterial effector proteins into host cells. These effector proteins have the capacity to modulate a large variety of cellular functions in a highly regulated manner. Here, we report that the phosphoinositide phosphatase SopB, a Salmonella Typhimurium type III secreted effector protein, diversifies its function by localizing to different cellular compartments in a ubiquitin-dependent manner. We show that SopB utilizes the same enzymatic activity to modulate actin-mediated bacterial internalization and Akt activation at the plasma membrane and vesicular trafficking and intracellular bacterial replication at the phagosome. Thus, by exploiting the host cellular machinery, Salmonella Typhimurium has evolved the capacity to broaden the functional repertoire of a virulence factor to maximize its ability to modulate cellular functions.

The biochemical properties of the Francisella pathogenicity island (FPI)-encoded proteins IglA, IglB, IglC, PdpB and DotU suggest roles in type VI secretion

The Francisella pathogenicity island (FPI) encodes proteins thought to compose a type VI secretion system (T6SS) that is required for the intracellular growth of Francisella novicida. In this work we used deletion mutagenesis and genetic complementation to determine that the intracellular growth of F. novicida was dependent on 14 of the 18 genes in the FPI. The products of the iglABCD operon were localized by the biochemical fractionation of F. novicida, and Francisella tularensis LVS. Sucrose gradient separation of water-insoluble material showed that the FPI-encoded proteins IglA, IglB and IglC were found in multiple fractions, especially in a fraction that did not correspond to a known membrane fraction. We interpreted these data to suggest that IglA, IglB and IglC are part of a macromolecular structure. Analysis of published structural data suggested that IglC is an analogue of Hcp, which is thought to form long nano-tubes. Thus the fractionation properties of IglA, IglB and IglC are consistent with the current model of the T6SS apparatus, which supposes that IglA and IglB homologues form an outer tube structure that surrounds an inner tube composed of Hcp (IglC) subunits. Fractionation of F. novicida expressing FLAG-tagged DotU (IcmH homologue) and PdpB (IcmF homologue) showed that these proteins localize to the inner membrane. Deletion of dotU led to the cleavage of PdpB, suggesting an interaction of these two proteins that is consistent with results obtained with other T6SSs. Our results may provide a mechanistic basis for many of the studies that have examined the virulence properties of Francisella mutants in FPI genes, namely that the observed phenotypes of the mutants are the result of the disruption of the FPI-encoded T6SS structure.

Salmonella-induced macrophage death: multiple mechanisms, different outcomes.

The facultative intracellular pathogen Salmonella enterica triggers programmed cell death in macrophages. The close examination of this phenomenon has revealed an unusually complex picture involving diverse mechanisms that lead to different types of programmed cell death. It appears that the outcome of the interaction of salmonella with macrophages depends on the relative contribution of two type III protein secretion systems, in conjunction with the stimulation of innate immunity outputs through conserved determinants collectively known as ‘pathogen‐associated molecular patterns’ (PAMPs). These interactions result in a breakdown of the balance between survival and pro‐apoptotic cellular pathways, which eventually leads to macrophage cell death. The relative significance for the infection process of the different types of macrophage cell death triggered by salmonella remains to be established

Combinations of two capsid regions controlling canine host range determine canine transferrin receptor binding by canine and feline parvoviruses.

ABSTRACT Feline panleukopenia virus (FPV) and its host range variant, canine parvovirus (CPV), can bind the feline transferrin receptor (TfR), while only CPV binds to the canine TfR. Introducing two CPV-specific changes into FPV (at VP2 residues 93 and 323) endowed that virus with the canine TfR binding property and allowed canine cell infection, although neither change alone altered either property. In CPV the reciprocal changes of VP2 residue 93 or 323 to the FPV sequences individually resulted in modest reductions in infectivity for canine cells. Changing both residues in CPV to the FPV amino acids blocked the canine cell infection, but that virus was still able to bind the canine TfR at low levels. This shows that both CPV-specific changes control canine TfR binding but that binding is not always sufficient to mediate infection.

Structures of host range-controlling regions of the capsids of canine and feline parvoviruses and mutants.

ABSTRACT Canine parvovirus (CPV) and feline panleukopenia virus (FPV) differ in their ability to infect dogs and dog cells. Canine cell infection is a specific property of CPV and depends on the ability of the virus to bind the canine transferrin receptor (TfR), as well as other unidentified factors. Three regions in the capsid structure, located around VP2 residues 93, 300, and 323, can all influence canine TfR binding and canine cell infection. These regions were compared in the CPV and FPV capsid structures that have been determined, as well as in two new structures of CPV capsids that contain substitutions of the VP2 Asn-93 to Asp and Arg, respectively. The new structures, determined by X-ray crystallography to 3.2 and 3.3 Å resolutions, respectively, clearly showed differences in the interactions of residue 93 with an adjacent loop on the capsid surface. Each of the three regions show small differences in structure, but each appears to be structurally independent of the others, and the changes likely act together to affect the ability of the capsid to bind the canine TfR and to infect canine cells. This emphasizes the complex nature of capsid alterations that change the virus-cell interaction to allow infection of cells from different hosts.

Residues in the Apical Domain of the Feline and Canine Transferrin Receptors Control Host-Specific Binding and Cell Infection of Canine and Feline Parvoviruses

ABSTRACT Canine parvovirus (CPV) and feline panleukopenia virus (FPV) capsids bind to the transferrin receptors (TfRs) of their hosts and use these receptors to infect cells. The binding is partially host specific, as FPV binds only to the feline TfR, while CPV binds to both the canine and feline TfRs. The host-specific binding is controlled by a combination of residues within a raised region of the capsid. To define the TfR structures that interact with the virus, we altered the apical domain of the feline or canine TfR or prepared chimeras of these receptors and tested the altered receptors for binding to FPV or CPV capsids. Most changes in the apical domain of the feline TfR did not affect binding, but replacing Leu221 with Ser or Asp prevented receptor binding to either FPV or CPV capsids, while replacing Leu221 with Lys resulted in a receptor that bound only to CPV but not to FPV. Analysis of recombinants of the feline and canine TfRs showed that sequences controlling CPV-specific binding were within the apical domain and that more than one difference between these receptors determined the CPV-specific binding of the canine TfR. Single changes within the canine TfR which removed a single amino acid insertion or which eliminated a glycosylation site gave that receptor the expanded ability to bind to FPV and CPV. In some cases, binding of capsids to mutant receptors did not result in infection, suggesting a structural role for the receptor in cell infection by the viruses.

Francisella Genes Required for Replication in Mosquito Cells

Abstract Francisella tularensis, a potential bioterrorism agent, is transmitted by arthropod vectors and causes tularemia in many mammals, including humans. Francisella novicida causes disease with similar pathology in mice. We show that F. novicida invades hemocyte-like cells of the Sua1B cell line derived from Anopheles gambiae and replicates vigorously within these cells. We used transposon knockouts of single genes of F. novicida to show that bacterial growth within these insect cells is dependent on virulence factors encoded in a bacterial pathogenicity island that has been linked to replication in mammalian macrophages. The virulence factors MglA, IglA, IglB, IglC, and IglD as well as PdpA and PdpB were necessary for efficient growth in insect cells, but PdpC and PdpD were not required. The Sua1B cell line presents a valuable model to study the interactions between this important pathogen and insect vectors.