Salmonella enterica, the cause of food poisoning and typhoid fever, induces actin cytoskeleton rearrangements and membrane ruffling to gain access into nonphagocytic cells, where it can replicate and avoid innate immune defenses. Here, we found that SopB, a phosphoinositide phosphatase that is delivered into host cells by a type III secretion system, was essential for the establishment of Salmonella's intracellular replicative niche. SopB mediated the formation of spacious phagosomes following bacterial entry and was responsible for maintaining high levels of phosphatidylinositol-three-phosphate [PtdIns(3)P] in the membrane of the bacteria-containing vacuoles. Absence of SopB caused a significant defect in the maturation of the Salmonella-containing vacuole and impaired bacterial intracellular growth.

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.Canine parvovirus (CPV) emerged in 1978 as the cause of new enteric and myocardial diseases in dogs. The new virus spread globally in a pandemic of disease during 1978 and has since remained endemic in dogs throughout the world (27, 43). The 1978 strain of CPV (termed CPV type 2) was a new virus infecting dogs since there is no serological or other evidence for infection of dogs by a related virus prior to the mid-1970s (27). Phylogenetic analysis shows that all CPV isolates were descended from a single ancestor which emerged during the mid-1970s, which was closely related to the long-known feline panleukopenia virus (FPV) which infects cats, mink, and raccoons but not dogs or cultured dog cells (43). FPV and CPV isolates differ by as little as 0.5% in DNA sequence, and the characteristic properties of CPV type 2 are controlled by a small number of changes in the capsid surface. Two differences between FPV and CPV changed VP2 residues 93 from Lys to Asn and 323 from Asp to Asn, and those changes alone could introduce the canine host range, a CPV-specific antigenic epitope, and a difference in the pH dependence of hemagglutination into FPV (9, 14). Despite the close relationship to FPV, CPV type 2 isolates did not replicate in cats (42,44), and this host range was determined at least in part by VP2 residues 80, 564, and 568 which are in close proximity in the capsid structure (41). Other mutations in the same structural region of CPV type 2 were selected by passage in cat cells (VP2 residue 300 from Ala to Asp), and these reduced the infection of canine cells, as did closely adjacent changes in in vitro prepared mutants (VP2 residue 299 Gly to Glu) (18,26).Host range-controlling residues are located on a raised region of the capsid that surrounds the threefold axis (the threefold spike) (9, 46). VP2 residues 93 and 323 are found near the top of that structure, whereas residues 299 and 300, and changes controlling feline host range, are all on a ridge ...

SUMMARY 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 traffic 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.

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 freezekhawed 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. 0

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 nanotubes. 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.

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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.

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.