Lewis G. Tilney

Dual roles of plcA in Listeria monocytogenes pathogenesis

The plcA gene of Listeria monocytogenes encodes a secreted phosphatidylinositol‐speciftc phospholi‐pase C (PI‐PLC). Recent studies have established that transposon mutations within plcA result in avirulence for mice and pleiotropic effects when examined in tissue‐culture models of infection. Genetic analysis reveals that many of the effects of the transposon insertions are due to loss of readthrough transcription from plcA into the downstream gene prfA, which encodes an essential transcription factor of numerous L. monocytogenes virulence genes. Construction of an in‐frame deletion within plcA had no effect on expression of prfA thus allowing direct assignment of a role of the PI‐PLC in pathogenesis. PI‐PLC was shown to play a significant role in mediating escape of L. monocytogenes from phagosomes of primary murine macrophages. Interestingly, this defect manifested itself in vivo in the liver but not in the spleen of infected mice.

The road less traveled: transport of Legionella to the endoplasmic reticulum

Phagosomes containing the bacterial pathogen Legionella pneumophila are transported to the ER after macrophage internalization. To modulate phagosome transport, Legionella use a specialized secretion system that injects bacterial proteins into eukaryotic cells. This review will focus on recent studies that have identified bacterial proteins and host processes that play a concerted role in transporting Legionella to the ER.

Actin filaments, stereocilia, and hair cells of the bird cochlea. III. The development and differentiation of hair cells and stereocilia.

The cochleae of chick embryos of 8 days of incubation until hatching (21 days) were examined by scanning electron microscopy. Unlike what one would expect from the literature, the total number of hair cells per cochlea (10,405 +/- 529) is already determined and visible in a 10-day embryo and the growth of the cochlea is a result of the growth in size and surface area of the hair cells. We also find that the hair cells differentiate simultaneously throughout the cochlea and have followed the differentiation of individual hair cells throughout development. During development we find that the total number, hexagonal packing, and orientation of the stereocilia in each hair cell is determined early and accurately (9- to 10-day embryos). The stereocilia then begin to elongate in all the cells of the cochlea at approximately 0.5 micron/day. By Day 12 the tallest stereocilia in each cell are 1.5-1.8 micron long, the mature length for cells at the proximal end of the cochlea. At this point all stereocilia cease elongating, but those along the inferior edge gradually increase in width from 0.11 micron to maximally 0.19 micron in 17-day embryos. When the stereocilia on the inferior edge reach their mature width, widening ceases and the elongation of stereocilia in the distal hair cells begins again. When these stereocilia have attained their mature lengths, they stop growing. Thus elongation and widening of stereocilia are separated in time. During this period, 11 to 13 days, the shape of the tufts at the proximal end of the cochlea changes. This occurs because stereocilia in the front of each tuft are absorbed while others at the sides appear de novo. This rearrangement converts a circular bundle of stereocilia to a rectangular bundle.

The Deaf Jerker Mouse Has a Mutation in the Gene Encoding the Espin Actin-Bundling Proteins of Hair Cell Stereocilia and Lacks Espins

The espins are actin-bundling proteins of brush border microvilli and Sertoli cell-spermatid junctions. We have determined that espins are also present in hair cell stereocilia and have uncovered a connection between the espin gene and jerker, a recessive mutation that causes hair cell degeneration, deafness, and vestibular dysfunction. The espin gene maps to the same region of mouse chromosome 4 as jerker. The tissues of jerker mice do not accumulate espin proteins but contain normal levels of espin mRNAs. The espin gene of jerker mice has a frameshift mutation that affects the espin C-terminal actin-bundling module. These data suggest that jerker mice are, in effect, espin null and that the jerker phenotype results from a mutation in the espin gene.

Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes.

ABSTRACT Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a host vacuolar compartment and grows rapidly in the cytosol. Listeriolysin O (LLO) is a secreted pore-forming protein essential for the escape of L. monocytogenes from the vacuole formed upon initial internalization. However, its role in intracellular growth and cell-to-cell spread events has not been testable by a genetic approach. In this study, purified six-His-tagged LLO (HisLLO) was noncovalently coupled to the surface of nickel-treated LLO-negative mutants. Bound LLO mediated vacuolar escape in approximately 2% of the mutants. After 5.5 h of growth, cytosolic bacteria were indistinguishable from wild-type bacteria with regard to formation of pseudopod-like extensions, here termed listeriopods, and spread to adjacent cells. However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles. Addition of bound LLO to mutants lacking LLO and two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuoles that are reminiscent of autophagocytic vacuoles. These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptake of bacteria into neighboring cells but that LLO is required for the escape ofL. monocytogenes from the double-membrane vacuole that forms upon cell-to-cell spread.

A plastid segregation defect in the protozoan parasite Toxoplasma gondii.

Apicomplexan parasites—including the causative agents of malaria (Plasmodium sp.) and toxoplasmosis (Toxoplasma gondii)—harbor a secondary endosymbiotic plastid, acquired by lateral genetic transfer from a eukaryotic alga. The apicoplast has attracted considerable attention, both as an evolutionary novelty and as a potential target for chemotherapy. We report a recombinant fusion (between a nuclear‐encoded apicoplast protein, the green fluorescent protein and a rhoptry protein) that targets to the apicoplast but grossly alters its morphology, preventing organellar segregation during parasite division. Apicoplast‐deficient parasites replicate normally in the first infectious cycle and can be isolated by fluorescence‐activated cell sorting, but die in the subsequent host cell, confirming the ‘delayed death’ phenotype previously described pharmacologically, and validating the apicoplast as essential for parasite viability.

Hair cell damage produced by acoustic trauma in the chick cochlea

Examination of pure-tone acoustic damage in the chick basilar papilla revealed that the location and extent of hair cell damage was a function of both the stimulus intensity and the age at which the chicks were exposed. Scanning electron microscopic evaluation of noise-exposed cochleae at post-hatching days 1, 10 and 30 permitted the identification of discrete regions of damage, including hair cells with stereocilia injuries as well as those lost from the epithelium. The hair cell damage was tonotopically distributed along the cochlea according to frequency. However, for each exposure frequency two distinct sites of damage were often produced, and their locations were correlated with stimulus intensity. At low intensities, a longitudinal strip of hair cell damage ran along the superior edge of the basilar papilla. As exposure intensity increased, a second damage site developed along the inferior edge of the basilar papilla, distal to the longitudinal strip. This second type of damage initially took the form of a series of laterally-oriented wedges, but at higher intensities, the wedges coalesced to form a large crescent-shaped patch of damage. The location of the damage sites for each frequency did not shift with age. However, there were differences in the extent and position of the damage which could be correlated with stimulus intensity and with changes in middle ear admittance during development [(1983) Development of Auditory and Vestibular Systems, pp. 3-25. Editor: R. Romand. Academic Press, New York]. These results suggest that developmental changes in the location and extent of hair cell damage depend on the effective stimulus intensity reaching the cochlea, rather than on alterations in the frequency coding of the hair cells.

Structure of actin-containing filaments from two types of non-muscle cells☆

Abstract Bundles of actin-containing filaments from the acrosomal process of horseshoe crab sperm and from sea urchin egg contain a second protein having a molecular weight of about 55,000. Electron micrographs of these filamentous bundles show features reminiscent of paracrystalline arrays of actin except that bundles from the sea urchin egg have distinctive transverse bands every 110 A. From optical diffraction patterns of the micrographs, we deduced very similar models for both structures. The models consist of hexagonal arrays of actin filaments cross-linked by the second protein. The pattern of transverse bands in bundles derived from the sea urchin eggs is accounted for by postulating that the second protein is bonded to actin only at positions where cross-linking can occur, rather than being bonded to every actin. The helical symmetry of the actin requires that the bonding contacts involved in the cross-linking be slightly different at different positions along the length of the bundle. The technique of image reconstruction was used to obtain a three-dimensional map of the bundles from the acrosomal process.

SUBSTRUCTURAL ANALYSIS OF THE MICROTUBULE AND ITS POLYMORPHIC FORMS

Microtubules are composed of 13 protofilaments which are aligned parallel to the long axis of the tubule, as can be seen in thin sections of material fixed in the presence of tannic acid. Evidence is presented based upon Markham rotations of the isolated flagellar axoneme that the bridges (spoke, nexin, dynein) must connect to certain specified protofilaments. By means of geometric considerations it is concluded that the bridges in a Centrohelida and Raphidiophrys also connect certain specified protofilaments. Two polymorphic forms of microtubules were also examined using tannic acid in the fixative, a 320 A tubule produced in Echinosphaerium by treatment with low temperature, and a vinblastine-induced microtubule crystal. In both forms, the arrangement of the subunits is not in the form of linear protofilaments. The vinblastine crystal in transverse section is made up of hexagonally packed tubules approximately 320 A in diameter. The wall of each tubule in turn is composed of two parallel protofilaments which spiral upwards, giving the appearance of a barber pole. The center-to-center separation of adjacent protofilaments is 180 A. We conclude that the tubulin heterodimer lies parallel to the axis of the protofilaments, exposing the colchicine binding site.

Changes in the organization of actin filaments in the stereocilia of noise-damaged lizard cochleae.

Alligator lizards exposed to 105 dB broadband noise for 24 h showed a 33 dB loss in hearing which was almost completely recovered 11 days after removal from the noise. Two lesions were found in the actin filament organization which could affect the rigidity of the stereocilia and thus could account for this hearing loss. These lesions preferentially affect the tallest stereocilia. The more common one is a depolymerization of the actin filaments at the base of the stereocilium where it makes contact with the cuticular plate. This results in a displacement and detachment of the stereocilium from its rootlet, thereby affecting the orientation of the tallest stereocilium. The other lesion involves a loss in crossbridges between adjacent actin filaments in the stereocilium. We demonstrate that such a loss will dramatically affect the rigidity of the stereocilia.