The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes.

Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-A pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37 degrees C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.

Possible risk factors for keel bone damage in organic laying hens.

Keel bone damage (KBD) in laying hens is an important welfare problem in both conventional and organic egg production systems. We aimed to identify possible risk factors for KBD in organic hens by analysing cross-sectional data of 107 flocks assessed in eight European countries. Due to partly missing data, the final multiple regression model was based on data from 50 flocks. Keel bone damage included fractures and/or deviations, and was recorded, alongside with other animal based measures, by palpation and visual inspection of at least 50 randomly collected hens per flock between 52 and 73 weeks of age. Management and housing data were obtained by interviews, inspection and by feed analysis. Keel bone damage flock prevalences ranged from 3% to 88%. Compiled on the basis of literature and practical experience, 26 potential associative factors of KBD went into an univariable selection by Spearman correlation analysis or Mann-Whitney U test (with P<0.1 level). The resulting nine factors were presented to stepwise forward linear regression modelling. Aviary v. floor systems, absence of natural daylight in the hen house, a higher proportion of underweight birds, as well as a higher laying performance were found to be significantly associated with a higher percentage of hens with KBD. The final model explained 32% of the variation in KBD between farms. The moderate explanatory value of the model underlines the multifactorial nature of KBD. Based on the results increased attention should be paid to an adequate housing design and lighting that allows the birds easy orientation and safe manoeuvring in the system. Furthermore, feeding management should aim at sufficient bird live weights that fulfil breeder weight standards. In order to achieve a better understanding of the relationships between laying performance, feed management and KBD further investigations are needed.

Stability of fear and sociality in two strains of laying hens.

1. This trial studied the effects of strain and age on tonic immobility (TI) duration, emergence time (ET) and social reinstatement time (SRT) in laying hens and investigated the consistency of individual behavioural characteristics over rearing and laying periods and the correlations between these behavioural traits. 2. One hundred chicks from each of ISA Brown (ISA) and Lohmann Tradition (LT) laying hens were reared from one day old in pens. At 3 weeks, birds of each line were divided into 4 groups. Twenty birds in one group of each line were marked individually for repeated testing and the other groups were assigned for single testing to test the habituation effect and possible age effects at a group level. 3. ISA birds had higher overall means for TI duration and latency to leave the start box. ISA also showed longer latency in SRT at week 28 than Lohmanns. TI duration increased from weeks 3 to 10 and then decreased to week 35 in both lines. The latency to explore the test area and to reinstate decreased from weeks 10 to 35. 4. Tonic immobility, exploratory and social reinstatement behaviours were consistent over time in both lines, as revealed by Kendall's W coefficient of concordance. 5. In social test situations, an inter-situational consistency was found, that is, birds emerged quickly from the start box and reinstated quickly with their companion. TI (non-social test) was negatively correlated with ET and SRT. Thus the two lines of laying hens respond differently in social and non-social tests.

[Importance of the rearing period for laying hens in alternative systems]. / Bedeutung der Aufzucht der Legehennen für alternative Haltungsformen.

Feather pecking and cannibalism are still major problems in alternative systems for laying hens. Literature and practical experience indicate that unfavourable rearing conditions might be important risk factors for the occurrence of these behavioural disturbances during the laying period. Typical rearing conditions of laying hens from 50 rearing units in Germany and Austria are presented. Obvious risk factors during rearing for feather pecking and cannibalism during the laying period were found. Most flocks were kept under high stocking density (mean: 15 pullets per m' useable area) and some flocks had access to litter only after the second week of life or access to raised perches after the fourth week of life. Plumage condition of pullets and laying hens varied widely in non-beak-trimmed as well as in beak-trimmed flocks. The percentage of pullets with damaged plumage was higher in beak-trimmed than in non-beak-trimmed flocks (medians: 53 % versus 30 %, p = 0,022). In laying hens there was a higher percentage of hens with plumage damage in non-beak-trimmed flocks compared to beak-trimmed flocks (medians: 23 % versus 50 %, p = 0,007). Data analysis will be continued, especially with regard to particular risk factors.

Hyperexpression of listeriolysin in the nonpathogenic species Listeria innocua and high yield purification.

Listeriolysin, the hemolysin of the pathogenic species Listeria monocytogenes, was expressed in the non-pathogenic species Listeria innocua. Coexpression of the positive regulatory factor prfA in the plasmid vector in conjunction with the structural gene hly increased the expression over 500-fold. Purification from supernatant fluids was achieved by two steps of ion exchange chromatography. The procedure resulted in over 60% yield of a hemolytically active, homogeneous 58 kDa protein which was used to produce monospecific antibodies. As shown by immunoblot the purified listeriolysin was free of p60, a highly immunogenic protein of similar size also produced by Listeria spp., which otherwise would interfere with immunoassays. Listeriolysin retained full activity for more than 6 months at -70 degrees C.

Influence of pop hole dimensions on the number of laying hens outside on the range.

The aim was to evaluate whether pop hole width is a factor influencing the number of laying hens on the range. Eight groups of 256 birds each were kept in 8 compartments in a deep litter system. Hens could leave each compartment through two equally-sized pop holes arranged evenly along the side of each compartment. Pop hole dimensions were varied every second week in each compartment in a random order from 30, 60, 90, 120 up to 150 x 30 cm (width x height). Range per hen (10 m(2)) were provided. The number of laying hens on range was counted hourly from 07:00 to 20:00 h. Pop hole width did not significantly influence the number of laying hens on the range. Our findings show that, within the limits of the dimensions investigated, other factors are more important than pop hole dimensions.

Neutralizing monoclonal antibodies against listeriolysin: mapping of epitopes involved in pore formation.

Six different mouse monoclonal antibodies (MAbs) and a specific rabbit polygonal antibody were raised against listeriolysin. Four of the MAbs also recognized seeligeriolysin, and five cross-reacted with ivanolysin. The hemolytic activity could be neutralized by the polygonal antibody as well as by five of the MAbs. None of the neutralizing antibodies interfered with the binding of listeriolysin to the cellular membrane. The epitopes recognized by the MAbs were localized by using overlapping synthetic peptides between positions 59 and 279, a region hitherto not implicated in mediating hemolytic activity.

The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton.

Listeria monocytogenes, a facultative intracellular pathogen, employs actin and other microfilament-associated proteins to move through the host cell cytoplasm. Isogenic mutants of L. monocytogenes lacking the surface-bound ActA polypeptide no longer interact with cytoskeletal elements and are, as a consequence, non-motile (Domann et al., 1992, EMBO J., 11, 1981-1990; Kocks et al., 1992, Cell, 68, 521-531). To investigate the interaction of ActA with the microfilament system in the absence of other bacterial factors, the listerial actA gene was expressed in eukaryotic cells. Immunofluorescence studies revealed that the complete ActA, including its C-terminally located bacterial membrane anchor, colocalized with mitochondria in transfected cells. When targeted to mitochondria, the ActA polypeptide recruited actin and alpha-actinin to these cellular organelles with concomitant reorganization of the microfilament system. Removal of the internal proline-rich repeat region of ActA completely abrogated interaction with cytoskeletal components. Our results identify the ActA polypeptide as a nucleator of the actin cytoskeleton and provide the first insights into the molecular nature of such controlling elements in microfilament organization.

Localization of the ActA polypeptide of Listeria monocytogenes in infected tissue culture cell lines: ActA is not associated with actin "comets".

The ActA protein of the gram-positive pathogen Listeria monocytogenes is a 90-kDa polypeptide required for interaction of the bacteria with components of the host cell microfilament system to generate intra- and intercellular movement. To study the localization, distribution, and expression of the ActA polypeptide in L. monocytogenes grown either in broth culture or in infected tissue culture cells, we first isolated ActA by monoclonal antibody-based immunoaffinity chromatography. Polyclonal rabbit antisera raised against purified ActA revealed that ActA was associated with the cell wall and exposed on the surface of the bacteria, readily accessible to ActA antibodies. In contrast, a C-terminally truncated ActA1 polypeptide expressed by the isogenic actA1 mutant was detected only in the supernatant fluids. Immunofluorescence microscopy and electron microscopic studies using immunogold labeling showed that ActA was present on the surface of the bacteria infecting PtK2 and J774 cells at all stages of the infection cycle and was not found to be associated with the actin "tail" of individual bacteria. For the isogenic actA1 mutant strain, which grew as microcolonies within infected cells, only diffuse staining of the secreted ActA1 polypeptide in the host cytoplasm was observed. The ActA polypeptide therefore appears to be required in the initiation of actin accumulation by the bacterium and is apparently not directly involved in the generation of the actin tail. Analysis of strains of several L. monocytogenes serotypes indicated microheterogeneity in the molecular weights of the ActA polypeptides of individual strains and led to the detection of a serotype 3a strain that does not produce ActA.

Detection of a prfA-independent promoter responsible for listeriolysin gene expression in mutant Listeria monocytogenes strains lacking the PrfA regulator.

Expression of listeriolysin, a major virulence factor of pathogenic Listeria monocytogenes, is positively regulated by the pleiotropic virulence regulator PrfA. In this study, we demonstrate that L. monocytogenes strains lacking the prfA regulator gene produce listeriolysin in small, albeit detectable, amounts when analyzed in a hemolysin assay and by immunoblots with listeriolysin-specific monoclonal antibodies. Transcriptional analysis revealed the existence of a PrfA-independent promoter that was responsible for the hemolytic activity expressed by these strains.

Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics.

Drosophila Enabled is required for proper formation of axonal structures and is genetically implicated in signaling pathways mediated by Drosophila AbI. We have identified two murine proteins, Mena and Evl, that are highly related to Enabled as well as VASP (Vasodilator-Stimulated Phosphoprotein). A conserved domain targets Mena to localized proteins containing a specific proline-rich motif. The association of Mena with the surface of the intracellular pathogen Listeria monocytogenes and the G-actin binding protein profilin suggests that this molecule may participate in bacterial movement by facilitating actin polymerization. Expression of neural-enriched isoforms of Mena in fibroblasts induces the formation of abnormal F-actin-rich outgrowths, supporting a role for this protein in microfilament assembly and cell motility.

IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formation.

Invasion of epithelial cells by Shigella flexneri involves entry and intercellular dissemination. Entry of bacteria into non-phagocytic cells requires the IpaA-D proteins that are secreted by the Mxi-Spa type III secretion machinery. Type III secretion systems are found in several Gram-negative pathogens and serve to inject bacterial effector proteins directly into the cytoplasm of host cells. In this study, we have analysed the IpgD protein of S. flexneri, the gene of which is located on the virulence plasmid at the 5' end of the mxi-spa locus. We have shown that IpgD (i) is stored in the bacterial cytoplasm in association with a specific chaperone, IpgE; (ii) is secreted by the Mxi-Spa type III secretion system in amounts similar to those of the IpaA-D proteins; (iii) is associated with IpaA in the extracellular medium; and (iv) is involved in the modulation of the host cell response after contact of the bacterium with epithelial cells. This suggests that IpgD is an effector that might be injected into host cells to manipulate cellular processes during infection.

Identification and purification of novel internalin-related proteins in Listeria monocytogenes and Listeria ivanovii.

Monoclonal antibodies were generated against a 30-kDa protein fraction derived from culture supernatants of a Listeria monocytogenes strain complemented with additional copies of the prfA regulator gene. Several of the antibodies reacted specifically with a hitherto unidentified, secreted 30-kDa polypeptide. By immunoblot analysis, the expression of this 30kDa polypeptide was found to be dependent on the presence of the PrfA regulator protein. Microsequencing of peptides derived from the partially purified 30-kDa protein revealed homologies to the InlA and InlB polypeptides of L. monocytogenes, which are required for the internalization of the bacteria into nonphagocytic cell lines. This prompted us to term the 30-kDa polypeptide internalin-related protein (Irp). Irp-specific monoclonal antibodies cross-reacted with a 24-kDa polypeptide present in culture supernatants of Listeria ivanovii, indicating the existence of an Irp-related protein in this pathogenic Listeria species.

Exploitation of microfilament proteins by Listeria monocytogenes: microvillus-like composition of the comet tails and vectorial spreading in polarized epithelial sheets.

Effective cell-to-cell spreading of the facultative intracellular pathogen Listeria monocytogenes requires the interaction between bacteria and the microfilament system of the host cell. By recruiting actin filaments into a 'comet tail' localized at one pole of the bacterial cell wall, Listeria become mobile and propel themselves through the cytoplasm. They create protrusions at the plasma membrane that can invaginate adjacent cells. In this work, we have analysed the structural composition of Listeria-recruited microfilaments in various epithelial cell lines by immunofluorescence microscopy. The microfilament-crosslinking proteins alpha-actinin, fimbrin and villin were localized around bacteria as soon as actin filaments could be detected on the bacterial surface. Surprisingly, the same was found for ezrin/radixin, proteins involved in linking microfilaments to the plasma membrane. We found that in a polarized cell line derived from brush border kidney epithelium (LLC-PK1), the actin filaments surrounding intracytoplasmic motile bacteria show the same immunoreactivity as the brush border-like microvilli, when analysed by a specific actin antibody. The successful invasion of polarized LLC-PK1 islets is vectorial, i.e. it progresses predominantly from the periphery of the islets towards the centre. Infection of the peripheral cells is sufficient for infiltration of the entire cellular islets, without any further contact with the extracellular milieu. This is in contrast to nonpolarized epithelial sheets, which can be invaded from the apical surface of any individual cell. The importance of active bacterial motility in this vectorial spreading is emphasized by our finding that an isogenic Listeria mutant that is unable to recruit actin filaments cannot colonize polarized epithelial layers but accumulates in the peripheral cells of the islets.

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.

A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family.

The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. We identify E/DFPPPPXD/E, a motif repeated 4-fold within the primary sequence of ActA, as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein-protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. Taken together we demonstrate that ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling.