Characterization of a Listeria monocytogenes Scott A isolate with high tolerance towards high hydrostatic pressure

An isolate of L. monocytogenes Scott A that is tolerant to high hydrostatic pressure (HHP), named AK01, was isolated upon a single pressurization treatment of 400 MPa for 20 min and was further characterized. The survival of exponential- and stationary-phase cells of AK01 in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer was at least 2 log units higher than that of the wild type over a broad range of pressures (150 to 500 MPa), while both strains showed higher HHP tolerance (piezotolerance) in the stationary than in the exponential phase of growth. In semiskim milk, exponential-phase cells of both strains showed lower reductions upon pressurization than in buffer, but again, AK01 was more piezotolerant than the wild type. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella, were elongated, and showed slightly lower maximum specific growth rates than the wild type at 8, 22, and 30°C. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H2O2 compared with the wild type. The difference in HHP tolerance between the piezotolerant strain and the wild-type strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wild type had identical in situ lipid melting curves as determined by Fourier transform infrared spectroscopy. The demonstrated occurrence of a piezotolerant isolate of L. monocytogenes underscores the need to further investigate the mechanisms underlying HHP resistance of food-borne microorganisms, which in turn will contribute to the appropriate design of safe, accurate, and feasible HHP treatments.

SEF17 fimbriae are essential for the convoluted colonial morphology of Salmonella enteritidis

Salmonella enteritidis isolated from poultry infections generated a convoluted colonial morphology after 48 h growth on colonisation factor antigen (CFA) agar at 25 degrees C. A mutant S. enteritidis defective for the elaboration of the SEF17 fimbrial antigen, in which the agf gene cluster was inactivated by insertion of an ampicillin resistance gene cassette, and other wild-type S. enteritidis transduced to this genotype failed to produce convoluted colonies. However, growth of SEF17(-) mutans at 25 degrees C on CFA agar supplemented with 0.001% Congo red resulted in partial recovery of the phenotype. Immunoelectron microscopy demonstrated that copious amounts of the SEF17 fimbrial antigen were present in the extracellular matrix of convoluted colonies of wild-type virulent S. enteritidis isolates. Bacteria were often hyperflagellated also. Immunoelectron microscopy of SEF17(-) mutants grown on CFA agar+0.001% Congo red demonstrated the elaboration of an as yet undefined fimbrial structure. Isolates of S. enteritidis which were described previously as avirulent and sensitive to environmental stress failed to express SEF17 or produce convoluted colonies. These data indicate an essential role for SEF17, and possibly for another fimbria and flagella, in the generation of the convoluted colonial phenotype. The relationship between virulence and colonial phenotype is discussed.

Colonisation of the chicken caecum by afimbriate and aflagellate derivatives of Salmonella enterica serotype Enteritidis

A semi-quantitative cloacal-swab method was used as an indirect measure of caecal colonisation of one-day old and five-day old chicks after oral dosing with wild-type Salmonella enterica serovar Enteritidis PT4 and,genetically defined isogenic derivatives lacking the ability to elaborate flagella or fimbriae. Birds of both ages were readily and persistently colonised by all strains although there war a decline in shedding by the older birds after about 21 days. There were no significant differences in shedding of wild-type or mutants in single-dose experiments. In competition experiments, in which five-day old birds were dosed orally with wild-type and mutants together, shedding of non-motile derivatives was significantly lower than wild-type, At 35 days post infection, birds were sacrificed and direct counts of mutants and wild-type from each caecum were determined. Whilst there appeared to be poor correlation between direct counts and the indirect swab method, the overall trends shown by these methods of assessment indicated that flagella and not fimbriae were important in caecal colonisation in these models. Crown Copyright (C) 1999 Published by Elsevier Science B.V.

The role of SEF14 and SEF17 fimbriae in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces

To gain an understanding of the role of fimbriae and flagella in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces, the extent of adherence of viable wild-type strains to a polystyrene microtitration plate was determined by a crystal violet staining assay, Elaboration of surface antigens by adherent bacteria was assayed by fimbriae- and flagella-specific ELISAs, Wild-type Enteritidis strains adhered well at 37 degrees C and 25 degrees C when grown in microtitration wells in Colonisation Factor Antigen broth, but not in other media tested, At 37 degrees C, adherent bacteria elaborated copious quantities of SEF14 fimbrial antigen, whereas at 25 degrees C adherent bacteria elaborated copious quantities of SEF17 fimbrial antigen. Non-fimbriate and non-flagellate knock-out mutant strains were also assessed in the adherence assay. Mutant strains unable to elaborate SEF14 and SEF17 fimbriae adhered poorly at 37 degrees C and 25 degrees C, respectively, but adherence was not abolished. Non-motile mutant strains showed reduced adherence whilst type-1, PEF and LPF fimbriae appeared not to contribute to adherence in this assay. These data indicate that SEF17 and SEF14 fimbriae mediate bacterial cell aggregation on inanimate surfaces under appropriate growth conditions.

Virulence factors of Escherichia coli serotypes associated with avian colisepticaemia

The current understanding of the pathogenesis of avian pathogenic Escherichia coli (APEC) in colisepticaemia is limited. This review discusses putative virulence determinants per se, such as a number of surface organelles including fimbriae and flagella; together with other factors such as iron sequestering mechanisms, which are involved in the survival of E. coli in the host rather than initiation of infection. It is concluded that avian colisepticaemia is a multi-factorial disease and that to date only a limited number of virulence factors of APEC have been thoroughly elucidated. Crown Copyright (C) 2002 Published by Elsevier Science Ltd. All rights reserved.

Co-ordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC)

BipA is a novel member of the ribosome binding GTPase superfamily and is widely distributed in bacteria and plants. We report here that it regulates -multiple cell surface- and virulence-associated -components in the enteropathogenic Escherichia coli (EPEC) strain E2348/69. The regulated components include bacterial flagella, the espC pathogenicity island and a type III secretion system specified by the locus of enterocyte effacement (LEE). BipA positively regulated the espC and LEE gene clusters through transcriptional control of the LEE-encoded regulator, Ler. Additionally, it affected the pattern of proteolysis of intimin, a key LEE-encoded adhesin specified by the LEE. BipA control of the LEE operated independently of the previously characterized regulators Per, integration host factor and H-NS. In contrast, it negatively regulated the flagella-mediated motility of EPEC and in a Ler-independent manner. Our results indicate that the BipA GTPase functions high up in diverse regulatory cascades to co-ordinate the expression of key pathogenicity islands and other virulence-associated factors in E. coli.

Membrane ruffling and invasion of human and avian cell lines is reduced for aflagellate mutants of Salmonella enterica serotype Enteritidis

Independent studies have demonstrated that flagella are associated with the invasive process of Salmonella enterica serotypes, and aflagellate derivatives of Salmonella enterica serotype Enteritidis are attenuated in murine and avian models of infection. One widely held view is that the motility afforded by flagella, probably aided by chemotactic responses, mediates the initial interaction between bacterium and host cell. The adherence and invasion properties of two S. Enteritidis wild-type strains and isogenic aflagellate mutants were assessed on HEp-2 and Div-1 cells that are of human and avian epithelial origin, respectively. Both aflagellate derivatives showed a significant reduction of invasion compared with wild type over the three hours of the assays. Complementation of the defective fliC allele recovered partially the wild-type phenotype. Examination of the bacterium-host cell interaction by electron and confocal microscopy approaches showed that wild-type bacteria induced ruffle formation and significant cytoskeletal rearrangements on HEp-2 cells within 5 minutes of contact. The aflagellate derivatives induced fewer ruffles than wild type. Ruffle formation on the Div-1 cell line was less pronounced than for HEp-2 cells for wild-type S. Enteritidis. Collectively, these data support the hypothesis that flagella play an active role in the early events of the invasive process.

Colonization of 8-week-old conventionally reared goats by Escherichia coli O157:H7 after oral inoculation

Enterohaemorrhagic Escherichia coli O157 : H7 infections of man have been associated with consumption of unpasteurized goat's milk and direct contact with kid goats on petting farms, yet little is known about colonization of goats with this organism. To assess the contribution of flagella and intimin of E coli O157 : H7 in colonization of the goat, 8-week-old conventionally reared goats were inoculated orally in separate experiments with 1 X 10(10) c.f.u. of a non-verotoxigenic strain of E coli O157: H7 (strain NCTC 12900 Nal(r)), an aflagellate derivative (DMB1) and an intimin-deficient derivative (DMB2). At 24 In after inoculation, the three E coli O157 : H7 strains were shed at approximately 5 X 1 04 c.f.u. (g faeces)(-1) from all animals. Significantly fewer intimin-deficient bacteria were shed only on days 2 (P = 0(.)003) and 4 (P = 0(.)014), whereas from day 7 to 29 there were no differences. Tissues from three animals inoculated with wild-type E coli O157 : H7 strain NCTC 12900 Nalr were sampled at 24,48 and 96 In after inoculation and the organism was cultured from the large intestine of all three animals and from the duodenum and ileum of the animal examined at 96 h. Tissues were examined histologically but attaching-effacing (AE) lesions were not observed at any intestinal site of the animals examined at 24 or 48 In. However, the animal examined at 96 h, which had uniquely shed approximately 1 x 10(7) E coli O157: H7 (g faeces)(-1) for the preceding 3 days, showed a heavy, diffuse infection with cryptosporidia. and abundant, multifocal AE lesions in the distal colon, rectum and at the recto-anal junction. These AE lesions were confirmed by immunohistochemistry to be associated with E coli O157: H7.

Variable and strain dependent colonisation of chickens by Escherichia coli O157

The prevalence of enterohaemorrhagic Escherichia coli (EHEC) O157 in poultry is considered minimal compared with other species, especially ruminants. However, deliberate inoculation studies have shown that poultry are readily and persistently infected by this organism but that the mechanism of colonisation is independent of intimin, a recognised factor in host-EHEC interactions in mammalian species, and may be dependent upon flagella. Few strains of EHEC O157 have been tested in poultry and here 1-day-old and 6-week-old chicks were inoculated with seven non-toxigenic E. coli O157 strains in separate experiments. Persistence was measured semi-quantitatively by bacteriological assessment of E. coli O157 cultured from cloacal swabs (shedding score). In the 1-day-old chick model that was monitored for 43 days, all seven strains established well after inoculation. In the 6-week-old chicken model, one strain established and gave consistently high shedding for the duration of the experiment (156 days). Whereas of the remaining six strains, two persisted for 113 days, two persisted for 43 days, one persisted for 22 days and one strain was never detected.

Efficacy of a live attenuated Escherichia coli O78:K80 vaccine in chickens and turkeys

A candidate live vaccine for avian pathogenic Escherichia coli (APEC) was constructed from a virulent field APEC O78 strain by mutation of the aroA gene. The mutant was highly similar to the parent wild-type strain in respect of colony morphology, motility, growth in suspension, hemagglutination, Congo Red binding, HEp-2 cell adhesion, and the elaboration of surface antigens type 1 fimbriae and flagella, although production of curli fimbriae was reduced marginally. The mutant proved avirulent when inoculated into 1-day-old chicks by spray application and when presented again in the drinking water at 7 days of age. Chickens and turkeys vaccinated with an O78 aroA mutant were protected against a challenge at 6 wk of age by virulent APEC strains.

Evolutionary resurrection of flagellar motility via rewiring of the nitrogen regulation system

A central process in evolution is the recruitment of genes to regulatory networks. We engineered immotile strains of the bacterium Pseudomonas fluorescens that lack flagella due to deletion of the regulatory gene fleQ. Under strong selection for motility, these bacteria consistently regained flagella within 96 hours via a two-step evolutionary pathway. Step 1 mutations increase intracellular levels of phosphorylated NtrC, a distant homologue of FleQ, which begins to commandeer control of the fleQ regulon at the cost of disrupting nitrogen uptake and assimilation. Step 2 is a switch-of-function mutation that redirects NtrC away from nitrogen uptake and towards its novel function as a flagellar regulator. Our results demonstrate that natural selection can rapidly rewire regulatory networks in very few, repeatable mutational steps.

Leishmania amazonensis META2 protein confers protection against heat shock and oxidative stress

The META cluster of Leishmania amazonensis contains both META1 and META2 genes, which are upregulated in metacyclic promastigotes and encode proteins containing the META domain. Previous studies defined META2 as a 48.0-kDa protein, which is conserved in other Leishmania species and in Trypanosoma brucei. In this work, we demonstrate that META2 protein expression is regulated during the Leishmania life cycle but constitutive in T. brucei. META2 protein is present in the cytoplasm and flagellum of L amazonensis promastigotes. Leishmania META2-null replacement mutants are more sensitive to oxidative stress and, upon heat shock, assume rounded morphology with shortened flagella. The increased susceptibility of null parasites to heat shock is reversed by extra-chromosomal expression of the META2 gene. Defective Leishmania promastigotes exhibit decreased ability to survive in macrophages. By contrast, META2 expression is decreased by 80% in RNAi-induced T. brucei bloodstream forms with no measurable effect on survival or resistance to heat shock. (C) 2010 Elsevier Inc. All rights reserved.

CD8(+) T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (G) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280-288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280-288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations. (C) 2009 Elsevier Ltd. All rights reserved.

Ultrastructure of spermiogenesis and spermatozoa of Gymnotus cf. anguillaris and Brachyhypopomus cf. pinnicaudatus (Teleostei : Gymnotiformes)

The ultrastructure of spermiogenic stages and spermatozoa of representatives of two gymnotiform families, Gymnotus cf. anguillaris (Gymnotidae) and Brachyhypopomus cf. pinnicaudatus (Hypopomidae) were studied. Spermiogenesis of both species is characterized by lateral development of the flagellum and formation of a nuclear fossa. Some differences were found between these species, such as whether (B. cf. pinnicaudatus) or not (G. cf. anguillaris) nuclear rotation occurs, permanence of the cytoplasmic channel, and type and localization of the nuclear fossa. In the G. cf anguillaris spermatozoon the nucleus is spherical with highly condensed chromatin. The nuclear fossa is shallow and lateral and is associated with the centriolar complex through stabilizing fibrils. The midpiece is short, with many vesicles, a cytoplasmic channel, and elongate mitochondria. In the B. cf. pinnicaudatus spermatozoon the ovoid nucleus is elongated lateral and posterior to the centriolar complex, and has highly condensed chromatin. The eccentric nuclear fossa is of the moderate type, and contains the entire centriolar complex. The midpiece is long, with numerous vesicles, elongate mitochondria, and no cytoplasmic channel. In both species the flagella are laterally disposed in relation to the nucleus and comprise of the classical 9 + 2 axoneme. Most of the characteristics found in the spermatozoa of these two species of Gymnotiformes are shared with species of Characiformes, whereas only a few are also found in Siluriformes. This suggests that Gymnotiformes and Characiformes may be more closely related than previously proposed. (C) 2007 Elsevier Ltd. All rights reserved.

Spermiogenesis and spermatozoa ultrastructure in five species of the Curimatidae with some considerations on spermatozoal ultrastructure in the Characiformes

A espermiogênese nas espécies Steindachnerina insculpta, Cyphocharax gillii, C. modestus, C. spilotus e Potamorhina altamazonica de Curimatidae é caracterizada pelo desenvolvimento lateral do flagelo, rotação do núcleo, formação excêntrica da fossa nuclear e cromatina compactada em fibras espessas. Estes espermatozóides exibem uma cabeça esférica contendo um núcleo com cromatina altamente condensada em fibras espessas com pequenas áreas eletronlúcidas, e sem acrossoma. A fossa nuclear é do tipo moderado e excêntrico, penetrada pelo complexo centriolar. A peça média é pequena, tem muitas vesículas alongadas e um curto canal citoplasmático. Mitocôndrias podem ser alongadas, ramificadas ou em forma de C, e são separadas do segmento inicial do axonema pelo canal citoplasmático. O flagelo contém a estrutura clássica do axonema (9+2) e tem um compartimento membranoso na região inicial; não possui expansões laterais (fins). Somente pequenas diferenças foram observadas entre as espécies e gêneros analisados de Curimatidae. A espermiogênese e os espermatozóides de Curimatidae têm muitas das características encontradas em quase todas as outras espécies de Characiformes. Por outro lado, a presença de um compartimento membranoso na região inicial do flagelo dos curimatídeos, uma estrutura comum nos espermatozóides de muitos cipriniformes, é desconhecida em outros characiformes. Discute-se sobre a espermiogênese e espermatozóides de Characiformes.