The Polar Legionella Icm/Dot T4SS Establishes Distinct Contact Sites with the Pathogen Vacuole Membrane.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen that survives inside phagocytic host cells by establishing a protected replication niche, termed the "Legionella-containing vacuole" (LCV). To form an LCV and subvert pivotal host pathways, L. pneumophila employs a type IV secretion system (T4SS), which translocates more than 300 different effector proteins into the host cell. The L. pneumophila T4SS complex has been shown to span the bacterial cell envelope at the bacterial poles. However, the interactions between the T4SS and the LCV membrane are not understood. Using cryo-focused ion beam milling, cryo-electron tomography, and confocal laser scanning fluorescence microscopy, we show that up to half of the intravacuolar L. pneumophila bacteria tether their cell pole to the LCV membrane. Tethering coincides with the presence and function of T4SSs and likely promotes the establishment of distinct contact sites between T4SSs and the LCV membrane. Contact sites are characterized by indentations in the limiting LCV membrane and localize juxtaposed to T4SS machineries. The data are in agreement with the notion that effector translocation occurs by close membrane contact rather than by an extended pilus. Our findings provide novel insights into the interactions of the L. pneumophila T4SS with the LCV membrane in situ. IMPORTANCE Legionnaires' disease is a life-threatening pneumonia, which is characterized by high fever, coughing, shortness of breath, muscle pain, and headache. The disease is caused by the amoeba-resistant bacterium L. pneumophila found in various soil and aquatic environments and is transmitted to humans via the inhalation of small bacteria-containing droplets. An essential virulence factor of L. pneumophila is a so-called "type IV secretion system" (T4SS), which, by injecting a plethora of "effector proteins" into the host cell, determines pathogen-host interactions and the formation of a distinct intracellular compartment, the "Legionella-containing vacuole" (LCV). It is unknown how the T4SS makes contact to the LCV membrane to deliver the effectors. In this study, we identify indentations in the host cell membrane in close proximity to functional T4SSs localizing at the bacterial poles. Our work reveals first insights into the architecture of Legionella-LCV contact sites.

A contractile injection system stimulates tubeworm metamorphosis by translocating a proteinaceous effector.

The swimming larvae of many marine animals identify a location on the sea floor to undergo metamorphosis based on the presence of specific bacteria. Although this microbe-animal interaction is critical for the life cycles of diverse marine animals, what types of biochemical cues from bacteria that induce metamorphosis has been a mystery. Metamorphosis of larvae of the tubeworm Hydroides elegans is induced by arrays of phage tail-like contractile injection systems, which are released by the bacterium Pseudoalteromonas luteoviolacea. Here we identify the novel effector protein Mif1. By cryo-electron tomography imaging and functional assays, we observe Mif1 as cargo inside the tube lumen of the contractile injection system and show that the mif1 gene is required for inducing metamorphosis. Purified Mif1 is sufficient for triggering metamorphosis when electroporated into tubeworm larvae. Our results indicate that the delivery of protein effectors by contractile injection systems may orchestrate microbe-animal interactions in diverse contexts.

Robust workflow and instrumentation for cryo-focused ion beam milling of samples for electron cryotomography.

Electron cryotomography is able to visualize macromolecular complexes in their cellular context, in a frozen-hydrated state, and in three dimensions. The method, however, is limited to relatively thin samples. Cryo-focused ion beam (FIB) milling is emerging as a powerful technique for sample thinning prior to cryotomography imaging. Previous cryo-FIB milling reports utilized custom-built non-standard equipment. Here we present a workflow and the required commercially available instrumentation to either implement the method de novo, or as an upgrade of pre-existing dual beam milling instruments. We introduce two alternative protocols and the respective sample holders for milling. The "bare grid holder" allows for milling on plain grids, having the advantage of enabling relatively shallow milling angles for wedge geometries. The "Autogrid holder" is designed for milling grids clamped into a mechanical support ring (Autogrid), resulting in increased stability for lamella geometries. We applied the workflow to prepare samples and record high-quality tomograms of diverse model organisms, including infected and uninfected HeLa cells, amoebae, yeast, multicellular cyanobacteria, Pseudomonas aeruginosa and Escherichia coli cells. The workflow will contribute to the dissemination of electron cryotomography of cryo-FIB milled samples in the biological sciences.

Imaging bacteria inside their host by cryo-focused ion beam milling and electron cryotomography.

Bacterium-host interactions are important for diverse ecological settings including pathogenicity and symbiosis. Electron cryotomography is a powerful method for studying the macromolecular complexes that mediate such interactions in situ. The main limitation of electron cryotomography is its restriction to relatively thin samples such as individual bacterial cells. Cryo-focused ion beam milling was recently proposed as a solution to the thickness limitation. This approach allows the artifact-free thinning of biological specimens for subsequent imaging in the transmission electron microscope. By enabling near-native imaging of bacteria inside their eukaryotic host, this combination of techniques promotes the integration of data from structural biology and infection biology. Therefore, electron cryotomography associated with cryo-focused ion beam milling holds great potential for establishing multiscale models of cell-cell interactions from the atomic, to the cellular and to the intercellular scale.

In situ architecture, function, and evolution of a contractile injection system.

Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail-like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Here we used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are more widespread than previously thought. Our insights form the basis for understanding T6SS key concepts and exploring T6SS diversity.

In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography.

The unique property of electron cryotomography (ECT) is its capability to resolve the structure of macromolecular machines in their cellular context. The integration of ECT data with high-resolution structures of purified subcomplexes and live-cell fluorescence light microscopy can generate pseudo-atomic models that lead to a mechanistic understanding across size and time scales. Recent advances in electron detection, sample thinning, data acquisition, and data processing have significantly enhanced the applicability and performance of ECT. Here we describe a detailed workflow for an ECT experiment, including cell culture, vitrification, data acquisition, data reconstruction, tomogram analysis, and subtomogram averaging. This protocol provides an entry point to the technique for students and researchers and indicates the many possible variations arising from specific target properties and the available instrumentation.

Stepwise metamorphosis of the tubeworm Hydroides elegans is mediated by a bacterial inducer and MAPK signaling.

Diverse animal taxa metamorphose between larval and juvenile phases in response to bacteria. Although bacteria-induced metamorphosis is widespread among metazoans, little is known about the molecular changes that occur in the animal upon stimulation by bacteria. Larvae of the tubeworm Hydroides elegans metamorphose in response to surface-bound Pseudoalteromonas luteoviolacea bacteria, producing ordered arrays of phage tail-like metamorphosis-associated contractile structures (MACs). Sequencing the Hydroides genome and transcripts during five developmental stages revealed that MACs induce the regulation of groups of genes important for tissue remodeling, innate immunity, and mitogen-activated protein kinase (MAPK) signaling. Using two MAC mutations that block P. luteoviolacea from inducing settlement or metamorphosis and three MAPK inhibitors, we established a sequence of bacteria-induced metamorphic events: MACs induce larval settlement; then, particular properties of MACs encoded by a specific locus in P. luteoviolacea initiate cilia loss and activate metamorphosis-associated transcription; finally, signaling through p38 and c-Jun N-terminal kinase (JNK) MAPK pathways alters gene expression and leads to morphological changes upon initiation of metamorphosis. Our results reveal that the intricate interaction between Hydroides and P. luteoviolacea can be dissected using genomic, genetic, and pharmacological tools. Hydroides' dependency on bacteria for metamorphosis highlights the importance of external stimuli to orchestrate animal development. The conservation of Hydroides genome content with distantly related deuterostomes (urchins, sea squirts, and humans) suggests that mechanisms of bacteria-induced metamorphosis in Hydroides may have conserved features in diverse animals. As a major biofouling agent, insight into the triggers of Hydroides metamorphosis might lead to practical strategies for fouling control.

Paratuberculose em caprinos e ovinos no Estado da Paraíba / Paratuberculosis in goats and sheep in the state of Paraiba

Mediante o teste de ELISA foi determinada a presença de anticorpos para Mycobacterium avium subsp. paratuberculosis (map) em amostras de soro de 734 caprinos e 392 ovinos, sem sinais clìnicos aparentes, provenientes de 14 Municipios do semiárido Paraibano. Em caprinos, a frequência média de anticorpos de 44,86± 22,91% e em ovinos foi de 52,96±31,49. Das 46 propriedades estudadas, 44 (95,65%) apresentaram pelo menos um animal soropositivo. Nos 14 municípios avaliados houve presença de animais sorologicamente positivos, variando de 20% a 70%. Em caprinos sem raça definida e mestiços a frequência (48,56%) foi significativamente inferior (P<0,0270) que a de caprinos de raças puras (57,24%). Em ovinos com escore de 1 a 3 a frequência (59,39%) foi significativamente maior (P=0,0034) que a frequência em ovinos com escore superior a 3 (42,42%). Não houve diferença significativa nas frequências em caprinos e ovinos de diferentes idades, em caprinos com diferente escore corporal e em ovinos de diferentes raças. No exame microbiológico, em cultivos em meio HEYM com micobactina J, foram observadas colônias semelhantes às do Map em 9 (6,58%) das 180 amostras de fezes, sendo uma de caprinos e 8 de ovinos. Na coloração de Ziehl Nieelsen as bactérias apresentavam características morfo-tintorias de Map. Os resultados obtidos indicam alta frequência de caprinos e ovinos infectados com Map no semiárido da Paraíba, tornando-se necessário desenvolver pesquisas sobre a epidemiologia e forma de controle da doença nas condições de criação da região.

Antibodies to paratuberculosis were determined by ELISA in serum samples of 734 goats and 392 sheep, without clinical signs, from 14 municipalities of the semiarid region of Paraíba state, Brazil. In goats, average frequency of antibodies was 44.86±22.91% and in sheep was 52.96±31.49%. All municipalities had affected animals with a frequency varying from 20% to 70%. In goats of unknown breed and crossbreds the prevalence (48.56%) was significantly lower (P=0.0270) than in pure breed goats (57.24%). In sheep with body scores of 1 to 3, the frequency (59.39%) was significantly higher (P=0.0034) than in sheep with body score from 3 to 5 (42.42%). No significant differences were observed in goats and sheep of different ages, in goats with different body score, and in sheep of different breeds. In the microbiologic examination, after 16 weeks of incubation in HEYM medium with mycobactin J, colonies similar to those of do Mycobacterium avium subsp. paratuber culosis were observed in cultures of 9 (6.58%) out of 180 fecal samples, being one from goats and 8 from sheep. In the Ziehl Nieelsen the bacteria had morphologic and staining characteristics similar to M. avium subsp. paratuberculosis. These results suggest that paratuberculosis is endemic with high prevalence in the semiarid region of Paraíba. It is necessary to develop research on the epidemiology and control measures of the disease in the conditions of this region. Also the risk for human beings should be determined, mainly in relation with milking goats.

Pododermatite infecciosa e outras doenças podais de caprinos e ovinos no semiárido Nordestino / Foot rot and other foot diseases of goat and sheep in the semiarid region of northeastern Brazil

This paper reports the occurrence and epidemiology of outbreaks of foot rot and other foot diseases in goats and sheep in the semiarid region of Paraíba, northeastern Brazil. Four farms were inspected for the presence of foot lesion in sheep and goats and for environmental conditions, general hygiene, pastures, and disease control measures. The prevalence of foot lesions was 19.41 percent (170/876) in sheep and 17.99 percent (52/289) in goats, ranging between 5.77 percent and 33.85 percent in different farms. Foot rot was the most common disease, affecting 12.1 percent of the animals examined (141/1165), but with significantly higher (p<0.05) prevalence in sheep (13.69 percent) than in goats (7.27 percent). The frequency of malignant foot rot was also significantly lower (p<0.05) in goats (9.53 percent) than in the sheep (40.83 percent). On one farm, Dorper sheep showed significantly higher (p<0.05) prevalence of foot rot (17.5 percent) than Santa Inês sheep (6.79 percent), and the number of digits affected was also higher in the former. Dichelobacter nodosus and Fusobacterium necrophorum were isolated from cases of foot rot. White line disease was found in 3.95 percent of the animals, sole ulcers in 1.29 percent, foot abscess in 1.03 percent and hoof overgrowth in 0.5 percent. The high rainfall at the time of occurrence, grazing in wetlands, clay soils with poor drainage, presence of numerous stony grounds, closure of the flocks in pens at night, and introduction of affected animals were considered predisposing factors for the occurrence of foot diseases.

Este trabalho relata a ocorrência e aspectos epidemiológicos de surtos de pododermatite infecciosa e outras afecções podais em caprinos e ovinos no semiárido paraibano. As propriedades foram inspecionadas quanto à presença de problemas podais e aspectos relacionados às condições ambientais, higiene, locais de pastejo e medidas de controle utilizadas. Nos ovinos a prevalência de lesões podais foi de 19,41 por cento (170/876) e nos caprinos de 17,99 por cento (52/289), variando entre 5,77 por cento e 33,85 por cento nas diferentes propriedades. A pododermatite infecciosa foi a doença mais frequente acometendo 12,1 por cento dos animais examinados (141/1165), sendo a prevalência nos ovinos (13,69 por cento) significativamente maior (p<0,05) do que a nos caprinos (7,27 por cento). A frequência de lesões malignas desta doença em caprinos (9,53 por cento) foi, também, significativamente menor (p<0,05) do que nos ovinos (40,83 por cento). Em uma das propriedades a prevalência de pododermatite infecciosa entre os ovinos Dorper (17,5 por cento) foi significativamente maior do que a nos ovinos da raça Santa Inês (6,79 por cento), tendo os animais pela pododermatite infecciosa. Dichelobacter nodosus e Fusobacterium necrophorum foram isolados de casos de pododermatite infecciosa. A doença da linha branca foi constatada em 3,95 por cento dos animais, a úlcera de sola em 1,29 por cento, o abscesso do pé em 1,03 por cento, e crescimento excessivo do casco em 0,5 por cento. Os altos índices pluviométricos na época de ocorrência, o pastejo em áreas úmidas, os solos argilosos com pouca drenagem, os terrenos pedregosos, o encerramento em currais durante a noite e a introdução de animais doentes foram considerados fatores predisponentes para a ocorrência dos surtos.

Tristeza parasitária bovina no Sertão da Paraíba / Cattle tick fever in the semiarid region of the Brazilian state of Paraiba

Descrevem-se 24 surtos de tristeza parasitária bovina no sertão paraibano, sendo 18 de anaplasmose por Anaplasma margimale, dois de babesiose por Babesia bigemina, dois por Babesia não identificada e dois por infecção mista de A. marginale e Babesia sp. Os surtos ocorreram entre agosto de 2007 a outubro de 2009, porém, com uma concentração dos surtos no final do período chuvoso e início do período seco de cada ano, sendo 22 em animais adultos e dois em bezerros de aproximadamente 11 meses. Dois surtos ocorreram em bovinos da raça Nelore, um em animais da raça Gir e os 21 restantes ocorreram em animais das raças Holandês, Pardo Suiço e mestiços das mesmas com zebuínos. Conclui-se que no sertão da Paraíba há áreas de instabilidade enzoótica, ocorrendo surtos de tristeza no final da época de chuvas, principalmente nas áreas de planaltos e serras da região da Borborema e em áreas úmidas como a Bacia do Rio do Peixe, Rio Piranhas e Rio Espinharas em que há a formação de microclimas favoráveis à sobrevivência do carrapato.

Twenty four outbreaks of cattle tick fever are reported in the semiarid region of Paraiba known as Sertão. Eighteen outbreaks were caused by Anaplasma. marginale, two by Babesia bigemina, and two by mixed infection of A. marginale and Babesia sp. In other two outbreaks of babesiosis the species of Babesia was not identified. Outbreaks occurred from August 2007 to October 2009, however with a concentration of the outbreaks at the end of the rainy period and beginning of the dry period in each year. Twenty two outbreaks affected adult cattle and two affected calves approximately 11 months-old. Three outbreaks affected Bos taurus indicus cattle, of the Nelore and Gir breeds. In 21 outbreaks Holstein, Brown Swiss and crossbred of these breeds with Bos taurus indicus cattle were affected. It is concluded that in the sertão of Paraíba there are areas of enzootic instability for cattle tick fever occurring outbreaks at the end of the rainy season, mainly in hilly areas of the Borborema region, and in wet areas of some river basins, including Rio do Peixe, Rio Piranhas and Rio Espinharas.

Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium meliloti.

BACKGROUND: The TolC protein from Sinorhizobium meliloti has previously been demonstrated to be required for establishing successful biological nitrogen fixation symbiosis with Medicago sativa. It is also needed in protein and exopolysaccharide secretion and for protection against osmotic and oxidative stresses. Here, the transcriptional profile of free-living S. meliloti 1021 tolC mutant is described as a step toward understanding its role in the physiology of the cell. RESULTS: Comparison of tolC mutant and wild-type strains transcriptomes showed 1177 genes with significantly increased expression while 325 had significantly decreased expression levels. The genes with an increased expression suggest the activation of a cytoplasmic and extracytoplasmic stress responses possibly mediated by the sigma factor RpoH1 and protein homologues of the CpxRA two-component regulatory system of Enterobacteria, respectively. Stress conditions are probably caused by perturbation of the cell envelope. Consistent with gene expression data, biochemical analysis indicates that the tolC mutant suffers from oxidative stress. This is illustrated by the elevated enzyme activity levels detected for catalase, superoxide dismutase and glutathione reductase. The observed increase in the expression of genes encoding products involved in central metabolism and transporters for nutrient uptake suggests a higher metabolic rate of the tolC mutant. We also demonstrated increased swarming motility in the tolC mutant strain. Absence of functional TolC caused decreased expression mainly of genes encoding products involved in nitrogen metabolism and transport. CONCLUSION: This work shows how a mutation in the outer membrane protein TolC, common to many bacterial transport systems, affects expression of a large number of genes that act in concert to restore cell homeostasis. This finding further underlines the fundamental role of this protein in Sinorhizobium meliloti biology.

Eficácia de dois agentes térmicos em dentes com coroa protética / Effectiviness of two thermal agents in crown teeth restorations

Os testes térmicos em dentes molares com coroa protética apresentam literatura escassa e resultados frente a metodologias a amostragem incosistentes. O propósito do estudo foi comparar a eficácia do gelo e do gás tetrafluoretano na determinação da vitalidade pulpar em 400 dentes molares humanos de 197 pacientes com idade variável entre 19 e 62 anos. As coroas protéticas eram confeccionadas em metalo-cerâmica, metalo-plática (provisório). Os agentes de resfriamento empregados foram aplicados diretamente na superfície vestibular do terço cervical da coroa. Conclui-se que o tetrafluoretano foi significativamente (p=0,01) mais eficaz em relação ao gelo em todos os dentes avalidos