Legionella pneumophila secretes a mitochondrial carrier protein during infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

Subcellular localization of legionella Dot/Icm effectors

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.

LtpD is a novel legionella pneumophila effector that binds phosphatidylinositol 3-phosphate and inositol monophosphatase IMPA1

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD_471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD_471-626 bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide- binding L. pneumophila effector that has a role in intracellular bacterial replication. © 2013, American Society for Microbiology.

Bacterial proteases from the intracellular vacuole niche ; protease conservation and adaptation for pathogenic advantage

Proteases with important roles for bacterial pathogens which specifically reside within intracellular vacuoles are frequently homologous to those which have important virulence functions for other bacteria. Research has identified that some of these conserved proteases have evolved specialised functions for intracellular vacuole residing bacteria. Unique proteases with pathogenic functions have also been described from Chlamydia, Mycobacteria, and Legionella. These findings suggest that there are further novel functions for proteases from these bacteria which remain to be described. This review summarises recent findings of novel protease functions from the intracellular human pathogenic bacteria which reside exclusively in vacuoles.

Health risk from the use of roof-harvested rainwater in Southeast Queensland, Australia, as potable or nonpotable water, determined using quantitative microbial risk assessment

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analysed for the zoonotic bacterial and protozoan pathogen using real-time binary PCR and quantitative PCR (qPCR). Quantitative Microbial Risk Assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from potable and non-potable uses of roof-harvested rainwater. Of the 214 samples tested, 10.7%, 9.8%, and 5.6%, and 0.4% samples were positive for Salmonella invA, Giardia lamblia β-giardin , Legionella pneumophila mip, and Campylobacter jejuni mapA genes. Cryptosporidium parvum could not be detected. The estimated numbers of viable Salmonella spp., G. lamblia β-giradin, and L. pneumophila genes ranged from 1.6 × 101 to 9.5 × 101 cells, 1.4 × 10-1 to 9.0 × 10-1 cysts, and 1.5 × 101 to 4.3 × 101 per 1000 ml of water, respectively. Six risk scenarios were considered from exposure to Salmonella spp., G. lamblia and L. pneumophila. For Salmonella spp., and G. lamblia, these scenarios were: (1) liquid ingestion due to drinking of rainwater on a daily basis (2) accidental liquid ingestion due to garden hosing twice a week (3) aerosol ingestion due to showering on a daily basis, and (4) aerosol ingestion due to hosing twice a week. For L. pneumophila, these scenarios were: (5) aerosol inhalation due to showering on a daily basis, and (6) aerosol inhalation due to hosing twice a week. The risk of infection from Salmonella spp., G. lamblia, and L. pneumophila associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting Salmonella spp. and G. lamblia via drinking exceeds this threshold value, and indicates that if undisinfected rainwater were ingested by drinking, then the gastrointestinal diseases of Salmonellosis and Giardiasis is expected to range from 5.0 × 100 to 2.8 × 101 (Salmonellosis) and 1.0 × 101 to 6.4 × 101 (Giardiasis) cases per 10,000 persons per year, respectively. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for potable use.

Chitosan-dextran sulphate nanocapsule drug delivery system as an effective therapeutic against intraphagosomal pathogen Salmonella

Objectives: The ability to target conventional drugs efficiently inside cells to kill intraphagosomal bacteria has been a major hurdle in treatment of infective diseases. We aimed to develop an efficient drug delivery system for combating infection caused by Salmonella, a well-known intracellular and intraphagosomal pathogen. Chitosan dextran sulphate (CD) nanocapsules were assessed for their efficiency in delivering drugs against Salmonella. Methods: The CD nanocapsules were prepared using the layer-by-layer method and loaded with ciprofloxacin or ceftriaxone. Antibiotic-loaded nanocapsules were analysed in vitro for their ability to enter epithelial and macrophage cells to kill Salmonella. In vivo pharmacokinetics and organ distribution studies were performed to check the efficiency of the delivery system. The in vivo antibacterial activity of free antibiotic and antibiotic loaded into nanocapsules was tested in a murine salmonellosis model. Results: In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection, CD nanocapsules were successfully employed for efficient targeting and killing of the intracellular pathogen at a dosage significantly lower than that of the free antibiotic. The increased retention time of ciprofloxacin in the blood and organs when it was delivered by CD nanocapsules compared with the conventional routes of administration may be the reason underlying the requirement for a reduced dosage and frequency of antibiotic administration. Conclusions: CD nanocapsules can be used as an efficient drug delivery system to treat intraphagosomal pathogens, especially Salmonella infection, This delivery system might be used effectively for other vacuolar pathogens including Mycobacteria, Brucella and Legionella.

Chitosan-dextran sulphate nanocapsule drug delivery system as an effective therapeutic against intraphagosomal pathogen Salmonella

Objectives: The ability to target conventional drugs efficiently inside cells to kill intraphagosomal bacteria has been a major hurdle in treatment of infective diseases. We aimed to develop an efficient drug delivery system for combating infection caused by Salmonella, a well-known intracellular and intraphagosomal pathogen. Chitosan dextran sulphate (CD) nanocapsules were assessed for their efficiency in delivering drugs against Salmonella. Methods: The CD nanocapsules were prepared using the layer-by-layer method and loaded with ciprofloxacin or ceftriaxone. Antibiotic-loaded nanocapsules were analysed in vitro for their ability to enter epithelial and macrophage cells to kill Salmonella. In vivo pharmacokinetics and organ distribution studies were performed to check the efficiency of the delivery system. The in vivo antibacterial activity of free antibiotic and antibiotic loaded into nanocapsules was tested in a murine salmonellosis model. Results: In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection, CD nanocapsules were successfully employed for efficient targeting and killing of the intracellular pathogen at a dosage significantly lower than that of the free antibiotic. The increased retention time of ciprofloxacin in the blood and organs when it was delivered by CD nanocapsules compared with the conventional routes of administration may be the reason underlying the requirement for a reduced dosage and frequency of antibiotic administration. Conclusions: CD nanocapsules can be used as an efficient drug delivery system to treat intraphagosomal pathogens, especially Salmonella infection, This delivery system might be used effectively for other vacuolar pathogens including Mycobacteria, Brucella and Legionella.

Development of a novel DNA microarray to detect bacterial pathogens in patients with chronic obstructive pulmonary disease (COPD)

A novel microarray was constructed with DNA PCR product probes targeting species specific functional genes of nine clinically significant respiratory pathogens, including the Gram-positive organisms (Streptococcus pneumoniae, Streptococcus pyogenes), the Gram-negative organisms (Chlamydia pneumoniae, Coxiella burnetii Haemophilus spp., Legionella pneumophila, Moraxella catarrhalis, and Pseudomonas aeruginosa), as well as the atypical bacterium, Mycoplasma pneumoniae. In a "proof-of-concept" evaluation of the developed microarray, the microarray was compared with real-time PCR from 14 sputum specimens from COPD patients. All of the samples positive for bacterial species in real-time PCR were also positive for the same bacterial species using the microarray. This study shows that a microarray using PCR probes is a potentially useful method to monitor the populations of bacteria in respiratory specimens and can be tailored to specific clinical needs such as respiratory infections of particular patient populations, including patients with cystic fibrosis and bronchiectasis. (C) 2010 Elsevier B.V. All rights reserved.

Burkholderia cepacia complex isolates survive intracellularly without replication within acidic vacuoles of Acanthamoeba polyphaga

We have previously demonstrated that isolates of the Burkholderia cepacia complex can survive intracellularly in murine macrophages and in free-living Acanthamoeba. In this work, we show that the clinical isolates B. vietnamiensis strain CEP040 and B. cenocepacia H111 survived but did not replicate within vacuoles of A. polyphaga. B. cepacia-containing vacuoles accumulated the fluid phase marker Lysosensor Blue and displayed strong blue fluorescence, indicating that they had low pH. In contrast, the majority of intracellular bacteria within amoebae treated with the V-ATPse inhibitor bafilomycin A1 localized in vacuoles that did not fluoresce with Lysosensor Blue. Experiments using bacteria fluorescently labelled with chloromethylfluorescein diacetate demonstrated that intracellular bacteria remained viable for at least 24 h. In contrast, Escherichia coli did not survive within amoebae after 2 h post infection. Furthermore, intracellular B. vietnamiensis CEP040 retained green fluorescent protein within the bacterial cytoplasm, while this protein rapidly escaped from the cytosol of phagocytized heat-killed bacteria into the vacuolar lumen. Transmission electron microscopy analysis confirmed that intracellular Burkholderia cells were structurally intact. In addition, both Legionella pneumophila- and B. vietnamiensis-containing vacuoles did not accumulate cationized ferritin, a compound that localizes within the lysosome. Thus, our observations support the notion that B. cepacia complex isolates can use amoebae as a reservoir in the environment by surviving without intracellular replication within an acidic vacuole that is distinct from the lysosomal compartment.

Optimização energética de um sistema de climatização industrial

O desenvolvimento deste trabalho teve como objectivo a optimização de um sistema de climatização industrial, constituído por quatro centrais de climatização adiabáticas, que apresentam limitações de capacidade de arrefecimento, controlo e eficiência. Inicialmente foi necessária a pesquisa bibliográfica e recolha de informação relativa à indústria têxtil e ao processo de arrefecimento evaporativo. Numa fase posterior foram recolhidos e analisados os diversos dados essenciais à compreensão do binómio edifício/sistema de climatização, para a obtenção de possíveis hipóteses de optimização. Da fase de recolha de informações e dados, destaca-se, também, a realização de análises à qualidade do ar interior (QAI). As optimizações seleccionadas como passíveis de implementação, foram estudadas e analisadas com o auxílio do software de simulação energética dinâmica DesignBuilder e os resultados obtidos foram devidamente trabalhados e ajustados de modo a permitir uma assimilação amigável e de fácil interpretação das suas vantagens e desvantagens, tendo ainda sido objecto de estudo de viabilidade económica. A optimização proposta reflecte uma melhoria substancial das condições interiores ao nível da temperatura e humidade relativa, resultando, ainda assim, numa redução de consumos energéticos na ordem dos 23 % (490.337 kWh), isto é, uma poupança anual de 42.169 € aos custos de exploração e com um período de retorno de 1 ano e 11 meses.

O jornalismo online da TVI24 e a sua relação com as fontes

Elaborado no âmbito do mestrado em Jornalismo da Faculdade de Ciências Sociais e Humanas – Universidade Nova de Lisboa, o presente relatório, intitulado “O Jornalismo Online da TVI24 e a sua relação com as fontes”, resulta dos conhecimentos que apreendi na componente não-lectiva e da experiência prática de três meses no site da TVI24, entre Setembro e Dezembro de 2014. Proponho reflectir sobre o jornalismo online, as suas potencialidades e, em particular, sobre a relação dos jornalistas com as fontes, a partir da respectiva revisão de literatura e de uma análise da cobertura dos acontecimentos relacionados com o vírus Ébola e a bactéria Legionella no que respeita às fontes utilizadas e ao aproveitamento das ferramentas digitais. Através da análise dos dados recolhidos, concluo que o jornalismo online é ainda marcado pelo sedentarismo, pelo contacto indirecto com as fontes e pelo pouco aproveitamento das ferramentas digitais.

Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant

The complex ecology of free-living amoebae (FLA) and their role in spreading pathogenic microorganisms through water systems have recently raised considerable interest. In this study, we investigated the presence of FLA and amoebae-resisting bacteria (ARB) at various stages of a drinking water plant fed with river water. We isolated various amoebal species from the river and from several points within the plant, mostly at early steps of water treatment. Echinamoeba- and Hartmannella-related amoebae were mainly recovered in the drinking water plant whereas Acanthamoeba- and Naegleria-related amoebae were recovered from the river water and the sand filtration units. Some FLA isolates were recovered immediately after the ozonation step, thus suggesting resistance of these microorganisms to this disinfection procedure. A bacterial isolate related to Mycobacterium mucogenicum was recovered from an Echinamoeba-related amoeba isolated from ozone-treated water. Various other ARB were recovered using co-culture with axenic Acanthamoeba castellanii, including mycobacteria, legionella, Chlamydia-like organisms and various proteobacteria. Noteworthy, a new Parachlamydia acanthamoebae strain was recovered from river water and from granular activated carbon (GAC) biofilm. As amoebae mainly multiply in sand and GAC filters, optimization of filter backwash procedures probably offers a possibility to better control these protists and the risk associated with their intracellular hosts

β-Lactam Monotherapy vs β-Lactam-Macrolide Combination Treatment in Moderately Severe Community-Acquired Pneumonia: A Randomized Noninferiority Trial.

IMPORTANCE: The clinical benefit of adding a macrolide to a β-lactam for empirical treatment of moderately severe community-acquired pneumonia remains controversial. OBJECTIVE: To test noninferiority of a β-lactam alone compared with a β-lactam and macrolide combination in moderately severe community-acquired pneumonia. DESIGN, SETTING, AND PARTICIPANTS: Open-label, multicenter, noninferiority, randomized trial conducted from January 13, 2009, through January 31, 2013, in 580 immunocompetent adult patients hospitalized in 6 acute care hospitals in Switzerland for moderately severe community-acquired pneumonia. Follow-up extended to 90 days. Outcome assessors were masked to treatment allocation. INTERVENTIONS: Patients were treated with a β-lactam and a macrolide (combination arm) or with a β-lactam alone (monotherapy arm). Legionella pneumophila infection was systematically searched and treated by addition of a macrolide to the monotherapy arm. MAIN OUTCOMES AND MEASURES: Proportion of patients not reaching clinical stability (heart rate <100/min, systolic blood pressure >90 mm Hg, temperature <38.0°C, respiratory rate <24/min, and oxygen saturation >90% on room air) at day 7. RESULTS: After 7 days of treatment, 120 of 291 patients (41.2%) in the monotherapy arm vs 97 of 289 (33.6%) in the combination arm had not reached clinical stability (7.6% difference, P = .07). The upper limit of the 1-sided 90% CI was 13.0%, exceeding the predefined noninferiority boundary of 8%. Patients infected with atypical pathogens (hazard ratio [HR], 0.33; 95% CI, 0.13-0.85) or with Pneumonia Severity Index (PSI) category IV pneumonia (HR, 0.81; 95% CI, 0.59-1.10) were less likely to reach clinical stability with monotherapy, whereas patients not infected with atypical pathogens (HR, 0.99; 95% CI, 0.80-1.22) or with PSI category I to III pneumonia (HR, 1.06; 95% CI, 0.82-1.36) had equivalent outcomes in the 2 arms. There were more 30-day readmissions in the monotherapy arm (7.9% vs 3.1%, P = .01). Mortality, intensive care unit admission, complications, length of stay, and recurrence of pneumonia within 90 days did not differ between the 2 arms. CONCLUSIONS AND RELEVANCE: We did not find noninferiority of β-lactam monotherapy in patients hospitalized for moderately severe community-acquired pneumonia. Patients infected with atypical pathogens or with PSI category IV pneumonia had delayed clinical stability with monotherapy. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00818610.