An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking.

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking.

Discovery of a radio emitting neutron star with an ultra-long spin period of 76 seconds.

The radio-emitting neutron star population encompasses objects with spin periods ranging from milliseconds to tens of seconds. As they age and spin more slowly, their radio emission is expected to cease. We present the discovery of an ultra-long period radio-emitting neutron star, PSR J0901-4046, with spin properties distinct from the known spin and magnetic-decay powered neutron stars. With a spin-period of 75.88 s, a characteristic age of 5.3 Myr, and a narrow pulse duty-cycle, it is uncertain how radio emission is generated and challenges our current understanding of how these systems evolve. The radio emission has unique spectro-temporal properties such as quasi-periodicity and partial nulling that provide important clues to the emission mechanism. Detecting similar sources is observationally challenging, which implies a larger undetected population. Our discovery establishes the existence of ultra-long period neutron stars, suggesting a possible connection to the evolution of highly magnetized neutron stars, ultra-long period magnetars, and fast radio bursts.

Gastrointestinal Parasites in Sheep from the Brazilian Pampa Biome: Prevalence and Associated Factors.

Parasitic infections are common in sheep farming worldwide. A cross-sectional study was carried out to determine the prevalence and associated risk factors among gastrointestinal parasites and sheep herds from the Brazilian Pampa biome, Rio Grande do Sul state, Brazil. Twenty-one farms were visited, where rectal faecal samples were individually collected from 309 animals. The sheep industry on the studied farms was characterised by small farms with a low level of technification, an extensive grazing system and meat production. Of all samples analysed, strongyle nematodes had the highest prevalence (77.02%), followed by Eimeria spp. (70.55%), Moniezia expansa (20.39%) and Strongyloides papillosus (17.48%). Mixed infection, between helminths and protozoans, was detected in 68.61% of samples. All herds were positive for strongyle and Eimeria spp. A predominance of Haemonchus spp. and Trichostrongylus spp. nematodes was observed in the herds. Younger animals were significantly more affected by Eimeria and M. expansa. In the semi-intensive and intensive systems, a higher frequency of Eimeria and strongyle infections was observed. Parasite infection was significantly reduced at low animal densities. The Brazilian Pampa region presents a high prevalence of gastrointestinal parasites among sheep; age, breeding system and stocking density were factors associated with parasite infection.

As infecções parasitárias são comuns na criação de ovinos em todo o mundo. Um estudo transversal foi realizado para determinar a prevalência e fatores de risco associados entre parasitoses gastrintestinais e rebanhos ovinos do bioma Pampa brasileiro, estado do Rio Grande do Sul, Brasil. Vinte e uma fazendas foram visitadas, onde foram coletadas amostras de fezes individuais da ampola retal de 309 animais. A produção ovina era caracterizada por pequenas propriedades com baixo nível de tecnificação, os animais eram criados em sistema extensivo e para produção de carne. De todas as amostras analisadas, os estrongilídeos (Nematoda: Strongylida) foram mais prevalentes (77,02%), seguido por Eimeria spp. (70,55%), Moniezia expansa (20,39%) e Strongyloides papillosus (17,48%). Infecção mista, entre helmintos e protozoários, foi detectada em 68,61% das amostras. Todos os rebanhos foram positivos para estrongilídeos e Eimeria spp. Foi observada predominância dos nematódeos Haemonchus spp. e Trichostrongylus nos rebanhos. Animais mais jovens foram significativamente mais afetados por Eimeria e M. expansa. Nos sistemas semi-intensivo e intensivo, observou-se maior frequência de infecções por Eimeria e estrongilídeos. A infecção parasitária foi significativamente menor em fazendas com baixa densidade animal. A região do Pampa brasileiro apresenta alta prevalência de parasitos gastrintestinais em ovinos; idade, sistema reprodutivo e densidade animal foram fatores associados à infecção parasitária.

Soil contamination by Ancylostoma spp. and Toxocara spp. eggs in elementary school playgrounds in the extreme south of Brazil.

Millions of people worldwide, and especially schoolchildren, may be infected by geohelminths due to their exposure to a contaminated environment. The aim of this study was to evaluate soil contamination by Ancylostoma spp. and Toxocara spp. eggs in recreation areas at elementary schools in Pelotas, state of Rio Grande do Sul, Brazil. Sand samples were collected from 22 schools and were processed using the centrifugal flotation method. Helminth eggs with zoonotic potential were found in 12 out of the 22 schools (54.5%). Contamination by Ancylostoma spp. and Toxocara spp. was observed in 36.4% (8/22) and 27.3% (6/22) of the soil samples collected at these schools, respectively. These findings of eggs show that the school communities are exposed to risks of zoonotic transmission.

Soil contamination by Ancylostoma spp. and Toxocara spp. eggs in elementary school playgrounds in the extreme south of Brazil / Contaminação do solo de praças de recreação de escolas de ensino fundamental por ovos de Ancylostoma spp. e Toxocara spp. no extremo sul do Brasil

Abstract Millions of people worldwide, and especially schoolchildren, may be infected by geohelminths due to their exposure to a contaminated environment. The aim of this study was to evaluate soil contamination by Ancylostoma spp. and Toxocara spp. eggs in recreation areas at elementary schools in Pelotas, state of Rio Grande do Sul, Brazil. Sand samples were collected from 22 schools and were processed using the centrifugal flotation method. Helminth eggs with zoonotic potential were found in 12 out of the 22 schools (54.5%). Contamination by Ancylostoma spp. and Toxocara spp. was observed in 36.4% (8/22) and 27.3% (6/22) of the soil samples collected at these schools, respectively. These findings of eggs show that the school communities are exposed to risks of zoonotic transmission.

Resumo Milhões de pessoas podem ser acometidas por geohelmintos, especialmente crianças em idade escolar, devido a sua maior exposição a ambientes contaminados. O objetivo deste estudo foi avaliar a contaminação do solo por ovos de Ancylostoma spp. e Toxocara spp. em áreas de recreação de escolas de ensino fundamental da cidade de Pelotas, Rio Grande do Sul, Brasil. Foram colhidas amostras de areia de 22 escolas e processadas pelo método de centrífugo-flutuação. Em 54,5% (12/22) das escolas houve registro da presença de ovos de helmintos com potencial zoonótico. A contaminação por Ancylostoma spp. e Toxocara spp. foi observada em 36,4% (8/22) e 27,3% (6/22) das amostras de solo das escolas, respectivamente. Existe a presença de ovos de Ancylostoma spp. e Toxocara spp., havendo risco de transmissão de zoonoses à comunidade escolar.

Cryptosporidium spp. in Columba livia Gmelin, 1789 (Columbiformes: Columbidae) free-living pigeons from urban areas in Pelotas, Rio Grande do Sul, Brazil.

Cryptosporidium spp. are parasites with zoonotic potential that cause intestinal diseases, generally intense diarrheal, on their hosts, which tend to be immunocompromised. Large populations of pigeons in urban environments can lead to greater human exposure to Cryptosporidium spp., as this bird is considered a potential reservoir and is able to transmit several pathogens. This study aimed in determining the occurrence of Cryptosporidium spp. oocysts in feces of free-living pigeons (Columba livia) found in urban areas in the city of Pelotas, Rio Grande do Sul, south of Brazil. Fecal samples (n = 50) were collected from young and adult pigeons captured in different locations in the urban area and the parasitological diagnosis was performed through Ritchie's modified technique and Kinyoun's technique. Among the 50 samples, 18% (IC95% 9.7-30.8) were positive for Cryptosporidium spp. with a low number of oocysts being detected on fecal smears. Our results confirmed the occurrence of Cryptosporidium spp. oocysts in feces of free-living pigeons from the urban area of the city of Pelotas. This is the first report of Cryptosporidium spp. oocysts in feces of pigeons in south Brazil. This data has epidemiological significance because the oocysts could be from zoonotic species, which consequently shows that humans might be exposed to infection.

The Landscape of Pseudomonas aeruginosa Membrane-Associated Proteins.

BACKGROUND: Pseudomonas aeruginosa cell envelope-associated proteins play a relevant role in infection mechanisms. They can contribute to the antibiotic resistance of the bacterial cells and be involved in the interaction with host cells. Thus, studies contributing to elucidating these key molecular elements are of great importance to find alternative therapeutics. METHODS: Proteins and peptides were extracted by different methods and analyzed by Multidimensional Protein Identification Technology (MudPIT) approach. Proteomic data were processed by Discoverer2.1 software and multivariate statistics, i.e., Linear Discriminant Analysis (LDA), while the Immune Epitope Database (IEDB) resources were used to predict antigenicity and immunogenicity of experimental identified peptides and proteins. RESULTS: The combination of 29 MudPIT runs allowed the identification of 10,611 peptides and 2539 distinct proteins. Following application of extraction methods enriching specific protein domains, about 15% of total identified peptides were classified in trans inner-membrane, inner-membrane exposed, trans outer-membrane and outer-membrane exposed. In this scenario, nine outer membrane proteins (OprE, OprI, OprF, OprD, PagL, OprG, PA1053, PAL and PA0833) were predicted to be highly antigenic. Thus, they were further processed and epitopes target of T cells (MHC Class I and Class II) and B cells were predicted. CONCLUSION: The present study represents one of the widest characterizations of the P. aeruginosa membrane-associated proteome. The feasibility of our method may facilitates the investigation of other bacterial species whose envelope exposed protein domains are still unknown. Besides, the stepwise prioritization of proteome, by combining experimental proteomic data and reverse vaccinology, may be useful for reducing the number of proteins to be tested in vaccine development.

Eimeria spp. infection in lambs from southern Brazil / Infecção por Eimeria spp. em cordeiros do sul do Brasil

Eimeria infections are common in the sheep industry worldwide. Lambs are more susceptible to coccidiosis, especially in stressful conditions, being infected by different species of the parasite. Eimeria crandallis and Eimeria ovinoidalis are considered the most pathogenic, causing reduced growth, dehydration, anorexia, and death. In this study, the frequency of Eimeria species was evaluated in lambs from the southern region of the Rio Grande do Sul state, Brazil. Fecal samples from 248 lambs, from 19 farms, were tested for the presence of oocysts. The positive samples were re-examined and the sporulated oocysts analyzed morphometrically to identify the presence of Eimeria species. In 100% of the evaluated farms, there were animals positive for the protozoan. The frequency of Eimeria species was: E. ovinoidalis (94.74%), E. crandallis (89.47%), E. granulosa (78.95%), E. parva (68.42%), E. ahsata (63.13%), E. punctata (42.11%), E. bakuensis (36.84%), E. faurei (10.53%), and E. pallida (5.26%). Mixed infection was found in 94.74% of the samples. This research describes, for the first time, the occurrence of E. crandallis and E. ovinoidalis infecting lambs in the study area. The wide distribution of this protozoan and the high frequency of pathogenic species show the importance and potential damage of sheep coccidiosis in herds from Rio Grande do Sul.(AU)

As infecções por Eimeria são comuns na ovinocultura mundial. Cordeiros são mais suscetíveis a coccidiose, especialmente em condições estressantes, sendo infectados por diferentes espécies do parasito. Eimeria crandallis e Eimeria ovinoidalis são consideradas as mais patogênicas, causando redução do crescimento, desidratação, anorexia e morte. Neste estudo, a prevalência de Eimeria spp. foi avaliada em cordeiros da região sul do Estado do Rio Grande do Sul, Brasil. Amostras fecais de 248 cordeiros, provenientes de 19 fazendas, foram testadas quanto à presença de oocistos. As amostras positivas foram reexaminadas e os oocistos esporulados analisados morfometricamente para identificação das espécies de Eimeria presentes. Em 100% das fazendas avaliadas houve animais positivos para o protozoário. A frequência das espécies de Eimeria foi: E. ovinoidalis (94.74%), E. crandallis (89.47%), E. granulosa (78.95%), E. parva (68.42%), E. ahsata (63.13%), E. punctata (42.11%), E. bakuensis (36.84%), E. faurei (10.53%) e E. pallida (5.26). Infecção mista foi encontrada em 94.74% das amostras. Este trabalho descreve pela primeira vez a ocorrência de E. crandallis e E. ovinoidalis infectando cordeiros na área de estudo. Este trabalho descreve pela primeira vez a ocorrência de E. crandallis e E. ovinoidalis infectando cordeiros na área de estudo. A ampla distribuição desse protozoário e a alta frequência das espécies patogênicas evidenciam a importância da coccidiose ovina e os danos potenciais nos rebanhos do Rio Grande do Sul.(AU)

Autonomous role of Wiskott-Aldrich syndrome platelet deficiency in inducing autoimmunity and inflammation.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by eczema, infections, and susceptibility to autoimmunity and malignancies. Thrombocytopenia is a constant finding, but its pathogenesis remains elusive. OBJECTIVE: To dissect the basis of the WAS platelet defect, we used a novel conditional mouse model (CoWas) lacking Wiskott-Aldrich syndrome protein (WASp) only in the megakaryocytic lineage in the presence of a normal immunologic environment, and in parallel we analyzed samples obtained from patients with WAS. METHODS: Phenotypic and functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic interaction network. Platelet immunogenicity was tested by using ELISAs and B-cell and platelet cocultures. RESULTS: CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis in vitro, but WASp-deficient platelets had short lifespan and high expression of activation markers. Proteomic analysis identified signatures compatible with defects in cytoskeletal reorganization and metabolism yet surprisingly increased antigen-processing capabilities. In addition, WASp-deficient platelets expressed high levels of surface and soluble CD40 ligand and were capable of inducing B-cell activation in vitro. WASp-deficient platelets were highly immunostimulatory in mice and triggered the generation of antibodies specific for WASp-deficient platelets, even in the context of a normal immune system. Patients with WAS also showed platelet hyperactivation and increased plasma soluble CD40 ligand levels correlating with the presence of autoantibodies. CONCLUSION: Overall, these findings suggest that intrinsic defects in WASp-deficient platelets decrease their lifespan and dysregulate immune responses, corroborating the role of platelets as modulators of inflammation and immunity.

Misidentification of transthyretin and immunoglobulin variants by proteomics due to methyl lysine formation in formalin-fixed paraffin-embedded amyloid tissue.

Proteomics is becoming the de facto gold standard for identifying amyloid proteins and is now used routinely in a number of centres. The technique is compound class independent and offers the added ability to identify variant and modified proteins. We re-examined proteomics results from a number of formalin-fixed paraffin-embedded amyloid samples, which were positive for transthyretin (TTR) by immunohistochemistry and proteomics, using the UniProt human protein database modified to include TTR variants. The amyloidogenic variant, V122I TTR, was incorrectly identified in 26/27 wild-type and non-V122I variant samples due to its close mass spectral similarity with the methyl lysine-modified WT peptide [126KMe]105-127 (p.[146 KMe]125-147) generated during formalin fixation. Similarly, the methyl lysine peptide, [50KMe]43-59, from immunoglobulin lambda light chain constant region was also misidentified as arising from a rare myeloma-derived lambda variant V49I. These processing-derived modifications are not present in fresh cardiac tissue, non-fixed fat nor serum and do not materially affect the identification of amyloid proteins. They could result in the incorrect assignment of a variant, and this may have consequences for the immediate family who will require genetic counselling and potentially early clinical intervention. As proteomics becomes a routine clinical test for amyloidosis, it becomes important to be aware of potentially confounding issues such as formalin-mediated lysine methylation, and how these may influence diagnosis and possibly treatment.

Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c-di-GMP production by the DgcQ protein of Escherichia coli.

Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N-carbamoyl-aspartate appears to be favoured by protein-protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N-carbamoyl-aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway.

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

Tuberculose em felinos domésticos (Felis catus) no sul do Rio Grande do Sul / Tuberculosis in domestic cats (Felis catus) in southern Rio Grande do Sul

Descrevem-se os aspectos clínico patológicos de quatro casos de tuberculose em felinos domésticos no Sul do Rio Grande do sul. Foi realizado um estudo retrospectivo dos casos diagnosticados como tuberculose em gatos necropsiados no Laboratório Regional de Diagnóstico da Universidade Federal de Pelotas (LRD/UFPel) no período de janeiro de 2000 a dezembro de 2014. Em dois casos a amplificação da sequência genética IS6110 específica para Mycobacterium tuberculosis demonstrou que a doença era de origem humana. Os outros dois foram positivos para Mycobacterium spp. Os sinais clínicos caracterizaram-se por emagrecimento progressivo, anorexia e dispneia. As alterações macroscópicas evidenciadas eram de caquexia, aumento de volume dos linfonodos submandibulares com áreas focais de aspecto caseoso ao corte. Nos pulmões havia áreas multifocais a coalescentes amareladas de aspecto caseoso, discretamente elevadas e firmes na superfície pleural. Histologicamente havia broncopneumonia e linfadenite granulomatosas. A coloração de Ziehl-Neelsen evidenciou a presença de bacilos álcool-ácidos resistentes. Houve imunomarcação positiva para micobactéria pela técnica de imuno-histoquímica. Alerta-se para a importância da tuberculose felina como um problema de saúde pública pois gatos infectados podem servir de fonte para a disseminação das micobactérias no ambiente, além de serem sentinelas para a ocorrência da enfermidade em seres humanos.(AU)

Clinical pathological aspects of four cases of tuberculosis in domestic cats in southern Rio Grande do Sul is described. A retrospective study was conducted of cats diagnosed with tuberculosis and necropsied at the Regional Diagnostic Laboratory of the Federal University of Pelotas (LRD/UFPel) from January 2000 to December 2014. In two cases, the amplification of genetic sequence IS6110 specific to Mycobacterium tuberculosis demonstrated that the disease was of human origin. The other two were positive for Mycobacterium spp. The clinical signs were characterized by progressive weight loss, anorexia, and dyspnea. The evident macroscopic changes were cachexia, swelling of submandibular lymph nodes with focal areas of caseous aspect on cut surface. The lungs had multifocal caseous areas at the pleural surface. Histologically, the cats had pneumonia and granulomatous lymphadenitis. The Ziehl-Neelsen staining revealed the presence of alcohol-acid resistant bacteria. There was a positive immunostaining for mycobacteria by immunohistochemistry technique. We alert to the importance of feline tuberculosis as a public health problem, because infected cats may serve as source for dissemination of mycobacteria in the environment, and be sentinels for the occurrence of the disease in humans.(AU)

Proteomics-based network analysis characterizes biological processes and pathways activated by preconditioned mesenchymal stem cells in cardiac repair mechanisms.

BACKGROUND: We have demonstrated that intramyocardial delivery of human mesenchymal stem cells preconditioned with a hyaluronan mixed ester of butyric and retinoic acid (MSCp+) is more effective in preventing the decay of regional myocardial contractility in a swine model of myocardial infarction (MI). However, the understanding of the role of MSCp+ in proteomic remodeling of cardiac infarcted tissue is not complete. We therefore sought to perform a comprehensive analysis of the proteome of infarct remote (RZ) and border zone (BZ) of pigs treated with MSCp+ or unconditioned stem cells. METHODS: Heart tissues were analyzed by MudPIT and differentially expressed proteins were selected by a label-free approach based on spectral counting. Protein profiles were evaluated by using PPI networks and their topological analysis. RESULTS: The proteomic remodeling was largely prevented in MSCp+ group. Extracellular proteins involved in fibrosis were down-regulated, while energetic pathways were globally up-regulated. Cardioprotectant pathways involved in the production of keto acid metabolites were also activated. Additionally, we found that new hub proteins support the cardioprotective phenotype characterizing the left ventricular BZ treated with MSCp+. In fact, the up-regulation of angiogenic proteins NCL and RAC1 can be explained by the increase of capillary density induced by MSCp+. CONCLUSIONS: Our results show that angiogenic pathways appear to be uniquely positioned to integrate signaling with energetic pathways involving cardiac repair. GENERAL SIGNIFICANCE: Our findings prompt the use of proteomics-based network analysis to optimize new approaches preventing the post-ischemic proteomic remodeling that may underlie the limited self-repair ability of adult heart.

Azithromycin Attenuates Pseudomonas-Induced Lung Inflammation by Targeting Bacterial Proteins Secreted in the Cultured Medium.

BACKGROUND: Pseudomonas aeruginosa airway infections are a major cause of morbidity and mortality in patients with cystic fibrosis (CF). Azithromycin improves the related clinical outcomes, but its mechanisms of action remain poorly understood. We tested the hypothesis that azithromycin downregulates P. aeruginosa-induced pro-inflammatory responses by modifying release of bacterial proteins. METHODS: We monitored inflammatory markers in lungs of CF mutant mice and their littermate controls in response to conditioned media (CM) collected from the reference P. aeruginosa PAO1 strain cultured in the presence or in the absence of azithromycin. A mass spectrometry-based proteomic approach was applied to examine whether the macrolide elicits a differential release of bacterial proteins. RESULTS: CM collected from azithromycin-untreated PAO1 cultures induced powerful pro-inflammatory neutrophil-dominated responses. Azithromycin attenuated the responses, mainly of macrophage chemoattractant protein-1, tumor necrosis factor-α, and interferon-γ, in CF but not in wild-type mice. Proteomic analysis showed that azithromycin upregulated an array of bacterial proteins including those associated with regulation of immune functions and with repair and resolution of inflammatory responses like the chaperone DnaK and the S-adenosylmethionine synthase, while it downregulated the extracellular heme acquisition protein HasA and the catalytic enzyme lysylendopeptidase. CONCLUSION: Supernatants collected from cultures of the bacterial strain PAO1 represent a novel experimental model to trigger in vivo lung inflammatory responses that should be closer to those obtained with live bacteria, but without bacterial infection. Combined with a bactericidal effect, complex regulation of bacterial innate immune and metabolic factors released in the cultured medium by the action of the macrolide can contribute to its anti-inflammatory effects.

Differential Proteomics Based on Multidimensional Protein Identification Technology to Understand the Biogenesis of Outer Membrane of Escherichia coli.

Cell envelope proteins in bacteria are typically difficult to characterize due to their low abundance, poor solubility, and the problematic isolation of pure surface fraction with only minimal contamination. Here we describe a method for cell membrane fractionation followed by mass spectrometry-based proteomics to analyze and determine protein abundance in bacterial membranes.

Interplay of the modified nucleotide phosphoadenosine 5'-phosphosulfate (PAPS) with global regulatory proteins in Escherichia coli: modulation of cyclic AMP (cAMP)-dependent gene expression and interaction with the HupA regulatory protein.

In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli.

Sulfate assimilation pathway intermediate phosphoadenosine 59-phosphosulfate acts as a signal molecule affecting production of curli fibres in Escherichia coli.

The enterobacterium Escherichia coli can utilize a variety of molecules as sulfur sources, including cysteine, sulfate, thiosulfate and organosulfonates. An intermediate of the sulfate assimilation pathway, adenosine 59-phosphosulfate (APS), also acts as a signal molecule regulating the utilization of different sulfur sources. In this work, we show that inactivation of the cysH gene, leading to accumulation of phosphoadenosine 59-phosphosulfate (PAPS), also an intermediate of the sulfate assimilation pathway, results in increased surface adhesion and cell aggregation by activating the expression of the curli-encoding csgBAC operon. In contrast, curli production was unaffected by the inactivation of any other gene belonging to the sulfate assimilation pathway. Overexpression of the cysH gene downregulated csgBAC transcription, further suggesting a link between intracellular PAPS levels and curli gene expression. In addition to curli components, the Flu, OmpX and Slp proteins were also found in increased amounts in the outer membrane compartment of the cysH mutant; deletion of the corresponding genes suggested that these proteins also contribute to surface adhesion and cell surface properties in this strain. Our results indicate that, similar to APS, PAPS also acts as a signal molecule, albeit with a distinct mechanism and role: whilst APS regulates organosulfonate utilization, PAPS would couple availability of sulfur sources to remodulation of the cell surface, as part of a more global effect on cell physiology.

Dissecting Escherichia coli outer membrane biogenesis using differential proteomics.

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality.