Publications by authors named "Suzana P Salcedo"

33 Publications

The TIR-domain containing effectors BtpA and BtpB from Brucella abortus impact NAD metabolism.

PLoS Pathog 2020 04 16;16(4):e1007979. Epub 2020 Apr 16.

Laboratory of Molecular Microbiology and Structural Biochemistry, Centre National de la Recherche Scientifique UMR5086, Université de Lyon, Lyon, France.

Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.
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http://dx.doi.org/10.1371/journal.ppat.1007979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188309PMC
April 2020

Acquisition of plasmids conferring carbapenem and aminoglycoside resistance and loss of surface-exposed macromolecule structures as strategies for the adaptation of CC104/CC15 strains to the clinical setting.

Microb Genom 2020 09 26;6(9). Epub 2020 Mar 26.

Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.

() is an emerging opportunistic pathogen associated to nosocomial infections. The rapid increase in multidrug resistance (MDR) among strains underscores the urgency of understanding how this pathogen evolves in the clinical environment. We conducted here a whole-genome sequence comparative analysis of three phylogenetically and epidemiologically related MDR strains from Argentinean hospitals, assigned to the CC104/CC15 clonal complex. While the Ab244 strain was carbapenem-susceptible, Ab242 and Ab825, isolated after the introduction of carbapenem therapy, displayed resistance to these last resource β-lactams. We found a high chromosomal synteny among the three strains, but significant differences at their accessory genomes. Most importantly, carbapenem resistance in Ab242 and Ab825 was attributed to the acquisition of a Rep_3 family plasmid carrying a gene. Other differences involved a genomic island carrying resistance to toxic compounds and a Tn element exclusive to Ab244 and Ab825, respectively. Also remarkably, 44 insertion sequences (ISs) were uncovered in Ab825, in contrast with the 14 and 11 detected in Ab242 and Ab244, respectively. Moreover, Ab825 showed a higher killing capacity as compared to the other two strains in the infection model. A search for virulence and persistence determinants indicated the loss or IS-mediated interruption of genes encoding many surface-exposed macromolecules in Ab825, suggesting that these events are responsible for its higher relative virulence. The comparative genomic analyses of the CC104/CC15 strains conducted here revealed the contribution of acquired mobile genetic elements such as ISs and plasmids to the adaptation of to the clinical setting.
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http://dx.doi.org/10.1099/mgen.0.000360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643966PMC
September 2020

Bioinformatic Analysis of the Type VI Secretion System and Its Potential Toxins in the Genus.

Front Microbiol 2019 1;10:2519. Epub 2019 Nov 1.

Laboratory of Molecular Microbiology and Structural Biochemistry, CNRS UMR 5086, University of Lyon, Lyon, France.

Several strains are important nosocomial pathogens, with as the species of greatest concern worldwide due to its multi-drug resistance and recent appearance of hyper-virulent strains in the clinical setting. colonization of the environment and the host is associated with a multitude of factors which remain poorly characterized. Among them, the secretion systems (SS) encoded by species confer adaptive advantages depending on the niche occupied. Different SS have been characterized in this group of microorganisms, including T6SS used by several species to outcompete other bacteria and in some strains for colonization. Therefore, to better understand the distribution of the T6SS in this genus we carried out an in-depth comparative genomic analysis of the T6SS in 191 sequenced strains. To this end, we analyzed the gene content, sequence similarity, synteny and operon structure of each T6SS loci. The presence of a single conserved T6SS-main cluster (T6SS-1), with two different genetic organizations, was detected in the genomes of several ecologically diverse species. Furthermore, a second main cluster (T6SS-2) was detected in a subgroup of 3 species of environmental origin. Detailed analysis also showed an impressive genetic versatility in T6SS-associated islands, carrying VgrG, PAAR and putative toxin-encoding genes. This study represents the first detailed intra-species comparative analysis of T6SS-associated genes in the r genus, that should contribute to the future experimental characterization of T6SS proteins and effectors.
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http://dx.doi.org/10.3389/fmicb.2019.02519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838775PMC
November 2019

Identification of a Contact-Dependent Growth Inhibition (CDI) System That Reduces Biofilm Formation and Host Cell Adhesion of DSM30011 Strain.

Front Microbiol 2019 30;10:2450. Epub 2019 Oct 30.

Cell Biology of Bacterial Pathogenicity Team, Laboratory of Molecular Microbiology and Structural Biochemistry, CNRS UMR 5086, University of Lyon, Lyon, France.

is a multidrug-resistant nosocomial opportunistic pathogen that is becoming a major health threat worldwide. In this study, we have focused on the DSM30011 strain, an environmental isolate that retains many virulence-associated traits. We found that its genome contains two loci encoding for contact-dependent growth inhibition (CDI) systems. These systems serve to kill or inhibit the growth of non-sibling bacteria by delivering toxins into the cytoplasm of target cells, thereby conferring the host strain a significant competitive advantage. We show that one of the two toxins functions as a DNA-damaging enzyme, capable of inducing DNA double-stranded breaks to the chromosome of strain. The second toxin has unknown catalytic activity but stops the growth of without bactericidal effect. In our conditions, only one of the CDI systems was highly expressed in the DSM30011 strain and was found to mediate interbacterial competition. Surprisingly, the absence of this CDI system promotes adhesion of DSM30011 to both abiotic and biotic surfaces, a phenotype that differs from previously described CDI systems. Our results suggest that a specific regulation mediated by this DSM30011 CDI system may result in changes in bacterial physiology that repress host cell adhesion and biofilm formation.
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http://dx.doi.org/10.3389/fmicb.2019.02450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831553PMC
October 2019

Unsaturated Fatty Acids Affect Quorum Sensing Communication System and Inhibit Motility and Biofilm Formation of Acinetobacter baumannii.

Int J Mol Sci 2018 Jan 10;19(1). Epub 2018 Jan 10.

Normandie University, Unirouen, 76000 Rouen, France.

The increasing threat of as a nosocomial pathogen is mainly due to the occurrence of multidrug-resistant strains that are associated with the real problem of its eradication from hospital wards. The particular ability of this pathogen to form biofilms contributes to its persistence, increases antibiotic resistance, and promotes persistent/device-related infections. We previously demonstrated that virstatin, which is a small organic compound known to decrease virulence of via an inhibition of T4-pili expression, displayed very promising activity to prevent biofilm development. Here, we examined the antibiofilm activity of mono-unsaturated chain fatty acids, palmitoleic (PoA), and myristoleic (MoA) acids, presenting similar action on virulence. We demonstrated that PoA and MoA (at 0.02 mg/mL) were able to decrease ATCC 17978 biofilm formation up to 38% and 24%, respectively, presented a biofilm dispersing effect and drastically reduced motility. We highlighted that these fatty acids decreased the expression of the regulator from the LuxIR-type quorum sensing (QS) communication system AbaIR and consequently reduced the -acyl-homoserine lactone production (AHL). This effect can be countered by addition of exogenous AHLs. Besides, fatty acids may have additional non-targeted effects, independent from QS. Atomic force microscopy experiments probed indeed that PoA and MoA could also act on the initial adhesion process in modifying the material interface properties. Evaluation of fatty acids effect on 22 clinical isolates showed a strain-dependent antibiofilm activity, which was not correlated to hydrophobicity or pellicle formation ability of the tested strains, and suggested a real diversity in cell-to-cell communication systems involved in biofilm formation.
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http://dx.doi.org/10.3390/ijms19010214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796163PMC
January 2018

The Environmental Acinetobacter baumannii Isolate DSM30011 Reveals Clues into the Preantibiotic Era Genome Diversity, Virulence Potential, and Niche Range of a Predominant Nosocomial Pathogen.

Genome Biol Evol 2017 09;9(9):2292-2307

Laboratory of Molecular Microbiology and Structural Biochemistry, CNRS UMR5086, University of Lyon, France.

Acinetobacter baumannii represents nowadays an important nosocomial opportunistic pathogen whose reservoirs outside the clinical setting are obscure. Here, we traced the origins of the collection strain A. baumannii DSM30011 to an isolate first reported in 1944, obtained from the enriched microbiota responsible of the aerobic decomposition of the resinous desert shrub guayule. Whole-genome sequencing and phylogenetic analysis based on core genes confirmed DSM30011 affiliation to A. baumannii. Comparative studies with 32 complete A. baumannii genomes revealed the presence of 12 unique accessory chromosomal regions in DSM30011 including five encompassing phage-related genes, five containing toxin genes of the type-6 secretion system, and one with an atypical CRISPRs/cas cluster. No antimicrobial resistance islands were identified in DSM30011 agreeing with a general antimicrobial susceptibility phenotype including folate synthesis inhibitors. The marginal ampicillin resistance of DSM30011 most likely derived from chromosomal ADC-type ampC and blaOXA-51-type genes. Searching for catabolic pathways genes revealed several clusters involved in the degradation of plant defenses including woody tissues and a previously unreported atu locus responsible of aliphatic terpenes degradation, thus suggesting that resinous plants may provide an effective niche for this organism. DSM30011 also harbored most genes and regulatory mechanisms linked to persistence and virulence in pathogenic Acinetobacter species. This strain thus revealed important clues into the genomic diversity, virulence potential, and niche ranges of the preantibiotic era A. baumannii population, and may provide an useful tool for our understanding of the processes that led to the recent evolution of this species toward an opportunistic pathogen of humans.
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http://dx.doi.org/10.1093/gbe/evx162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604120PMC
September 2017

Relatedness of wildlife and livestock avian isolates of the nosocomial pathogen Acinetobacter baumannii to lineages spread in hospitals worldwide.

Environ Microbiol 2017 10 9;19(10):4349-4364. Epub 2017 Oct 9.

Faculty of Biological Sciences, University of Zielona Góra, Prof. Z. Szafrana Street 1, 65-561 Zielona Góra, Poland.

The natural habitats and potential reservoirs of the nosocomial pathogen Acinetobacter baumannii are poorly defined. Here, we put forth and tested the hypothesis of avian reservoirs of A. baumannii. We screened tracheal and rectal swab samples from livestock (chicken, geese) and wild birds (white stork nestlings) and isolated A. baumannii from 3% of sampled chicken (n = 220), 8% of geese (n = 40) and 25% of white stork nestlings (n = 661). Virulence of selected avian A. baumannii isolates was comparable to that of clinical isolates in the Galleria mellonella infection model. Whole genome sequencing revealed the close relationship of an antibiotic-susceptible chicken isolate from Germany with a multidrug-resistant human clinical isolate from China and additional linkages between livestock isolates and human clinical isolates related to international clonal lineages. Moreover, we identified stork isolates related to human clinical isolates from the United States. Multilocus sequence typing disclosed further kinship between avian and human isolates. Avian isolates do not form a distinct clade within the phylogeny of A. baumannii, instead they diverge into different lineages. Further, we provide evidence that A. baumannii is constantly present in the habitats occupied by storks. Collectively, our study suggests A. baumannii could be a zoonotic organism that may disseminate into livestock.
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http://dx.doi.org/10.1111/1462-2920.13931DOI Listing
October 2017

Protein-Protein Interactions: Pull-Down Assays.

Methods Mol Biol 2017 ;1615:247-255

Molecular Microbiology and Structural Biochemistry, CNRS UMR 5086, Université Lyon 1, Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, 69 367, Lyon Cedex 07, France.

Determining protein partners is an essential step toward understanding protein function and identifying relevant biological pathways. Many methods exist for investigating protein-protein interactions. The pull-down assay is an in vitro technique used to detect physical interactions between two or more proteins and an invaluable tool for confirming a predicted protein-protein interaction or identifying novel interacting partners. This method typically involves the use of affinity purification with various wash and elution steps. In this chapter, we describe how an interaction between two purified bacterial proteins or between bacterial and eukaryotic proteins can be detected by pull-down experiments.
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http://dx.doi.org/10.1007/978-1-4939-7033-9_20DOI Listing
March 2018

A  TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors.

EMBO J 2017 07 8;36(13):1869-1887. Epub 2017 May 8.

Laboratory of Molecular Microbiology and Structural Biochemistry, Centre National de la Recherche Scientifique, University of Lyon, Lyon, France

Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/interleukin 1 receptor (TIR) domain-containing protein (PumA) of the multi-drug resistant PA7 strain. We found that PumA is essential for virulence and inhibits NF-κB, a property transferable to non-PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll-like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin-associated protein 1 (UBAP1), a component of the endosomal-sorting complex required for transport I (ESCRT-I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.
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http://dx.doi.org/10.15252/embj.201695343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494471PMC
July 2017

The influence of two-partner secretion systems on the virulence of Acinetobacter baumannii.

Virulence 2017 08 24;8(6):653-654. Epub 2017 Jan 24.

a Molecular Microbiology and Structural Biochemistry , University of Lyon and CNRS , Lyon , France.

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http://dx.doi.org/10.1080/21505594.2017.1283465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626234PMC
August 2017

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

J Vis Exp 2016 08 5(114). Epub 2016 Aug 5.

Focal Area Infection Biology, Biozentrum, University of Basel;

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.
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http://dx.doi.org/10.3791/54263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5091747PMC
August 2016

Functional Characterization of Pseudomonas Contact Dependent Growth Inhibition (CDI) Systems.

PLoS One 2016 25;11(1):e0147435. Epub 2016 Jan 25.

Laboratoire d'Ingénierie des Systèmes Macromoléculaires, CNRS UMR7255, Université Aix Marseille, Marseille, France.

Contact-dependent inhibition (CDI) toxins, delivered into the cytoplasm of target bacterial cells, confer to host strain a significant competitive advantage. Upon cell contact, the toxic C-terminal region of surface-exposed CdiA protein (CdiA-CT) inhibits the growth of CDI- bacteria. CDI+ cells express a specific immunity protein, CdiI, which protects from autoinhibition by blocking the activity of cognate CdiA-CT. CdiA-CT are separated from the rest of the protein by conserved peptide motifs falling into two distinct classes, the "E. coli"- and "Burkholderia-type". CDI systems have been described in numerous species except in Pseudomonadaceae. In this study, we identified functional toxin/immunity genes linked to CDI systems in the Pseudomonas genus, which extend beyond the conventional CDI classes by the variability of the peptide motif that delimits the polymorphic CdiA-CT domain. Using P. aeruginosa PAO1 as a model, we identified the translational repressor RsmA as a negative regulator of CDI systems. Our data further suggest that under conditions of expression, P. aeruginosa CDI systems are implicated in adhesion and biofilm formation and provide an advantage in competition assays. All together our data imply that CDI systems could play an important role in niche adaptation of Pseudomonadaceae.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147435PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725963PMC
July 2016

Differential Role of the T6SS in Acinetobacter baumannii Virulence.

PLoS One 2015 24;10(9):e0138265. Epub 2015 Sep 24.

Bases Moléculaires et Structurales des Systèmes Infectieux, CNRS UMR 5086, Université Lyon 1, Institut de Biologie et Chimie des Protéines, Lyon, France.

Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138265PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4581634PMC
June 2016

BtpB, a novel Brucella TIR-containing effector protein with immune modulatory functions.

Front Cell Infect Microbiol 2013 8;3:28. Epub 2013 Jul 8.

Aix-Marseille Univ UM 2, Centre d'Immunologie de Marseille-Luminy Marseille, France.

Several bacterial pathogens have TIR domain-containing proteins that contribute to their pathogenesis. We identified a second TIR-containing protein in Brucella spp. that we have designated BtpB. We show it is a potent inhibitor of TLR signaling, probably via MyD88. BtpB is a novel Brucella effector that is translocated into host cells and interferes with activation of dendritic cells. In vivo mouse studies revealed that BtpB is contributing to virulence and control of local inflammatory responses with relevance in the establishment of chronic brucellosis. Together, our results show that BtpB is a novel Brucella effector that plays a major role in the modulation of host innate immune response during infection.
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http://dx.doi.org/10.3389/fcimb.2013.00028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703528PMC
December 2013

Internal affairs: investigating the Brucella intracellular lifestyle.

FEMS Microbiol Rev 2012 May 22;36(3):533-62. Epub 2012 Mar 22.

Faculté de Sciences de Luminy, Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, UM 2, Marseille Cedex, France.

Bacteria of the genus Brucella are Gram-negative pathogens of several animal species that cause a zoonotic disease in humans known as brucellosis or Malta fever. Within their hosts, brucellae reside within different cell types where they establish a replicative niche and remain protected from the immune response. The aim of this article is to discuss recent advances in the field in the specific context of the Brucella intracellular 'lifestyle'. We initially discuss the different host cell targets and their relevance during infection. As it represents the key to intracellular replication, the focus is then set on the maturation of the Brucella phagosome, with particular emphasis on the Brucella factors that are directly implicated in intracellular trafficking and modulation of host cell signalling pathways. Recent data on the role of the type IV secretion system are discussed, novel effector molecules identified and how some of them impact on trafficking events. Current knowledge on Brucella gene regulation and control of host cell death are summarized, as they directly affect intracellular persistence. Understanding how Brucella molecules interplay with their host cell targets to modulate cellular functions and establish the intracellular niche will help unravel how this pathogen causes disease.
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http://dx.doi.org/10.1111/j.1574-6976.2012.00334.xDOI Listing
May 2012

In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system.

Cell Microbiol 2011 Aug 24;13(8):1261-74. Epub 2011 Jun 24.

Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, Universidad Nacional de San Martín, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.

Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.
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http://dx.doi.org/10.1111/j.1462-5822.2011.01618.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139020PMC
August 2011

Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2.

Cell Microbiol 2011 Jul 30;13(7):1044-58. Epub 2011 May 30.

URBM, University of Namur (FUNDP), Belgium.

Bacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown translocated effectors. To identify such effectors, we used a high-throughput yeast two-hybrid screen to identify interactions between putative human phagosomal proteins and predicted Brucella spp. proteins. We identified a specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-β-lactamase-RicA fusion was translocated from Brucella abortus to RAW264.7 macrophages during infection. This translocation was not detectable in a strain deleted for the virB operon, coding for the type IV secretion system. However, RicA secretion in a bacteriological culture was still observed in a ΔvirB mutant. In HeLa cells, a ΔricA mutant recruits less GTP-locked myc-Rab2 on its Brucella-containing vacuoles, compared with the wild-type strain. We observed altered kinetics of intracellular trafficking and faster proliferation of the B. abortusΔricA mutant in HeLa cells, compared with the wild-type control. Altogether, the data reported here suggest RicA as the first reported effector with a proposed function for B. abortus.
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http://dx.doi.org/10.1111/j.1462-5822.2011.01601.xDOI Listing
July 2011

Nontypable Haemophilus influenzae: an intracellular phase within epithelial cells might contribute to persistence.

Microbiology (Reading) 2011 Jan;157(Pt 1):1-2

Dpto de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), Madrid-28040, Spain.

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http://dx.doi.org/10.1099/mic.0.046722-0DOI Listing
January 2011

Coxiella burnetii, the agent of Q fever, replicates within trophoblasts and induces a unique transcriptional response.

PLoS One 2010 Dec 14;5(12):e15315. Epub 2010 Dec 14.

Unité de Recherche sur les Maladies Infectieuses Tropicales et Emergentes, CNRS-IRD UMR 6236, IFR 48, Université de la Méditerranée, Marseille, France.

Q fever is a zoonosis caused by Coxiella burnetii, an obligate intracellular bacterium typically found in myeloid cells. The infection is a source of severe obstetrical complications in humans and cattle and can undergo chronic evolution in a minority of pregnant women. Because C. burnetii is found in the placentas of aborted fetuses, we investigated the possibility that it could infect trophoblasts. Here, we show that C. burnetii infected and replicated in BeWo trophoblasts within phagolysosomes. Using pangenomic microarrays, we found that C. burnetii induced a specific transcriptomic program. This program was associated with the modulation of inflammatory responses that were shared with inflammatory agonists, such as TNF, and more specific responses involving genes related to pregnancy development, including EGR-1 and NDGR1. In addition, C. burnetii stimulated gene networks organized around the IL-6 and IL-13 pathways, which both modulate STAT3. Taken together, these results revealed that trophoblasts represent a protective niche for C. burnetii. The activation program induced by C. burnetii in trophoblasts may allow bacterial replication but seems unable to interfere with the development of normal pregnancy. Such pathophysiologocal processes should require the activation of immune placental cells associated with trophoblasts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015315PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001886PMC
December 2010

Contrasting roles of macrophages and dendritic cells in controlling initial pulmonary Brucella infection.

Eur J Immunol 2010 Dec;40(12):3458-71

Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Marseille, France.

Control of pulmonary pathogens constitutes a challenging task as successful immune responses need to be mounted without damaging the lung parenchyma. Using immunofluorescence microscopy and flow cytometry, we analyzed in the mouse the initial innate immune response that follows intranasal inoculation of Brucella abortus. Bacteria were absent from parenchymal dendritic cells (DC) but present in alveolar macrophages in which they replicated. When the number of alveolar macrophages was reduced prior to Brucella infection, small numbers of pulmonary DC were infected and a massive recruitment of TNF-α- and iNOS-producing DC ensued. Coincidentally, Brucella disseminated to the lung-draining mediastinal lymph nodes (LN) where they replicated in both migratory DC and migratory alveolar macrophages. Together, these results demonstrate that alveolar macrophages are critical regulators of the initial innate immune response against Brucella within the lungs and show that pulmonary DC and alveolar macrophages play rather distinct roles in the control of microbial burden.
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http://dx.doi.org/10.1002/eji.201040497DOI Listing
December 2010

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice.

Vaccine 2010 Aug 19;28(34):5627-34. Epub 2010 Jun 19.

Department of Biochemistry and Immunology, Federal University of Minas Gerais, Pampulha, Belo Horizonte, MG, Brazil.

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortusDeltawbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. DeltawbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence.
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http://dx.doi.org/10.1016/j.vaccine.2010.06.023DOI Listing
August 2010

The Brucella abortus phosphoglycerate kinase mutant is highly attenuated and induces protection superior to that of vaccine strain 19 in immunocompromised and immunocompetent mice.

Infect Immun 2010 May 1;78(5):2283-91. Epub 2010 Mar 1.

Departamento de Bioquimica e Imunologia, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Pampulha, Belo Horizonte, MG, Brazil.

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that can function as virulence factors to better understand the host-pathogen interplay. Herein, we identified the gene encoding the phosphoglycerate kinase (PGK) of B. abortus strain 2308. To test the role of PGK in Brucella pathogenesis, a pgk deletion mutant was constructed. Replacement of the wild-type pgk by recombination was demonstrated by Southern and Western blot analyses. The B. abortus Delta pgk mutant strain exhibited extreme attenuation in bone marrow-derived macrophages and in vivo in BALB/c, C57BL/6, 129/Sv, and interferon regulatory factor-1 knockout (IRF-1 KO) mice. Additionally, at 24 h postinfection the Delta pgk mutant was not found within the same endoplasmic reticulum-derived compartment as the wild-type bacteria, but, instead, over 60% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP1. Furthermore, the B. abortus Delta pgk deletion mutant was used as a live vaccine. Challenge experiments revealed that the Delta pgk mutant strain induced protective immunity in 129/Sv or IRF-1 KO mice that was superior to the protection conferred by commercial strain 19 or RB51. Finally, the results shown here demonstrated that Brucella PGK is critical for full bacterial virulence and that a Delta pgk mutant may serve as a potential vaccine candidate in future studies.
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http://dx.doi.org/10.1128/IAI.01433-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863508PMC
May 2010

The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

PLoS Pathog 2009 Jun 26;5(6):e1000487. Epub 2009 Jun 26.

Aix Marseille Université, Centre d'Immunologie de Marseille-Luminy, UMR6546, Marseille, France.

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the "Brucella-containing vacuole" (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.
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http://dx.doi.org/10.1371/journal.ppat.1000487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2695806PMC
June 2009

Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1.

PLoS Pathog 2008 Feb;4(2):e21

Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, Faculté de Sciences de Luminy, Marseille, France.

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.
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http://dx.doi.org/10.1371/journal.ppat.0040021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2233671PMC
February 2008

The translocated Salmonella effector proteins SseF and SseG interact and are required to establish an intracellular replication niche.

Infect Immun 2006 Dec 2;74(12):6965-72. Epub 2006 Oct 2.

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.

The facultative intracellular pathogen Salmonella enterica causes a variety of diseases, including gastroenteritis and typhoid fever. Inside epithelial cells, Salmonella replicates in vacuoles, which localize in the perinuclear area in close proximity to the Golgi apparatus. Among the effector proteins translocated by the Salmonella pathogenicity island 2-encoded type III secretion system, SifA and SseG have been shown necessary but not sufficient to ensure the intracellular positioning of Salmonella vacuoles. Hence, we have investigated the involvement of other secreted effector proteins in this process. Here we show that SseF interacts functionally and physically with SseG but not SifA and is also required for the perinuclear localization of Salmonella vacuoles. The observations show that the intracellular positioning of Salmonella vacuoles is a complex phenomenon resulting from the combined action of several effector proteins.
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http://dx.doi.org/10.1128/IAI.00648-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1698088PMC
December 2006

Synthesis of phosphatidylcholine, a typical eukaryotic phospholipid, is necessary for full virulence of the intracellular bacterial parasite Brucella abortus.

Cell Microbiol 2006 Aug;8(8):1322-35

Departamento de Microbiología, Universidad de Navarra, Pamplona, Spain.

Phosphatidylcholine (PC) is a typical eukaryotic phospholipid absent from most prokaryotes. Thus, its presence in some intracellular bacteria is intriguing as it may constitute host mimicry. The role of PC in Brucella abortus was examined by generating mutants in pcs (BApcs) and pmtA (BApmtA), which encode key enzymes of the two bacterial PC biosynthetic routes, the choline and methyl-transferase pathways. In rich medium, BApcs and the double mutant BApcspmtA but not BApmtA displayed reduced growth, increased phosphatidylethanolamine and no PC, showing that Pcs is essential for PC synthesis under these conditions. In minimal medium, the parental strain, BApcs and BApmtA showed reduced but significant amounts of PC suggesting that PmtA may also be functional. Probing with phage Tb, antibiotics, polycations and serum demonstrated that all mutants had altered envelopes. In macrophages, BApcs and BApcspmtA showed reduced ability to evade fusion with lysosomes and establish a replication niche. In mice, BApcs showed attenuation only at early times after infection, BApmtA at later stages and BApcspmtA throughout. The results suggest that Pcs and PmtA have complementary roles in vivo related to nutrient availability and that PC and the membrane properties that depend on this typical eukaryotic phospholipid are essential for Brucella virulence.
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http://dx.doi.org/10.1111/j.1462-5822.2006.00712.xDOI Listing
August 2006

Pathogen-endoplasmic-reticulum interactions: in through the out door.

Nat Rev Immunol 2006 Feb;6(2):136-47

Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, Connecticut 065362, USA.

A key determinant for the survival of intracellular pathogens is their ability to subvert the cellular processes of the host to establish a compartment that allows replication. Although most microorganisms internalized by host cells are efficiently cleared following fusion with lysosomes, many pathogens have evolved mechanisms to escape this degradation. In this Review, we provide insight into the molecular processes that are targeted by pathogens that interact with the endoplasmic reticulum and thereby subvert the immune response, ensure their survival intracellularly and cause disease. We also discuss how the endoplasmic reticulum 'strikes back' and controls microbial growth.
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http://dx.doi.org/10.1038/nri1775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7097709PMC
February 2006

Bacterial interactions with the eukaryotic secretory pathway.

Curr Opin Microbiol 2005 Feb;8(1):92-8

Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Univ, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cedex 9, France.

Pathogenic bacteria exploit a wide variety of host cellular processes to adhere to, invade, replicate within and damage host cells. One such process is the eukaryotic secretory pathway, in which proteins and lipids are modified and transported from the endoplasmic reticulum through the Golgi network to the plasma membrane and other cellular destinations. Certain bacteria secrete toxins that utilise this transport pathway to reach their cellular targets. Some intracellular pathogens, including Legionella, Brucella and Chlamydia, engage other steps of the pathway to establish intracellular replicative organelles. Recent work has implicated specific virulence proteins of enterohaemorrhagic Escherichia coli and Salmonella enterica in secretory pathway interactions.
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http://dx.doi.org/10.1016/j.mib.2004.12.007DOI Listing
February 2005