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1.  A pathogen’s journey in the host cell 
Bioarchitecture  2012;2(2):38-42.
Manipulation of the actin cytoskeleton is a commonly used process by which bacterial pathogens and viruses are able to neutralize host defense mechanisms and subvert them in order to replicate in a hostile environment. Diverse bacteria display a wide array of mechanisms of regulation of microfilaments to enter, move within or exit the host cell. A less studied subject is how pathogens may co-opt the actin cytoskeleton to disturb vesicle trafficking pathways, namely phagolysosomal fusion, and avoid degradation. In fact, although actin plays a role in endosomal trafficking and phagosome maturation, the knowledge on the exact mechanisms and additional players is still scarce. Recently, we found that the Legionella pneumophila virulence factor VipA is an actin nucleator, associates with actin filaments and early endosomes during infection, and interferes in yeast organelle trafficking pathways, suggesting it may be linking actin dynamics to endosome biogenesis. Further studies on this protein, together with work on other bacterial effectors, may help shed light in the role of actin in endosomal maturation.
PMCID: PMC3383720  PMID: 22754628
Legionella pneumophila; Type IV Secretion System; VipA; actin; effector; multivesicular body; organelle trafficking
2.  Towards Novel Amino Acid-Base Contacts in Gene Regulatory Proteins: AraR – A Case Study 
PLoS ONE  2014;9(11):e111802.
AraR is a transcription factor involved in the regulation of carbon catabolism in Bacillus subtilis. This regulator belongs to the vast GntR family of helix-turn-helix (HTH) bacterial metabolite-responsive transcription factors. In this study, AraR-DNA specific interactions were analysed by an in vitro missing-contact probing and validated using an in vivo model. We show that amino acid E30 of AraR, a highly conserved residue in GntR regulators, is indirectly responsible for the specificity of amino acid-base contacts, and that by mutating this residue it will be possible to achieve new specificities towards DNA contacts. The results highlight the importance in DNA recognition and binding of highly conserved residues across certain families of transcription factors that are located in the DNA-binding domain but not predicted to specifically contact bases on the DNA. These new findings not only contribute to a more detailed comprehension of AraR-operator interactions, but may also be useful for the establishment of a framework of rules governing protein-DNA recognition.
doi:10.1371/journal.pone.0111802
PMCID: PMC4218819  PMID: 25364981
3.  The Legionella pneumophila Effector VipA Is an Actin Nucleator That Alters Host Cell Organelle Trafficking 
PLoS Pathogens  2012;8(2):e1002546.
Legionella pneumophila, the causative agent of Legionnaires' disease, invades and replicates within macrophages and protozoan cells inside a vacuole. The type IVB Icm/Dot secretion system is necessary for the translocation of effector proteins that modulate vesicle trafficking pathways in the host cell, thus avoiding phagosome-lysosome fusion. The Legionella VipA effector was previously identified by its ability to interfere with organelle trafficking in the Multivesicular Body (MVB) pathway when ectopically expressed in yeast. In this study, we show that VipA binds actin in vitro and directly polymerizes microfilaments without the requirement of additional proteins, displaying properties distinct from other bacterial actin nucleators. Microscopy studies revealed that fluorescently tagged VipA variants localize to puncta in eukaryotic cells. In yeast these puncta are associated with actin-rich regions and components of the Multivesicular Body pathway such as endosomes and the MVB-associated protein Bro1. During macrophage infection, native translocated VipA associated with actin patches and early endosomes. When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets. Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization. These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways. VipA constitutes a novel type of actin nucleator that may contribute to the intracellular lifestyle of Legionella by altering cytoskeleton dynamics to target host cell pathways.
Author Summary
Legionella pneumophila is a facultative intracellular bacterium that can cause an often fatal type of pneumonia known as Legionnaires' disease. In nature, L. pneumophila is found in both fresh water and soil where it parasitizes free-living protists. Upon inhalation of contaminated aerosols, L. pneumophila invades and replicates in alveolar macrophages, leading to inflammation and development of the disease. Legionella uses a type IVB secretion system to translocate effector proteins into the host cell that modify its trafficking pathways and prevent fusion of the newly formed phagosome with the lysosome. One of these effectors is VipA, which, when expressed in yeast interferes with the Multivesicular Body (MVB) pathway. We found that VipA protein binds actin and nucleates its polymerization without additional host factors. VipA localizes in puncta in eukaryotic cells, and these colocalize with actin-rich regions and endosomes. We demonstrate that the ability to disrupt the MVB is associated with the capacity to bind actin. Thus VipA may contribute to the intracellular lifestyle of L. pneumophila by targeting the cytoskeleton in order to disrupt normal vacuolar trafficking pathways in host cells.
doi:10.1371/journal.ppat.1002546
PMCID: PMC3285593  PMID: 22383880
4.  Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon 
Nucleic Acids Research  2007;35(14):4755-4766.
In the absence of arabinose, the AraR transcription factor represses the expression of genes involved in the utilization of arabinose, xylose and galactose in Bacillus subtilis. AraR exhibits a chimeric organization: the N-terminal DNA-binding region belongs to the GntR family and the C-terminal effector-binding domain is homologous to the GalR/LacI family. Here, the AraR–DNA-binding interactions were characterized in vivo and in vitro. The effect of residue substitutions in the AraR N-terminal domain and of base-pair exchanges into an AraR–DNA-binding operator site were examined by assaying for AraR-mediated regulatory activity in vivo and DNA-binding activity in vitro. The results showed that residues K4, R45 and Q61, located in or near the winged-helix DNA-binding motif, were the most critical amino acids required for AraR function. In addition, the analysis of the various mutations in an AraR palindromic operator sequence indicated that bases G9, A11 and T16 are crucial for AraR binding. Moreover, an AraR mutant M34T was isolated that partially suppressed the effect of mutations in the regulatory cis-elements. Together, these findings extend the knowledge on the nature of AraR nucleoprotein complexes and provide insight into the mechanism that underlies the mode of action of AraR and its orthologues.
doi:10.1093/nar/gkm509
PMCID: PMC1950556  PMID: 17617643
5.  Functional Domains of the Bacillus subtilis Transcription Factor AraR and Identification of Amino Acids Important for Nucleoprotein Complex Assembly and Effector Binding†  
Journal of Bacteriology  2006;188(8):3024-3036.
The Bacillus subtilis AraR transcription factor represses at least 13 genes required for the extracellular degradation of arabinose-containing polysaccharides, transport of arabinose, arabinose oligomers, xylose, and galactose, intracellular degradation of arabinose oligomers, and further catabolism of this sugar. AraR exhibits a chimeric organization comprising a small N-terminal DNA-binding domain that contains a winged helix-turn-helix motif similar to that seen with the GntR family and a larger C-terminal domain homologous to that of the LacI/GalR family. Here, a model for AraR was derived based on the known crystal structures of the FadR and PurR regulators from Escherichia coli. We have used random mutagenesis, deletion, and construction of chimeric LexA-AraR fusion proteins to map the functional domains of AraR required for DNA binding, dimerization, and effector binding. Moreover, predictions for the functional role of specific residues were tested by site-directed mutagenesis. In vivo analysis identified particular amino acids required for dimer assembly, formation of the nucleoprotein complex, and composition of the sugar-binding cleft. This work presents a structural framework for the function of AraR and provides insight into the mechanistic mode of action of this modular repressor.
doi:10.1128/JB.188.8.3024-3036.2006
PMCID: PMC1446991  PMID: 16585763

Results 1-5 (5)