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1.  Brucella Evades Macrophage Killing via VirB-dependent Sustained Interactions with the Endoplasmic Reticulum 
The intracellular pathogen Brucella is the causative agent of brucellosis, a worldwide zoonosis that affects mammals, including humans. Essential to Brucella virulence is its ability to survive and replicate inside host macrophages, yet the underlying mechanisms and the nature of the replicative compartment remain unclear. Here we show in a model of Brucella abortus infection of murine bone marrow–derived macrophages that a fraction of the bacteria that survive an initial macrophage killing proceed to replicate in a compartment segregated from the endocytic pathway. The maturation of the Brucella-containing vacuole involves sustained interactions and fusion with the endoplasmic reticulum (ER), which creates a replicative compartment with ER-like properties. The acquisition of ER membranes by replicating Brucella is independent of ER-Golgi COPI-dependent vesicular transport. A mutant of the VirB type IV secretion system, which is necessary for intracellular survival, was unable to sustain interactions and fuse with the ER, and was killed via eventual fusion with lysosomes. Thus, we demonstrate that live intracellular Brucella evade macrophage killing through VirB-dependent sustained interactions with the ER. Moreover, we assign an intracellular function to the VirB system, as being required for late maturation events necessary for the biogenesis of an ER-derived replicative organelle.
doi:10.1084/jem.20030088
PMCID: PMC2194179  PMID: 12925673
macrophage; Brucella; endoplasmic reticulum; type IV secretion; trafficking
2.  In Vivo Identification and Characterization of CD4+ Cytotoxic T Cells Induced by Virulent Brucella abortus Infection 
PLoS ONE  2013;8(12):e82508.
CD4+ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4+ T cells (CD4+ CTL) during Brucella abortus infection. These CD4+ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4+ CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4+ T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4+ CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.
doi:10.1371/journal.pone.0082508
PMCID: PMC3868576  PMID: 24367519
3.  A Toxin-Antitoxin Module of Salmonella Promotes Virulence in Mice 
PLoS Pathogens  2013;9(12):e1003827.
Toxin-antitoxin (TA) modules are widely prevalent in both bacteria and archaea. Originally described as stabilizing elements of plasmids, TA modules are also widespread on bacterial chromosomes. These modules promote bacterial persistence in response to specific environmental stresses. So far, the possibility that TA modules could be involved in bacterial virulence has been largely neglected, but recent comparative genomic studies have shown that the presence of TA modules is significantly associated with the pathogenicity of bacteria. Using Salmonella as a model, we investigated whether TA modules help bacteria to overcome the stress conditions encountered during colonization, thereby supporting virulence in the host. By bioinformatics analyses, we found that the genome of the pathogenic bacterium Salmonella Typhimurium encodes at least 11 type II TA modules. Several of these are conserved in other pathogenic strains but absent from non-pathogenic species indicating that certain TA modules might play a role in Salmonella pathogenicity. We show that one TA module, hereafter referred to as sehAB, plays a transient role in virulence in perorally inoculated mice. The use of a transcriptional reporter demonstrated that bacteria in which sehAB is strongly activated are predominantly localized in the mesenteric lymph nodes. In addition, sehAB was shown to be important for the survival of Salmonella in these peripheral lymphoid organs. These data indicate that the transient activation of a type II TA module can bring a selective advantage favouring virulence and demonstrate that TA modules are engaged in Salmonella pathogenesis.
doi:10.1371/journal.ppat.1003827
PMCID: PMC3868539  PMID: 24385907
4.  Lipopolysaccharides with Acylation Defects Potentiate TLR4 Signaling and Shape T Cell Responses 
PLoS ONE  2013;8(2):e55117.
Lipopolysaccharides or endotoxins are components of Gram-negative enterobacteria that cause septic shock in mammals. However, a LPS carrying hexa-acyl lipid A moieties is highly endotoxic compared to a tetra-acyl LPS and the latter has been considered as an antagonist of hexa-acyl LPS-mediated TLR4 signaling. We investigated the relationship between the structure and the function of bacterial LPS in the context of human and mouse dendritic cell activation. Strikingly, LPS with acylation defects were capable of triggering a strong and early TLR4-dependent DC activation, which in turn led to the activation of the proteasome machinery dampening the pro-inflammatory cytokine secretion. Upon activation with tetra-acyl LPS both mouse and human dendritic cells triggered CD4+ T and CD8+ T cell responses and, importantly, human myeloid dendritic cells favored the induction of regulatory T cells. Altogether, our data suggest that LPS acylation controlled by pathogenic bacteria might be an important strategy to subvert adaptive immunity.
doi:10.1371/journal.pone.0055117
PMCID: PMC3563657  PMID: 23390517
5.  In search of Brucella abortus Type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system 
Cellular microbiology  2011;13(8):1261-1274.
SUMMARY
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.
doi:10.1111/j.1462-5822.2011.01618.x
PMCID: PMC3139020  PMID: 21707904
6.  What have we learned from brucellosis in the mouse model? 
Veterinary Research  2012;43(1):29.
Brucellosis is a zoonosis caused by Brucella species. Brucellosis research in natural hosts is often precluded by practical, economical and ethical reasons and mice are widely used. However, mice are not natural Brucella hosts and the course of murine brucellosis depends on bacterial strain virulence, dose and inoculation route as well as breed, genetic background, age, sex and physiological statu of mice. Therefore, meaningful experiments require a definition of these variables. Brucella spleen replication profiles are highly reproducible and course in four phases: i), onset or spleen colonization (first 48 h); ii), acute phase, from the third day to the time when bacteria reach maximal numbers; iii), chronic steady phase, where bacterial numbers plateaus; and iv), chronic declining phase, during which brucellae are eliminated. This pattern displays clear physiopathological signs and is sensitive to small virulence variations, making possible to assess attenuation when fully virulent bacteria are used as controls. Similarly, immunity studies using mice with known defects are possible. Mutations affecting INF-γ, TLR9, Myd88, Tγδ and TNF-β favor Brucella replication; whereas IL-1β, IL-18, TLR4, TLR5, TLR2, NOD1, NOD2, GM-CSF, IL/17r, Rip2, TRIF, NK or Nramp1 deficiencies have no noticeable effects. Splenomegaly development is also useful: it correlates with IFN-γ and IL-12 levels and with Brucella strain virulence. The genetic background is also important: Brucella-resistant mice (C57BL) yield lower splenic bacterial replication and less splenomegaly than susceptible breeds. When inoculum is increased, a saturating dose above which bacterial numbers per organ do not augment, is reached. Unlike many gram-negative bacteria, lethal doses are large (≥ 108 bacteria/mouse) and normally higher than the saturating dose. Persistence is a useful virulence/attenuation index and is used in vaccine (Residual Virulence) quality control. Vaccine candidates are also often tested in mice by determining splenic Brucella numbers after challenging with appropriate virulent brucellae doses at precise post-vaccination times. Since most live or killed Brucella vaccines provide some protection in mice, controls immunized with reference vaccines (S19 or Rev1) are critical. Finally, mice have been successfully used to evaluate brucellosis therapies. It is concluded that, when used properly, the mouse is a valuable brucellosis model.
doi:10.1186/1297-9716-43-29
PMCID: PMC3410789  PMID: 22500859
7.  The Brucella abortus Phosphoglycerate Kinase Mutant Is Highly Attenuated and Induces Protection Superior to That of Vaccine Strain 19 in Immunocompromised and Immunocompetent Mice ▿  
Infection and Immunity  2010;78(5):2283-2291.
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 Δpgk 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 Δpgk 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 Δpgk deletion mutant was used as a live vaccine. Challenge experiments revealed that the Δpgk 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 Δpgk mutant may serve as a potential vaccine candidate in future studies.
doi:10.1128/IAI.01433-09
PMCID: PMC2863508  PMID: 20194591
8.  The Virulence Protein SopD2 Regulates Membrane Dynamics of Salmonella-Containing Vacuoles 
PLoS Pathogens  2010;6(7):e1001002.
Salmonella enterica serovar Typhimurium is a Gram-negative bacterial pathogen causing gastroenteritis in humans and a systemic typhoid-like illness in mice. The capacity of Salmonella to cause diseases relies on the establishment of its intracellular replication niche, a membrane-bound compartment named the Salmonella-containing vacuole (SCV). This requires the translocation of bacterial effector proteins into the host cell by type three secretion systems. Among these effectors, SifA is required for the SCV stability, the formation of Salmonella-induced filaments (SIFs) and plays an important role in the virulence of Salmonella. Here we show that the effector SopD2 is responsible for the SCV instability that triggers the cytoplasmic release of a sifA− mutant. Deletion of sopD2 also rescued intra-macrophagic replication and increased virulence of sifA− mutants in mice. Membrane tubular structures that extend from the SCV are the hallmark of Salmonella-infected cells. Until now, these unique structures have not been observed in the absence of SifA. The deletion of sopD2 in a sifA− mutant strain re-established membrane trafficking from the SCV and led to the formation of new membrane tubular structures, the formation of which is dependent on other Salmonella effector(s). Taken together, our data demonstrate that SopD2 inhibits the vesicular transport and the formation of tubules that extend outward from the SCV and thereby contributes to the sifA− associated phenotypes. These results also highlight the antagonistic roles played by SopD2 and SifA in the membrane dynamics of the vacuole, and the complex actions of SopD2, SifA, PipB2 and other unidentified effector(s) in the biogenesis and maintenance of the Salmonella replicative niche.
Author Summary
Salmonella typhimurium is a bacterial pathogen that causes diseases ranging from gastroenteritis to typhoid fever. This bacterium survives inside eukaryotic cells within a membrane-bound compartment, namely the Salmonella-containing vacuole. Salmonella injects proteins, named effectors, into the infected cell. These effectors change the biology of the infected cell and collectively support Salmonella replication and virulence. The effector SifA plays a key role in the bacterial vacuole stability and in the formation of membrane tubules that extend from the vacuole. Absence of SifA leads to the disruption of the vacuolar membrane and, therefore to the release of bacteria in the cytosolic compartment. Consequently, this mutant presents significant replication and virulence defects. Here, we show that an additional Salmonella effector, SopD2, is responsible for the membrane instability of the sifA− vacuole. In addition, we demonstrate that SopD2 acts as an inhibitor of vesicle transport from the vacuole and that it down-modulates the formation of tubular structures. These findings describe a role for SopD2 as an antagonist of SifA in terms of vacuolar membrane dynamics.
doi:10.1371/journal.ppat.1001002
PMCID: PMC2904799  PMID: 20664790
9.  Helicobacter pylori Impairs Murine Dendritic Cell Responses to Infection 
PLoS ONE  2010;5(5):e10844.
Background
Helicobacter pylori, a human pathogen associated with chronic gastritis, peptic ulcer and gastric malignancies, is generally viewed as an extracellular microorganism. Here, we show that H. pylori replicates in murine bone marrow derived-dendritic cells (BMDCs) within autophagosomes.
Methodology/Principal Findings
A 10-fold increase of CFU is found between 2 h and 6 h p.i. in H. pylori-infected BMDCs. Autophagy is induced around the bacterium and participates at late time points of infection for the clearance of intracellular H. pylori. As a consequence of infection, LC3, LAMP1 and MHC class II molecules are retained within the H. pylori-containing vacuoles and export of MHC class II molecules to cell surface is blocked. However, formalin-fixed H. pylori still maintain this inhibitory activity in BMDC derived from wild type mice, but not in from either TLR4 or TLR2-deficient mice, suggesting the involvement of H. pylori-LPS in this process. TNF-alpha, IL-6 and IL-10 expression was also modulated upon infection showing a TLR2-specific dependent IL-10 secretion. No IL-12 was detected favoring the hypothesis of a down modulation of DC functions during H. pylori infection. Furthermore, antigen-specific T cells proliferation was also impaired upon infection.
Conclusions/Significance
H. pylori can infect and replicate in BMDCs and thereby affects DC-mediated immune responses. The implication of this new finding is discussed for the biological life cycle of H. pylori in the host.
doi:10.1371/journal.pone.0010844
PMCID: PMC2877707  PMID: 20523725
10.  Transcriptome Analysis of the Brucella abortus BvrR/BvrS Two-Component Regulatory System 
PLoS ONE  2010;5(4):e10216.
Background
The two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions.
Methodology/Principal Findings
A total of 127 differentially expressed genes were found: 83 were over expressed and 44 were less expressed in the bvrR mutant. Two operons, the phosphotransferase system and the maltose transport system, were down-regulated. Several genes involved in cell envelope or outer membrane biogenesis were differentially expressed: genes for outer membrane proteins (omp25a, omp25d), lipoproteins, LPS and fatty acid biosynthesis, stress response proteins, chaperones, flagellar genes, and twelve genes encoding ABC transport systems. Ten genes related with carbon metabolism (pckA and fumB among others) were up-regulated in the bvrR mutant, and denitrification genes (nirK, norC and nosZ) were also regulated. Notably, seven transcriptional regulators were affected, including VjbR, ExoR and OmpR that were less expressed in the bvrR mutant. Finally, the expression of eleven genes which have been previously related with Brucella virulence was also altered.
Conclusions/Significance
All these data corroborate the impact of BvrR/BvrS on cell envelope modulation, confirm that this system controls the carbon and nitrogen metabolism, and suggest a cross-talk among some regulators to adjust the Brucella physiology to the shift expected to occur during the transit from the extracellular to the intracellular niche.
doi:10.1371/journal.pone.0010216
PMCID: PMC2858072  PMID: 20422049
11.  The Glyceraldehyde-3-Phosphate Dehydrogenase and the Small GTPase Rab 2 Are Crucial for Brucella Replication 
PLoS Pathogens  2009;5(6):e1000487.
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 ι, 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.
Author Summary
A key determinant for intracellular pathogenic bacteria to ensure their virulence within host cells is their ability to bypass the endocytic pathway and to reach a safe replication niche. Brucella bacteria reach the endoplasmic reticulum (ER) to create their replicating niche called the Brucella-containing vacuole (BCV). The ER is a suitable strategic place for pathogenic Brucella. Bacteria can be hidden from host cell defences to persist within the host, and can take advantage of the membrane reservoir delivered by the ER to replicate. Interactions between BCV and the ER lead to the presence of ER proteins on the BCV membrane. Currently, no other proteins (eukaryotic or prokaryotic) have yet been associated with the BCV membrane. Here we show that non-ER related proteins are also present on the BCV membrane, in particular, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 known to be located on secretory vesicles that traffic between the ER and the Golgi apparatus. GAPDH and the small GTPase Rab 2 are involved in Brucella replication at late post-infection. Similarly, integrity of secretory vesicle trafficking is also necessary for Brucella replication. Here, we show that recruitment of the two eukaryotic proteins GAPDH and Rab 2 on BCV membranes is necessary for the establishment of the replicative niche by sustaining interactions between the ER and secretory membrane vesicles.
doi:10.1371/journal.ppat.1000487
PMCID: PMC2695806  PMID: 19557163
12.  Nanobacteria Are Mineralo Fetuin Complexes 
PLoS Pathogens  2008;4(2):e41.
“Nanobacteria” are nanometer-scale spherical and ovoid particles which have spurred one of the biggest controversies in modern microbiology. Their biological nature has been severely challenged by both geologists and microbiologists, with opinions ranging from considering them crystal structures to new life forms. Although the nature of these autonomously replicating particles is still under debate, their role in several calcification-related diseases has been reported. In order to gain better insights on this calciferous agent, we performed a large-scale project, including the analysis of “nanobacteria” susceptibility to physical and chemical compounds as well as the comprehensive nucleotide, biochemical, proteomic, and antigenic analysis of these particles. Our results definitively ruled out the existence of “nanobacteria” as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call “nanons.” The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor.
Author Summary
In the last decade, the exact nature of nanobacteria was one of the most controversial of scientific questions. An audacious theory proposed the existence of nanobacteria, initially discovered in Italian hot spring deposits, as a new life form responsible for a wide range of diseases in humans, thus qualifying them as new agents of emerging infectious diseases. The community of microbiologists remained therefore skeptical about the fact that such structures, 100 times smaller than bacteria and highly resistant to heat and other treatments that would normally kill the latter, could be living entities fully capable of self-replication. Other scientists wondered if they might be an unusual form of crystal rather than micro-organisms. The comprehensive characterization of nanobacteria was the focus of our study. Our results definitively ruled out the existence of nanobacteria as living entities and revealed that they correspond to self-propagating mineral-fetuin complexes that we called “nanons.”
doi:10.1371/journal.ppat.0040041
PMCID: PMC2242841  PMID: 18282102
13.  Mimivirus Giant Particles Incorporate a Large Fraction of Anonymous and Unique Gene Products▿ †  
Journal of Virology  2006;80(23):11678-11685.
Acanthamoeba polyphaga mimivirus is the largest known virus in both particle size and genome complexity. Its 1.2-Mb genome encodes 911 proteins, among which only 298 have predicted functions. The composition of purified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approach allowing the identification of 114 proteins. Besides the expected major structural components, the viral particle packages 12 proteins unambiguously associated with transcriptional machinery, 3 proteins associated with DNA repair, and 2 topoisomerases. Other main functional categories represented in the virion include oxidative pathways and protein modification. More than half of the identified virion-associated proteins correspond to anonymous genes of unknown function, including 45 “ORFans.” As demonstrated by both Western blotting and immunogold staining, some of these “ORFans,” which lack any convincing similarity in the sequence databases, are endowed with antigenic properties. Thus, anonymous and unique genes constituting the majority of the mimivirus gene complement encode bona fide proteins that are likely to participate in well-integrated processes.
doi:10.1128/JVI.00940-06
PMCID: PMC1642625  PMID: 16971431
14.  The Translocated Salmonella Effector Proteins SseF and SseG Interact and Are Required To Establish an Intracellular Replication Niche▿  
Infection and Immunity  2006;74(12):6965-6972.
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.
doi:10.1128/IAI.00648-06
PMCID: PMC1698088  PMID: 17015457
15.  Bactericidal and Antiendotoxic Properties of Short Cationic Peptides Derived from a Snake Venom Lys49 Phospholipase A2 
The activities of short synthetic, nonhemolytic peptides derived from the C-terminal region of myotoxin II, a catalytically inactive phospholipase A2 homologue present in the venom of the snake Bothrops asper, have been shown to reproduce the bactericidal activity of the parent protein. They combine cationic and hydrophobic-aromatic amino acids, thus functionally resembling the antimicrobial peptides of innate defenses. This study evaluated the antimicrobial and antiendotoxic properties of a 13-mer derivative peptide of the C-terminal sequence from positions 115 to 129 of myotoxin II, named pEM-2. This peptide (KKWRWWLKALAKK) showed bactericidal activity against both gram-positive and gram-negative bacteria. In comparison to previously described peptide variants derived from myotoxin II, the toxicity of pEM-2 toward eukaryotic cells in culture was significantly reduced, being similar to that of lactoferricin B but lower than that of polymyxin B. The all-d enantiomer of pEM-2 [pEM-2 (d)] retained the same bactericidal potency of its l-enantiomeric counterpart, but it showed an enhanced ability to counteract the lethal activity of an intraperitoneal lipopolysaccharide challenge in mice, which correlated with a significant reduction of the serum tumor necrosis factor alpha levels triggered by this endotoxin. Lethality induced by intraperitoneal infection of mice with Escherichia coli or Salmonella enterica serovar Typhimurium was reduced by the administration of pEM-2 (d). These results demonstrate that phospholipase A2-derived peptides may have the potential to counteract microbial infections and encourage further evaluations of their actions in vivo.
doi:10.1128/AAC.49.4.1340-1345.2005
PMCID: PMC1068596  PMID: 15793109
16.  The Cytoplasmic Tail of Invariant Chain Regulates Endosome Fusion and Morphology 
Molecular Biology of the Cell  2002;13(6):1846-1856.
The major histocompatibility complex class II associated invariant chain (Ii) has been shown to inhibit endocytic transport and to increase the size of endosomes. We have recently found that this property has a significant impact on antigen processing and presentation. Here, we show in a cell-free endosome fusion assay that expression of Ii can increase fusion after phosphatidylinositol 3-kinase activity is blocked by wortmannin. In live cells wortmannin was also not able to block formation of the Ii-induced enlarged endosomes. The effects of Ii on endosomal transport and morphology depend on elements within the cytoplasmic tail. Data from mutagenesis analysis and nuclear magnetic resonance-based structure calculations of the Ii cytoplasmic tail demonstrate that free negative charges that are not involved in internal salt bridges are essential for both interactions between the tails and for the formation of enlarged endosomes. This correlation indicates that it is interactions between the Ii cytoplasmic tails that are involved in endosome fusion. The combined data from live cells, cell-free assays, and molecular dynamic simulations suggest that Ii molecules on different vesicles can promote endosome docking and fusion and thereby control endosomal traffic of membrane proteins and endosomal content.
doi:10.1091/mbc.01-10-0478
PMCID: PMC117608  PMID: 12058053
17.  Survival of Tropheryma whipplei, the Agent of Whipple's Disease, Requires Phagosome Acidification 
Infection and Immunity  2002;70(3):1501-1506.
Tropheryma whipplei was established as the agent of Whipple's disease in 2000, but the mechanisms by which it survives within host cells are still unknown. We show here that T. whipplei survives within HeLa cells by controlling the biogenesis of its phagosome. Indeed, T. whipplei colocalized with lysosome-associated membrane protein 1, a membrane marker of late endosomal and lysosomal compartments, but not with cathepsin D, a lysosomal hydrolase. This defect in phagosome maturation is specific to live organisms, since heat-killed bacilli colocalized with cathepsin D. In addition, T. whipplei survived within HeLa cells by adapting to acidic pH. The vacuoles containing T. whipplei were acidic (pH 4.7 ± 0.3) and acquired vacuolar ATPase, responsible for the acidic pH of late phagosomes. The treatment of HeLa cells with pH-neutralizing reagents, such as ammonium chloride, N-ethylmaleimide, bafilomycin A1, and chloroquine, increased the intravacuolar pH and promoted the killing of T. whipplei. The ability of T. whipplei to survive in an acidic environment and to interfere with phagosome-lysosome fusion is likely critical for its prolonged persistence in host cells during the course of Whipple's disease. Our results suggest that manipulating the intravacuolar pH may provide a new approach for the treatment of Whipple's disease.
doi:10.1128/IAI.70.3.1501-1506.2002
PMCID: PMC127739  PMID: 11854238
18.  Trafficking of Shigella Lipopolysaccharide in Polarized Intestinal Epithelial Cells  
The Journal of Cell Biology  1999;145(4):689-698.
Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395–4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.
PMCID: PMC2133196  PMID: 10330399
Shigella; lipopolysaccharide; transcytosis; trafficking; epithelial
19.  The Outer Membrane of Brucella ovis Shows Increased Permeability to Hydrophobic Probes and Is More Susceptible to Cationic Peptides than Are the Outer Membranes of Mutant Rough Brucella abortus Strains 
Infection and Immunity  1999;67(11):6181-6186.
The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-l-lysine, and poly-l-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells.
PMCID: PMC97012  PMID: 10531286
20.  Interaction of Brucella abortus Lipopolysaccharide with Major Histocompatibility Complex Class II Molecules in B Lymphocytes 
Infection and Immunity  1999;67(8):4048-4054.
Lipopolysaccharide (LPS), a major amphiphilic molecule located at the outer membrane of gram-negative bacteria, is a potent antigen known to induce specific humoral immune responses in infected mammals. LPS has been described as a polyclonal activator of B lymphocytes, triggering the secretion of antibodies directed against distinct sugar epitopes of the LPS chain. But, how LPS is handled by B cells remains to be fully understood. This task appears to be essential for a better knowledge of the anti-LPS humoral immune response. In this study, we examine the internalization of LPS and its interaction with antigen-presenting major histocompatibility complex (MHC) class II molecules in murine and human B-cell lines. By use of immunofluorescence, we observe that structurally different LPSs from Brucella and Shigella strains accumulate in an intracellular compartment enriched in MHC class II molecules. By use of immunoprecipitation, we illustrate that only Brucella abortus LPS associates with MHC class II molecules in a haplotype-independent manner. Taken together, these results raise the possibility that B. abortus LPS may play a role in T-cell activation.
PMCID: PMC96700  PMID: 10417173
21.  Brucella abortus Transits through the Autophagic Pathway and Replicates in the Endoplasmic Reticulum of Nonprofessional Phagocytes 
Infection and Immunity  1998;66(12):5711-5724.
Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At ∼1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61β but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61β- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic B. abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER.
PMCID: PMC108722  PMID: 9826346
22.  Virulent Brucella abortus Prevents Lysosome Fusion and Is Distributed within Autophagosome-Like Compartments 
Infection and Immunity  1998;66(5):2387-2392.
Virulent and attenuated Brucella abortus strains attach to and penetrate nonprofessional phagocytic HeLa cells. Compared to pathogenic Brucella, the attenuated strain 19 hardly replicates within cells. The majority of the strain 19 bacteria colocalized with the lysosome marker cathepsin D, suggesting that Brucella-containing phagosomes had fused with lysosomes, in which they may have degraded. The virulent bacteria prevented lysosome-phagosome fusion and were found distributed in the perinuclear region within compartments resembling autophagosomes.
PMCID: PMC108212  PMID: 9573138
23.  Brucella Cyclic β-1,2-Glucan Plays a Critical Role in the Induction of Splenomegaly in Mice 
PLoS ONE  2014;9(7):e101279.
Brucella, the etiological agent of animal and human brucellosis, is a bacterium with the capacity to modulate the inflammatory response. Cyclic β-1,2-glucan (CβG) is a virulence factor key for the pathogenesis of Brucella as it is involved in the intracellular life cycle of the bacteria. Using comparative studies with different CβG mutants of Brucella, cgs (CβG synthase), cgt (CβG transporter) and cgm (CβG modifier), we have identified different roles for this polysaccharide in Brucella. While anionic CβG is required for bacterial growth in low osmolarity conditions, the sole requirement for a successful Brucella interaction with mammalian host is its transport to periplasmic space. Our results uncover a new role for CβG in promoting splenomegaly in mice. We showed that CβG-dependent spleen inflammation is the consequence of massive cell recruitment (monocytes, dendritics cells and neutrophils) due to the induction of pro-inflammatory cytokines such as IL-12 and TNF-α and also that the reduced splenomegaly response observed with the cgs mutant is not the consequence of changes in expression levels of the characterized Brucella PAMPs LPS, flagellin or OMP16/19. Complementation of cgs mutant with purified CβG increased significantly spleen inflammation response suggesting a direct role for this polysaccharide.
doi:10.1371/journal.pone.0101279
PMCID: PMC4077732  PMID: 24983999
24.  Intracellular Bacteria Interfere with Dendritic Cell Functions: Role of the Type I Interferon Pathway 
PLoS ONE  2014;9(6):e99420.
Dendritic cells (DCs) orchestrate host defenses against microorganisms. In infectious diseases due to intracellular bacteria, the inefficiency of the immune system to eradicate microorganisms has been attributed to the hijacking of DC functions. In this study, we selected intracellular bacterial pathogens with distinct lifestyles and explored the responses of monocyte-derived DCs (moDCs). Using lipopolysaccharide as a control, we found that Orientia tsutsugamushi, the causative agent of scrub typhus that survives in the cytosol of target cells, induced moDC maturation, as assessed by decreased endocytosis activity, the ability to induce lymphocyte proliferation and the membrane expression of phenotypic markers. In contrast, Coxiella burnetii, the agent of Q fever, and Brucella abortus, the agent of brucellosis, both of which reside in vacuolar compartments, only partly induced the maturation of moDCs, as demonstrated by a phenotypic analysis. To analyze the mechanisms used by C. burnetii and B. abortus to alter moDC activation, we performed microarray and found that C. burnetii and B. abortus induced a specific signature consisting of TLR4, TLR3, STAT1 and interferon response genes. These genes were down-modulated in response to C. burnetii and B. abortus but up-modulated in moDCs activated by lipopolysaccharide and O. tsutsugamushi. This transcriptional alteration was associated with the defective interferon-β production. This study demonstrates that intracellular bacteria specifically affect moDC responses and emphasizes how C. burnetii and B. abortus interfere with moDC activation and the antimicrobial immune response. We believe that comparing infection by several bacterial species may be useful for defining new pathways and biomarkers and for developing new treatment strategies.
doi:10.1371/journal.pone.0099420
PMCID: PMC4051653  PMID: 24915541
25.  Microarray-Based Identification of Differentially Expressed Genes in Intracellular Brucella abortus within RAW264.7 Cells 
PLoS ONE  2013;8(8):e67014.
Brucella spp. is a species of facultative intracellular Gram-negative bacteria that induces abortion and causes sterility in domesticated mammals and chronic undulant fever in humans. Important determinants of Brucella’s virulence and potential for chronic infection include the ability to circumvent the host cell’s internal surveillance system and the capability to proliferate within dedicated and non-dedicated phagocytes. Hence, identifying genes necessary for intracellular survival may hold the key to understanding Brucella infection. In the present study, microarray analysis reveals that 7.82% (244/3334) of all Brucella abortus genes were up-regulated and 5.4% (180/3334) were down-regulated in RAW264.7 cells, compared to free-living cells in TSB. qRT-PCR verification further confirmed a >5-fold up-regulation for fourteen genes. Functional analysis classified araC, ddp, and eryD as to partake in information storage and processing, alp, flgF and virB9 to be involved in cellular processes, hpcd and aldh to play a role in metabolism, mfs and nikC to be involved in both cellular processes and metabolism, and four hypothetical genes (bruAb1_1814, bruAb1_0475, bruAb1_1926, and bruAb1_0292) had unknown functions. Furthermore, we constructed a B. abortus 2308 mutant Δddp where the ddp gene is deleted in order to evaluate the role of ddp in intracellular survival. Infection assay indicated significantly higher adherence and invasion abilities of the Δddp mutant, however it does not survive well in RAW264.7 cells. Brucella may survive in hostile intracellular environment by modulating gene expression.
doi:10.1371/journal.pone.0067014
PMCID: PMC3737221  PMID: 23950864

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