RESUMEN
Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.
Asunto(s)
Brucella , Ochrobactrum , Ochrobactrum/clasificación , Ochrobactrum/genética , Ochrobactrum/patogenicidad , Ochrobactrum/fisiología , Brucella/clasificación , Brucella/genética , Brucella/patogenicidad , Brucella/fisiología , Terminología como Asunto , Filogenia , Brucelosis/tratamiento farmacológico , Brucelosis/microbiología , Humanos , Infecciones Oportunistas/microbiologíaRESUMEN
Peyer's patches (PPs) are secondary lymphoid organs in contact with the external environment via the intestinal lumen, thus combining antigen sampling and immune response initiation sites. Therefore, they provide a unique opportunity to study the entire process of phagocyte differentiation and activation in vivo. Here, we deciphered the transcriptional and spatial landscape of PP phagocyte populations from their emergence in the tissue to their final maturation state at homeostasis and under stimulation. Activation of monocyte-derived Lysozyme-expressing dendritic cells (LysoDCs) differs from that of macrophages by their upregulation of conventional DC (cDC) signature genes such as Ccr7 and downregulation of typical monocyte-derived cell genes such as Cx3cr1. We identified gene sets that distinguish PP cDCs from the villus ones and from LysoDCs. We also identified key immature, early, intermediate, and late maturation markers of PP phagocytes. Finally, exploiting the ability of the PP interfollicular region to host both villous and subepithelial dome emigrated cDCs, we showed that the type of stimulus, the subset, but also the initial location of cDCs shape their activation profile and thus direct the immune response. Our study highlights the importance of targeting the right phagocyte subset at the right place and time to manipulate the immune response.
Asunto(s)
Células Dendríticas , Ganglios Linfáticos Agregados , Fagocitos , Macrófagos , Sistema Mononuclear FagocíticoRESUMEN
The intracellular pathogens of the genus Brucella are phylogenetically close to Ochrobactrum, a diverse group of free-living bacteria with a few species occasionally infecting medically compromised patients. A group of taxonomists recently included all Ochrobactrum organisms in the genus Brucella based on global genome analyses and alleged equivalences with genera such as Mycobacterium. Here, we demonstrate that such equivalencies are incorrect because they overlook the complexities of pathogenicity. By summarizing Brucella and Ochrobactrum divergences in lifestyle, structure, physiology, population, closed versus open pangenomes, genomic traits, and pathogenicity, we show that when they are adequately understood, they are highly relevant in taxonomy and not unidimensional quantitative characters. Thus, the Ochrobactrum and Brucella differences are not limited to their assignments to different "risk-groups", a biologically (and hence, taxonomically) oversimplified description that, moreover, does not support ignoring the nomen periculosum rule, as proposed. Since the epidemiology, prophylaxis, diagnosis, and treatment are thoroughly unrelated, merging free-living Ochrobactrum organisms with highly pathogenic Brucella organisms brings evident risks for veterinarians, medical doctors, and public health authorities who confront brucellosis, a significant zoonosis worldwide. Therefore, from taxonomical and practical standpoints, the Brucella and Ochrobactrum genera must be maintained apart. Consequently, we urge researchers, culture collections, and databases to keep their canonical nomenclature.
RESUMEN
Salmonella enterica serotype Typhimurium is a bacterium that causes gastroenteritis and diarrhea in humans. The genome of S. Typhimurium codes for diverse virulence factors, among which are the toxin-antitoxin (TA) systems. SehAB is a type II TA, where SehA is the toxin and SehB is the antitoxin. It was previously reported that the absence of the SehB antitoxin affects the growth of S. Typhimurium. In addition, the SehB antitoxin can interact directly with the SehA toxin neutralizing its toxic effect as well as repressing its own expression. We identified conserved residues on SehB homologous proteins. Point mutations were introduced at both N- and C-terminal of SehB antitoxin to analyze the effect of these changes on its transcription repressor function, on its ability to form homodimers and on the virulence of S. Typhimurium. All changes in amino acid residues at both the N- and C-terminal affected the repressor function of SehB antitoxin and they were required for DNA-binding activity. Mutations in the amino acid residues at the N-terminal showed a lower capacity for homodimer formation of the SehB protein. However, none of the SehB point mutants were affected in the interaction with the SehA toxin. In terms of virulence, the eight single-amino acid mutations were attenuated for virulence in the mouse model. In agreement with our results, the eight amino acid residues of SehB antitoxin were required for its repressor activity, affecting both homodimerization and DNA-binding activity, supporting the notion that both activities of SehB antitoxin are required to confer virulence to Salmonella enterica.
RESUMEN
Cyclic ß-1,2-D-glucans (CßG) are natural bionanopolymers present in the periplasmic space of many Proteobacteria. These molecules are sugar rings made of 17 to 25 D-glucose units linked exclusively by ß-1,2-glycosidic bonds. CßG are important for environmental sensing and osmoadaptation in bacteria, but most importantly, they play key roles in complex host-cell interactions such as symbiosis, pathogenesis, and immunomodulation. In the last years, the identification and characterisation of the enzymes involved in the synthesis of CßG allowed to know in detail the steps necessary for the formation of these sugar rings. Due to its peculiar structure, CßG can complex large hydrophobic molecules, a feature possibly related to its function in the interaction with the host. The capabilities of the CßG to function as molecular boxes and to solubilise hydrophobic compounds are attractive for application in the development of drugs, in food industry, nanotechnology, and chemistry. More importantly, its excellent immunomodulatory properties led to the proposal of CßG as a new class of adjuvants for vaccine development.
Asunto(s)
Interacciones Huésped-Patógeno , Proteobacteria/fisiología , Proteobacteria/patogenicidad , Simbiosis , beta-Glucanos/química , beta-Glucanos/metabolismo , Vías Biosintéticas , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Brucellosis is a zoonotic bacterial infection that may persist for long periods causing relapses in antibiotic-treated patients. The ability of Brucella to develop chronic infections is linked to their capacity to invade and replicate within the mononuclear phagocyte system, including the bone marrow (BM). Persistence of Brucella in the BM has been associated with hematological complications such as neutropenia, thrombocytopenia, anemia, and pancytopenia in human patients. In the mouse model, we observed that the number of Brucella abortus in the BM remained constant for up to 168 days of postinfection. This persistence was associated with histopathological changes, accompanied by augmented numbers of BM myeloid GMP progenitors, PMNs, and CD4+ lymphocytes during the acute phase (eight days) of the infection in the BM. Monocytes, PMNs, and GMP cells were identified as the cells harboring Brucella in the BM. We propose that the BM is an essential niche for the bacterium to establish long-lasting infections and that infected PMNs may serve as vehicles for dispersion of Brucella organisms, following the Trojan horse hypothesis. Monocytes are solid candidates for Brucella reservoirs in the BM.
Asunto(s)
Médula Ósea/microbiología , Brucella abortus/fisiología , Brucelosis/inmunología , Linfocitos T CD4-Positivos/inmunología , Monocitos/inmunología , Células Progenitoras Mieloides/fisiología , Neutrófilos/inmunología , Animales , Autofagia , Bovinos , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1ß by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.
Asunto(s)
Brucella abortus/inmunología , Brucelosis/inmunología , Lipopolisacáridos/inmunología , Mortalidad Prematura , Neutrófilos/citología , Brucella abortus/aislamiento & purificación , Muerte Celular , Citocinas/metabolismo , Humanos , Inmunidad Innata/inmunología , Leucocitos/metabolismoRESUMEN
Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, Brucella abortus is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-γ and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular Brucella infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity.
Asunto(s)
Brucella abortus/patogenicidad , Brucelosis/inmunología , Inmunidad Innata/inmunología , Neutrófilos/inmunología , Células TH1/inmunología , Inmunidad Adaptativa , Animales , Brucelosis/virología , Citocinas/metabolismo , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Neutrófilos/virología , Células TH1/virologíaRESUMEN
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-ß favor Brucella replication; whereas IL-1ß, 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.
Asunto(s)
Brucella/fisiología , Brucella/patogenicidad , Brucelosis/inmunología , Brucelosis/microbiología , Modelos Animales de Enfermedad , Ratones , Animales , Brucelosis/genética , Brucelosis/fisiopatología , Humanos , VirulenciaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Brucella abortus/metabolismo , Brucella abortus/patogenicidad , Macrófagos/microbiología , Factores de Virulencia/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Fusión Artificial Génica , Proteínas Bacterianas/genética , Brucella abortus/genética , Brucelosis/microbiología , Línea Celular , Biología Computacional/métodos , Modelos Animales de Enfermedad , Genes Reporteros , Genómica/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Confocal , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Virulencia/genéticaRESUMEN
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.
Asunto(s)
Vacuna contra la Brucelosis/inmunología , Brucella abortus/enzimología , Brucelosis/inmunología , Transferasas de Hidroximetilo y Formilo/metabolismo , Macrófagos/inmunología , Animales , Células de la Médula Ósea/inmunología , Brucella abortus/genética , Brucella abortus/inmunología , Brucella abortus/patogenicidad , Brucelosis/microbiología , Transferasas de Hidroximetilo y Formilo/genética , Interleucina-12/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Factor de Necrosis Tumoral alfa/inmunología , Vacunas Atenuadas/inmunología , VirulenciaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Vacuna contra la Brucelosis/inmunología , Brucella abortus/enzimología , Brucella abortus/patogenicidad , Brucelosis/prevención & control , Fosfoglicerato Quinasa/deficiencia , Animales , Vacuna contra la Brucelosis/genética , Brucella abortus/inmunología , Brucelosis/inmunología , Células Cultivadas , Recuento de Colonia Microbiana , ADN Bacteriano/química , ADN Bacteriano/genética , Femenino , Eliminación de Gen , Humanos , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Bazo/microbiología , Análisis de Supervivencia , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
BACKGROUND: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some alpha-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In contrast to Brucella abortus, Ochrobactrum anthropi did not replicate within professional and non-professional phagocytes and, whereas neutrophils had a limited action on B. abortus, they were essential to control O. anthropi infections. O. anthropi triggered proinflammatory responses markedly lower than Salmonella enterica but higher than B. abortus. In macrophages and dendritic cells, the corresponding lipopolysaccharides reproduced these grades of activation, and binding of O. anthropi lipopolysaccharide to the TLR4 co-receptor MD-2 and NF-kappaB induction laid between those of B. abortus and enteric bacteria lipopolysaccharides. These differences correlate with reported variations in lipopolysaccharide core sugars, sensitivity to bactericidal peptides and outer membrane permeability. CONCLUSIONS/SIGNIFICANCE: The results suggest that Brucellaceae ancestors carried molecules not readily recognized by innate immunity, so that non-drastic variations led to the emergence of stealthy intracellular parasites. They also suggest that some critical envelope properties, like selective permeability, are profoundly altered upon modification of pathogen-associated molecular patterns, and that this represents a further adaptation to the host. It is proposed that this adaptive trend is relevant in other intracellular alpha-Proteobacteria like Bartonella, Rickettsia, Anaplasma, Ehrlichia and Wolbachia.
Asunto(s)
Brucella abortus/metabolismo , Brucella abortus/patogenicidad , Inmunidad Innata , Ochrobactrum/metabolismo , Ochrobactrum/patogenicidad , Virulencia , Animales , Brucella abortus/genética , Células Dendríticas/microbiología , Femenino , Inflamación , Lipopolisacáridos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Ochrobactrum/genéticaRESUMEN
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.
Asunto(s)
Bothrops , Venenos de Crotálidos/enzimología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Neurotoxinas/química , Fragmentos de Péptidos/farmacología , Fosfolipasas A/química , Animales , Línea Celular , Endotoxemia/tratamiento farmacológico , Endotoxemia/microbiología , Endotoxinas/metabolismo , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/metabolismo , Fosfolipasas A2 Grupo II , Humanos , Lipopolisacáridos/metabolismo , Ratones , Mioblastos , Pruebas de Neutralización , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Peritonitis/tratamiento farmacológico , Peritonitis/microbiología , Fosfolipasas A2 , Proteínas de Reptiles , Salmonella typhimurium/efectos de los fármacosRESUMEN
Brucella abortus is an intracellular pathogen that relies on unconventional virulence factors to infect hosts. In non-professional phagocytes, Rho GTPases-activation by the Escherichia coli cytotoxic necrotizing factor (CNF) promoted massive Brucella entrance by membrane ruffling, a mechanism that differs from the common mode of entrance used by this bacterium in non-treated cells. Cytotoxic necrotizing factor treatment, however, did not alter the intracellular route followed by the wild type or non-virulent defined mutants. In contrast, expression of a constitutively active Rab5Q79L GTPase did not alter cell-invasion by Brucella but hampered its ability to reach the endoplasmic reticulum. The CNF-induced Brucella super-infection did not reduce the ability of host cells to synthesize DNA and progress through the cell cycle. Furthermore, CNF-treatment increased the isolation of Brucella-containing compartments by a factor of 15. These results demonstrate that in non-professional phagocytic cells, Brucella manipulates two different sets of GTPases during its biogenesis, being internalization and intracellular trafficking two consecutive but independent processes. Besides, CNF-induced super-infection demonstrates that Brucella does not interfere with crucial cellular processes and has shown its potential as tool to characterize the intracellular compartments occupied by this bacterium.