Entry and Elimination of Marine Mammal Brucella spp. by Hooded Seal (Cystophora cristata) Alveolar Macrophages In Vitro

A high prevalence of Brucella pinnipedialis serology and bacteriology positive animals has been found in the Northeast Atlantic stock of hooded seal ( Cystophora cristata ); however no associated gross pathological changes have been identified. Marine mammal brucellae have previously displayed different infection patterns in human and murine macrophages. To investigate if marine mammal Brucella spp. are able to invade and multiply in cells originating from a presumed host species, we infected alveolar macrophages from hooded seal with a B . pinnipedialis hooded seal isolate. Hooded seal alveolar macrophages were also challenged with B . pinnipedialis reference strain (NCTC 12890) from harbor seal ( Phoca vitulina ), B . ceti reference strain (NCTC 12891) from harbor porpoise ( Phocoena phocoena ) and a B . ceti Atlantic white-sided dolphin ( Lagenorhynchus acutus ) isolate (M83/07/1), to evaluate possible species-specific differences. Brucella suis 1330 was included as a positive control. Alveolar macrophages were obtained by post mortem bronchoalveolar lavage of euthanized hooded seals. Phenotyping of cells in the lavage fluid was executed by flow cytometry using the surface markers CD14 and CD18. Cultured lavage cells were identified as alveolar macrophages based on morphology, expression of surface markers and phagocytic ability. Alveolar macrophages were challenged with Brucella spp. in a gentamicin protection assay. Following infection, cell lysates from different time points were plated and evaluated quantitatively for colony forming units. Intracellular presence of B . pinnipedialis hooded seal isolate was verified by immunocytochemistry. Our results show that the marine mammal brucellae were able to enter hooded seal alveolar macrophages; however, they did not multiply intracellularly and were eliminated within 48 hours, to the contrary of B. suis that showed the classical pattern of a pathogenic strain. In conclusion, none of the four marine mammal strains tested were able to establish a persistent infection in primary alveolar macrophages from hooded seal.     

Marine mammal Brucella isolates with different genomic characteristics display a differential response when infecting human macrophages in culture

Marine mammal Brucella strains with different genomic characteristics according to distribution of IS711 elements in their genomes were analysed for their intracellular behaviour in human THP-1 macrophage-like cells. Seven different groups of marine mammal strains were identified including a human isolate from New Zealand presumably from marine origin. Entry and intracellular survival of strains representative of these groups in THP-1 human macrophage-like cells were analysed at several times of infection. Three patterns of infection were identified. The Brucella strain isolated from the human case from New Zealand, and two other groups of strains belonging to B. ceti or B. pinnipedialis were able to infect THP-1 macrophage cells to the same extent as the virulent strains B. suis 1330 or B. melitensis 16M. Three other groups of strains belonging to B. ceti or B. pinnipedialis were able to enter the cells as classical virulent strains but were eliminated after 48 h. The last group was composed only of strains isolated from hooded seals (Cystophora cristata) and was even unable to enter and infect THP-1 macrophage cells. Thus, several groups of marine mammal Brucella strains appear to be non-infectious for human macrophages.     

Marine Mammal Brucella Reference Strains Are Attenuated in a BALB/c Mouse Model

Brucellosis is a zoonosis of worldwide distribution with numerous animal host species. Since the novel isolation of Brucella spp. from marine mammals in 1994 the bacteria have been isolated from various marine mammal hosts. The marine mammal reference strains Brucella pinnipedialis 12890 (harbour seal, Phoca vitulina) and Brucella ceti 12891 (harbour porpoise, Phocoena phocoena) were included in genus Brucella in 2007, however, their pathogenicity in the mouse model is pending. Herein this is evaluated in BALB/c mice with Brucella suis 1330 as a control. Both marine mammal strains were attenuated, however, B. ceti was present at higher levels than B. pinnipedialis in blood, spleen and liver throughout the infection, in addition B. suis and B. ceti were isolated from brains and faeces at times with high levels of bacteraemia. In B. suis-infected mice serum cytokines peaked at day 7. In B. pinnipedialis-infected mice, levels were similar, but peaked predominantly at day 3 and an earlier peak in spleen weight likewise implied an earlier response. The inflammatory response induced pathology in the spleen and liver. In B. ceti-infected mice, most serum cytokine levels were comparable to those in uninfected mice, consistent with a limited inflammatory response, which also was indicated by restricted spleen and liver pathology. Specific immune responses against all three strains were detected in vitro after stimulation of splenocytes from infected mice with the homologous heat-killed brucellae. Antibody responses in vivo were also induced by the three brucellae. The immunological pattern of B. ceti in combination with persistence in organs and limited pathology has heretofore not been described for other brucellae. These two marine mammal wildtype strains show an attenuated pattern in BALB/c mice only previously described for Brucella neotomea.     

Cholesterol,ganglioside GM1 and class A scavenger receptor contribute to infection by Brucella ovis and Brucella canis in murine macrophages

The establishment of infection by Brucella ovis and Brucella canis in J774.A1 macrophages was found to be dependent upon cholesterol and ganglioside GM₁, two components of lipid rafts. This process also required a class A scavenger receptor of macrophages, and was not inhibited by smooth and rough lipopolysaccharides from Brucella spp. In response to infection, both bacteria induced a weak degree of macrophage activation. These results demonstrate that B. ovis and B. canis use cell surface receptors common to smooth Brucella spp. for macrophage infection, thus limiting macrophage activation and favouring intracellular multiplication and/or the survival of both bacteria.     

Phenotypic and molecular characterisation of <Emphasis Type="Italic">Brucella</Emphasis> isolates from marine mammals

Background  

Bacteria of the genus Brucella are the causative organisms of brucellosis in animals and man. Previous characterisation of Brucella strains originating from marine mammals showed them to be distinct from the terrestrial species and likely to comprise one or more new taxa. Recently two new species comprising Brucella isolates from marine mammals, B. pinnipedialis and B. ceti, were validly published. Here we report on an extensive study of the molecular and phenotypic characteristics of marine mammal Brucella isolates and on how these characteristics relate to the newly described species.     

A Brucella Virulence Factor Targets Macrophages to Trigger B-cell Proliferation

Brucella spp. and Trypanosoma cruzi are two intracellular pathogens that have no evolutionary common origins but share a similar lifestyle as they establish chronic infections for which they have to circumvent the host immune response. Both pathogens have a virulence factor (prpA in Brucella and tcPrac in T. cruzi) that induces B-cell proliferation and promotes the establishment of the chronic phase of the infectious process. We show here that, even though PrpA promotes B-cell proliferation, it targets macrophages in vitro and is translocated to the cytoplasm during the intracellular replication phase. We observed that PrpA-treated macrophages induce the secretion of a soluble factor responsible for B-cell proliferation and identified nonmuscular myosin IIA (NMM-IIA) as a receptor required for binding and function of this virulence factor. Finally, we show that the Trypanosoma cruzi homologue of PrpA also targets macrophages to induce B-cell proliferation through the same receptor, indicating that this virulence strategy is conserved between a bacterial and a protozoan pathogen.     

Comparison of PCR-RFLP and PFGE for determining the clonality of Brucella isolates from human and livestock specimens

Brucellosis is an important zoonotic disease caused by different species of genus Brucella that are pathogenic for humans and a variety of animals. Accurate detection of Brucella spp. infection is important for control of disease. The aim of this study was to comparison of molecular genotyping of Brucella strains by Pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction -Restriction Fragment Length Polymorphism (PCR-RFLP) techniques.Twenty- seven Brucella spp. were isolated from human and animal samples. The isolates identified by conventional microbiological methods and confirmed using PCR for amplification of omp2a gene. Molecular typing of Brucella strains carried out by PCR-RFLP after PstI and PFGE of chromosomal DNA after XbaI enzyme digestion. The omp2a gene PCR Products with different patterns of PCR-RFLP were sequenced.The omp2a gene amplification of all human and animal Brucella isolates were positive for 1100 bp fragment. By PCR-RFLP analysis two genotypes/patterns for human isolates and four genotypes for animal isolates were obtained. In PFGE analysis totally, 7 common clones/clusters and 3 single clones were obtained.The results of this study showed the PFGE method is the more reliable and useful assay for molecular typing of Brucella strains and is more preferred to PCR-RFLP in determination of genetic similarity among human and animal Brucella isolates. The presented data showed PCR-RFLP analysis was not able to differentiate between B. melitensis biovars and vaccine strain.     

Transcriptome analysis of HeLa cells response to Brucella melitensis infection: a molecular approach to understand the role of the mucosal epithelium in the onset of the Brucella pathogenesis

Brucella spp. infect hosts primarily by adhering and penetrating mucosal surfaces, however the initial molecular phenomena of this host:pathogen interaction remain poorly understood. We hypothesized that characterizing the epithelial-like human HeLa cell line molecular response to wild type Brucella melitensis infection would help to understand the role of the mucosal epithelium at the onset of the Brucella pathogenesis. RNA samples from B. melitensis-infected HeLa cells were taken at 4 and 12 h of infection and hybridized in a cDNA microarray. The analysis using a dynamic Bayesian network modeling approach (DBN) identified several pathways, biological processes, cellular components and molecular functions altered due to infection at 4 h p.i., but almost none at 12 h p.i. The in silico modeling results were experimentally tested by knocking down the expression of MAPK1 by siRNA technology. MAPK1-siRNA transfected cell cultures decreased the internalization and impaired the intracellular replication of the pathogen in HeLa cells after 4 h p.i. DBN analysis provides important insights into the role of the epithelial cells response to Brucella infection and guide research to novel mechanisms identification.     

Glutamate Decarboxylase-Dependent Acid Resistance in Brucella spp.: Distribution and Contribution to Fitness under Extremely Acidic Conditions

Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative.     

Characterization of Novel Brucella Strains Originating from Wild Native Rodent Species in North Queensland,Australia

We report on the characterization of a group of seven novel Brucella strains isolated in 1964 from three native rodent species in North Queensland, Australia, during a survey of wild animals. The strains were initially reported to be Brucella suis biovar 3 on the basis of microbiological test results. Our results indicated that the rodent strains had microbiological traits distinct from those of B. suis biovar 3 and all other Brucella spp. To reinvestigate these rodent strains, we sequenced the 16S rRNA, recA, and rpoB genes and nine housekeeping genes and also performed multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA). The rodent strains have a unique 16S rRNA gene sequence compared to the sequences of the classical Brucella spp. Sequence analysis of the recA, rpoB, and nine housekeeping genes reveals that the rodent strains are genetically identical to each other at these loci and divergent from any of the currently described Brucella sequence types. However, all seven of the rodent strains do exhibit distinctive allelic MLVA profiles, although none demonstrated an amplicon for VNTR 07, whereas the other Brucella spp. did. Phylogenetic analysis of the MLVA data reveals that the rodent strains form a distinct clade separate from the classical Brucella spp. Furthermore, whole-genome sequence comparison using the maximal unique exact matches index (MUMi) demonstrated a high degree of relatedness of one of the seven rodent Brucella strains (strain NF 2653) to another Australian rodent Brucella strain (strain 83-13). Our findings strongly suggest that this group of Brucella strains isolated from wild Australian rodents defines a new species in the Brucella genus.Brucella species are facultative intracellular Gram-negative members of the Alphaproteobacteria class capable of causing brucellosis in a range of animal hosts, including domesticated livestock, wildlife, marine mammals, and humans (1, 5, 7, 29, 32, 33, 36, 47). Brucellosis is the most prevalent zoonotic disease worldwide, causing spontaneous abortion and fetal death in animals and severe flu-like symptoms, focal complications, and often, chronic disease in humans (7, 11, 22, 27, 40, 41, 49, 50). Brucella species are typically transmitted to humans through consumption of unpasteurized dairy products or exposure to fluids or tissues from infected animals (45, 49). Animals are primary hosts of all Brucella spp., which include Brucella abortus (cattle), B. canis (dogs), B. melitensis (goats, cows, and sheep), B. suis (swine), B. ovis (rams), and B. neotomae (desert rats) (3, 7, 8). Recently, three additional Brucella species have been recognized: B. pinnipedialis (seals), B. ceti (dolphins) (5), and B. microti. B. microti was initially isolated from the common vole in the Czech Republic (33, 35). In the mid-1980s, DNA-DNA hybridization studies demonstrated a very high level of genetic similarity (98.5%) among the Brucella spp., which led to the adoption of a monospecies concept for the Brucella genus, with all the species at that time renamed as biovars of B. melitensis (46). However, 20 years later, the use of a phylogenetic-evolutionary approach to Brucella taxonomy was accepted. By that approach, host preferences, virulence, and pathogenicity were considered important criteria in the delineation of Brucella species, and consequently, the multispecies taxonomy was restored to the Brucella genus (28). With the development of more advanced molecular typing methodologies such as multilocus sequence analysis (MLSA) (48), multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) (23), and techniques interrogating single nucleotide polymorphisms (SNPs) (17, 47), Brucella spp. can be quickly genotyped and strains can be readily examined for their phylogenetic and evolutionary relationships (15).Recently, we reported on two unusual human brucellosis cases, one of which led to the identification of a novel species, Brucella inopinata, whose type strain is strain BO1 and which was associated with a breast implant infection in a patient in Oregon (12, 36). The second brucellosis case involved an atypical Brucella strain (strain BO2) isolated from the lung biopsy fluid of a patient with chronic destructive pneumonia in Australia (44). Because both patients denied common risk factors associated with human brucellosis, the primary hosts of these strains remain unknown. However, nucleotide sequence analysis of the outer membrane proteins (omp2a and omp2b) of both strain BO2 and strain BO1^T demonstrated close clustering to an atypical B. suis strain (strain 83-210) isolated from a rodent in Australia (30, 44). Further genetic analysis of the 16S rRNA genes from strains BO1^T and BO2 and the 16S rRNA gene from atypical Brucella strain 83-13 (available at http://www.broadinstitute.org), which was isolated from a rodent in Australia and briefly described by Corbel and Brinley-Morgan in 1984 (9), showed that strain 83-13 yielded notable genetic similarity to the novel human isolates, which led us to speculate that atypical human Brucella strains BO1^T and BO2 may have an animal reservoir in rodents from Australia.Rodent brucellosis is self-limiting and is mostly associated with wild rodents that cohabitate among domestic livestock presumably infected with classical Brucella spp. (27). Over 22 different wild rodent species worldwide have been reported to be susceptible to Brucella infection, as demonstrated by serology and/or culture (41). Earlier field studies in Argentina, Venezuela, and Denmark reported on the prevalence of B. suis in hares, opossums, and rats and B. abortus in ferrets and capybaras (10, 11, 27). However, two rodent-specific Brucella spp. have been identified, including B. neotomae, isolated from the desert wood rat (Neotomae lepida) in Utah (39), and most recently, B. microti, isolated from the common vole (Microtus arvalis) in the Czech Republic (35). In the early 1960s, Cook et al. reported on the isolation and biochemical identification of seven Brucella suis biovar 3 strains from three known species of wild native rats from Australia (6). We reinvestigated the microbiological characteristics of these seven rodent B. suis biovar 3 strains and performed genetic analyses with respect to the microbiological characteristics and genetics of the classical and atypical Brucella species. In this report, we describe this group of Brucella strains isolated from wild rodents in Australia, which confer unique microbiological and molecular characteristics distinct from those of any of the currently described species.     

Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.     

Smooth Brucella strains invade and replicate in human lung epithelial cells without inducing cell death

Inhalation is a common route for Brucella infection. We investigated whether Brucella species can invade and replicate within alveolar (A549) and bronchial (Calu-6 and 16HBE14o-) human epithelial cells. The number of adherent and intracellular bacteria was higher for rough strains (Brucella canis and Brucella abortus RB51) than for smooth strains (B. abortus 2308 and Brucella suis 1330). Only smooth strains exhibited efficient intracellular replication (1.5–3.5 log increase at 24 h p.i.). A B. abortus mutant with defective expression of the type IV secretion system did not replicate. B. abortus internalization was inhibited by specific inhibitors of microfilaments, microtubules and PI3-kinase activity. As assessed with fluorescent probes, B. abortus infection did not affect the viability of A549 and 16HBE14o- cells, but increased the percentage of injured cells (both strains) and dead cells (RB51) in Calu-6 cultures. LDH levels were increased in supernatants of Calu-6 and 16HBE14o- cells infected with B. abortus RB51, and to a lower extent in Calu-6 infected with B. abortus 2308. No apoptosis was detected by TUNEL upon infection with smooth or rough B. abortus. This study shows that smooth brucellae can infect and replicate in human respiratory epithelial cells inducing minimal or null cytotoxicity.     

Direct and monocyte-induced innate immune response of human lung epithelial cells to Brucella abortus infection

Although Brucella frequently infects humans through inhalation, its interaction with pulmonary cells has been overlooked. We examined whether human lung epithelial cells produce proinflammatory mediators in response to Brucella infection. Infection with smooth or rough strains of Brucella abortus induced the secretion of IL-8 and GM-CSF by the bronchial epithelial cell lines Calu-6 and 16HBE14o-, but not by the alveolar epithelial cell line A549. Infected Calu-6 cells also produced low levels of MCP-1. Since monocyte-derived cytokines may induce chemokine secretion in epithelial cells, cocultures of human monocytes (THP-1 cell line) and respiratory epithelial cells were used to study such interaction. IL-8 and MCP-1 levels in B. abortus-infected THP-1:A549 and THP-1:Calu-6 cocultures, and MCP-1 levels in THP-1:16HBE14o- cocultures, were higher than those detected in infected epithelial monocultures. Conditioned medium from infected monocytes induced the secretion of IL-8 and/or MCP-1 by A549 and Calu-6 cells, and these effects were mainly mediated by IL-1 (in A549 cells) or TNF-α (in Calu-6 cells). Conversely, culture supernatants from Brucella-infected bronchial epithelial cells induced MCP-1 production by monocytes, an effect largely mediated by GM-CSF. This study shows that human lung epithelial cells mount a proinflammatory response to Brucella, either directly or after interaction with Brucella-infected monocytes.     

Serum-mediated Immune Cellular Responses to Brucella melitensis IV. Infection of Macrophages Under Anaerobic Conditions

Immune mechanisms active against Brucella were studied under conditions of oxygen deficiency. B. melitensis grew in rabbit serum-Tyrode medium flooded with N₂ and CO₂ gas mixtures. Immune sera from rabbits injected with B. melitensis strain Rev I possessed growth-inhibitory activity that operated in anaerobic environments against Rev I and virulent strain 6015. When mixed with macrophages, immune sera mediated even greater inhibition of bacterial growth and slowed the spread of infection throughout the tissue culture. Although under anaerobic conditions the rate of phagocytosis was reduced, the macrophages in immune serum killed significant percentages of Brucella, suggesting that an antibacterial mechanism had been activated. Sonic extracts of macrophages prepared and tested under anaerobic conditions depressed the growth rate of strain Rev I. The extracts, however, exhibited no immediate killing capacity when tested in Tyrode solution. A factor from serum was required for depression of the growth rate.     

Identification of Brucella by MALDI-TOF mass spectrometry. Fast and reliable identification from agar plates and blood cultures

Background

MALDI-TOF mass spectrometry (MS) is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany) and their usefulness for identifying brucellae from culture plates and blood cultures.

Methodology/Principal Findings

We created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis), and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct.

Conclusions/Significance

MALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles.     

布鲁氏菌入侵相关基因IalB缺失株的构建及生物学特性分析

入侵相关基因(invasion-associated locus B, ialB)的同源基因在布鲁氏菌所属的根瘤菌目中是广泛保守的,但其在布鲁氏菌中的功能研究几乎为空白。根据有限的报道资料,猜测ialB的功能可能与布鲁氏菌入侵细胞以及适应胞内环境胁迫有关。【目的】探究ialB在布鲁氏菌中的生物学功能,揭示其在布鲁氏菌黏附和入侵细胞以及胞内存活中的作用。【方法】以猪种布鲁氏菌S2株为亲本,运用同源重组的方法构建布鲁氏菌ialB缺失株ΔialB,并通过表达质粒转化的方法构建其回补株CΔialB,比较3种菌株的生长特性、对体外应激的敏感性;通过扫描电镜观察ialB缺失对布鲁氏菌形态的影响,通过实时荧光定量PCR检测3种菌株极性延长相关基因的表达;通过免疫荧光和平板计数的方法分析ialB缺失对布鲁氏菌黏附、入侵RAW264.7细胞以及胞内存活的影响。【结果】成功构建了菌株ΔialB和CΔialB;ΔialB与布鲁氏菌S2株相比,生长受限,活力降低,在酸应激、高渗应激、低渗应激、多黏菌素B应激条件下存活率降低,在氧化应激条件下存活率上升;而且,ΔialB的菌体形态发生改变,菌体变短,直径增加,极...     

Biochemical and functional analysis of TIR domain containing protein from Brucella melitensis

Toll/interleukin-1 like receptors are evolutionarily conserved proteins in eukaryotes that play crucial role in pathogen recognition and innate immune responses. Brucella are facultative intracellular bacterial pathogens causing brucellosis in animal and human hosts. Brucella behave as a stealthy pathogen by evading the immune recognition or suppressing the TLR signaling cascades. Brucella encode a TIR domain containing protein, TcpB, which suppresses NF-κB activation as well as pro-inflammatory cytokine secretion mediated by TLR2 and TLR4 receptors. TcpB targets the TIRAP mediated pathway to suppress TLR signaling. With the objective of detailed characterization, we have over expressed and purified TcpB from Brucella melitensis in native condition. The purified protein exhibited lipid-binding properties and cell permeability. NF-κB inhibition property of endogenous TcpB has also been demonstrated. The data provide insight into the mechanism of action of TcpB in the intracellular niche of Brucella.     

Interactions of attenuated Mycobacterium tuberculosis phoP mutant with human macrophages

Background

Mycobacterium tuberculosis phoP mutant SO2 derived from a clinical isolate was shown to be attenuated in mouse bone marrow-derived macrophages and in vivo mouse infection model and has demonstrated a high potential as attenuated vaccine candidate against tuberculosis.

Methodology/Principal Findings

In this study, we analyze the adhesion and the intracellular growth and trafficking of SO2 in human macrophages. Our results indicate an enhanced adhesion to phagocitic cells and impaired intracellular replication of SO2 in both monocyte-derived macrophages and human cell line THP-1 in comparison with the wild type strain, consistent with murine model. Intracellular trafficking analysis in human THP-1 cells suggest that attenuation of SO2 within macrophages could be due to an impaired ability to block phagosome-lysosome fusion compared with the parental M. tuberculosis strain. No differences were found between SO2 and the wild-type strains in the release and mycobacterial susceptibility to nitric oxide (NO) produced by infected macrophages.

Conclusions/Significance

SO2 has enhanced ability to bind human macrophages and differs in intracellular trafficking as to wild-type M. tuberculosis. The altered lipid profile expression of the phoP mutant SO2 and its inability to secrete ESAT-6 is discussed.