RESUMO
The purpose of this study was to elucidate the roles of peptidoglycan-associated lipoprotein (Pal protein) in the proliferation of Brucella in macrophage and bacterial virulence, and to evaluate the immune effect of Pal protein to Salmonella enteritidis. Murine macrophage-like cell line Raw264.7 was stimulated by recombinant Pal protein, and the expression of TNF-α and IFN-γ were up-regulated, but not it of IL-1ß and IL-6. The macrophages infection and in vitro simulated stress assays showed that deletion of pal gene reduced the proliferation of Brucella in macrophages, the survival in acidic, oxidative and polymyxin B-contained environment. The mice infection assay showed that mice challenged with the pal mutant strain were found to have more severe splenomegaly, but less bacterial load. After oral immunization of mice, Pal protein induced a higher titer of mucosal and humoral antibody (IgA and IgG) against heat-killed Salmonella enteritidis, and a stronger Th1 cellular immune response. The challengte experiments showed Pal protein elevated the survival rate and reduced the bacterial load of spleens in immunized mice. In conclusion, our results revealed the important roles of pal gene in Brucella virulence, and Pal protein was a potentially valuable adjuvant against mucosal pathogens, such as Salmonella enteritidis.
Assuntos
Brucella , Camundongos , Animais , Salmonella enteritidis/genética , Virulência , Macrófagos , Proliferação de CélulasRESUMO
The ability to resist the killing effects of host antimicrobial peptides (AMPs) plays a vital role in the virulence of pathogens. The Brucella melitensis NI genome has a gene cluster that encodes ABC transport. In this study, we constructed yejA1, yejA2, yejB, yejE, yejF, and whole yej operon deletion mutants, none of which exhibited discernible growth defect in TSB or minimal medium. Unlike their parental strain, the mutants showed a significantly increased sensitivity to acidic stress. The NIΔyejE and NIΔyejABEF mutants were also more sensitive than B. melitensis NI to polymyxin B, and the expression of yej operon genes was induced by polymyxin B. Moreover, cell and mouse infection assays indicated that NIΔyejE and NIΔyejABEF have restricted invasion and replication abilities inside macrophages and are rapidly cleared from the spleens of infected mice. These findings indicate that the ABC transporter YejABEF is required for the virulence of Brucella, suggesting that resistance to host antimicrobials is a key mechanism for Brucella to persistently survive in vivo. This study provided insights that led us to further investigate the potential correlation of AMP resistance with the mechanisms of immune escape and persistent infection by pathogens.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Bactérias/genética , Brucella melitensis/genética , Óperon , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Brucella melitensis/efeitos dos fármacos , Brucella melitensis/patogenicidade , Brucelose/metabolismo , Brucelose/microbiologia , Linhagem Celular , Farmacorresistência Bacteriana/genética , Feminino , Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos Endogâmicos BALB C , Virulência/genéticaRESUMO
Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of Brucella to survive and multiply in the hostile environment of host macrophages is essential for its virulence. The cold shock protein CspA plays an important role in the virulence of B. melitensis. To analyze the genes regulated by CspA, the whole transcriptomes of B. melitensis NIΔcspA and its parental wild-type strain, B. melitensis NI, were sequenced and analyzed using the Solexa/Illumina sequencing platform. A total of 446 differentially expressed genes were identified, including 324 up-regulated and 122 down-regulated genes. Numerous genes identified are involved in amino acid, fatty acid, nitrogen, and energy metabolism. Interestingly, all genes involved in the type IV secretion system and LuxR-type regulatory protein VjbR were significantly down-regulated in NIΔcspA. In addition, an effector translocation assay confirmed that the function of T4SS in NIΔcspA is influenced by deletion of the cspA gene. These results revealed the differential phenomena associated with virulence and metabolism in NIΔcspA and NI, providing important information for understanding detailed CspA-regulated interaction networks and Brucella pathogenesis.
Assuntos
Proteínas de Bactérias/genética , Brucella melitensis/genética , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Western Blotting , Brucella melitensis/metabolismo , Brucella melitensis/patogenicidade , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Camundongos , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Virulência/genéticaRESUMO
Copper is a potent antimicrobial agent. Multiple mechanisms of copper tolerance are utilized by some pathogenic bacteria. BMEII0580, which is significantly similar to the multicopper oxidase from Escherichia coli, was predicted to be the probable blue copper protein YacK precursor in Brucella melitensis 16M, and was designated as Brucella multicopper oxidase (BmcO). A bioinformatics analysis indicated that the typical motifs of multicopper oxidases are present in BmcO. BmcO, the expression of which was up-regulated by copper, could catalyze the oxidation of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), dimethoxyphenol (DMP) and para-phenylenediamine (pPD), which are widely used as substrates for multicopper oxidase. Additionally, BmcO exhibited ferroxidase activity, which indicated that it might play an important role in the Fe(2+) uptake of B. melitensis. Importantly, the mutant strain 16MΔbmcO was more sensitive to copper than the wild-type strain B. melitensis 16M as well as its complementation strain 16MΔbmcO(bmcO). The infection assays of cells showed that similar bacterial numbers of B. melitensis 16M, 16MΔbmcO and 16MΔbmcO(bmcO) strains were recovered from the infected macrophages. This result indicated that BmcO was not essential for B. melitensis intracellular growth. In conclusion, our results confirm that BmcO is a multicopper oxidase and contributes to the copper tolerance of B. melitensis 16M.
Assuntos
Brucella melitensis/efeitos dos fármacos , Brucella melitensis/enzimologia , Cobre/farmacologia , Farmacorresistência Bacteriana/genética , Oxirredutases/metabolismo , Antibacterianos/farmacologia , Brucella melitensis/genética , Brucella melitensis/crescimento & desenvolvimento , Brucelose/microbiologia , Ceruloplasmina/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Teste de Complementação Genética , Ferro/metabolismo , Macrófagos/microbiologia , Oxirredutases/genética , Deleção de SequênciaRESUMO
Brucella melitensis is a facultative intracellular pathogen. The regM gene encodes a sensory transduction protein kinase in B. melitensis 16M, and genes orthologous to regM have been found to exist in many bacterial species. However, little is known about the regulation function of this gene in Brucella. In order to characterize this gene, we constructed a marked deletion mutant of regM as well as its complemented strain. The mutant was less able to withstand acid and hyperosmotic conditions than wild-type strain but shown no significant difference with wild-type strain when challenged by elevated temperature and hypotonic conditions. In addition, inactivation of regM did not affect virulence in B. melitensis 16M in macrophage and mice infection models.
Assuntos
Brucella melitensis/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/metabolismo , Estresse Fisiológico , Ácidos/toxicidade , Animais , Brucella melitensis/efeitos dos fármacos , Brucella melitensis/genética , Brucella melitensis/efeitos da radiação , Brucelose/microbiologia , Brucelose/patologia , Modelos Animais de Doenças , Deleção de Genes , Teste de Complementação Genética , Temperatura Alta , Macrófagos/microbiologia , Camundongos , Pressão Osmótica , Proteínas Quinases/genética , VirulênciaRESUMO
A total of 129 sRNA candidates were identified in Brucella abortus 2308 in our previous work, and one candidate with potential to regulate expression of hemH gene was further analyzed in this study. We found that the novel sRNA can inhibit the expression of hemH and called it BsrH (Brucella sRNA regulating HemH). The expression level of BsrH was tested in four different stress conditions. A significant upregulation was detected during the growth in acidic and Brucella minimal media, as well as in the presence of hydroxyl peroxide, while iron deficiency caused the opposite effect. As expected, BsrH strongly affected the survival ratio of the Brucella cells under iron-limitation conditions, though overexpression of BsrH did not affect Brucella virulence. Thus, we conclude that BsrH plays a regulatory role in bacterial heme biosynthesis and can be considered as the first Brucella sRNA involved in stress responses.
Assuntos
Brucella abortus/genética , Brucella abortus/patogenicidade , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Virulência/genética , Animais , Proteínas de Bactérias , Brucelose/microbiologia , Biologia Computacional , Células HeLa , Humanos , Ferro/metabolismo , Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Brucella melitensis possesses an operon with two components: the response regulator OtpR and a putative cAMP-dependent protein kinase regulatory subunit encoded by the BMEI0067 gene. In the previous study, the function of OtpR has been studied, while little is known about the function of the BMEI0067 gene. Using a bioinformatics approach, we showed that the BMEI0067 gene encodes an additional putative cAMP-binding protein, which we refer to as CbpB. Structural modeling predicted that CbpB has a cAMP-binding protein (CAP) domain and is structurally similar to eukaryotic protein kinase A regulatory subunits. Here, we report the characterization of CbpB, a cAMP-binding protein in Brucella melitensis, showed to be involved in mouse persistent infections. ∆cbpB::km possessed cell elongation, bubble-like protrusions on cell surface and its resistance to environmental stresses (temperature, osmotic stress and detergent). Interestingly, comparative real-time qPCR assays, the cbpB mutation resulted in significantly different expression of aqpX and several penicillin-binding proteins and cell division proteins in Brucella. Combined, these results demonstrated characterization of CbpB in B. melitensis and its key role for intracellular multiplication.
Assuntos
Proteínas de Bactérias/fisiologia , Brucella melitensis/enzimologia , Brucelose/microbiologia , Parede Celular/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Animais , Brucella melitensis/efeitos dos fármacos , Brucella melitensis/patogenicidade , Linhagem Celular , Parede Celular/efeitos dos fármacos , Detergentes/farmacologia , Feminino , Macrófagos/microbiologia , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Dodecilsulfato de Sódio/farmacologia , VirulênciaRESUMO
Brucella melitensis is a facultative intracellular pathogen that mainly resides within macrophages. The mechanisms employed by Brucella to adapt to harsh intracellular environments and survive within host macrophages are not clearly understood. Here, we constructed a cspA gene deletion mutant, NIΔcspA, that did not exhibit any discernible growth defect at a normal culture temperature (37 °C) or at a low temperature (15 °C). However, expression of the cspA gene in Brucella was induced by cold, acidic, and oxidative conditions, as determined via quantitative reverse transcription PCR. Unlike its parental strain, B. melitensis NI, the NIΔcspA mutant showed an increased sensitivity to acidic and H2 O2 stresses, especially during the mid-log-phase, and these stress conditions would presumably be encountered by bacteria during intracellular infections. Moreover, macrophage and mouse infection assays indicated that the NIΔcspA mutant fails to replicate in cultured J774.A1 murine macrophages and is rapidly cleared from the spleens of experimentally infected BALB/c mice. These findings suggest that the Brucella cspA gene makes an essential contribution to virulence in vitro and in vivo, most likely by allowing brucellae to adapt appropriately to the harsh environmental conditions encountered within host macrophages.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella melitensis/metabolismo , Brucella melitensis/patogenicidade , Brucelose/veterinária , Doenças dos Bovinos/microbiologia , Proteínas e Peptídeos de Choque Frio/metabolismo , Adaptação Fisiológica , Animais , Proteínas de Bactérias/genética , Brucella melitensis/genética , Brucelose/microbiologia , Bovinos , Proteínas e Peptídeos de Choque Frio/genética , Feminino , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , VirulênciaRESUMO
MucR is a transcriptional regulator in many bacterial pathogens and is required for virulence in mice and macrophages, resistance to stress responses, and modification of the cell envelope in Brucella spp. To determine why the mucR deleted mutant is attenuated in vivo and in vitro, we performed RNA-seq analysis using Brucella melitensis RNA obtained from B. melitensis 16M and 16MΔmucR grown under the same conditions. We found 442 differentially expressed genes; 310 were over expressed, and 132 were less expressed in 16MΔmucR. Many genes identified are involved in metabolism, cell wall/envelope biogenesis, replication, and translation. Notably, genes involved in type IV secretion system and quorum sensing system were down-regulated in 16MΔmucR. In addition, genes involved in tolerance to acid and iron-limitation were also affected and experimentally verified in this study. The effects of MucR on Brucella survival and persistence in mice and macrophages were related to type IV secretion system, quorum sensing system, and stress tolerance, which also provide added insight to the MucR regulon.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Brucella melitensis/fisiologia , Brucelose/microbiologia , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , Brucella melitensis/genética , Brucella melitensis/patogenicidade , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Regulon , Estresse Fisiológico , Fatores de Transcrição/genética , VirulênciaRESUMO
Smooth Brucella spp. inhibit macrophage apoptosis, whereas rough Brucella mutants induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current study, a random mutagenesis approach was used to create a mutant bank consisting of 11,354 mutants by mariner transposon mutagenesis using Brucella melitensis rough mutant 16M delta manBA as the parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (release of 10% or less of the lactate dehydrogenase). The absence of cytotoxicity during infection with these mutants was independent of demonstrable defects in in vitro bacterial growth or uptake and survival in macrophages. Interrupted genes in 51 mutants were identified by DNA sequence analysis, and the mutations included interruptions in virB encoding the type IV secretion system (T4SS) (n = 36) and in vjbR encoding a LuxR-like regulatory element previously shown to be required for virB expression (n = 3), as well as additional mutations (n = 12), one of which also has predicted roles in virB expression. These results suggest that the T4SS is associated with Brucella cytotoxicity in macrophages. To verify this, deletion mutants were constructed in B. melitensis 16M by removing genes encoding phosphomannomutase/phosphomannoisomerase (delta manBA) and the T4SS (delta virB). As predicted, deletion of virB from 16M delta manBA and 16M resulted in a complete loss of cytotoxicity in rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme multiplicities of infection (>1,000). Taken together, these results demonstrate that Brucella cytotoxicity in macrophages is T4SS dependent.
Assuntos
Proteínas de Bactérias/biossíntese , Brucella melitensis/genética , Brucella melitensis/patogenicidade , Deleção de Genes , Macrófagos/microbiologia , Animais , Apoproteínas , Proteínas de Bactérias/genética , Brucella melitensis/metabolismo , Linhagem Celular , Regulação Bacteriana da Expressão Gênica , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , NecroseRESUMO
BACKGROUND: Random gene inactivation used to identify cellular functions associated with virulence and survival of Brucella spp has relied heavily upon the use of the transposon Tn5 that integrates at G/C base pairs. Transposons of the mariner family do not require species-specific host factors for efficient transposition, integrate nonspecifically at T/A base pairs, and, at a minimum, provide an alternative approach for gene discovery. In this study, plasmid vector pSC189, containing both the hyperactive transposase C9 and transposon terminal inverted repeats flanking a kanamycin resistance gene, were used to deliver Himar1 transposable element into the B. melitensis genome. Conjugation was performed efficiently and rapidly in less than one generation in order to minimize the formation of siblings while assuring the highest level of genome coverage. RESULTS: Although previously identified groups or classes of genes required for virulence and survival were represented in the screen, additional novel identifications were revealed and may be attributable to the difference in insertion sequence biases of the two transposons. Mutants identified using a fluorescence-based macrophage screen were further evaluated using gentamicin-based protection assay in macrophages, survival in the mouse splenic clearance model and growth in vitro to identify mutants with reduced growth rates. CONCLUSION: The identification of novel genes within previously described groups was expected, and nearly two-thirds of the 95 genes had not been previously reported as contributing to survival and virulence using random Tn5-based mutagenesis. The results of this work provide added insight with regard to the regulatory elements, nutritional demands and mechanisms required for efficient intracellular growth and survival of the organism.
Assuntos
Proteínas de Bactérias/genética , Brucella melitensis/crescimento & desenvolvimento , Brucella melitensis/patogenicidade , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Transposases/genética , Animais , Proteínas de Bactérias/metabolismo , Brucella melitensis/genética , Brucella melitensis/metabolismo , Brucelose/microbiologia , Linhagem Celular , Conjugação Genética , Regulação Bacteriana da Expressão Gênica , Humanos , Macrófagos/microbiologia , Camundongos , Mutagênese , Baço/microbiologia , VirulênciaRESUMO
Previous studies suggest that smooth Brucella organisms inhibit macrophage apoptosis. In contrast, necrotic cell death of macrophages infected with rough Brucella organisms in vitro has been reported, which may in part explain the failure of some rough organisms to thrive. To characterize these potential macrophage killing mechanisms, J774.A1 murine macrophages were infected with Brucella abortus S2308-derived rough mutant CA180. Electron microscopic analysis and polyethylene glycol protection assays revealed that the cells were killed as a result of necrosis and oncosis. This killing was shown to be unaffected by treatment with carbenicillin, an inhibitor of bacterial cell wall biosynthesis and, indirectly, replication. In contrast, chloramphenicol treatment of macrophages infected at multiplicities of infection exceeding 10,000 prevented cell death, despite internalization of large numbers of bacteria. Similarly, heat-killed and gentamicin-killed CA180 did not induce cytopathic effects in the macrophage. These results suggested that killing of infected host cells requires active bacterial protein synthesis. Cytochalasin D treatment revealed that internalization of the bacteria was necessary to initiate killing. Transwell experiments demonstrated that cell death is not mediated by a diffusible product, including tumor necrosis factor alpha and nitric oxide, but does require direct contact between host and pathogen. Furthermore, macrophages preinfected with B. abortus S2308 or pretreated with B. abortus O polysaccharide did not prevent rough CA180-induced cell death. In conclusion, Brucella rough mutant infection induces necrotic and oncotic macrophage cell death that requires bacterial protein synthesis and direct interaction of bacteria with the target cells.
Assuntos
Proteínas de Bactérias/biossíntese , Brucella abortus/patogenicidade , Macrófagos/patologia , Biossíntese de Proteínas , Animais , Apoptose , Brucella abortus/metabolismo , Linhagem Celular , L-Lactato Desidrogenase/metabolismo , Ativação de Macrófagos , Camundongos , Necrose , Fosfotransferases (Fosfomutases)/fisiologiaRESUMO
The neuraminidase (NA) genes of 12 H9N2 influenza virus strains isolated from diseased chickens in different farms in mainland China during 1995-2002 were amplified and sequenced. Amino acids at hemadsorbing (HB) site of these isolates are different from those of A/quail/Hong Kong/G1/97-like viruses and A/chicken/Korea/96-like viruses. Neuraminidases of the 12 strains had a deletion of 3 amino acid residues at positions 63-65 as compared to that of A/turkey/Wisconsin/189/66, while those of Korea and Pakistan H9N2 isolates had no deletion. Phylogenetic analyses showed NA gene of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. NA gene of the H9N2 viruses isolated in Korea and Pakistan belonged to lineage different from those of the 12 isolates. The present results indicate that the NA of H9N2 strains isolated in mainland China during the past 8 years were well preserved and the geographical distribution play a significant role in the evolution of the H9N2 influenza viruses.