Summary
Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric ...subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin, to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, as a flagellin‐deficient mutant of B. melitensis wasfound to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunological stand‐off between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.
Abstract
Chronic bacterial diseases such as Brucellosis may result from the pathogen’s ability to evade the initial immune response. Moreover, since Brucella spp. is known to target phagocytes, we ...investigated its effects on macrophage (mø) phenotype and induction of anti-inflammatory responses. Brucella did not induce alternative activation of mø in cultured bone marrow-derived mø (BMM) or in splenic CD11b+ cells isolated from mice during early infection. However, upregulation of interleukin 10 (IL-10) in serum, spleen and splenic mø from B. abortus infected mice was observed as early as 3 days post infection. Infection of BMM from MyD88-/-, TLR2-/-, TLR4-/-, TLR2/4-/- and TLR9-/- mice demonstrated that IL-10 induction was MyD88 dependent and was partially dependent on pathogen recognition by TLR2 and TLR4. Functionally, in vitro IL-10-/- BMM infection resulted in significantly higher pro-inflammatory cytokine production and decreased bacterial intracellular survival. In vivo 9 days infection of IL-10-/- mice confirmed a lower ability of B. abortus to survive in absence of IL-10, as well as higher induction of pro-inflammatory cytokines, resulting in increased pathology in liver and spleen of infected mice. Taken together, our results suggest that early IL-10 induction by infected mø contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby leading to enhanced bacterial survival and persistent infection.
Evasion of host immune responses is a prerequisite for chronic bacterial diseases;however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen ...Brucella abortus prevents immune activation of macrophages by inducing CD4.sup.+CD25.sup.+ T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-κB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25.sup.+CD4.sup.+ T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and ...sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Our investigations using macrophages show that B. abortus and B. melitensis activate the inflammasome via the type IV secretion system (T4SS). In vitro, activation leads to cleavage of caspase-1 and ...secretion of active IL-1β and IL-18 into the supernatant of LPS pre-treated bone marrow derived macrophages (BMDM). Infection of BMDM with B. abortus mutants in vceA and vceC, two effectors of the T4SS identified by our lab, resulted in small, variable differences in IL-1β secretion compared to infection with WT B. abortus. This suggests that those effectors have either little or redundant roles in inflammasome activation. To determine the mechanism of IL-1β secretion by infected BMDM, we infected Nlrp3-/-, Nlrc4-/-, and Asc -/- BMDM with B. abortus. Nlrp3 -/- and Nlrc4-/- macrophages secreted a similar amount of IL-1β as WT macrophages. However, secretion was completely abolished in Asc-/- macrophages. Thus, inflammasome activation was ASC-dependent, but vceA- and vceC-independent. To further elucidate the mechanism of inflammasome activation by Brucella spp, we determined the role of flagellin in inflammasome activation by B. melitensis. A flagellin mutant of B. melitensis induced less IL-1β secretion than WT B. melitensis. Using a flagellin over-expressing strain, we determined that a functioning T4SS was required for flagellin dependent IL-1β secretion. Infection of a variety of primary and immortalized BMDM revealed that NLRP3, NLRC4, NLRP6, NLRP12, and AIM2 are not involved in the inflammasome activation induced by flagellin over-expressing B. melitenisis. However, like T4SS-dependent inflammasome activation, flagellin was also sensed by an ASC dependent mechanism. Since inflammasome activation by B. abortus is completely ASC-dependent, we infected Asc-/- mice to determine the role of the inflammasome in persistence and transmission. Bacterial burden in the spleen and liver of Asc-/- mice was the same as in WT mice at 3, 7, 14, and 56 days post-infection. However, early in infection Asc-/- exhibited less inflammation compared to WT mice. This was evident by observing significantly smaller spleens as a percentage of body weight as compared to WT mice at 7 and 14 days post-infection. Additionally, Asc-/- mice expressed less IFNγ in the liver at 7 days post-infection. Inflammation is implicated in causing abortion in the pregnant mouse model (Kim et al., 2005). Since ASC induces inflammation and flagellin activates the inflammasome via ASC, we hypothesized that flagellin induced inflammation would be responsible for abortion in the pregnant mouse. To test this hypothesis, we infected pregnant mice with WT, ΔfliC, and ΔflgCfliEflgG B. abortus. We expected ΔfliC to induce less inflammation and abortion. Since flagellin induced inflammasome activation requires a T4SS, and ΔflgCfliEflgG lacks only the basal body of flagella and should still make and translocate flagellin, it should behave like WT B. abortus. Surprisingly, both mutants induced more reabsorption and inflammation in fetal and placental tissue than WT B. abortus. Additionally, the numbers of both bacteria were recovered at a slightly higher level than WT, although the difference was not statistically significant. These results suggest that flagellin or flagella influence abortion independent of inflammasome activation. To directly determine if the inflammasome contributes to abortion in the pregnant mouse model, we infected pregnant WT and Asc-/- mice with B. abortus. We found that the fetal reabsorption and decreased fetal weight caused by B. abortus was almost completely absent in Asc-/- mice. (Abstract shortened by UMI.)
Brucella ovis is a major cause of reproductive failure in rams and it is one of the few well-described Brucella species that is not zoonotic. Previous work showed that a B. ovis mutant lacking a ...species-specific ABC transporter ( Delta abcBA) was attenuated in mice and was unable to survive in macrophages. The aim of this study was to evaluate the role of this ABC transporter during intracellular survival of B. ovis. In HeLa cells, B. ovis WT was able to survive and replicate at later time point (48 hpi), whereas an Delta abcBA mutant was attenuated at 24 hpi. The reduced survival of the Delta abcBA mutant was associated with a decreased ability to exclude the lysosomal marker LAMP1 from its vacuolar membrane, suggesting a failure to establish a replicative niche. The Delta abcBA mutant showed a reduced abundance of the Type IV secretion system (T4SS) proteins VirB8 and VirB11 in both rich and acid media, when compared to WT B. ovis. However, mRNA levels of virB1, virB8, hutC, and vjbR were similar in both strains. These results support the notion that the ABC transporter encoded by abcEDCBA or its transported substrate acts at a post-transcriptional level to promote the optimal expression of the B. ovis T4SS within infected host cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MRSA enterocolitis is under-recognized in the setting of PCR testing. In this case report, we describe risk factors, the importance of stool culture, and the third published case of MRSA ...enterocolitis in a patient with leukemia. In addition, we performed a retrospective analysis of all stool cultures at our institution that have grown Staphylococcus aureus, and we describe an additional five cases. We also report the diagnostic yield of organisms detected by culture, but not on the FilmArray panel. While rare, these cases demonstrate that MRSA in stool may indicate a severe and potentially life-threatening infection, particularly in immunocompromised persons.
A novel approach was developed for the coencapsulation of an anti-HIV drug (tenofovir) and a latency-breaking agent (vorinostat), using magnetically guided layer-by-layer (LbL) assembled nanocarriers ...for the treatment of neuroAIDS. Ultrasmall iron oxide (Fe3O4) nanoparticles (10±3 nm) were synthesized and characterized. The LbL technique was used to achieve a sustained release profile, and application of 2 bilayers (tenofovir+dextran sulphate2+vorinostat) to magnetic nanoparticles resulted in a 2.8 times increase in drug (tenofovir) loading and also resulted in an increase in the drug release period by 30-fold, with 100% drug release in sustained manner over a period of 5 days with the simultaneous stimulation of latent HIV expression. Nanoformulation showed a good blood-brain barrier transmigration ability (37.95%±1.5%) with good in vitro antiviral efficacy (~33% reduction of p24 level) over a period of 5 days after HIV infection in primary human astrocytes, with good cell viability (>90%). Hence, LbL arrangements of drugs on magnetic nanoparticles provides sustained release and, therefore, may improve the patient's adherence to therapy and lead to better compliance.
The neurological complications of AIDS (neuroAIDS) during the infection of human immunodeficiency virus (HIV) are symptomized by non-specific, multifaceted neurological conditions and therefore, ...defining a specific diagnosis/treatment mechanism(s) for this neuro-complexity at the molecular level remains elusive. Using an in silico based integrated gene network analysis we discovered that HIV infection shares convergent gene networks with each of twelve neurological disorders selected in this study. Importantly, a common gene network was identified among HIV infection, Alzheimer's disease, Parkinson's disease, multiple sclerosis, and age macular degeneration. An mRNA microarray analysis in HIV-infected monocytes showed significant changes in the expression of several genes of this in silico derived common pathway which suggests the possible physiological relevance of this gene-circuit in driving neuroAIDS condition. Further, this unique gene network was compared with another in silico derived novel, convergent gene network which is shared by seven major neurological disorders (Alzheimer's disease, Parkinson's disease, Multiple Sclerosis, Age Macular Degeneration, Amyotrophic Lateral Sclerosis, Vascular Dementia, and Restless Leg Syndrome). These networks differed in their gene circuits; however, in large, they involved innate immunity signaling pathways, which suggests commonalities in the immunological basis of different neuropathogenesis. The common gene circuits reported here can provide a prospective platform to understand how gene-circuits belonging to other neuro-disorders may be convoluted during real-time neuroAIDS condition and it may elucidate the underlying-and so far unknown-genetic overlap between HIV infection and neuroAIDS risk. Also, it may lead to a new paradigm in understanding disease progression, identifying biomarkers, and developing therapies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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