The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManBcore proteins have been fully characterized using NMR spectroscopy. The results revealed that ...disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (β-d-Glcp-(1→4)-α-Kdop-(2→4)β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)β-d-GlcpN-(1→6)-β-d-GlcpN-(1→3)-α-d-Manp-(1→5)-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P), in addition to components lacking one of the terminal β-d-GlcpN and/or the β-d-Glcp residues (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of manBcore gives rise to a deep-rough pentasaccharide core (β-d-Glcp-(1→4)-α-Kdop-(2→4)-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal β-d-Glcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcore proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)β-d-GlcpN-(1→6)-β-d-GlcpN-(1→3)-α-d-Manp-(1→5) structure in virulence.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
BackgroundEconomical and effective vaccines against Streptococcus pneumoniae (pneumococcus) are needed for implementation in poorer countries where the disease burden is highest. Here, we evaluated ...Lactococcus lactis intracellularly producing the pneumococcal surface protein A (PspA) as a mucosal vaccine in conferring protection against pneumococcal disease MethodsMice were intranasally (inl) immunized with the lactococcal vaccine. Control groups were also immunized with similar amounts of recombinant PspA administered inl or subcutaneously with alum. PspA-specific antibodies in serum samples and lung lavage fluids were measured before challenge in intraperitoneal sepsis and inl respiratory-infection models of pneumococcal disease ResultsThe lactococcal vaccine afforded better protection against respiratory challenge with pneumococcus than did vaccination with purified antigen given inl or by injection with alum. This finding was associated with a shift toward a Th1-mediated immune response characterized by reduced antibody titers to the PspA antigen. In the sepsis model, the lactococcal vaccine afforded resistance to disease on a par with that obtained with the injected vaccine, demonstrating its efficacy against different forms of pneumococcal disease ConclusionGiven the safety profile of L. lactis there is considerable potential to develop a pneumococcal vaccine for use in humans and to broaden this approach to combat other major pathogens
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Whooping cough is a severe, highly contagious disease of the human respiratory tract, caused by
. The pathogenicity requires several virulence factors, including
toxin (PTX), a key component of ...current available vaccines. Current vaccines do not induce mucosal immunity. Tissue-resident memory T cells (Trm) are among the first lines of defense against invading pathogens and are involved in long-term protection. However, the factors involved in Trm establishment remain unknown. Comparing two
strains expressing PTX (WT) or not (ΔPTX), we show that the toxin is required to generate both lung CD4
and CD8
Trm. Co-administering purified PTX with ΔPTX is sufficient to generate these Trm subsets. Importantly, adoptive transfer of lung CD4
or CD8
Trm conferred protection against
in naïve mice. Taken together, our data demonstrate for the first time a critical role for PTX in the induction of mucosal long-term protection against
.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
•Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype.•Platelet cancer cell interactions appear to be mediated by 5 key genes which have established roles in ...metastasis.•Targeting these mediators of metastasis could improve outcomes for cancer patients.
Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions.
The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model.
Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR.
Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The lipopolysaccharide (LPS) is a major virulence factor of Brucella, a facultative intracellular pathogenic Gram-negative bacterium. Brucella LPS exhibits a low toxicity and its atypical structure ...was postulated to delay the host immune response, favouring the establishment of chronic disease. Here we carried out an in-depth in vitro and in vivo characterisation of the immunomodulatory effects of Brucella LPS on different dendritic cell (DC) subpopulations. By using LPSs from bacteria that share some of Brucella LPS structural features, we demonstrated that the core component of B. melitensis wild-type (Bm-wt) LPS accounts for the low activation potential of Brucella LPS in mouse GM-CSF-derived (GM-) DCs. Contrary to the accepted dogma considering Brucella LPS a poor TLR4 agonist and DC activator, Bm-wt LPS selectively induced expression of surface activation markers and cytokine secretion from Flt3-Ligand-derived (FL-) DCs in a TLR4-dependent manner. It also primed in vitro T cell proliferation by FL-DCs. In contrast, modified LPS with a defective core purified from Brucella carrying a mutated wadC gene (Bm-wadC), efficiently potentiated mouse and human DC activation and T cell proliferation in vitro. In vivo, Bm-wt LPS promoted scant activation of splenic DC subsets and limited recruitment of monocyte- DC like cells in the spleen, conversely to Bm-wadC LPS. Bm-wadC live bacteria drove high cytokine secretion levels in sera of infected mice. Altogether, these results illustrate the immunomodulatory properties of Brucella LPS and the enhanced DC activation ability of the wadC mutation with potential for vaccine development targeting Brucella core LPS structure.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
CD4(+) T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization ...of murine cytotoxic CD4(+) T cells (CD4(+) CTL) during Brucella abortus infection. These CD4(+) CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4(+) CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4(+) T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4(+) CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by ...innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the
homologs of
, and
, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance.
, which encodes a putative ethanolamine transferase, carries a frame-shift in
but not in other
spp. and phylogenetic neighbors like the opportunistic pathogen
Consistent with the genomic evidence, a
mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while
carrying
displayed increased resistance.
encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and
Although we found no evidence of lipid A dephosphorylation, a
mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene
putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except
and is intact in
. Free-lipid analysis revealed that
corresponded to
, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in
and
, while
carrying the
of
and
synthesized hydroxylated OLs. Interestingly, mutants in
, or
were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in
and
but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of
LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life.
The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor ...improvement. A synthetic mgl gene encoding methionine-γ-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade L-methionine as well as other sulfur-containing compounds such as L-cysteine, L-cystathionine, and L-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.
Group B streptococci (GBS) usually behave as commensal organisms that asymptomatically colonize the gastrointestinal and urogenital tracts of adults. However, GBS are also pathogens and the leading ...bacterial cause of life-threatening invasive disease in neonates. While the events leading to transmission and disease in neonates remain unclear, GBS carriage and level of colonization in the mother have been shown to be significant risk factors associated with invasive infection. Surface antigens represent ideal vaccine targets for eliciting antibodies that can act as opsonins and/or inhibit colonization and invasion. Using a genetic screen for exported proteins in GBS, we identified a gene, designated lrrG, that encodes a novel LPXTG anchored surface antigen containing leucine-rich repeat (LRR) motifs found in bacterial invasins and other members of the LRR protein family. Southern blotting showed that lrrG was present in all GBS strains tested, representing the nine serotypes, and revealed the presence of an lrrG homologue in Streptococcus pyogenes. Recombinant LrrG protein was shown in vitro to adhere to epithelial cells in a dose-dependent manner, suggesting that it may function as an adhesion factor in GBS. More importantly, immunization with recombinant LrrG elicited a strong immunoglobulin G response in CBA/ca mice and protected against lethal challenge with virulent GBS. The data presented in this report suggest that this conserved protein is a highly promising candidate antigen for use in a GBS vaccine.
Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological ...approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit α,γ-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H₂S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.
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