Mycobacteria counteract a TLR-mediated nitrosative defense mechanism in a zebrafish infection model.

Pulmonary tuberculosis (TB), caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb), is a major world health problem. The production of reactive nitrogen species (RNS) is a potent cytostatic and cytotoxic defense mechanism against intracellular pathogens. Nevertheless, the protective role of RNS during Mtb infection remains controversial. Here we use an anti-nitrotyrosine antibody as a readout to study nitration output by the zebrafish host during early mycobacterial pathogenesis. We found that recognition of Mycobacterium marinum, a close relative of Mtb, was sufficient to induce a nitrosative defense mechanism in a manner dependent on MyD88, the central adaptor protein in Toll like receptor (TLR) mediated pathogen recognition. However, this host response was attenuated by mycobacteria via a virulence mechanism independent of the well-characterized RD1 virulence locus. Our results indicate a mechanism of pathogenic mycobacteria to circumvent host defense in vivo. Shifting the balance of host-pathogen interactions in favor of the host by targeting this virulence mechanism may help to alleviate the problem of infection with Mtb strains that are resistant to multiple drug treatments

Interactions between host and intestinal crypt-resided biofilms are controlled by epithelial fucosylation

Summary: As highly organized consortia of bacteria, biofilms have long been implicated in aggravating inflammation. However, our understanding regarding in vivo host-biofilm interactions in the complex tissue environments remains limited. Here, we show a unique pattern of crypt occupation by mucus-associated biofilms during the early stage of colitis, which is genetically dependent on bacterial biofilm-forming capacity and restricted by host epithelial α1,2-fucosylation. α1,2-Fucosylation deficiency leads to markedly augmented crypt occupation by biofilms originated from pathogenic Salmonella Typhimurium or indigenous Escherichia coli, resulting in exacerbated intestinal inflammation. Mechanistically, α1,2-fucosylation-mediated restriction of biofilms relies on interactions between bacteria and liberated fucose from biofilm-occupied mucus. Fucose represses biofilm formation and biofilm-related genes in vitro and in vivo. Finally, fucose administration ameliorates experimental colitis, suggesting therapeutic potential of fucose for biofilm-related disorders. This work illustrates host-biofilm interactions during gut inflammation and identifies fucosylation as a physiological strategy for restraining biofilm formation

Surface expression patterns of defined glycan antigens change during Schistosoma mansoni cercarial transformation and development of schistosomula

Contains fulltext : 152760.pdf (publisher's version ) (Closed access)During the complex lifecycle of Schistosoma mansoni, a large variety of glycans is expressed. To many of these glycans, antibodies are induced by the infected host and some might be targets for vaccines or diagnostic tests. Spatial changes in glycan expression during schistosome development are largely unexplored. To study the surface-exposed glycans during the important initial stages of infection, we analyzed the binding of a panel of anti-glycan monoclonal antibodies (mAbs) to cercariae and schistosomula up to 72 h after transformation by immunofluorescence microscopy. The mAb specificity toward their natural targets was studied using a microarray containing a wide range of schistosomal N-glycans, O-glycans and glycosphingolipid glycans. With the exception of GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDN-F), mono- and multifucosylated GalNAcbeta1-4GlcNAc (LDN)-motifs were exposed at the surface of all developmental stages studied. Multifucosylated LDN-motifs were present on cercarial glycocalyx-derived O-glycans as well as cercarial glycolipids. In contrast, the Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis X) and LDN-F-motifs, also expressed on cercarial glycolipids, and in addition on a range of cercarial N- and O-glycans, became surface expressed only after transformation of cercariae to schistosomula. In line with the documented shedding of the O-glycan-rich cercarial glycocalyx after transformation these observations suggest that surface accessible multifucosylated LDN-motifs are mostly expressed by O-glycans in cercariae, but principally by glycosphingolipids in schistosomula. We hypothesize that these temporal changes in surface exposure of glycan antigens are relevant to the interaction with the host during the initial stages of infection with schistosomes and discuss the potential of these glycan antigens as intervention targets

Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system

Streptococcal Lancefield polysaccharides are critical cell wall determinants for human Group IIA secreted phospholipase A2 to exert its bactericidal effects

Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the bactericidal enzyme hGIIA to exert its bactericidal function

Interference with Lipoprotein Maturation Sensitizes Methicillin-Resistant Staphylococcus aureus to Human Group IIA-Secreted Phospholipase A2 and Daptomycin

Methicillin-resistant Staphylococcus aureus (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA-secreted phospholipase A2 (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including S. aureus. To determine hGIIA-resistance mechanisms of MRSA, we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of lspA, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the lspA mutant was associated with enhanced activity of hGIIA on the cell membrane. Moreover, lspA deletion increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. MRSA wild type could be sensitized to hGIIA and daptomycin killing through exposure to LspA-specific inhibitors globomycin and myxovirescin A1. Analysis of >26,000 S. aureus genomes showed that LspA is highly sequence-conserved, suggesting universal application of LspA inhibition. The role of LspA in hGIIA resistance was not restricted to MRSA since Streptococcus mutans and Enterococcus faecalis were also more hGIIA-susceptible after lspA deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological interference with LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically applied antibiotics

Tyrosine nitration is a quantifiable measure of nitration output during early Mm infection.

<p>(A) Confocal micrographs of DAF-FM DA stained embryos at 1 day post infection (dpi) in the caudal vein region. Neutrophils are shown by <i>lyz</i> driven dsRed. Upper panels show an example of an uninfected embryo while lower panels show an infected embryo. N.b. The bright stripe of DAF-FM DA staining in the upper panels is the notochord, which has variable brightness when focusing on the caudal vein region due to differences in embryo mounting. (B) Confocal micrographs of 1 dpi embryos stained with iNOS antibody (dim red) when infected with Mm (bright red, white arrow) or non-infected. Neutrophils are shown by <i>mpx</i>:GFP. (C) Fluorescence micrographs of confocal images stitched together to show the location of anti-nitrotyrosine staining in a whole-mount wild type zebrafish embryo of 3 dpf. (D) Confocal fluorescence micrographs of 1 dpi embryos showing the co-localization of <i>mpx:</i>GFP and anti-nitrotyrosine antibody staining in the absence or presence of Mm infection. (E) Corrected fluorescence intensity measurements of antibody stainings in the absence or presence of Mm. Upper panel shows the levels for the iNOS antibody shown in (B). Data shown are mean ± SEM, n = 45 cells from 15 embryos combined from 3 independent experiments. Lower panel shows the levels for the anti-nitrotyrosine antibody shown in D). Data shown are mean ± SEM, n = 58–99 cells from 15 embryos combined from 3 independent experiments. (F) Confocal micrographs showing co-localization of nitrotyrosine with compartments of neutrophils with myeloperoxidase activity (shown by TSA staining, in the absence of infection at 2 dpf. In the <i>mpx^−/−</i> mutant there is no myeloperoxidase activity and a corresponding decrease in nitrotyrosine.</p

Mm infection increased tyrosine nitration levels at 12 hpi.

<p>(A) Example fluorescence confocal z-stacks of the caudal vein region of embryos stained with anti-nitrotyrosine antibody, imaged at 4, 8 or 12 hpi in the presence or absence of Mm infection. (B) Corrected fluorescence intensity levels of anti-nitrotyrosine antibody confocal z-stacks of equal size at 4, 8 or 12 hours after Mm infection relative to the control group per time point. Data shown are mean ± SEM, n = 50–85 cells from 15 embryos combined from 3 independent experiments.</p