Pairing Bacteroides vulgatus LPS Structure with Its Immunomodulatory Effects on Human Cellular Models.

The gut microbiota guide the development of the host immune system by setting a systemic threshold for immune activation. Lipopolysaccharides (LPSs) from gut bacteria are able to trigger systemic and local proinflammatory and immunomodulatory responses, and this capability strongly relies on their fine structures. Up to now, only a few LPS structures from gut commensals have been elucidated; therefore, the molecular motifs that may be important for LPS-mammalian cell interactions at the gut level are still obscure. Here, we report on the full structure of the LPS isolated from one of the prominent species of the genus Bacteroides, Bacteroides vulgatus. The LPS turned out to consist of a particular chemical structure based on hypoacylated and mono-phosphorylated lipid A and with a galactofuranose-containing core oligosaccharide and an O-antigen built up of mannose and rhamnose. The evaluation of the immunological properties of this LPS on human in vitro models revealed a very interesting capability to produce anti-inflammatory cytokines and to induce a synergistic action of MD-2/TLR4- and TLR2-mediated signaling pathways.

Role of a fluid-phase PRR in fighting an intracellular pathogen: PTX3 in Shigella infection.

Shigella spp. are pathogenic bacteria that cause bacillary dysentery in humans by invading the colonic and rectal mucosa where they induce dramatic inflammation. Here, we have analyzed the role of the soluble PRR Pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity. Mice that had been intranasally infected with S. flexneri were rescued from death by treatment with recombinant PTX3. In vitro PTX3 exerts the antibacterial activity against Shigella, impairing epithelial cell invasion and contributing to the bactericidal activity of serum. PTX3 is produced upon LPS-TLR4 stimulation in accordance with the lipid A structure of Shigella. In the plasma of infected patients, the level of PTX3 amount only correlates strongly with symptom severity. These results signal PTX3 as a novel player in Shigella pathogenesis and its potential role in fighting shigellosis. Finally, we suggest that the plasma level of PTX3 in shigellosis patients could act as a biomarker for infection severity.

Bradyrhizobium Lipid A: Immunological Properties and Molecular Basis of Its Binding to the Myeloid Differentiation Protein-2/Toll-Like Receptor 4 Complex.

Lipopolysaccharides (LPS) are potent activator of the innate immune response through the binding to the myeloid differentiation protein-2 (MD-2)/toll-like receptor 4 (TLR4) receptor complexes. Although a variety of LPSs have been characterized so far, a detailed molecular description of the structure-activity relationship of the lipid A part has yet to be clarified. Photosynthetic Bradyrhizobium strains, symbiont of Aeschynomene legumes, express distinctive LPSs bearing very long-chain fatty acids with a hopanoid moiety covalently linked to the lipid A region. Here, we investigated the immunological properties of LPSs isolated from Bradyrhizobium strains on both murine and human immune systems. We found that they exhibit a weak agonistic activity and, more interestingly, a potent inhibitory effect on MD-2/TLR4 activation exerted by toxic enterobacterial LPSs. By applying computational modeling techniques, we also furnished a plausible explanation for the Bradyrhizobium LPS inhibitory activity at atomic level, revealing that its uncommon lipid A chemical features could impair the proper formation of the receptorial complex, and/or has a destabilizing effect on the pre-assembled complex itself.

Dynamic density shaping of photokinetic E. coli.

Many motile microorganisms react to environmental light cues with a variety of motility responses guiding cells towards better conditions for survival and growth. The use of spatial light modulators could help to elucidate the mechanisms of photo-movements while, at the same time, providing an efficient strategy to achieve spatial and temporal control of cell concentration. Here we demonstrate that millions of bacteria, genetically modified to swim smoothly with a light controllable speed, can be arranged into complex and reconfigurable density patterns using a digital light projector. We show that a homogeneous sea of freely swimming bacteria can be made to morph between complex shapes. We model non-local effects arising from memory in light response and show how these can be mitigated by a feedback control strategy resulting in the detailed reproduction of grayscale density images.

The Deep-Sea Polyextremophile Halobacteroides lacunaris TB21 Rough-Type LPS: Structure and Inhibitory Activity towards Toxic LPS.

The structural characterization of the lipopolysaccharide (LPS) from extremophiles has important implications in several biomedical and therapeutic applications. The polyextremophile Gram-negative bacterium Halobacteroideslacunaris TB21, isolated from one of the most extreme habitats on our planet, the deep-sea hypersaline anoxic basin Thetis, represents a fascinating microorganism to investigate in terms of its LPS component. Here we report the elucidation of the full structure of the R-type LPS isolated from H. lacunaris TB21 that was attained through a multi-technique approach comprising chemical analyses, NMR spectroscopy, and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. Furthermore, cellular immunology studies were executed on the pure R-LPS revealing a very interesting effect on human innate immunity as an inhibitor of the toxic Escherichia coli LPS.

The Helicobacter cinaedi antigen CAIP participates in atherosclerotic inflammation by promoting the differentiation of macrophages in foam cells.

Recent studies have shown that certain specific microbial infections participate in atherosclerosis by inducing inflammation and immune reactions, but how the pathogens implicated in this pathology trigger the host responses remains unknown. In this study we show that Helicobacter cinaedi (Hc) is a human pathogen linked to atherosclerosis development since at least 27% of sera from atherosclerotic patients specifically recognize a protein of the Hc proteome, that we named Cinaedi Atherosclerosis Inflammatory Protein (CAIP) (n = 71). CAIP appears to be implicated in this pathology because atheromatous plaques isolated from atherosclerotic patients are enriched in CAIP-specific T cells (10%) which, in turn, we show to drive a Th1 inflammation, an immunopathological response typically associated to atherosclerosis. Recombinant CAIP promotes the differentiation and maintenance of the pro-inflammatory profile of human macrophages and triggers the formation of foam cells, which are a hallmark of atherosclerosis. This study identifies CAIP as a relevant factor in atherosclerosis inflammation linked to Hc infection and suggests that preventing and eradicating Hc infection could reduce the incidence of atherosclerosis.

The Lipid†A from Rhodopseudomonas palustris Strain BisA53 LPS Possesses a Unique Structure and Low Immunostimulant Properties.

The search for novel lipid A analogues from any biological source that can act as antagonists, displaying inhibitory activity towards the production of pro-inflammatory cytokines, or as immunomodulators in mammals, is a very topical issue. To this aim, the structure and immunological properties of the lipopolysaccharide lipid A from the purple nonsulfur bacterium Rhodopseudomonas palustris strain BisA53 have been determined. This lipid A displays a unique structural feature, with a non-phosphorylated skeleton made up of the tetrasaccharide Manp-α-(1→4)-GlcpN3N-ß-1→6-GlcpN3N-α-(1→1)-α-GalpA, and four primary amide-linked 14:0(3-OH) and, as secondary O-acyl substituents, a 16:0 and the very long-chain fatty acid 26:0(25-OAc), appended on the GlcpN3N units. This lipid A architecture is definitely rare, so far identified only in the genus Bradyrhizobium. Immunological tests on both murine bone-marrow-derived and human monocyte-derived macrophages revealed an extremely low immunostimulant capability of this LPS lipid A.

The Orchestra and Its Maestro: Shigella's Fine-Tuning of the Inflammasome Platforms.

Shigella spp. are the causative agents of bacillary dysentery, leading to extensive mortality and morbidity worldwide. These facultative intracellular bacteria invade the epithelium of the colon and the rectum, inducing a severe inflammatory response from which the symptoms of the disease originate. Shigella are human pathogens able to manipulate and subvert the innate immune system surveillance. Shigella dampens inflammasome activation in epithelial cells. In infected macrophages, inflammasome activation and IL-1ß and IL-18 release lead to massive neutrophil recruitment and greatly contribute to inflammation. Here, we describe how Shigella hijacks and finely tunes inflammasome activation in the different cell populations involved in pathogenesis: epithelial cells, macrophages, neutrophils, DCs, and B and T lymphocytes. Shigella emerges as a "sly" pathogen that switches on/off the inflammasome mechanisms in order to optimize the interaction with the host and establish a successful infection.

Determination of the structure of the O-antigen and the lipid A from the entomopathogenic bacterium Pseudomonas entomophila lipopolysaccharide along with its immunological properties.

The structure and the immunology of the lipopolysaccharide (LPS) of Pseudomonas entomophila, an entomopathogenic bacterium isolated from the fruit fly Drosophila melanogaster, was characterized. The O-antigen portion was established and resulted to be built up of a repetitive unit constituted by four monosaccharide residues, all L configured, all deoxy at C-6 and with an acetamido function at C-2: →3)-α-l-FucNAc-(1→4)-α-l-FucNAc-(1→3)-α-l-FucNAc-(1→3)-ß-l-QuiNAc-(1→ The structural analysis of lipid A, showed a mixture of different species. The diphosphorylated glucosamine backbone carries six fatty acids consistent with the composition C10:0 3(OH), C12:0 2(OH) and C12:0 3(OH), whereas other species differs by the number of phosphates and/or of fatty acids. The immunology experiments demonstrated that the LPS structure of P. entomophila displayed a low ability to engage the TLR4-mediated signaling correlated to a significant antagonistic activity toward hexa-acylated LPS structures.

Thermophiles as potential source of novel endotoxin antagonists: the full structure and bioactivity of the lipo-oligosaccharide from Thermomonas hydrothermalis.

Thermomonas hydrothermalis is a Gram-negative thermophilic bacterium that is able to live at 50 °C. This ability is attributed to chemical modifications, involving those to bacterial cell-wall components, such as proteins and (glyco)lipids. As the main component of the outer membrane of Gram-negative bacteria, lipopolysaccharides (LPSs) are exposed to the environment, thus they can undergo structural chemical changes to allow thermophilic bacteria to live at their optimal growth temperature. Furthermore, as one of the major target of the eukaryotic innate immune system, LPS elicits host immune response in a structure-dependent mode; thus the uncommon chemical features of thermophilic bacterial LPSs might exert a different biological action on the innate immune system-an antagonistic effect, as shown in studies of LPS structure-activity relationship in the ongoing research into antagonist LPS candidates. Here, we report the complete structural and biological activity analysis of the lipo-oligosaccharide isolated from Thermomonas hydrothermalis, achieved by a multidisciplinary approach (chemical analysis, NMR, MALDI MS and cellular immunology). We demonstrate a tricky and interesting structure combined with a very interesting effect on human innate immunity.

Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation.

LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1ß in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.

Characterization of SfPgdA, a Shigella flexneri peptidoglycan deacetylase required for bacterial persistence within polymorphonuclear neutrophils.

Peptidoglycan deacetylases protect the Gram-positive bacteria cell wall from host lysozymes by deacetylating peptidoglycan. Sequence analysis of the genome of Shigella flexneri predicts a putative polysaccharide deacetylase encoded by the plasmidic gene orf185, renamed here SfpgdA. We demonstrated a peptidoglycan deacetylase (PGD) activity with the purified SfPgdA in vitro. To investigate the role SfPgdA in virulence, we constructed a SfpgdA mutant and studied its phenotype in vitro. The mutant showed an increased sensitivity to lysozyme compared to the parental strain. Moreover, the mutant was rapidly killed by polymorphonuclear neutrophils (PMNs). Specific substitution of histidines residues 120 and 125, located within the PGD catalytic domain, by phenylalanine abolished SfPgdA function. SfPgdA expression is controlled by PhoP. Mutation of phoP increases sensitivity to lysozyme compared to the SfpgdA mutant. Here, we confirmed that SfPgdA expression is enhanced under low magnesium concentration and not produced by the phoP mutant. Ectopic expression of SfPgdA in the phoP mutant restored lysozyme resistance and parental bacterial persistence within PMNs. Together our results indicate that PG deacetylation mechanism likely contributes to Shigella persistence by subverting detection by the host immune system.

Dampening Host Sensing and Avoiding Recognition in Pseudomonas aeruginosa Pneumonia.

Pseudomonas aeruginosa is an opportunistic pathogen and causes a wide range of acute and chronic infections. P. aeruginosa infections are kept in check by an effective immune surveillance in the healthy host, while any imbalance or defect in the normal immune response can manifest in disease. Invasive acute infection in the immunocompromised patients is mediated by potent extracellular and cell bound bacterial virulence factors. Life-threatening chronic infection in cystic fibrosis patients is maintained by pathogenic variants that contribute to evade detection and clearance by the immune system. Here, we reviewed the molecular basis of receptor-mediated recognition of P. aeruginosa and their role in initiating inflammation and the colonization. In addition, the consequence of the P. aeruginosa genetic adaptation for the antibacterial defence and the maintaining of chronic infection are discussed.

Pseudomonas aeruginosa exploits lipid A and muropeptides modification as a strategy to lower innate immunity during cystic fibrosis lung infection.

Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1beta cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF.

Adhesion molecules and cytokine profile in ileal tissue of sheep infected with Mycobacterium avium subsp. paratuberculosis.

Sheep develop clinical diseases after 3-5 years after infection with Mycobacterium avium subsp. paratuberculosis (MAP). Clinical symptoms of paratuberculosis include persistent diarrhea and weight loss due to a chronic inflammation of the small intestine. Tissue alterations in the areas of the ileo-cecal junction are often observed. Here, we investigate the molecular processes underlying tissue damages in intestinal mucosa of 14 sheep showing either tuberculoid or lepromatous form of MAP enteritis. We found that E-cadherins, alpha-catenin and beta1-integrins were present at significant low levels in tissues of sheep affected by lepromatous form and that this pattern was associated with high expression of TGF-beta, IL-10, IL-1beta, and TNF-alpha and with a modest increase of CD4+ and CD25+ T cells. Tissues of sheep with the tuberculoid form showed high expression of IFNgamma, IL-12, and MCP-1 and a significant presence of CD4+ and CD25+ T cells. Finally, anti-transglutaminase (tTG) IgG1 antibodies were detected in sera of infected animal belonging to both groups, as already described for human inflammatory bowel diseases. Our results further stress the similarities in the clinical and histological features between ruminant paratuberculosis and human intestinal inflammatory diseases.

Intracellular bacteriolysis triggers a massive apoptotic cell death in Shigella-infected epithelial cells.

Infected epithelial cells, which act as a first barrier against pathogens, seldom undergo apoptosis. Rather, infected epithelial cells undergo a slow cell death that displays hallmarks of necrosis. Here, we demonstrate that rapid intracellular lysis of Shigella flexneri, provoked by either the use of a diaminopimelic acid auxotroph mutant or treatment of infected cells with antibiotics of the beta-lactam family, resulted in a massive and rapid induction of apoptotic cell death. This intracellular bacteriolysis-mediated apoptotic death (IBAD) was characterized by the specific involvement of the mitochondrial-dependent cytochrome c/Apaf-1 axis that resulted in the activation of caspases-3, -6 and -9. Importantly, Bcl-2 family members and the NF-kappaB pathway seemed to be critical modulators of IBAD. Finally, we identified that IBAD was also triggered by Salmonella enterica serovar Typhimurium but not by the Gram-positive bacteria, Listeria monocytogenes. Together, our results demonstrate that, contrary to previous findings, epithelial cells are intrinsically able to mount an efficient apoptotic cell death response following infection. Indeed, apoptosis in normal circumstances is masked by powerful anti-apoptotic mechanisms, which are overcome in IBAD. Our results also uncover an unexpected consequence of the treatment of infected cells with certain classes of antibiotics.

IpgB1 and IpgB2, two homologous effectors secreted via the Mxi-Spa type III secretion apparatus, cooperate to mediate polarized cell invasion and inflammatory potential of Shigella flexenri.

Type III secretion systems (T3SS) are present in many pathogenic gram-negative bacteria and mediate the translocation of bacterial effector proteins into host cells. Here, we report the phenotypic characterization of S. flexneri ipgB1 and ipgB2 mutants, in which the genes encoding the IpgB1 and IpgB2 effectors have been inactivated, either independently or simultaneously. Like IpgB1, we found that IpgB2 is secreted by the T3SS and its secretion requires the Spa15 chaperone. Upon infection of semi-confluent HeLa cells, the ipgB2 mutant exhibited the same invasive capacity as the wild-type strain and the ipgB1 mutant was 50% less invasive. Upon infection of polarised Caco2-cells, the ipgB2 mutant did not show a significant defect in invasion and the ipgB1 mutant was slightly more invasive than the wild-type strain. Entry of the ipgB1 ipgB2 mutant in polarized cells was reduced by 70% compared to the wild-type strain. Upon infection of the cornea in Guinea pigs, the ipgB2 mutant exhibited a wild-type phenotype, the ipgB1 mutant was hypervirulent and elicited a more pronounced proinflammatory response, while the ipgB1 ipgB2 mutant was highly attenuated. The attenuated phenotype of the ipgB1 ipgB2 mutant was confirmed using a murine pulmonary model of infection and histopathology and immunochemistry studies.

Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its delta galU mutant: glycine residue location in the inner core of the lipopolysaccharide.

Shigella flexneri is a gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections. Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multidisciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a L,D-Hep-(1 -->7)-L,D-Hep-(1 -->3)-L,D-Hep-(1 -->5)-[Kdo-(2 -->4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T delta galU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.

Muramylpeptide shedding modulates cell sensing of Shigella flexneri.

Bacterial infections trigger the activation of innate immunity through the interaction of pathogen-associated molecular patterns (PAMPs) with pattern recognition molecules (PRMs). The nucleotide-binding oligomerization domain (Nod) proteins are intracellular PRMs that recognize muramylpeptides contained in peptidoglycan (PGN) of bacteria. It is still unclear how Nod1 physically interacts with PGN, a structure internal to the Gram-negative bacterial envelope. To contribute to the understanding of this process, we demonstrate that, like Escherichia coli, Bordetella pertussis and Neisseria gonorrheae, the Gram-negative pathogen Shigella spontaneously releases PGN fragments and that this process can be increased by inactivating either ampG or mppA, genes involved in PGN recycling. Both Shigella mutants, but especially the strain carrying the mppA deletion, trigger Nod1-mediated NF-kappaB activation to a greater extent than the wild-type strain. Likewise, muramylpeptides spontaneously shed by Shigella are able per se to trigger a Nod1-mediated response consistent with the relative amount. Finally, we found that qualitative changes in muramylpeptide shedding can alter in vivo host responses to Shigella infection. Our findings support the idea that muramylpeptides released by pathogens during infection could modulate the immune response through Nod proteins and thereby influence the outcome of disease.

Mucosal lymphoid infiltrate dominates colonic pathological changes in murine experimental shigellosis.

BACKGROUND: Shigella species are invasive human pathogens that cause acute rectocolitis by triggering a dysregulated inflammatory reaction in the colonic and rectal mucosa. Because mice are naturally resistant to shigellosis, there is no mouse model that mimics human disease. We explore the susceptibility of intestinal flora-depleted mice to shigellosis after intragastric infection with Shigella strains. METHODS: Mice given 5 g/L streptomycin as a beverage were infected intragastrically with 1 x 108 cfu of either invasive or noninvasive Shigella strains. RESULTS: We found that invasive Shigella strains persist up to 30 days in feces, whereas the persistence of noninvasive Shigella strains was reduced. Colonization primarily involves the colon and the cecum and, to a lesser extent, the ileum. The hallmark of inflammation in the intestinal tissue is a dramatic expansion of the lymphoid follicles, in which a high apoptotic index is recorded. CONCLUSIONS: We provide a murine model in which shigellae are able to reach their natural tissue target: the colon. Moreover, the absence of polymorphonuclear leukocyte recruitment and of epithelial cell lesions reveal some aspects of shigellosis that are usually hidden by the prevalence of this cell population. This novel model may contribute to the identification of new targets for vaccines and therapies.