IgG in cervicovaginal mucus traps HSV and prevents vaginal Herpes infections

IgG is the predominant immunoglobulin in cervicovaginal mucus (CVM), yet how immunoglobulin G (IgG) in mucus can protect against infections is not fully understood. IgG diffuses rapidly through cervical mucus, slowed only slightly by transient adhesive interactions with mucins. We hypothesize that this almost unhindered diffusion allows IgG to accumulate rapidly on pathogen surfaces, and the resulting IgG array forms multiple weak adhesive crosslinks to mucus gel that effectively trap (immobilize) pathogens, preventing them from initiating infections. Here, we report that herpes simplex virus serotype 1 (HSV-1) readily penetrated fresh, pH-neutralized ex vivo samples of CVM with low or no detectable levels of anti-HSV-1 IgG but was trapped in samples with even modest levels of anti-HSV-1 IgG. In samples with little or no endogenous anti-HSV-1 IgG, addition of exogenous anti-HSV-1 IgG, affinity-purified from intravenous immunoglobulin, trapped virions at concentrations below those needed for neutralization and with similar potency as endogenous IgG. Deglycosylating purified anti-HSV-1 IgG, or removing its Fc component, markedly reduced trapping potency. Finally, a non-neutralizing IgG against HSV-gG significantly protected mice against vaginal infection, and removing vaginal mucus by gentle lavage abolished protection. These observations suggest that IgG-Fc has a glycan-dependent “muco-trapping” effector function that may provide exceptionally potent protection at mucosal surfaces.     

Divergent Mechanisms of Interaction of Helicobacter pylori and Campylobacter jejuni with Mucus and Mucins

Helicobacter pylori and Campylobacter jejuni colonize the stomach and intestinal mucus, respectively. Using a combination of mucus-secreting cells, purified mucins, and a novel mucin microarray platform, we examined the interactions of these two organisms with mucus and mucins. H. pylori and C. jejuni bound to distinctly different mucins. C. jejuni displayed a striking tropism for chicken gastrointestinal mucins compared to mucins from other animals and preferentially bound mucins from specific avian intestinal sites (in order of descending preference: the large intestine, proximal small intestine, and cecum). H. pylori bound to a number of animal mucins, including porcine stomach mucin, but with less avidity than that of C. jejuni for chicken mucin. The strengths of interaction of various wild-type strains of H. pylori with different animal mucins were comparable, even though they did not all express the same adhesins. The production of mucus by HT29-MTX-E12 cells promoted higher levels of infection by C. jejuni and H. pylori than those for the non-mucus-producing parental cell lines. Both C. jejuni and H. pylori bound to HT29-MTX-E12 mucus, and while both organisms bound to glycosylated epitopes in the glycolipid fraction of the mucus, only C. jejuni bound to purified mucin. This study highlights the role of mucus in promoting bacterial infection and emphasizes the potential for even closely related bacteria to interact with mucus in different ways to establish successful infections.     

Oral vaccination with Salmonella enterica as a cruzipain-DNA delivery system confers protective immunity against Trypanosoma cruzi

To stimulate both local and systemic immune responses against Trypanosoma cruzi, Salmonella enterica serovar Typhimurium aroA was exploited as a DNA delivery system for cruzipain (SCz). In a murine model we compared SCz alone (GI) or coadministered with Salmonella carrying a plasmid encoding granulocyte-macrophage colony-stimulating factor (GII), as well as protocols in which SCz priming was followed by boosting with recombinant cruzipain (rCz) admixed with either CpG-ODN (GIII) or MALP-2, a synthetic derivative of a macrophage-activating lipopeptide of 2 kDa from Mycoplasma fermentans (GIV). The results showed that protocols that included four oral doses of SCz (GI) elicited mainly a mucosal response characterized by immunoglobulin A (IgA) secretion and proliferation of gut-associated lymphoid tissue cells, with weak systemic responses. In contrast, the protocol that included a boost with rCz plus CpG (GIII) triggered stronger systemic responses in terms of Cz-specific serum IgG titers, splenocyte proliferation, gamma interferon (IFN-γ) secretion, and delayed-type hypersensitivity response. Trypomastigote challenge of vaccinated mice resulted in significantly lower levels of parasitemia compared to controls. Protection was abolished by depletion of either CD4⁺ or CD8⁺ T cells. Parasite control was also evident from the reduction of tissue damage, as revealed by histopathologic studies and serum levels of enzymes that are markers of muscle injury in chronic Chagas' disease (i.e., creatine kinase, aspartate aminotransferase, and lactate dehydrogenase). Enhanced release of IFN-γ and interleukin-2 was observed in GI and GII upon restimulation of splenocytes in the nonparasitic phase of infection. Our results indicate that Salmonella-mediated delivery of Cz-DNA by itself promotes the elicitation of an immune response that controls T. cruzi infection, thereby reducing parasite loads and subsequent damage to muscle tissues.     

Identification of Three Highly Attenuated Salmonella typhimurium Mutants That Are More Immunogenic and Protective in Mice than a Prototypical aroA Mutant

A panel of Salmonella typhimurium 14028s mutants, which were previously shown to be highly attenuated in the BALB/c mouse model of infection, were analyzed for their potential as live Salmonella oral-vaccine candidates. A prototypical aroA mutant was chosen as a basis of comparison. From the panel of mutants initially chosen for this study, three mutants with comparable levels of attenuation elicited higher Salmonella-specific serum immunoglobulin G (IgG) and/or mucosal secretory-IgA antibody titers than the aroA vaccine strain. The three mutants, CL288, CL401, and CL554, also elicited a better protective immune response than the aroA control strain, after a single oral dose of 1 × 10⁹ to 2 × 10⁹ bacteria.     

Peyer's patches are required for intestinal immunoglobulin A responses to Salmonella spp

Previous studies have shown that Peyer's patches (PP) are not required for intestinal immunoglobulin A (IgA) responses to orally administered soluble protein. However, the roles of PP in regulation of mucosal immune responses against bacterial antigen remain to be clarified. In the present study, we generated several gut-associated lymphoreticular tissue-null mice by treatment with anti-interleukin-7 receptor antibody, the fusion protein of lymphotoxin β receptor and IgG Fc, and/or tumor necrosis factor receptor p55 and IgG Fc. These mice were then immunized with recombinant Salmonella expressing the C fragment of the tetanus toxin (rSalmonella-Tox C). Orally immunized PP-null mice as well as isolated lymphoid follicle (ILF)-null, PP/ILF-null, and PP/ILF/mesenteric lymph node-null mice induced identical levels of tetanus toxoid (TT)-specific systemic IgG responses to those of control mice. However, PP-null mice, but not ILF-null mice, failed to induce TT-specific intestinal IgA antibodies. Analysis of TT-specific CD4⁺ T-cell responses showed a reduction of gamma interferon (IFN-γ) synthesis in the intestinal lamina propriae of PP-null mice given oral rSalmonella-Tox C. In contrast, TT-specific IFN-γ responses in the spleen and delayed-type hypersensitivity responses were intact in those immunized mice. Interestingly, Salmonella lipopolysaccharide (LPS)-specific fecal IgA responses were not elicited in PP-null mice, while serum IgG anti-LPS antibodies were identical to those of control mice. These results suggest that while none of the gut-associated lymphoreticular tissues are required for the induction of systemic immune responses, PP are an essential lymphoid tissue for induction and regulation of intestinal IgA immunity against orally administered rSalmonella.     

Herpes simplex virus-binding IgG traps HSV in human cervicovaginal mucus across the menstrual cycle and diverse vaginal microbial composition

IgG possesses an important yet little recognized effector function in mucus. IgG bound to viral surface can immobilize otherwise readily diffusive viruses to the mucin matrix, excluding them from contacting target cells and facilitating their elimination by natural mucus clearance mechanisms. Cervicovaginal mucus (CVM) is populated by a microbial community, and its viscoelastic and barrier properties can vary substantially not only across the menstrual cycle, but also in women with distinct microbiota. How these variations impact the “muco-trapping” effector function of IgGs remains poorly understood. Here we obtained multiple fresh, undiluted CVM specimens (n = 82 unique specimens) from six women over time, and employed high-resolution multiple particle tracking to quantify the mobility of fluorescent Herpes Simplex Viruses (HSV-1) in CVM treated with different HSV-1-binding IgG. The IgG trapping potency was then correlated to the menstrual cycle, and the vaginal microbial composition was determined by 16 s rRNA. In the specimens studied, both polyclonal and monoclonal HSV-1-binding IgG appeared to consistently and effectively trap HSV-1 in CVM obtained at different times of the menstrual cycle and containing a diverse spectrum of commensals, including G. vaginalis-dominant microbiota. Our findings underscore the potential broad utility of this “muco-trapping” effector function of IgG to reinforce the vaginal mucosal defense, and motivates further investigation of passive immunization of the vagina as a strategy to protect against vaginally transmitted infections.     

The Mucin Muc2 Limits Pathogen Burdens and Epithelial Barrier Dysfunction during Salmonella enterica Serovar Typhimurium Colitis

Salmonella enterica serovar Typhimurium is a model organism used to explore the virulence strategies underlying Salmonella pathogenesis. Although intestinal mucus is the first line of defense in the intestine, its role in protection against Salmonella is still unclear. The intestinal mucus layer is composed primarily of the Muc2 mucin, a heavily O-glycosylated glycoprotein. The core 3-derived O-glycans of Muc2 are synthesized by core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT). Mice lacking these glycans still produce Muc2 but display a thinner intestinal mucus barrier. We began our investigations by comparing Salmonella-induced colitis and mucus dynamics in Muc2-deficient (Muc2^−/−) mice, C3GnT^−/− mice, and wild-type C57BL/6 (WT) mice. Salmonella infection led to increases in luminal Muc2 secretion in WT and C3GnT^−/− mice. When Muc2^−/− mice were infected with Salmonella, they showed dramatic susceptibility to infection, carrying significantly higher cecal and liver pathogen burdens, and developing significantly higher barrier disruption and higher mortality rates, than WT mice. We found that the exaggerated barrier disruption in infected Muc2^−/− mice was invA dependent. We also tested the susceptibility of C3GnT^−/− mice and found that they carried pathogen burdens similar to those of WT mice but developed exaggerated barrier disruption. Moreover, we found that Muc2^−/− mice were impaired in intestinal alkaline phosphatase (IAP) expression and lipopolysaccharide (LPS) detoxification activity in their ceca, potentially explaining their high mortality rates during infection. Our data suggest that the intestinal mucus layer (Muc2) and core 3 O-glycosylation play critical roles in controlling Salmonella intestinal burdens and intestinal epithelial barrier function, respectively.     

Aeromonas salmonicida Binds Differentially to Mucins Isolated from Skin and Intestinal Regions of Atlantic Salmon in an N-Acetylneuraminic Acid-Dependent Manner

Aeromonas salmonicida subsp. salmonicida infection, also known as furunculosis disease, is associated with high morbidity and mortality in salmonid aquaculture. The first line of defense the pathogen encounters is the mucus layer, which is predominantly comprised of secreted mucins. Here we isolated and characterized mucins from the skin and intestinal tract of healthy Atlantic salmon and studied how A. salmonicida bound to them. The mucins from the skin, pyloric ceca, and proximal and distal intestine mainly consisted of mucins soluble in chaotropic agents. The mucin density and mucin glycan chain length from the skin were lower than were seen with mucin from the intestinal tract. A. salmonicida bound to the mucins isolated from the intestinal tract to a greater extent than to the skin mucins. The mucins from the intestinal regions had higher levels of sialylation than the skin mucins. Desialylating intestinal mucins decreased A. salmonicida binding, whereas desialylation of skin mucins resulted in complete loss of binding. In line with this, A. salmonicida also bound better to mammalian mucins with high levels of sialylation, and N-acetylneuraminic acid appeared to be the sialic acid whose presence was imperative for binding. Thus, sialylated structures are important for A. salmonicida binding, suggesting a pivotal role for sialylation in mucosal defense. The marked differences in sialylation as well as A. salmonicida binding between the skin and intestinal tract suggest interorgan differences in the host-pathogen interaction and in the mucin defense against A. salmonicida.     

Oral Immunization with a Salmonella typhimurium Vaccine Vector Expressing Recombinant Enterotoxigenic Escherichia coli K99 Fimbriae Elicits Elevated Antibody Titers for Protective Immunity

Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mortality in piglets and newborn calves. In an effort to develop a safe and effective vaccine for the prevention of F5⁺ ETEC infections, a balanced lethal asd⁺ plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd⁺. Introduction of this plasmid into an attenuated Salmonella typhimurium Δaro Δasd strain, H683, resulted in strain AP112, which stably expresses E. coli K99 fimbriae. A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks. IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer’s patches (PP), and spleens of vaccinated and control BALB/c mice. This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen. These antibodies were important for protective immunity. One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a control Salmonella vector. These results confirm that oral Salmonella vaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity.     

Flagellin-deficient outer membrane vesicles as adjuvant induce cross-protection of Salmonella Typhimurium outer membrane proteins against infection by heterologous Salmonella serotypes

Salmonella enteric serovar infections result in high morbidity and mortality worldwide. Cross-protective vaccines are an effective strategy in controlling salmonellosis caused by multiple serotypes. In our previous study, outer membrane vesicles (OMVs) derived from flagellin-deficient Salmonella Typhimurium (S. Typhimurium) were proven effective in mediating cross-protection against infection by multiple Salmonella serotypes; OMVs also exhibit potent adjuvant effects. In this study, we further investigated the adjuvant capacities of flagellin-deficient S. Typhimurium OMVs. Our finding showed that outer membrane proteins (OMPs) in combination with flagellin-deficient S. Typhimurium OMVs could function as adjuvants and invoke stronger humoral, cellular, mucosal, and cross-protective immune responses compared to conventional aluminum (alum). Furthermore, as an adjuvant, OMVs could induce significantly higher cellular immune responses and display enhanced cross-protection for OMPs against wild-type virulent Salmonella Choleraesuis and Salmonella Enteritidis challenge. In summary, OMVs function as a potent adjuvant with the capability of conferring greater cross-protection against infection by multiple Salmonella serotypes, and may be of great value as an effective vaccine adjuvant in enteric diseases.     

IFNγ expression by an attenuated strain of Salmonella enterica serovar Typhimurium improves vaccine efficacy in susceptible TLR4-defective C3H/HeJ mice

C3H/HeJ mice carry a mutated allele of TLR4 gene (TLR4 ^d ) and thus are hyporesponsive to the lethal effects of lipopolysaccharide (LPS). Characteristically, however, the mice are also hypersusceptible to infections, particularly by Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) and are known to be difficult to vaccinate against virulent exposure. This is observed despite the expression of wild-type allele of Nramp1 gene, another important determinant of Salmonella susceptibility. In contrast, C3H/HeN mice (TLR4 ⁿ Nramp1 ⁿ ) express a functional TLR4 protein and are resistant to infection, even by virulent strains of S. typhimurium. In the present study, we describe the immune system-enhancing properties of an attenuated strain of S. typhimurium engineered to express murine IFN-γ. This strain (designated GIDIFN) was able to modulate immune responses following systemic inoculation by upregulating the production of inflammatory mediators (IL-6 and IL-12) and anti-bacterial effector molecules (nitric oxide; NO). Consequently, this led to a more effective control of bacterial proliferation in systemic target organs in both C3H/HeJ and C3H/HeN mice. Although evidence for the enhancement in immune responses could be observed as early as few hours post-inoculation, sustained improvements required 2–3 days to manifest. Vaccination of C3H/HeJ mice with GIDIFN strain, even at low doses, conferred a significantly higher degree of protection against challenge with virulent Salmonella in susceptible C3H/HeJ mice. Our data demonstrate that IFNγ-expressing Salmonella are immunogenic and confer excellent protection against virulent challenge in susceptible C3H/HeJ mice; in addition they may be used as an effective mucosal delivery vectors against virulent infection and for boosting immune responses in immunodeficient hosts.     

The Levels of Brachyspira hyodysenteriae Binding to Porcine Colonic Mucins Differ between Individuals,and Binding Is Increased to Mucins from Infected Pigs with De Novo MUC5AC Synthesis

Brachyspira hyodysenteriae colonizes the pig colon, resulting in mucohemorrhagic diarrhea and growth retardation. Fecal mucus is a characteristic feature of swine dysentery; therefore, we investigated how the mucin environment changes in the colon during infection with B. hyodysenteriae and how these changes affect this bacterium''s interaction with mucins. We isolated and characterized mucins, the main component of mucus, from the colon of experimentally inoculated and control pigs and investigated B. hyodysenteriae binding to these mucins. Fluorescence microscopy revealed a massive mucus induction and disorganized mucus structure in the colon of pigs with swine dysentery. Quantitative PCR (qPCR) and antibody detection demonstrated that the mucus composition of pigs with swine dysentery was characterized by de novo expression of MUC5AC and increased expression of MUC2 in the colon. Mucins from the colon of inoculated and control pigs were isolated by two steps of isopycnic density gradient centrifugation. The mucin densities of control and inoculated pigs were similar, whereas the mucin quantity was 5-fold higher during infection. The level of B. hyodysenteriae binding to mucins differed between pigs, and there was increased binding to soluble mucins isolated from pigs with swine dysentery. The ability of B. hyodysenteriae to bind, measured in relation to the total mucin contents of mucus in sick versus healthy pigs, increased 7-fold during infection. Together, the results indicate that B. hyodysenteriae binds to carbohydrate structures on the mucins as these differ between individuals. Furthermore, B. hyodysenteriae infection induces changes to the mucus niche which substantially increase the amount of B. hyodysenteriae binding sites in the mucus.     

The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system

The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine, and colon. The large highly glycosylated gel-forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine, mucus limits the number of bacteria that can reach the epithelium and the Peyer's patches. In the large intestine, the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells secrete not only the MUC2 mucin but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate-keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103⁺ type. In addition to the gel-forming mucins, the transmembrane mucins MUC3, MUC12, and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization, suggesting that enterocytes might control and report epithelial microbial challenge. There is communication not only from the epithelial cells to the immune system but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.     

rIL-22 as an adjuvant enhances the immunogenicity of rGroEL in mice and its protective efficacy against S. Typhi and S. Typhimurium

Salmonella infection, ranging from mild, self-limiting diarrhea to severe gastrointestinal, septicemic disease and enteric fever, is a global health problem both in humans and animals. Rapid development of microbial drug resistance has led to a need for efficacious and affordable vaccines against Salmonella. Microbial heat shock proteins (HSPs), including HSP60 and HSP70, are the dominant antigens that promote the host immune response. Co-administration of these antigens with cytokines, such as IL-22, which plays an important role in antimicrobial defense, can enhance the immune response and protection against pathogens. Therefore, the aim of the present study was to determine the immunogenicity of rGroEL (Hsp60) of S. Typhi, alone or administered in combination with murine rIL-22, and its protective efficacy against lethal infection with Salmonella, in mice. There was appreciable stimulation of the humoral and cell-mediated immune responses in mice immunized with rGroEL alone. However, co-administration of rGroEL with rIL-22 further boosted the antibody titers (IgG, IgG1 and IgG2a), T-cell proliferative responses and the secretion of both Th1 and Th2 cytokines. Additionally, rGroEL alone accorded 65%–70% protection against lethal challenge with S. Typhi and S. Typhimurium, which increased to 90% when co-administered with rIL-22.     

The adherence to Salmonella enteritidis of IgG, IgM and β1C-globulins from guinea-pig serum

The adherence of globulins from guinea-pig serum to Salmonella enteritidis was determined when these bacteria were sensitized in vitro: (a) in serum from normal guinea-pigs lacking detectable bactericidal antibody, (b) in serum from normal guinea-pigs possessing low titre of bactericidal antibody, and (c) in serum from guinea-pigs actively immunized against this Salmonella. Sensitized bacteria were washed free of contaminating serum constituents and used to immunize rabbits. Guinea-pig globulins that adhered to the bacteria and to which the rabbit responded by the production of antibody were identified by immunoelectrophoresis. IgM, but no IgG, attached to bacteria sensitized in serum lacking bactericidins. Both IgG and IgM attached to bacteria sensitized in immune serum or in serum containing `normal' bactericidins. β_1C-globulin attached to bacteria in all three cases.     

Core 1– and 3–derived O-glycans collectively maintain the colonic mucus barrier and protect against spontaneous colitis in mice

Core 1– and 3–derived mucin-type O-glycans are primary components of the mucus layer in the colon. Reduced mucus thickness and impaired O-glycosylation are observed in human ulcerative colitis. However, how both types of O-glycans maintain mucus barrier function in the colon is unclear. We found that C1galt1 expression, which synthesizes core 1 O-glycans, was detected throughout the colon, whereas C3GnT, which controls core 3 O-glycan formation, was most highly expressed in the proximal colon. Consistent with this, mice lacking intestinal core 1–derived O-glycans (IEC C1galt1^−/−) developed spontaneous colitis primarily in the distal colon, whereas mice lacking both intestinal core 1– and 3–derived O-glycans (DKO) developed spontaneous colitis in both the distal and proximal colon. DKO mice showed an early onset and more severe colitis than IEC C1galt1^−/− mice. Antibiotic treatment restored the mucus layer and attenuated colitis in DKO mice. Mucins from DKO mice were more susceptible to proteolysis than wild-type mucins. This study indicates that core 1– and 3–derived O-glycans collectively contribute to the mucus barrier by protecting it from bacterial protease degradation and suggests new therapeutic targets to promote mucus barrier function in colitis patients.     

Vaccination with a Single CD4 T Cell Peptide Epitope from a Salmonella Type III-Secreted Effector Protein Provides Protection against Lethal Infection

Salmonella infections affect millions worldwide and remain a significant cause of morbidity and mortality. It is known from mouse studies that CD4 T cells are essential mediators of immunity against Salmonella infection, yet it is not clear whether targeting CD4 T cell responses directly with peptide vaccines against Salmonella can be effective in combating infection. Additionally, it is not known whether T cell responses elicited against Salmonella secreted effector proteins can provide protective immunity against infection. In this study, we investigated both of these possibilities using prime-boost immunization of susceptible mice with a single CD4 T cell peptide epitope from Salmonella secreted effector protein I (SseI), a component of the Salmonella type III secretion system. This immunization conferred significant protection against lethal oral infection, equivalent to that conferred by whole heat-killed Salmonella bacteria. Surprisingly, a well-characterized T cell epitope from the flagellar protein FliC afforded no protection compared to immunization with an irrelevant control peptide. The protective response appeared to be most associated with polyfunctional CD4 T cells raised against the SseI peptide, since no antibodies were produced against any of the peptides and very little CD8 T cell response was observed. Overall, this study demonstrates that eliciting CD4 T cell responses against components of the Salmonella type III secretion system can contribute to protection against infection and should be considered in the design of future Salmonella subunit vaccines.     

Effects of Enteromyxum leei (Myxozoa) infection on gilthead sea bream (Sparus aurata) (Teleostei) intestinal mucus: glycoprotein profile and bacterial adhesion

The intestinal myxosporean parasite Enteromyxum leei causes severe desquamative enteritis in gilthead sea bream (Sparus aurata) (Teleostei) that impairs nutrient absorption causing anorexia and cachexia. In fish, as in terrestrial vertebrates, intestinal goblet cells are responsible for the adherent mucus secretion overlying epithelial cells, which constitutes a first line of innate immune defense against offending microorganisms but serves also as substrate and nutrient source for the commensal microflora. The secreted intestinal mucus of parasitized (n?=?6) and unexposed (n?=?8) gilthead sea bream was isolated, concentrated, and subjected to downward gel chromatography. Carbohydrate and protein contents (via PAS and Bradford stainings), terminal glycosylation (via lectin ELISA), and Aeromonas hydrophila and Vibrio alginolyticus adhesion were analyzed for the isolated intestinal mucins. Parasitized fish, compared with unexposed fish, presented intestinal mucus mucins with a lower glycoprotein content and glycosylation degree at the anterior and middle intestine, whereas both glycoprotein content and glycosylation degree increased at the posterior intestine section, though only significantly for the total carbohydrate content. Additionally, a slight molecular size increase was detected in the mucin glycoproteins of parasitized fish. Terminal glycosylation of the mucus glycoproteins in parasitized fish pointed to an immature mucin secretion (N-acetyl-α-d-galactosamine increase, α-l-fucose, and neuraminic-acid-α-2-6-galactose reduction). Bacterial adhesion to large-sized mucus glycoproteins (>2,000 kDa) of parasitized fish was significantly lower than in unexposed fish.