Odilia L. C. Wijburg

Innate secretory antibodies protect against natural Salmonella typhimurium infection

The production of IgA is induced in an antigen-unspecific manner by commensal flora. These secretory antibodies (SAbs) may bind multiple antigens and are thought to eliminate commensal bacteria and self-antigens to avoid systemic recognition. In this study, we addressed the role of “innate” SAbs, i.e., those that are continuously produced in normal individuals, in protection against infection of the gastrointestinal tract. We used polymeric immunoglobulin receptor (pIgR−/−) knock-out mice, which are unable to bind and actively transport dimeric IgA and pentameric IgM to the mucosae, and examined the role of innate SAbs in protection against the invasive pathogen Salmonella typhimurium. In vitro experiments suggested that innate IgA in pIgR−/− serum bound S. typhimurium in a cross-reactive manner which inhibited epithelial cell invasion. Using a “natural” infection model, we demonstrated that pIgR−/− mice are profoundly sensitive to infection with S. typhimurium via the fecal-oral route and, moreover, shed more bacteria that readily infected other animals. These results imply an important evolutionary role for innate SAbs in protecting both the individual and the herd against infections, and suggest that the major role of SAbs may be to prevent the spread of microbial pathogens throughout the population, rather than protection of local mucosal surfaces.

MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices.

Influenza A virus facilitates Streptococcus pneumoniae transmission and disease

Streptococcus pneumoniae (the pneumococcus) kills ~1.6 million people annually. Pneumococcal infections predominantly manifest as pneumonia, sepsis, meningitis, and otitis media. S. pneumoniae is also a member of the normal nasopharyngeal flora, colonizing up to 80% of children. Infection with influenza A virus (IAV) has been associated with both pneumococcal disease and transmission. However, to date no animal model has been available to investigate the role of IAV in the spread of S. pneumoniae. Here we investigate pneumococcal‐influenza synergism with a particular focus on the role of IAV on pneumococcal transmission. Infant mice were colonized with S. pneumoniae and subsequently infected with IAV 3 d later. Using this novel model we show increased pneumococcal colonization and disease in the presence of IAV. Notably, in vivo imaging showed that IAV was essential for the transmission of S. pneumoniae from colonized (“index”) mice to their naive cohoused littermates (“contacts”). Transmission occurred only when all mice were infected with IAV and was prevented when an IAV‐neutralizing antibody was used to inhibit IAV replication in either index mice or contact mice. Together, these data provide novel insights into pneumococcal‐influenza synergism and may indicate a previously unappreciated role of IAV in the spread of S. pneumoniae. —Diavatopoulos, D. A, Short, K. R., Price, J. T., Wilksch, J. J., Brown, L. E., Briles, D. E., Strugnell, R. A, Wijburg, O. L. Influenza A virus facilitates Streptococcus pneumoniae transmission and disease. FASEB J. 24, 1789–1798 (2010). www.fasebj.org

NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8 + T cells

Memory T cells exert antigen-independent effector functions, but how these responses are regulated is unclear. We discovered an in vivo link between flagellin-induced NLRC4 inflammasome activation in splenic dendritic cells (DCs) and host protective interferon-γ (IFN-γ) secretion by noncognate memory CD8+ T cells, which could be activated by Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that CD8α+ DCs were particularly efficient at sensing bacterial flagellin through NLRC4 inflammasomes. Although this activation released interleukin 18 (IL-18) and IL-1β, only IL-18 was required for IFN-γ production by memory CD8+ T cells. Conversely, only the release of IL-1β, but not IL-18, depended on priming signals mediated by Toll-like receptors. These findings provide a comprehensive mechanistic framework for the regulation of noncognate memory T cell responses during bacterial immunity.

Role of the Polymeric Ig Receptor in Mucosal B Cell Homeostasis

Secretory IgA (SIgA) is the most characteristic component of the mucosal immune system and has long been considered the major protective factor that prevents pathogens from invading hosts through the mucosae. Recent studies, however, have suggested that complete immunity against a range of mucosal bacterial and viral pathogens can be achieved in the absence of IgA. Therefore, to further dissect the role of SIgA, we generated mice deficient in the polymeric Ig receptor (pIgR−/− mice). As a result of an inability to transport dimeric IgA to the secretions, pIgR−/− mice are deficient in SIgA and accumulate circulating dimeric IgA, with serum levels 100-fold greater than those observed in normal mice. Examination of lamina propria mononuclear cells showed that pIgR−/− mice had ∼3 times as many IgA-secreting cells as C57BL/6 mice. Further analysis showed that these cells displayed the differentiated IgA+ B220− phenotype and accounted for a 2-fold increase in the number of lamina propria blast cells in the pIgR−/− mice. Subsequent experiments showed that OVA-specific CD4+ T cell expansion following OVA feeding was not elevated in pIgR−/− mice. Furthermore, no differences in CD8+ T cell tolerance or induction of influenza virus-specific CD8+ T cells were detected in pIgR−/− mice compared with controls. Therefore, while SIgA is clearly involved in maintaining some parameters of mucosal homeostasis in the intestine, the mechanisms associated with its barrier function and the clinical consequences of its deficiency are yet to be identified.

The multi-copper-ion oxidase CueO of Salmonella enterica serovar Typhimurium is required for systemic virulence

ABSTRACT Salmonella enterica serovar Typhimurium possesses a multi-copper-ion oxidase (multicopper oxidase), CueO (also known as CuiD), a periplasmic enzyme known to be required for resistance to copper ions. CueO from S. Typhimurium was expressed as a recombinant protein in Escherichia coli, and the purified protein exhibited a high cuprous oxidase activity. We have characterized an S. Typhimurium cueO mutant and confirmed that it is more sensitive to copper ions. Using a murine model of infection, it was observed that the cueO mutant was significantly attenuated, as indicated by reduced recovery of bacteria from liver and spleen, although there was no significant difference in recovery from Peyers patches and mesenteric lymph nodes. However, the intracellular survival of the cueO mutant in unprimed or gamma-interferon-primed murine macrophages was not statistically different from that of wild-type Salmonella, suggesting that additional host factors are involved in clearance of the cueO mutant. Unlike a cueO mutant from E. coli, the S. Typhimurium cueO mutant did not show greater sensitivity to hydrogen peroxide and its sensitivity to copper ions was not affected by siderophores. Similarly, the S. Typhimurium cueO mutant was not rescued from copper ion toxicity by addition of the branched-chain amino acids and leucine.

Resistance to Celiac Disease in Humanized HLA-DR3-DQ2-Transgenic Mice Expressing Specific Anti-Gliadin CD4+ T Cells

Celiac disease is a chronic inflammatory enteropathy caused by cellular immunity to dietary gluten. More than 90% of patients carry HLA-DQ2 encoded by HLA-DQA1*05 and DQB1*02, and gluten-specific CD4+ T cells from intestinal biopsies of these patients are HLA-DQ2-restricted, produce Th1 cytokines and preferentially recognize gluten peptides deamidated by tissue transglutaminase. We generated mice lacking murine MHC class II genes that are transgenic for human CD4 and the autoimmunity and celiac disease-associated HLA-DR3-DQ2 haplotype. Immunization with the α-gliadin 17-mer that incorporates the overlapping DQ2-α-I and DQ2-α-II epitopes immunodominant in human celiac disease generates peptide-specific HLA-DQ2-restricted CD4+ T cells. When exposed to dietary gluten, naive or gliadin-primed mice do not develop pathology. Coincident introduction of dietary gluten and intestinal inflammation resulted in low-penetrance enteropathy and tissue transglutaminase-specific IgA. Two further strains of transgenic mice expressing HLA-DR3-DQ2 and human CD4, one with a NOD background and another TCR transgenic having over 90% of CD4+ T cells specific for the DQ2-α-II epitope with a Th1 phenotype, were also healthy when consuming gluten. These humanized mouse models indicate that gluten ingestion can be tolerated without intestinal pathology even when HLA-DQ2-restricted CD4+ T cell immunity to gluten is established, thereby implicating additional factors in controlling the penetrance of celiac disease.

Secondary Acylation of Klebsiella pneumoniae Lipopolysaccharide Contributes to Sensitivity to Antibacterial Peptides

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055ΔlpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055ΔlpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.

Vaccine-induced protection against gastrointestinal bacterial infections in the absence of secretory antibodies

Secretory IgA (SIgA) is widely held to be responsible for the defense of the mucosae against pathogenics and other potentially harmful agents. In this study, polymeric Ig receptor (pIgR) knockout mice, which lack secretory antibodies (SAb), were used to investigate the role of vaccine‐elicited SAb in protection against gastrointestinal bacterial infections. An essential role for specific SAb in protection against Vibrio cholerae was evident from experiments showing that vaccinated pIgR–/– mice, but not vaccinated C57BL/6 mice, were susceptible to cholera toxin challenge. Vaccination of C57BL/6 mice with Salmonella typhimurium elicited strong antigen‐specific, mucosal responses, which blocked in vitro invasion of epithelia. However, vaccinated C57BL/6 and pIgR–/– mice were equally resistant to challenge infection with virulent S. typhimurium. Finally, we investigated the importance of SIgA in protection against recurrent infections with Citrobacter rodentium. Although higher numbers of bacteria were detected early after challenge infection in feces of vaccinated pIgR–/– mice compared with vaccinated C57BL/6 mice, both mouse strains showed complete clearance after 9 days. These results suggested that, in immune animals, SIgA is crucial for the protection of gastrointestinal surfaces against secreted bacterial toxins, may inhibit early colonization by C. rodentium, but is not essential for protection against re‐infection with S. typhimurium or C. rodentium.

Dual role for macrophages in vivo in pathogenesis and control of murine Salmonella enterica var. Typhimurium infections.

Salmonella spp. are regarded as facultative intracellular bacterial pathogens which are found inside macrophages (Mϕ) after i.  v. infection. It is generally assumed that Mϕ restrict the replication of the bacteria during infection. In this study we examined the in vivo activities of Mϕ during experimental S. typhimurium infections, using a selective liposome‐based Mϕ elimination technique. Unexpectedly, elimination of Mϕ prior to infection with virulent S. typhimurium decreased morbidity and mortality, suggesting that Mϕ mediate the pathology caused by S. typhimurium. Removal of Mϕ during vaccination with attenuated S. typhimurium did not affect protection against challenge with virulent S. typhimurium, suggesting that Mϕ are not required for the induction of protective immunity and that other cells must function as antigen‐presenting cell to elicit T cell‐mediated protection. However, Mϕ appeared to be important effectors of protection against challenge infection since elimination of Mϕ from vaccinated mice prior to challenge infection with virulent S. typhimurium significantly decreased protection. These results enhance our understanding of the control of S. typhimurium growth in vivo, and moreover suggest that Mϕ play a major role in the pathology of virulent S. typhimurium infections. As such, these cells may present a novel target for therapeutic intervention.