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.

The mucosal secretory immune system provides an important primary defence against disease, as studies of humans with mucosal humoral immunodeficiencies suggest that the absence of secretory immunoglobulin A leads to an increase in mucosal infections. However, the infection risks posed do not seem to provide the evolutionary drive to retain constitutive secretion of often 'hard won' protein, suggesting that secretory antibodies may have some other important function (or functions). This Review examines the evidence that secretory antibodies provide an important defence against infection in specific animal models and explores complementary explanations for the evolution of the secretory immune system.

Streptococcus pneumoniae (the pneumococcus) kills approximately 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.

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.

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.

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. B5055⌬lpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055⌬lpxM 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.Klebsiella pneumoniae is a common cause of nosocomial pneumonia, septicemia, and urinary tract infections; a recent US study found the bacterium to be the third most commonly isolated organism from intensive care wards and the most common species identified in blood cultures (1), with a similar trend reported among European hospitals (2). This rising prevalence combined with the extensive spread of antibiotic-resistant strains, especially extended spectrum ␤-lactamase (ESBL)-producing strains, will drive the search for alternative treatments and/or an effective vaccine against the bacterium.K. pneumoniae is known to express a number of virulence determinants, including a thick polysaccharide capsule. The capsule protects the bacterium from phagocytosis by polymorphonuclear leukocytes (3) and is thought to prevent killing of the bacterium by bactericidal serum factors (4). Other, well studied virulence factors of K. pneumoniae include the pili/fimbriae and lipopolysaccharide (LPS).6 LPS consists of an outer membrane-embedded lipid A molecule, partially conserved inner core carbohydrate structure, attached to an outer core (of which there are at least two variants, conferred by the waa operon (5)), and a repeated polysaccharide or O-antigen (O-Ag). The O-Ag region specifically is thought to play a role in resistance to complement killing (6) and to contribute to bacteremia and lethality during murine pneumonia infections (7) although this contribution is still disputed (8). The comparatively small number of Klebsiella LPS or O-types (9 serotypes) compared with capsule or K-types (Ͼ77 serotypes) suggests that LPS could be utilized for vaccination. However any LPSbased vaccines would need to be detoxified b...

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 Peyer's 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.

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.