Streptococcus pneumoniae infection of host epithelial cells via polymeric immunoglobulin receptor transiently induces calcium release from intracellular stores

The pneumococcal surface protein C (PspC) is a major adhesin of Streptococcus pneumoniae (pneumococci) that interacts in a human-specific manner with the ectodomain of the human polymeric immunoglobulin receptor (pIgR) produced by respiratory epithelial cells. This interaction promotes bacterial colonization and bacterial internalization by initiating host signal transduction cascades. Here, we examined alterations of intracellular calcium ([Ca(2+)](i)) levels in epithelial cells during host cell infections with pneumococci via the PspC-hpIgR mechanism. The release of [Ca(2+)](i) from intracellular stores in host cells was significantly increased by wild-type pneumococci but not by PspC-deficient pneumococci. The increase in [Ca(2+)](i) was dependent on phospholipase C as pretreatment of cells with a phospholipase C-specific inhibitor U73122 abolished the increase in [Ca(2+)](i). In addition, we demonstrated the effect of [Ca(2+)](i) on pneumococcal internalization by epithelial cells. Uptake of pneumococci was significantly increased after pretreatment of epithelial cells with the cell-permeable calcium chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-tetraacetoxymethyl ester or use of EGTA as an extracellular Ca(2+)-chelating agent. In contrast, thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)ATPase, which increases [Ca(2+)](i) in a sustained fashion, significantly reduced pIgR-mediated pneumococcal invasion. Importantly, pneumococcal adherence to pIgR-expressing cells was not altered in the presence of inhibitors as demonstrated by immunofluorescence microscopy. In conclusion, these results demonstrate that pneumococcal infections induce mobilization of [Ca(2+)](i) from intracellular stores. This may constitute a defense response of host cells as the experimental reduction of intracellular calcium levels facilitates pneumococcal internalization by pIgR-expressing cells, whereas elevated calcium levels diminished bacterial internalization by host epithelial cells.     

Lung-specific overexpression of CC chemokine ligand (CCL) 2 enhances the host defense to Streptococcus pneumoniae infection in mice: role of the CCL2-CCR2 axis

Mononuclear phagocytes are critical components of the innate host defense of the lung to inhaled bacterial pathogens. The monocyte chemotactic protein CCL2 plays a pivotal role in inflammatory mononuclear phagocyte recruitment. In this study, we tested the hypothesis that increased CCL2-dependent mononuclear phagocyte recruitment would improve lung innate host defense to infection with Streptococcus pneumoniae. CCL2 transgenic mice that overexpress human CCL2 protein in type II alveolar epithelial cells and secrete it into the alveolar air space showed a similar proinflammatory mediator response and neutrophilic alveolitis to challenge with S. pneumoniae as wild-type mice. However, CCL2 overexpressing mice showed an improved pneumococcal clearance and survival compared with wild-type mice that was associated with substantially increased lung mononuclear phagocyte subset accumulations upon pneumococcal challenge. Surprisingly, CCL2 overexpressing mice developed bronchiolitis obliterans upon pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block the CCL2-CCR2 axis in CCL2 overexpressing mice, though completely abrogating bronchiolitis obliterans, led to progressive pneumococcal pneumonia. Collectively, these findings demonstrate the importance of the CCL2-CCR2 axis in the regulation of both the resolution/repair and remodelling processes after bacterial challenge and suggest that overwhelming innate immune responses may trigger bronchiolitis obliterans formation in bacterial lung infections.     

Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response

Outer membrane vesicles (OMVs) derived from pathogenic Gram-negative bacteria are an important vehicle for delivery of effector molecules to host cells, but the production of OMVs from Klebsiella pneumoniae, an opportunistic pathogen of both nosocomial and community-acquired infections, and their role in bacterial pathogenesis have not yet been determined. In the present study, we examined the production of OMVs from K. pneumoniae and determined the induction of the innate immune response against K. pneumoniae OMVs. Klebsiella pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. Proteomic analysis revealed that 159 different proteins were associated with K. pneumoniae OMVs. Klebsiella pneumoniae OMVs did not inhibit cell growth or induce cell death. However, these vesicles induced expression of proinflammatory cytokine genes such as interleukin (IL)-1β and IL-8 in epithelial cells. An intratracheal challenge of K. pneumoniae OMVs in neutropenic mice resulted in severe lung pathology similar to K. pneumoniae infection. In conclusion, K. pneumoniae produces OMVs like other pathogenic Gram-negative bacteria and K. pneumoniae OMVs are a molecular complex that induces the innate immune response.     

Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells

Yersinia enterocolitica is a major cause of human yersiniosis, with enterocolitis being a typical manifestation. These bacteria can cross the intestinal mucosa, and invade eukaryotic cells by binding to host β₁ integrins, a process mediated by the bacterial effector protein invasin. This study examines the role of hypoxia on the internalization of Y. enterocolitica into intestinal epithelial cells, since the gastrointestinal tract has been shown to be physiologically deficient in oxygen levels (hypoxic), especially in cases of infection and inflammation. We show that hypoxic pre-incubation of Caco-2 cells resulted in significantly decreased bacterial internalization compared to cells grown under normoxia. This phenotype was absent after functionally blocking host β₁ integrins as well as upon infection with an invasin-deficient Y. enterocolitica strain. Furthermore, downstream phosphorylation of the focal adhesion kinase was also reduced under hypoxia after infection. In good correlation to these data, cells grown under hypoxia showed decreased protein levels of β₁ integrins at the apical cell surface whereas the total protein level of the hypoxia inducible factor (HIF-1) alpha was elevated. Furthermore, treatment of cells with the HIF-1 α stabilizer dimethyloxalylglycine (DMOG) also reduced invasion and decreased β₁ integrin protein levels compared to control cells, indicating a potential role for HIF-1α in this process. These results suggest that hypoxia decreases invasin-integrin-mediated internalization of Y. enterocolitica into intestinal epithelial cells by reducing cell surface localization of host β₁ integrins.     

Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections

A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K. pneumoniae. S. pneumoniae and S. pyogenes A produced by intranasal inoculation. Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections. Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S. cerevisiae cells increases resistance to infection. It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.     

Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections

Abstract A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K. pneumoniae, S. pneumoniae and S. pyogenes A produced by intranasal inoculation. Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections. Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S. cerevisiae cells increases resistance to infection. It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.     

Mast cell IL-6 improves survival from Klebsiella pneumonia and sepsis by enhancing neutrophil killing

The pleiotropic cytokine IL-6 has favorable and harmful effects on survival from bacterial infections. Although many innate immune cells produce IL-6, little is known about relevant sources in vivo and the nature of its contributions to host responses to severe bacterial infections. To examine these roles, we subjected mast cell-specific IL-6-deficient mice to the cecal ligation and puncture model of septic peritonitis, finding that survival in these mice is markedly worse than in controls. Following intranasal or i.p. inoculation with Klebsiella pneumoniae, IL-6 (-/-) mice are less likely to survive than wild-type controls and at the time of death have higher numbers of bacteria but not inflammatory cells in lungs and peritoneum. Similarly, mast cell-specific IL-6-deficient mice have diminished survival and higher numbers of K. pneumoniae following i.p. infection. Neutrophils lacking IL-6 have greater numbers of live intracellular K. pneumonia, suggesting impaired intracellular killing contributes to reduced clearance in IL-6(-/-) mice. These results establish that mast cell IL-6 is a critical mediator of survival following K. pneumoniae infection and sepsis and suggest that IL-6 protects from death by augmenting neutrophil killing of bacteria.     

Staphylococcus saprophyticus ATCC 15305 is internalized into human urinary bladder carcinoma cell line 5637

Invasion of bacteria into nonphagocytic host cells is an important pathogenicity factor for escaping the host defence system. Gram-positive organisms, for example Staphylococcus aureus and Listeria monocytogenes, are invasive in nonphagocytic cells, and this mechanism is discussed as an important part of the infection process. Uropathogenic Escherichia coli and Staphylococcus saprophyticus can cause acute and recurrent urinary tract infections as well as bloodstream infections. Staphylococcus saprophyticus shows strong adhesion to human urinary bladder carcinoma and Hep2 cells and expresses the 'Microbial Surface Components Recognizing Adhesive Matrix molecule' (MSCRAMM)-protein SdrI with collagen-binding activity. MSCRAMMs are responsible for adhesion and collagen binding in S. aureus and are discussed as an important pathogenicity factor for invasion. To investigate internalization in S. aureus, several fluorescence activated cell sorting (FACS) assays have been described recently. We used a previously described FACS assay, with slight modifications, in addition to an antibiotic protection assay and transmission electron microscopy to show that S. saprophyticus ATCC 15305 and the wild-type strain 7108 were internalized into the human urinary bladder carcinoma cell line 5637. The discovery of the internalization of S. saprophyticus may be an important step for understanding the pathogenicity of recurrent infections caused by this organism.     

Dynamin2- and endothelial nitric oxide synthase-regulated invasion of bladder epithelial cells by uropathogenic Escherichia coli

Invasion of bladder epithelial cells by uropathogenic Escherichia coli (UPEC) contributes to antibiotic-resistant and recurrent urinary tract infections (UTIs), but this process is incompletely understood. In this paper, we provide evidence that the large guanosine triphosphatase dynamin2 and its partner, endothelial nitric oxide (NO) synthase (NOS [eNOS]), mediate bacterial entry. Overexpression of dynamin2 or treatment with the NO donor S-nitrosothiols increases, whereas targeted reduction of endogenous dynamin2 or eNOS expression with ribonucleic acid interference impairs, bacterial invasion. Exposure of mouse bladder to small molecule NOS inhibitors abrogates infection of the uroepithelium by E. coli, and, concordantly, bacteria more efficiently invade uroepithelia isolated from wild-type compared with eNOS(-/-) mice. E. coli internalization promotes rapid phosphorylation of host cell eNOS and NO generation, and dynamin2 S-nitrosylation, a posttranslational modification required for the bacterial entry, also increases during E. coli invasion. These findings suggest that UPEC escape urinary flushing and immune cell surveillance by means of eNOS-dependent dynamin2 S-nitrosylation and invasion of host cells to cause recurrent UTIs.     

MyD88 is pivotal for the early inflammatory response and subsequent bacterial clearance and survival in a mouse model of Chlamydia pneumoniae pneumonia

Chlamydia pneumoniae is the causative agent of respiratory tract infections and a number of chronic diseases. Here we investigated the involvement of the common TLR adaptor molecule MyD88 in host responses to C. pneumoniae-induced pneumonia in mice. MyD88-deficient mice were severely impaired in their ability to mount an acute early inflammatory response toward C. pneumoniae. Although the bacterial burden in the lungs was comparable 5 days after infection, MyD88-deficient mice exhibited only minor signs of pneumonia and reduced expression of inflammatory mediators. MyD88-deficient mice were unable to up-regulate proinflammatory cytokines and chemokines, demonstrated delayed recruitment of CD8+ and CD4+ T cells to the lungs, and were unable to clear the pathogen from their lungs at day 14. At day 14 the MyD88-deficent mice developed a severe, chronic lung inflammation with elevated IL-1beta and IFN-gamma leading to increased mortality, whereas wild-type mice as well as TLR2- or TLR4-deficient mice recovered from acute pneumonia and did not show delayed bacterial clearance. Thus, MyD88 is essential to recognize C. pneumoniae infection and initiate a prompt and effective immune host response against this organism leading to clearance of bacteria from infected lungs.     

Chlamydia pneumoniae inhibits activated human T lymphocyte proliferation by the induction of apoptotic and pyroptotic pathways

Chlamydia pneumoniae is an omnipresent obligate intracellular bacterial pathogen that infects numerous host species. C. pneumoniae infections of humans are a common cause of community acquired pneumonia but have also been linked to chronic diseases such as atherosclerosis, Alzheimer's disease, and asthma. Persistent infection and immune avoidance are believed to play important roles in the pathophysiology of C. pneumoniae disease. We found that C. pneumoniae organisms inhibited activated but not nonactivated human T cell proliferation. Inhibition of proliferation was pathogen specific, heat sensitive, and multiplicity of infection dependent and required chlamydial entry but not de novo protein synthesis. Activated CD4(+) and CD8(+) T cells were equally sensitive to C. pneumoniae antiproliferative effectors. The C. pneumoniae antiproliferative effect was linked to T cell death associated with caspase 1, 8, 9, and IL-1β production, indicating that both apoptotic and pyroptotic cellular death pathways were activated after pathogen-T cell interactions. Collectively, these findings are consistent with the conclusion that C. pneumoniae could induce a local T cell immunosuppression and inflammatory response revealing a possible host-pathogen scenario that would support both persistence and inflammation.     

Identification of a glycosaminoglycan binding region of the alpha C protein that mediates entry of group B Streptococci into host cells

Group B Streptococcus (GBS) frequently colonizes the human gastrointestinal and gynecological tracts and less frequently causes deep tissue infections. The transition between colonization and infection depends upon the ability of the organism to cross epithelial barriers. The alpha C protein (ACP) on the surface of GBS contributes to this process. A virulence factor in mouse models of infection, and prototype for a family of Gram-positive bacterial surface proteins, ACP facilitates GBS entry into human cervical epithelial cells and movement across cell layers. ACP binds to host cell surface glycosaminoglycan (GAG). From crystallography, we have identified a cluster of basic residues (BR2) that is a putative GAG binding area in Domain 2, near the junction of the N-terminal domain of ACP and the first of a series of tandem amino acid repeats. D2-R, a protein construct including this region, binds to cells similarly to full-length ACP. We now demonstrate that the predicted charged BR2 residues confer GAG binding; site-directed mutagenesis of these residues (Arg(172), Arg(185), or Lys(196)) eliminates cell-binding activity of construct D2-R. In addition, we have constructed a GBS strain expressing a variant ACP with a charge-neutralizing substitution at residue 185. This strain enters host cells less effectively than does the wild-type strain and similarly to an ACP null mutant strain. The point mutant strain transcytoses similarly to the wild-type strain. These data indicate that GAG-binding activity underlies ACP-mediated cellular entry of GBS. GBS entry into host cells and transcytosis of host cells may occur by distinct mechanisms.     

Invasive phenotype and apoptosis induction of Plesiomonas shigelloides P-1 strain to Caco-2 cells

AIMS: The mechanism of the host cell invasion of Plesiomonas shigelloides and its capability to induce apoptosis were investigated. METHODS AND RESULTS: We performed a time course experiment on the bacterial adherence and invasion of the P. shigelloides P-1 strain into Caco-2 cells using an invasion assay and flow cytometry. The adherence of P. shigelloides to the Caco-2 cells was almost completed within 10 min after the infection. Thereafter, P. shigelloides starts internalization within the Caco-2 cells, which was completed within 60 min after the infection. Based on the invasion assay using nocodazole, cytochalasin D, and genistein, it became clear that the mechanism of the internalization depended on the signal transduction followed by the rearrangement of the cytoskeletal protein. Based on the DNA laddering and TUNEL methods, the cytotoxicity of the Caco-2 cells by the invasion of P. shigelloides occurred through the induction of apoptosis. CONCLUSIONS: This work demonstrated that the mechanism of invasion of P. shigelloides into Caco-2 cells and the invasion of P. shigelloides induces apoptotic cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the virulence factor, which may be important for understanding of the pathogenesis of P. shigelloides.     

The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.     

Ablation of leptin receptor-mediated ERK activation impairs host defense against Gram-negative pneumonia

The adipocyte-derived hormone leptin plays an important role in regulation of energy homeostasis and the innate immune response against bacterial infections. Leptin's actions are mediated by signaling events initiated by phosphorylation of tyrosine residues on the long form of the leptin receptor. We recently reported that disruption of leptin receptor-mediated STAT3 activation augmented host defense against pneumococcal pneumonia. In this report, we assessed leptin receptor-mediated ERK activation, a pathway that was ablated in the l/l mouse through a mutation of the tyrosine 985 residue in the leptin receptor, to determine its role in host defense against bacterial pneumonia in vivo and in alveolar macrophage (AM) antibacterial functions in vitro. l/l mice exhibited increased mortality and impaired pulmonary bacterial clearance after intratracheal challenge with Klebsiella pneumoniae. The synthesis of cysteinyl-leukotrienes was reduced and that of PGE(2) enhanced in AMs in vitro and the lungs of l/l mice after infection with K. pneumoniae in vivo. We also observed reduced phagocytosis and killing of K. pneumoniae in AMs from l/l mice that was associated with reduced reactive oxygen intermediate production in vitro. cAMP, known to suppress phagocytosis, bactericidal capacity, and reactive oxygen intermediate production, was also increased 2-fold in AMs from l/l mice. Pharmacologic blockade of PGE(2) synthesis reduced cAMP levels and overcame the defective phagocytosis and killing of bacteria in AMs from l/l mice in vitro. These results demonstrate that leptin receptor-mediated ERK activation plays an essential role in host defense against bacterial pneumonia and in leukocyte antibacterial effector functions.     

Cathelicidin-related antimicrobial peptide is required for effective lung mucosal immunity in Gram-negative bacterial pneumonia

Cathelicidins are a family of endogenous antimicrobial peptides that exert diverse immune functions, including both direct bacterial killing and immunomodulatory effects. In this study, we examined the contribution of the murine cathelicidin, cathelicidin-related antimicrobial peptide (CRAMP), to innate mucosal immunity in a mouse model of Gram-negative pneumonia. CRAMP expression is induced in the lung in response to infection with Klebsiella pneumoniae. Mice deficient in the gene encoding CRAMP (Cnlp(-/-)) demonstrate impaired lung bacterial clearance, increased bacterial dissemination, and reduced survival in response to intratracheal K. pneumoniae administration. Neutrophil influx into the alveolar space during K. pneumoniae infection was delayed early but increased by 48 h in CRAMP-deficient mice, which was associated with enhanced expression of inflammatory cytokines and increased lung injury. Bone marrow chimera experiments indicated that CRAMP derived from bone marrow cells rather than structural cells was responsible for antimicrobial effects in the lung. Additionally, CRAMP exerted bactericidal activity against K. pneumoniae in vitro. Similar defects in lung bacterial clearance and delayed early neutrophil influx were observed in CRAMP-deficient mice infected with Pseudomonas aeruginosa, although this did not result in increased bacterial dissemination, increased lung injury, or changes in lethality. Taken together, our findings demonstrate that CRAMP is an important contributor to effective host mucosal immunity in the lung in response to Gram-negative bacterial pneumonia.