Grace Soong

Staphylococcus aureus protein A induces airway epithelial inflammatory responses by activating TNFR1

Staphylococcus aureus is a major human pathogen that is associated with diverse types of local and systemic infection characterized by inflammation dominated by polymorphonuclear leukocytes. Staphylococci frequently cause pneumonia, and these clinical isolates often have increased expression of protein A, suggesting that this protein may have a role in virulence. Here we show that TNFR1, a receptor for tumor-necrosis factor-α (TNF-α) that is widely distributed on the airway epithelium, is a receptor for protein A. We also show that the protein A–TNFR1 signaling pathway has a central role in the pathogenesis of staphylococcal pneumonia.

TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

Toll-like receptors (TLRs) mediate host responses to bacterial gene products. As the airway epithelium is potentially exposed to many diverse inhaled bacteria, TLRs involved in defense of the airways must be broadly responsive, available at the exposed apical surface of the cells, and highly regulated to prevent activation following trivial encounters with bacteria. We demonstrate that TLR2 is enriched in caveolin-1-associated lipid raft microdomains presented on the apical surface of airway epithelial cells after bacterial infection. These receptor complexes include myeloid differentiation protein (MyD88), interleukin-1 receptor-activated kinase-1, and TNF receptor-associated factor 6. The signaling capabilities of TLR2 are amplified through its association with the asialoganglioside gangliotetraosylceramide (Gal beta 1,2GalNAc beta 1,4Gal beta 1,4Glc beta 1,1Cer), which has receptor function itself for many pulmonary pathogens. Ligation of either TLR2 or asialoGM1 by ligands with specificity for either receptor, by Pseudomonas aeruginosa, or by Staphylococcus aureus stimulates IL-8 production through activation of NF-kappa B, as mediated by TLR2 and MyD88. Thus, TLR2 in association with asialo-glycolipids presented within the context of lipid rafts provides a broadly responsive signaling complex at the apical surfaces of airway cells to initiate the host response to potential bacterial infection.

Bacterial neuraminidase facilitates mucosal infection by participating in biofilm production

Many respiratory pathogens, including Hemophilus influenzae, Streptococcus pneumoniae, and Pseudomonas aeruginosa, express neuraminidases that can cleave alpha2,3-linked sialic acids from glycoconjugates. As mucosal surfaces are heavily sialylated, neuraminidases have been thought to modify epithelial cells by exposing potential bacterial receptors. However, in contrast to neuraminidase produced by the influenza virus, a role for bacterial neuraminidase in pathogenesis has not yet been clearly established. We constructed a mutant of P. aeruginosa PAO1 by deleting the PA2794 neuraminidase locus (Delta2794) and tested its virulence and immunostimulatory capabilities in a mouse model of infection. Although fully virulent when introduced i.p., the Delta2794 mutant was unable to establish respiratory infection by i.n. inoculation. The inability to colonize the respiratory tract correlated with diminished production of biofilm, as assessed by scanning electron microscopy and in vitro assays. The importance of neuraminidase in biofilm production was further demonstrated by showing that viral neuraminidase inhibitors in clinical use blocked P. aeruginosa biofilm production in vitro as well. The P. aeruginosa neuraminidase has a key role in the initial stages of pulmonary infection by targeting bacterial glycoconjugates and contributing to the formation of biofilm. Inhibiting bacterial neuraminidases could provide a novel mechanism to prevent bacterial pneumonia.

The NanA Neuraminidase of Streptococcus pneumoniae Is Involved in Biofilm Formation

ABSTRACT Streptococcus pneumoniae remains a major cause of bacteremia, pneumonia, and otitis media despite vaccines and effective antibiotics. The neuraminidase of S. pneumoniae, which catalyzes the release of terminal sialic acid residues from glycoconjugates, is involved in host colonization in animal models of infection and may provide a novel target for preventing pneumococcal infection. We demonstrate that the S. pneumoniae neuraminidase (NanA) cleaves sialic acid and show that it is involved in biofilm formation, suggesting an additional role in pathogenesis, and that it shares this property with the neuraminidase of Pseudomonas aeruginosa even though we show that the two enzymes are phylogenetically divergent. Using an in vitro model of biofilm formation incorporating human airway epithelial cells, we demonstrate that small-molecule inhibitors of NanA block biofilm formation and may provide a novel target for preventative therapy. This work highlights the role played by the neuraminidase in pathogenesis and represents an important step in drug development for prevention of colonization of the respiratory tract by this important pathogen.

Staphylococcus aureus activates type I IFN signaling in mice and humans through the Xr repeated sequences of protein A.

The activation of type I IFN signaling is a major component of host defense against viral infection, but it is not typically associated with immune responses to extracellular bacterial pathogens. Using mouse and human airway epithelial cells, we have demonstrated that Staphylococcus aureus activates type I IFN signaling, which contributes to its virulence as a respiratory pathogen. This response was dependent on the expression of protein A and, more specifically, the Xr domain, a short sequence-repeat region encoded by DNA that consists of repeated 24-bp sequences that are the basis of an internationally used epidemiological typing scheme. Protein A was endocytosed by airway epithelial cells and subsequently induced IFN-beta expression, JAK-STAT signaling, and IL-6 production. Mice lacking IFN-alpha/beta receptor 1 (IFNAR-deficient mice), which are incapable of responding to type I IFNs, were substantially protected against lethal S. aureus pneumonia compared with wild-type control mice. The profound immunological consequences of IFN-beta signaling, particularly in the lung, may help to explain the conservation of multiple copies of the Xr domain of protein A in S. aureus strains and the importance of protein A as a virulence factor in the pathogenesis of staphylococcal pneumonia.

Streptococcus pneumoniae DNA Initiates Type I Interferon Signaling in the Respiratory Tract

ABSTRACT The mucosal epithelium is the initial target for respiratory pathogens of all types. While type I interferon (IFN) signaling is traditionally associated with antiviral immunity, we demonstrate that the extracellular bacterial pathogen Streptococcus pneumoniae activates the type I IFN cascade in airway epithelial and dendritic cells. This response is dependent upon the pore-forming toxin pneumolysin. Pneumococcal DNA activates IFN-β expression through a DAI/STING/TBK1/IRF3 cascade. Tlr4−/−, Myd88−/−, Trif−/−, and Nod2−/− mutant mice had no impairment of type I IFN signaling. Induction of type I IFN signaling contributes to the eradication of pneumococcal carriage, as IFN-α/β receptor null mice had significantly increased nasal colonization with S. pneumoniae compared with that of wild-type mice. These studies suggest that the type I IFN cascade is a central component of the mucosal response to airway bacterial pathogens and is responsive to bacterial pathogen-associated molecular patterns that are capable of accessing intracellular receptors. IMPORTANCE The bacterium Streptococcus pneumoniae is a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clear S. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen. The bacterium Streptococcus pneumoniae is a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clear S. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen.

Staphylococcus aureus agr and sarA functions are required for invasive infection but not inflammatory responses in the lung.

ABSTRACT Staphylococcus aureus strains lacking agr- and sarA-dependent gene products or specific MSCRAMM (microbial surface components recognizing adhesive matrix molecules) adhesins were compared for the ability to activate inflammatory responses in the lung. The mutants were evaluated for virulence in a mouse model of pneumonia and by quantifying their ability to stimulate interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in respiratory epithelial cells. In a neonatal mouse, only strains with intact agr and sarA loci were consistently associated with invasive, fatal pulmonary infection (P < 0.001) and sarA was specifically required to cause bacteremia (P < 0.001). The agr and/or sarA mutants were, nonetheless, fully capable of producing pneumonia and were as proficient as the wild-type strain in stimulating epithelial IL-8 expression, a polymorphonuclear leukocyte chemokine, in airway cells. In contrast, agr and especially sarA mutants induced less epithelial GM-CSF expression, and MSCRAMM mutants lacking fibronectin binding proteins or clumping factor A, a ligand for fibrinogen, were unable to stimulate epithelial GM-CSF production. The ability to induce IL-8 expression was independent of the adherence properties of intact bacteria, indicating that shed and/or secreted bacterial components activate epithelial responses. While conserved staphylococcal components such as peptidoglycan are sufficient to evoke inflammation and cause pneumonia, the agr and sarA loci of S. aureus are critical for the coordination of invasive infection of the lungs.

The Type III Toxins of Pseudomonas aeruginosa Disrupt Epithelial Barrier Function

The type III secreted toxins of Pseudomonas aeruginosa are important virulence factors associated with clinically important infection. However, their effects on bacterial invasion across mucosal surfaces have not been well characterized. One of the most commonly expressed toxins, ExoS, has two domains that are predicted to affect cytoskeletal integrity, including a GTPase-activating protein (GAP) domain, which targets Rho, a major regulator of actin polymerization; and an ADP-ribosylating domain that affects the ERM proteins, which link the plasma membrane to the actin cytoskeleton. The activities of these toxins, and ExoS specifically, on the permeability properties of polarized airway epithelial cells with intact tight junctions were examined. Strains expressing type III toxins altered the distribution of the tight junction proteins ZO-1 and occludin and were able to transmigrate across polarized airway epithelial monolayers, in contrast to DeltaSTY mutants. These effects on epithelial permeability were associated with the ADP-ribosylating domain of ExoS, as bacteria expressing plasmids lacking expression of the ExoS GAP activity nonetheless increased the permeation of fluorescent dextrans, as well as bacteria, across polarized airway epithelial cells. Treatment of epithelial cells with cytochalasin D depolymerized actin filaments and increased permeation across the monolayers but did not eliminate the differential effects of wild-type and toxin-negative mutants on the epithelial cells, suggesting that additional epithelial targets are involved. Confocal imaging studies demonstrated that ZO-1, occludin, and ezrin undergo substantial redistribution in human airway cells intoxicated by ExoS, -T, and -Y. These studies support the hypothesis that type III toxins enhance P. aeruginosas invasive capabilities by interacting with multiple eukaryotic cytoskeletal components.

Immunostimulatory Properties of the Emerging Pathogen Stenotrophomonas maltophilia

ABSTRACT Stenotrophomonas maltophilia is a multiple-antibiotic-resistant opportunistic pathogen that is being isolated with increasing frequency from patients with health-care-associated infections and especially from patients with cystic fibrosis (CF). While clinicians feel compelled to treat infections involving this organism, its potential for virulence is not well established. We evaluated the immunostimulatory properties and overall virulence of clinical isolates of S. maltophilia using the well-characterized opportunistic pathogen Pseudomonas aeruginosa PAO1 as a control. The properties of CF isolates were examined specifically to see if they have a common phenotype. The immunostimulatory properties of S. maltophilia were studied in vitro by stimulating airway epithelial and macrophage cell lines. A neonatal mouse model of pneumonia was used to determine the rates of pneumonia, bacteremia, and mortality, as well as the inflammatory response elicited by S. maltophilia infection. Respiratory and nonrespiratory S. maltophilia isolates were highly immunostimulatory and elicited significant interleukin-8 expression by airway epithelial cells, as well as tumor necrosis factor alpha (TNF-α) expression by macrophages. TNF-α signaling appears to be important in the pathogenesis of S. maltophilia infection as less than 20% of TNFR1 null mice (compared with 100% of wild-type mice) developed pneumonia and bacteremia following intranasal inoculation. The S. maltophilia isolates were weakly invasive, and low-level bacteremia with no mortality was observed. Despite the lack of invasiveness of S. maltophilia, the immunostimulatory properties of this organism and its induction of TNF-α expression specifically indicate that it is likely to contribute significantly to airway inflammation.

Bacterial Induction of TNF-α Converting Enzyme Expression and IL-6 Receptor α Shedding Regulates Airway Inflammatory Signaling

Airway epithelial cells have a major role in initiating inflammation in response to bacterial pathogens. Through the immediate induction of CXCL8 and cytokine expression, polymorphonuclear cells are mobilized and activated to eradicate the infecting organisms. However, the influx of polymorphonuclear cells and the effects of their toxic exoproducts impede respiratory function. We postulated that respiratory epithelial cells must also participate in the regulation of their own proinflammatory signaling. Both Staphylococcus aureus and Pseudomonas aeruginosa were found to potently activate IL-6 expression immediately upon contact with epithelial cells, and by 1 h induced TNF-α converting enzyme (TACE) transcription. By 4 h of bacterial exposure, TACE colocalized with IL-6Rα on the apical surface of airway cells, and by 24 h, soluble IL-6Rα accumulated in the cell culture supernatant. Epithelial IL-6 and soluble IL-6Rα were shown to participate in trans-signaling, interacting with membrane-associated gp130 to activate CCL-2 expression and inhibit additional CXCL8 production. Thus, bacteria are physiological activators of TACE expression, which provides a mechanism to regulate inflammatory signaling that is initiated by airway epithelial cells.