Uptake of Streptococcus pneumoniae by respiratory epithelial cells.

Although Streptococcus pneumoniae is the leading cause of community-acquired pneumonia in humans, the mechanism whereby the organism penetrates lung tissue is poorly understood. In the present study we have examined the capacity of pneumococci to penetrate A549 cells, a human lung alveolar carcinoma (type II pneumocyte) cell line. Not all clinical S. pneumoniae isolates initially tested were capable of penetration of the cells, as judged by resistance to extracellular antibiotics. The presence of a polysaccharide capsule also significantly reduced the capacity to both adhere to and penetrate A549 cells. Electron micrographs showed the presence of pneumococci enclosed within vacuoles of intact A549 cells, but bacteria were also seen free in the cytoplasm of damaged cells. Ongoing bacterial DNA, RNA, or protein synthesis was not essential for uptake of pneumococci by A549 cells, and uptake was not diminished by pretreatment of the pneumococci with trypsin. However, inhibition of A549 microfilament assembly with cytochalasin D abolished the phenomenon.     

Enhanced CXC chemokine responses of human colonic epithelial cells to locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli

There is increasing evidence that by facilitating translocation of Shiga toxin (Stx) across the intestinal epithelium and by transporting bound toxin to remote sites such as the renal endothelium, polymorphonuclear leukocytes (PMNs) play a key role in the pathogenesis of Shiga-toxigenic Escherichia coli (STEC) disease. Plasma levels of PMN-attracting CXC chemokines such as interleukin-8 (IL-8) also appear to correlate in humans with the severity of disease. Thus, the capacity of STEC strains to elicit CXC chemokine responses in intestinal epithelial cells may be a crucial step in pathogenesis. Accordingly, we attempted to determine which STEC factors are responsible for CXC chemokine induction in human colonic epithelial cells. Infection of Hct-8 cells with locus for enterocyte effacement (LEE)-negative STEC strains isolated from patients with severe STEC disease resulted in up-regulation of IL-8, macrophage inflammatory protein 2alpha (MIP-2alpha), MIP-2beta, and ENA-78 mRNA significantly higher and earlier than that elicited by several LEE-positive STEC strains, including the O157:H7 strain EDL933. Similarly, levels of IL-8 protein in LEE-negative STEC-infected Hct-8 culture supernatants were significantly higher than in LEE-positive STEC-infected culture supernatants. The difference in responses could not be attributed to the expression or nonexpression of LEE genes, the presence or absence of an STEC megaplasmid, or differences in O serogroups or in the type or amount of Stx produced. Interestingly, however, several of the LEE-negative STEC strains eliciting the strongest chemokine responses belonged to flagellar serotype H21. Incubation of Hct-8 cells with isolated H21 flagellin elicited IL-8 and MIP-2alpha responses similar to those seen in the presence of the most potent LEE-negative STEC strains. Deletion of the fliC gene, but not the stx(2) gene, largely abolished the capacity of O113:H21 LEE-negative STEC strain 98NK2 to elicit IL-8 and MIP-2alpha responses in Hct-8 cells. Taken together, these data suggest that although Stx is capable of inducing CXC chemokine responses, the elevated responses seen in cells infected with certain STEC strains are largely attributable to the production of flagellin.     

A sandwich adhesion on Streptococcus pneumoniae attaching to human oropharyngeal epithelial cells in vitro

Streptococcus pneumoniae attach to human pharyngeal epithelial cells through the specific interaction of bacterial surface adhesins with glycoconjugate receptors. The present study defines the adhesin as a molecule bridging between an anchoring site in the bacterial cell wall and the epithelial cell receptor. The nature of the adhesin was defined in three ways: First, the attachment of whole bacteria was reduced by trypsin, periodate and heat. Second, heat treatment of whole bacteria was shown to release material, which was able to reconstitute the adherence. The heat extract bound to epithelial cells, as shown by fluorescence labelling, and agglutinated latex beads covalently coupled with receptor oligosaccharide. Active material could be extracted by heat from both high and low adhering strains, but could reconstitute only attaching strains. Third, the bacterial component binding the adhesin was localized to protoplasts and cell wall fractions obtained by mechanical or deoxycholate induced lysis of pneumococci. Isolated pneumococcal surface components, which did not inhibit attachment, included peptidoglycan, C polysaccharide, Forssmann antigen, capsular polysaccharide and a phenol extract produced in analogy to streptococcal lipoteichoic acid. The procedure used to extract the adhesin was previously used to prepare the competence factor. The competence deficient mutant RA7- attached poorly compared to the competent R6 parent. The possible relatedness of attachment to competence is discussed.     

Immune responses to novel pneumococcal proteins pneumolysin,PspA, PsaA,and CbpA in adenoidal B cells from children

Studies of mice suggest that pneumococcal proteins, including PspA, pneumolysin, PsaA, and CbpA, are promising vaccine candidates. To determine whether these proteins are good mucosal immunogens in humans, adenoidal lymphocytes from 20 children who had adenoidectomies were isolated and tested by ELISpot for antigen-specific antibody-secreting cells (ASCs). Cells were also cultured for 7 days in the presence of a concentrated culture supernatant (CCS) from a type 14 strain of pneumococcus which contained secreted pneumococcal proteins, including PspA, pneumolysin, PsaA, and CbpA, and then tested by ELISpot. ELISpot assays done on freshly isolated cells detected ASCs to all four antigens in most children studied. However, there were differences both between antigens and between isotypes. The densities of immunoglobulin G (IgG) ASCs against both PsaA and CbpA were significantly higher than those of ASCs for PspA and PdB (pneumolysin toxoid B) (P < 0.001). For all antigens, the numbers of IgA ASCs tended to be lower than those of both IgG and IgM ASCs. The numbers of anti-CbpA and -PsaA IgA ASCs were higher than those of anti-PdB IgA ASCs (P < 0.01). Concentrations of IgA antibodies to PspA and PsaA in saliva correlated with the numbers of IgA ASCs to PspA and PsaA in freshly isolated adenoidal cells, but no such correlation was found between salivary IgG antibody concentrations and IgG ASCs to the four antigens in adenoidal cells. In cultured cells, anti-PspA, -PsaA, and -CbpA IgG ASCs proliferated significantly, but only two of eight samples showed >2-fold increases in anti-CbpA and -PspA IgA ASCs after CCS stimulation. The results suggest that CbpA, PsaA, and PspA may be good upper respiratory mucosal antigens in children. Adenoids may be important inductive sites for memory IgG responses and important sources of salivary IgA. Some protein antigens may also prime for mucosal IgA memory. These data support the effort to explore mucosal immunization against pneumococcal infection.     

The role of CXC chemokine receptor 2 in Pseudomonas aeruginosa corneal infection

Pseudomonas is one of the leading causes of contact lens-related microbial keratitis. Despite the use of antibiotics, the host inflammatory response continues to cause damage to the cornea, which may lead to blindness. CXCR2-binding chemokines have been implicated in the pathogenesis of Pseudomonas keratitis, and the exact role of this receptor remains to be elucidated. Corneas of CXCR2 knockout and wild-type mice (Cmkar 2-/- and Cmkar 2+/+) were scratched, and 2x10(6) CFU/mL Pseudomonas 6294 or 6206 was added to corneas. Twenty-four hours postinfection, mice were killed, and eyes were harvested for enumeration of bacteria, myeloperoxidase (MPO) levels, and inflammatory mediators. Cmkar 2-/- had 20- to 100-fold more bacteria than Cmkar 2+/+ mice. There were no differences in MPO levels between gene knockout and Cmkar 2+/+ mice. Histology revealed PMN were restricted to the limbal area. Levels of CXCR2 chemokines (keratinocyte-derived chemokine and MIP-2) were elevated significantly in gene knockout mice. A lack of CXCR2 leads to an inability to control bacterial numbers as a result of the inability of PMN to reach the site of infection in the avascular cornea. These results imply that CXCR2 is critical to the extravasation of neutrophils into the avascular cornea.     

Adherence of nontypeable Streptococcus pneumoniae to human conjunctival epithelial cells

Conjunctivitis outbreaks have occurred in the US in which nontypeable (NT) Streptococcus pneumoniae (Pnc) strains have been identified as the etiologic agent; however, the pathogenesis of Pnc conjunctivitis has not been extensively evaluated. Here we assessed the adhesive and invasive properties of 13 NT US conjunctivitis outbreak strains (cPnc) using an immortalized human conjunctival epithelial cell (HCjE) line expressing high or low levels of mucin as a surrogate for in vivo ocular surface events. Studies reveal differential binding efficiencies (up to 18-fold) among cPnc strains to HCjE cells and reduced or little adherence efficiency to high mucin-expressing (HME-HCjE). Additionally, in the presence of exogenous mucin there is considerable inhibition (20% to approximately 100%) of bacterial binding to the HCjE cells. Invasion assays suggest that the cPnc are internalized in HCjE, and less in HME-HCjE cells. Microarray analysis of cPnc isolates revealed an up-regulation of Pnc neuraminidases, and treatment of HME-HCjE cells with exogenous neuraminidase resulted in a 2-13-fold enhancement in cPnc binding. The results indicate that mucin acts as a protective barrier in vitro and that neuraminidases, which can degrade mucin, may be contributing factors leading to bacterial adherence, a first step in the pathogenesis of this transmissible infection.     

Regions of PspA/EF3296 best able to elicit protection against Streptococcus pneumoniae in a murine infection model

Pneumococcal surface protein A (PspA) can elicit protection against Streptococcus pneumoniae in mouse infection models. PspA is classified by serology and amino acid sequence into two major families that are divided by sequence into five clades. The most variable portion of the molecule is the alpha-helical domain, which comprises the N-terminal half of PspA. Prior studies of a family 1 PspA protein observed that protective antibodies are reactive with epitopes in the alpha-helical domain and that most cross-protective epitopes mapped to the 108 most C-terminal amino acids of the alpha-helical region. In these studies, we have used six overlapping recombinant fragments of family 2, clade 3 PspA/EF3296 to map the protection-eliciting regions of its alpha-helical domain. The three fragments, which included the 104 most C-terminal amino acids of the alpha-helical domain (314 to 418), could each elicit protection against EF3296. A fragment comprising amino acids 75 to 305 failed to elicit significant protection. A fragment containing amino acids 1 to 115 elicited protection against EF3296 in BALB/c mice but not in CBA/N mice. All three fragments containing amino acids 314 to 418 were able to elicit cross-protection against pneumococci expressing PspA proteins of clades 2, 3, 4, and 5. Cross-protection elicited by these three fragments was easier to demonstrate in CBA/N mice than in BALB/c mice. The 1-to-115 fragment, however, elicited some cross-protection against clades 2 and 4 in BALB/c mice but not in CBA/N mice. These studies provide support for the importance of the C-terminal 104 and N-terminal 115 amino acids of the alpha-helical region of PspA in the elicitation of cross-protection.     

Development of antibodies to PspA families 1 and 2 in children after exposure to Streptococcus pneumoniae

Pneumococcal surface protein A (PspA) is an important virulence factor of Streptococcus pneumoniae. PspA exists as two major families, which include variable but serologically cross-reactive proteins. Previous studies with a family 1 PspA antigen suggested that children develop low concentrations of anti-PspA after pneumococcal carriage or infection. In this study, antibody to PspA families 1 and 2 was measured by an enzyme immunoassay of the serum and saliva of children with a history of culture-proven pneumococcal colonization and/or acute otitis media and in the serum and saliva of adults. The PspA families of the pneumococcal strains isolated from children were determined. The majority of the children had high serum and salivary anti-PspA concentrations to the PspA family they had encountered and low concentrations to the other, whereas adults had high antibody concentrations to both PspA families, both in serum and in saliva. The results suggest that children have a relatively family-specific antibody response to the PspA family they have been exposed to and that any PspA vaccine for children should contain members of both major PspA families.     

Immune responses to specific antigens of Streptococcus pneumoniae and Moraxella catarrhalis in the respiratory tract

Streptococcus pneumoniae and Moraxella catarrhalis are two common respiratory pathogens, colonizing as many as 54 and 72% of children, respectively, by 1 year of age. The immune responses to surface protein A of S. pneumoniae (PspA) and the high-molecular-weight outer membrane protein of M. catarrhalis (UspA) in the sera of various age groups in the general population and in the nasopharynges of 30 children monitored from birth through 1 year of age were evaluated. Immunoglobulin G (IgG) was the dominant serum antibody to PspA and UspA. Whereas the serum antibody response to PspA peaked in childhood, the antibody response to UspA peaked in adulthood. In the first 2 years of life, comparable amounts of IgM and IgG antibodies to both proteins were observed. In older persons, IgG antibodies to both antigens predominated over IgM antibodies. The levels of IgA antibody to these antigens in serum remained low during the first 2 years of life. The levels of IgM antibody to the two antigens in serum exceeded the levels of IgA antibody to the same two antigens throughout life. Although IgA was the dominant antibody to PspA and UspA in airway secretions, it was detected in a minority of the children (3 of 15 for PspA and 0 of 15 for UspA). Even the majority of the children previously colonized with these pathogens lacked antibody to them in their secretions.     

Effect of Streptococcus pneumoniae on human respiratory epithelium in vitro.

A total of 11 of 15 Streptococcus pneumoniae culture filtrates and all five bacterial autolysates produced by cell death in the stationary phase caused slowed ciliary beating and disruption of the surface integrity of human respiratory epithelium in organ culture. This effect was inhibited by cholesterol and was heat labile and reduced by standing at room temperature but was stable at -40 degrees C. The activity was detected at the late stationary phase of culture and was associated with the presence of hemolytic activity. Gel filtration of a concentrated culture filtrate and autolysate both yielded a single fraction of approximately 50 kilodaltons which slowed ciliary beating and were the only fractions with hemolytic activity. Rabbit antiserum to pneumolysin, a sulfhydryl-activated hemolytic cytotoxin released by S. pneumoniae during autolysis, neutralized the effect of the culture filtrate on respiratory epithelium. Both native and recombinant pneumolysin caused ciliary slowing and epithelial disruption. Electron microscopy showed a toxic effect of pneumolysin on epithelial cells: cytoplasmic blebs, mitochondrial swelling, cellular extrusion, and cell death, but no change in ciliary ultrastructure. Recombinant pneumolysin (10 micrograms/ml) caused ciliary slowing in the absence of changes in cell ultrastructure. Release of pneumolysin in the respiratory tract during infection may perturb host defenses, allowing bacterial proliferation and spread.     

Relationship between surface accessibility for PpmA, PsaA, and PspA and antibody-mediated immunity to systemic infection by Streptococcus pneumoniae

Antibodies to capsular polysaccharide (PS) are protective against systemic infection by Streptococcus pneumoniae, but the large number of pneumococcal serogroups and the age-related immunogenicity of pure PS limit the utility of PS-based vaccines. In contrast, cell wall-associated proteins from different capsular serotypes can be cross-reactive and immunogenic in all age groups. Therefore, we evaluated three pneumococcal proteins with respect to relative accessibility to antibody, in the context of intact pneumococci, and their ability to elicit protection against systemic infection by encapsulated S. pneumoniae. Sequences encoding pneumococcal surface adhesin A (PsaA), putative protease maturation protein A (PpmA), and the N-terminal region of pneumococcal surface protein A (PspA) from S. pneumoniae strain A66.1 were cloned and expressed in Escherichia coli. The presence of genes encoding PsaA, PpmA, and PspA in 11 clinical isolates was examined by PCR, and the expression of these proteins by each strain was examined by Western blotting with antisera raised to the respective recombinant proteins. We used flow cytometry to demonstrate that PspA was readily detectable on the surface of the pneumococcal strains analyzed, whereas PsaA and PpmA were not. Consistent with these observations, mice with passively or actively acquired antibodies to PspA or type 3 PS were equivalently protected from homologous systemic challenge with type 3 pneumococci, whereas mice with passively or actively acquired antibodies to PsaA or PpmA were not effectively protected. These experiments support the hypothesis that the extent of protection against systemic pneumococcal infection is influenced by target antigen accessibility to circulating host antibodies.     

Adenovirus infection enhances in vitro adherence of Streptococcus pneumoniae.

Viruses are thought to facilitate bacterial infections of the respiratory tract, but the mechanisms are poorly understood. The present study analyzed the effect of adenovirus on bacterial adherence to human respiratory tract epithelial cells. The human lung carcinoma cell line A549 was infected with adenovirus of types 1, 2, 3, 4, 5, and 9. At a multiplicity of infection of 75 particles per cell, cytopathic effects occurred in 75 to 100% of the cells within 48 h. The virus-infected cells were harvested at various times after infection and analyzed for the ability to bind strains of Haemophilus influenzae and Streptococcus pneumoniae. Adenovirus (types 1, 2, 3, and 5) commonly causing respiratory tract infections increased the binding of adherent S. pneumoniae strains to the cells. This effect was not seen for other adenovirus types. Adenovirus infection did not change the adherence of cells of poorly adhering strains of S. pneumoniae or H. influenzae. The increase in adherence of S. pneumoniae could be inhibited by the DNA synthesis inhibitor cytosine arabinofuranoside, which is known to block the late phase of the adenovirus infection. When electron microscopy was used, there was no evidence that virus particles bound directly to bacteria. Adherence was not affected by pretreatment of the cells with virus particles or viral proteins. This suggested that adenovirus infection upregulated receptors for S. pneumoniae. The increased attachment may be one mechanism by which viruses precondition the respiratory mucosa for bacterial infection.     

S-carboxymethylcysteine inhibits the attachment of Streptococcus pneumoniae to human pharyngeal epithelial cells

Streptococcus pneumoniae causes respiratory and other invasive infections. Increased resistance of this bacterium to antibiotics necessitates new approaches to the treatment of infections. Attachment of bacteria to human pharyngeal epithelial cells is the initial step in the pathogenesis of infection and S-carboxymethylcysteine (S-CMC) can modulate the attachment of Moraxella catarrhalis and nontypable Haemophilus influenzae to epithelial cells. Unlike these two, S. pneumoniae is gram-positive and has a well-defined capsule. Here we examined the effects of S-CMC on the attachment and detachment of S. pneumoniae to human pharyngeal epithelial cells in vitro. Treatment of these cells with S-CMC significantly reduced the number of attached S. pneumoniae. S-CMC also resulted in a significant increase in the detachment of already attached S. pneumoniae to epithelial cells. In addition, treatment of S. pneumoniae with S-CMC significantly reduced their ability to attach to epithelial cells, but not the number of viable bacteria. Our study shows that S-CMC modulates the attachment of S. pneumoniae to human pharyngeal epithelial cells by acting both on cells and bacteria.     

PspA protects Streptococcus pneumoniae from killing by apolactoferrin, and antibody to PspA enhances killing of pneumococci by apolactoferrin [corrected

Lactoferrin is an important component of innate immunity through its sequestration of iron, bactericidal activity, and immune modulatory activity. Apolactoferrin (ALF) is the iron-depleted form of lactoferrin and is bactericidal against pneumococci and several other species of bacteria. We observed that lactoferricin (LFN), an 11-amino-acid peptide from the N terminus of lactoferrin, is bactericidal for Streptococcus pneumoniae. Strains of S. pneumoniae varied in their susceptibility to ALF. Lactoferrin is bound to the pneumococcal surface by pneumococcal surface protein A (PspA). Using mutant PspA(-) pneumococci of four different strains, we observed that PspA offers significant protection against killing by ALF. Knockout mutations in genes for two other choline-binding proteins (PspC and PcpA) did not affect killing by ALF. PspA did not have to be attached to the bacterial surface to inhibit killing, because the soluble recombinant N-terminal half of PspA could prevent killing by both ALF and LFN. An 11-amino-acid fragment of PspA was also able to reduce the killing by LFN. Antibody to PspA enhanced killing by lactoferrin. These findings suggested that the binding of ALF to PspA probably blocks the active site(s) of ALF that is responsible for killing.     

Morphology of adenovirus type-3 infection of human respiratory epithelial cells in vitro

The adenovirus is a non-enveloped DNA virus which may lead to severe diseases of the respiratory tract. In order to study the influence of virus infection on primary cultured peribronchial submucosal gland cells, we performed in vitro infection with human adenovirus type 3. Peribronchial submucosal glands are the main source of tracheobronchial mucus and, therefore, play a major pathophysiological role in common pulmonary diseases such as bronchial asthma, chronic obstructive pulmonary disease and cystic fibrosis. The success of infection was verified by means of immunofluorescence and transmission electron microscopy. Infection follows a certain timetable with a climax of paracristalline intranuclear virus inclusions after 48 h of infection. Virus particles could be detected in the nucleus as well as in peripheral and perinuclear cytoplasmatic vacuoles. The release of virus capsids from the nucleus could be visualized using transmission electron microscopy and immunofluorescence with antibodies against hexon proteins. Two different kinds of mechanisms of transition of newly synthesized virus capsids from the nucleus into the cytoplasm could be identified. Due to an increasing cytopathic effect, viruses spread from cytoplasm after longer terms of infection. Cytopathic effects and cytoskeleton aspects under this virus infection could be characterized using immunofluorescence with several monoclonal antibodies against different cytokeratins.     

In vitro models of acute and long-term continuous infection of human respiratory epithelial cells with Chlamydophila pneumoniae have opposing effects on host cell apoptosis

Persistent infection with the obligate intracellular pathogen Chlamydophila pneumoniae has been implicated in the pathogenesis of many chronic diseases, but its mechanism remains unclear. Many pathogens have been found to modulate cellular apoptosis in order to survive and multiply. Chlamydial species were shown to both induce and inhibit host cell apoptosis depending on the experimental conditions. We utilized in vitro models of acute and long-term continuous (LTC) infection with the same cell line (HEp-2) and chlamydial isolate (TW-183) used in both models. Host cell apoptosis in infected and uninfected cells was assessed by fluorescence microscopy and flow cytometry. While acute infection induced apoptosis 72 h post-infection, LTC-infected cells had low rates of apoptosis and showed resistance to different exogenous inducers of apoptosis (sorbitol, serum withdrawal, hydrogen peroxide) when compared to uninfected cells. Chronicity of infection appears to be a critical factor in the modulation of host cell apoptosis by C. pneumoniae. Induction of apoptosis may help to propagate the infection, while inhibition of apoptosis could help protect the organism in chronic infection.