E-cadherin is a receptor for the common protein pneumococcal surface adhesin A (PsaA) of Streptococcus pneumoniae

Streptococcus pneumoniae (Pnc) binds to nasopharyngeal (NP) epithelial cells in the first steps of nasopharyngeal carriage and colonization through bacterial adhesins. The pneumococcal surface adhesin A (PsaA) has previously been reported to play a significant role in pneumococcal adherence and colonization. Identification of a receptor for PsaA on human epithelium will aid in understanding the pathogenesis of this bacterium. Using recombinant PsaA covalently bound to fluorescent spheres (fluospheres), we show PsaA binds to NP cells through interaction with the human cellular receptor, E-cadherin. SDS-PAGE silver stain analysis demonstrates binding of PsaA to E-cadherin. Recombinant human E-cadherin binds to and blocks PsaA-coated fluospheres and whole transparent bacteria from adhering to NP cells, but does not block a Pnc PsaA(-) mutant. Recombinant E-selectin and human alpha(5)beta(1) integrin did not bind to or block PsaA-coated fluosphere adherence to NP cells. Likewise, if NP cells were preincubated with anti-E-cadherin antibody, there was a significant decrease (46%, P=0.05) in PsaA-coated fluosphere adherence to the cells. Additionally, when using E-cadherin transfected cells, we observed PsaA-coated fluospheres bind more efficiently to cells which express E-cadherin. This work identifies E-cadherin as a receptor on human epithelial cells for the pneumococcal surface adhesin, PsaA.     

Toll-like receptors 2 and 4 do not contribute to clearance of Chlamydophila pneumoniae in mice, but are necessary for the release of monokines

Activation of immune cells by Chlamydophila pneumoniae in vitro has been shown to be toll-like receptor (TLR2)-dependent, but TLR4 is also involved to a minor extent. To investigate the role of TLR2 and TLR4 in vivo, a murine model of C. pneumoniae infection was established. Mice were infected intranasally with a low inoculum of 106 C. pneumoniae elementary bodies (EB) and spreading of bacteria was monitored by real-time PCR. The bronchoalveolar lavage (BAL) showed maximal bacterial load on the day of infection and the lung 2 days later. By day 95, C. pneumoniae were eradicated completely. In serum, anti-C. pneumoniae IgG became detectable on day 18 by microimmunofluorescence test. The course of infection was mild with no apparent symptoms, lack of acute phase response and no induction of tumor necrosis factor-alpha and interleukin-6 in BAL, lung supernatants or blood. Infection of TLR2-/- and C3H/HeJ mice revealed no differences in clearance of bacteria and serological responses compared to wild-type controls, even if a dose of 10(7) EB was used. Intracellular replication of C. pneumoniae in the lungs was proven by the efficacy of antibiotic treatment. These findings indicate that in vivo TLR2 and TLR4 are not important for the development of antibodies and elimination of C. pneumoniae.     

Nitric oxide exerts distinct effects in local and systemic infections with Streptococcus pneumoniae

Nitric oxide (NO) is known to be involved in the immune response against a range of organisms. Little is known about the effects of nitric oxide in pneumococcal infections. We have now investigated the role of nitric oxide in local and systemic infections caused by Streptococcus pneumoniae in NOS2 deficient mice. Although a deficiency in NO does not affect survival of mice during pneumococcal pneumonia, NO does control pneumococcal viability within the lung airways and tissue. Bronchoalveolar lavage fluid (BALF) from NOS2-deficient mice contained significantly elevated TNF activity, IFNgamma and total protein during mid/late infection. Incubation of S. pneumoniae with the NO donor SNAP revealed a direct anti-pneumococcal effect for NO in vitro. Deficiency in NOS2 did not affect bacteraemia following intranasal infection. In contrast NOS2-deficient mice were significantly less susceptible to intravenous infection with S. pneumoniae than were wild type mice and were able to control pneumococcal viability within the bloodstream. Our results indicate that NO is required within the lungs for anti-bacterial activity during the pneumococcal pneumonia but during Gram-positive bacteraemia NO is associated with increased bacterial loads and reduced survival.     

Exposure of Thomsen-Friedenreich antigen in Streptococcus pneumoniae infection is dependent on pneumococcal neuraminidase A

Pneumococcal hemolytic uremic syndrome is recognized in a small portion of otherwise healthy children who have or have recently had Streptococcus pneumoniae infections, including severe pneumonia, meningitis, and bacteremia. As in other types of hemolytic uremic syndrome (HUS), pneumococcal HUS is characterized by microangiopathic hemolytic anemia, and thrombocytopenia, usually with extensive kidney damage. Although not demonstrated in vivo, the pathogenesis of pneumococcal HUS has been attributed to the action pneumococcal neuraminidase exposing the usually cryptic Thomsen-Friedenreich antigen (T-antigen) on red blood cells (RBC), and kidney glomeruli. We evaluated the effect of pneumococcal infection on desialylation of RBC and glomeruli during pneumococcal infections in mice. Following intravenous infection with capsular type 19F pneumococci, CFU levels exceeding 1000 CFU/mL blood by the third day were significantly more likely to result in exposed T-antigen on RBC than lower levels of bacteremia. In a pneumonia model, significantly more T-antigen was exposed on RBC in mice treated with penicillin than in those receiving mock treatment. Utilizing mutant pneumococci, we demonstrated that neuraminidase A but not neuraminidase B was necessary for exposure of T-antigen on RBC in vivo. Thus, pneumococcal neuraminidase A is necessary for the exposure of T-antigen in vivo and treatment with penicillin increases this effect. Interestingly, NanA(-) pneumococci were found in the blood in higher numbers and caused more deaths than wild type, NanB(-), or the NanA(-)/NanB(-) pneumococci.     

DC-SIGN specifically recognizes Streptococcus pneumoniae serotypes 3 and 14

The Gram-positive bacterium Streptococcus pneumoniae is the leading causative pathogen in community-acquired pneumonia. The ever-increasing frequency of antibiotic-resistant S. pneumoniae strains severely hampers effective treatments. Thus, a better understanding of the mechanisms involved in the pathogenesis of pneumococcal disease is needed; in particular, of the initial interactions that take place between the host and the bacterium. Recognition of pathogens by dendritic cells is one of the most crucial steps in the induction of an immune response. For efficient pathogen recognition, dendritic cells express various kinds of receptors, including the DC-specific C-type lectin DC-SIGN. Pathogens such as Mycobacterium tuberculosis and HIV target DC-SIGN to escape immunity. Here the in vitro binding of DC-SIGN with S. pneumoniae was investigated. DC-SIGN specifically interacts with S. pneumoniae serotype 3 and 14 in contrast to other serotypes such as 19F. While the data described here suggest that DC-SIGN interacts with S. pneumoniae serotype 14 through a ligand expressed by the capsular polysaccharide, the binding to S. pneumoniae serotype 3 appears to depend on an as yet unidentified ligand. Despite the binding capacity of the capsular polysaccharide of S. pneumoniae 14 to DC-SIGN, no immunomodulatory effects on the dendritic cells were observed. The immunological consequences of the serotype-specific capacity to interact with DC-SIGN should be further explored and might result in new insights in the development of new and more potent vaccines.     

Role of Toll-like receptor 2 in innate resistance to Group B Streptococcus

The Gram-positive bacterium Group B Streptococcus (GBS) is an important cause of serious neonatal and adult infections. Toll-like receptor 2 (TLR2) recognizes components of the cell wall of Gram-positive bacteria and is critical for defense against certain invasive pathogens. In GBS, penicillin-binding protein 1a (PBP1a), encoded by ponA, is required for virulence. PBPs participate in cell wall synthesis and in previous studies; the absence of PBP1a was shown to result in subtle changes in the cell wall ultrastructure. Here, we examine the role of TLR2 in defense against GBS infection and the impact of mutation of ponA on TLR2-mediated host responses. We demonstrate TLR2-recognition of both wild-type (WT) GBS and the ponA mutant in vitro. TLR2(-/-) mice were significantly more susceptible than WT mice to infection with either strain of GBS, indicating a crucial role for TLR2 in defense against GBS. Additionally, the ponA mutant was severely attenuated for virulence in both strains of mice. The mutation in ponA did not affect cytokine expression by WT or TLR2(-/-) mice. These data indicate that TLR2 is required for host defense against GBS and this response is unaffected by the absence of PBP1a and the resultant changes in cell wall ultrastructure.     

Mosaic genes and mosaic chromosomes-genomic variation in Streptococcus pneumoniae

The genome sequences of two strains of Streptococcus pneumoniae, one of the major human pathogens, are currently available: that of the nonencapsulated laboratory strain R6, the origin of which dates back to the early 20th century, and of the serotype 4 TIGR strain isolated recently. The two genomes are not only different in size (2 versus 2.16 Mb) but differ also by approximately 10% of their genes, many of which being organized in large clusters. Their strain-specific genes and gene clusters are described here. The R6 genome contains 69 kb organized in six large regions that are absent from the TIGR strain, which in turn contains an extra 157kb in twelve clusters compared to R6. In addition, the TIGR strain contains 13 clusters of 4 kb and larger that are not shared by a variety of genetically different S. pneumoniae strains. Many regions bear signs of gene transfer events such as the presence of insertion sequences, transposable elements, and putative site-specific integrases/recombinases. Three strain-specific regions are devoted to genes encoding proteins with the cell wall anchor motif LPXTG which are important for the interaction with host cells and appear to be highly variable, similar to cell wall-associated choline-binding proteins.     

The contribution of efflux pumps to quinolone resistance in Streptococcus pneumoniae clinical isolates

Active efflux has been involved in fluoroquinolone resistance in Streptococcus pneumoniae. While only one efflux machinery has been well described (PmrA), the eventual existence of other pumps than PmrA has been suggested. The actual role of active quinolone efflux in producing ciprofloxacin resistance was examined by means of several methods. Strains HUB 2375 and HUB 3073 are clinical isolates resistant to fluoroquinolones. Since no mutations in the quinolone resistance-determining regions of these strains were found, resistance to fluoroquinolones should be attributed only to efflux. Strain R6 was transformed by DNA from both HUB 2375 and HUB 3073. Selected transformants were chosen for knockout of pmrA genes by polymerase chain reaction ligation mutagenesis in order to study the actual role and the degree of involvement of PmrA in determining ciprofloxacin resistance. Results strongly supported the hypothesis that efflux pumps other than PmrA are involved in fluoroquinolone resistance in clinical strains of pneumococci.     

The lack of Pneumococcal surface protein C (PspC) increases the susceptibility of Streptococcus pneumoniae to the killing by microglia

Microglial cells, the resident phagocytes in the brain, share many phenotypical and functional characteristics with peripheral macrophages, suggesting that they may participate in an innate immune response against microorganisms invading the central nervous system (CNS). In this study, we demonstrate that the microglial cells constitutively exhibit antibacterial activity in vitro against Streptococcus pneumoniae. By using a Pneumococcal surface protein C (PspC)-deleted strain and its wild-type counterpart, we found that the extent of such an activity is significantly influenced by the presence of a PspC molecule on the bacterial surface. The PspC– mutant FP20 is indeed more susceptible than the PspC+ strain HB565 to microglial killing. Interestingly, this phenomenon is observed when using a medium supplemented with heat-inactivated foetal bovine serum (FBS). Electron microscopy studies indicate that the microglial cells interact more efficiently with PspC– than with PspC+ pneumococci. Moreover, upon infection with the PspC– mutant, microglial cells produce levels of TNF-, MIP-2, IL-10 and nitric oxide, significantly higher than those observed with PspC+ bacteria. These findings indicate that the lack of PspC significantly enhances the susceptibility of S. pneumoniae to both bactericidal activity and secretory response by the microglial cells, suggesting that this molecule may play an important role in the invasion of CNS by pneumococcus.     

Fatal necrotizing fasciitis due to Streptococcus pneumoniae: a case report

Necrotizing fasciitis is known to be a highly lethal infection of deep-seated subcutaneous tissue and superficial fascia. Reports of necrotizing fasciitis due to Streptococcus pneumoniae are exceedingly rare. We report a case of necrotizing fasciitis in a 62-yr-old man with liver cirrhosis and diabetes mellitus. He presented with painful swelling of left leg and right hand. On the day of admission, compartment syndrome was aggravated and the patient underwent surgical exploration. Intra-operative findings revealed necrotizing fasciitis and cultures of two blood samples and wound aspirates showed S. pneumoniae. The patient died despite debridement and proper antimicrobial treatment. To the best of our knowledge, this is the first case of fatal necrotizing fasciitis with meningitis reported in Korea. We also review and discuss the literature on pneumococcal necrotizing fasciitis.     

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.     

Macrolide use identified as risk factor for macrolide-resistant Streptococcus pneumoniae in a 17-center case-control study

The objective of the case-control study presented here was to examine the risk factors for macrolide-resistant Streptococcus pneumoniae. As part of a 44-center U.S. surveillance study, 1,817 unique isolates of S. pneumoniae were collected from November 2002 through April 2003. Seventy-five randomly selected macrolide-resistant isolates (cases) were each matched with one susceptible control. Macrolide use in the 6 weeks prior to sample collection was reported for seven cases and one control. The final conditional logistic regression model identified two statistically significant variables: a history of alcohol abuse was protective, while macrolide use in the 6 weeks prior to sample collection was a significant risk factor for macrolide-resistant S. pneumoniae. Macrolide resistance was associated with use of any antibiotic during the prior 6 weeks, and was most strongly associated with previous macrolide use.The results of this study were presented at the 45th ICAAC Meeting in December 2005.     

Streptococcus pneumoniae type determination by multiplex polymerase chain reaction

The purpose of this study was to develop pneumococcal typing by multiplex PCR and compare it with conventional serotyping by quellung reaction. Pneumococcal strains used in this study included 77 isolates from clinical specimens collected from children at Seoul National University Children's Hospital from 2006 to 2010. These strains were selected as they represented 26 different serotypes previously determined by quellung reaction. Molecular type was determined by 8 sequential multiplex PCR assays. Bacterial DNA extracted from cultured colonies was used as a template for PCR, and primers used in this study were based on cps operon sequences. Types 6A, 6B, 6C, and 6D were assigned based on the presence of wciN(β) and/or wciP genes in 2 simplex PCRs and sequencing. All 77 isolates were successfully typed by multiplex PCR assays. Determined types were as follows: 1, 3, 4, 5, 6A, 6B, 6C, 6D, 7C, 7F, 9V, 10A, 11A, 12F, 13, 14, 15A, 15B/15C, 19A, 19F, 20, 22F, 23A, 23F, 34, 35B, and 37. The results according to the PCR assays were in complete concordance with those determined by conventional quellung reaction. The multiplex PCR assay is highly reliable and potentially reduces reliance upon conventional serotyping.     

Comparison of the pulmonary response against lethal and non-lethal intranasal challenges with two different pneumococcal strains

The differences between the immune response elicited during a self-limiting and a life-threatening lung infection with Streptococcus pneumoniae was analyzed in a mouse model of intranasal challenge using two different pneumococcal strains. M10, a serotype 11A strain, induced an early response within the first 12 h after the challenge, which was characterized by the early local secretion of TNF-α and IL-6, followed by a sharp and rapid neutrophil influx. Bacterial loads in the lungs already started to fall at 12 h after the challenge and no pneumococci could be recovered after 36 h, at the time point when the animals started to show improvement in disease symptoms. ATCC6303, a serotype 3 strain, on the other hand, showed only a late increase in local TNF-α and IL-6 levels, when bacterial growth already seems to be out of control. Although cell influx was also observed, neutrophil rise was not as marked as with M10 (type 11A). Pneumococcal loads increased constantly and bacteria started to be recovered from the blood at 30 h after the challenge. After this time point, animals showed worsening of symptoms and became lethargic. The resolution of the acute infection could be thus correlated with the early induction of proinflammatory cytokines, which could be due to the presence of a thinner polysaccharide capsule in M10 (type 11A), rendering bacterial components capable of activating the innate immune response more accessible.     

Vaccine-induced human antibodies to PspA augment complement C3 deposition on Streptococcus pneumoniae

Pneumococcal surface protein (PspA) is a virulence factor expressed by all clinical isolates of Streptococcus pneumoniae. PspAs are variable in structure and have been grouped into clades and cross-reacting families based on sequence similarities and immunologic cross-reactivity. At least 98% of PspAs are found in PspA families 1 or 2. PspA has been shown to interfere with complement deposition on pneumococci, thus reducing opsonization and clearance of bacteria by the host immune system. Prior studies using pooled human sera have shown that PspA interferes with C3 deposition on a single strain of S. pneumoniae, WU2, and that mouse antibody to PspA can enhance the deposition of C3 on WU2. The present studies have demonstrated that these previous findings are representative of most normal human sera and each of seven different strains of S. pneumoniae. It was observed that PspAs of PspA families 1 and 2 could inhibit C3 deposition in the presence of immunoglobulin present in all but 3 of 22 normal human sera. These studies have also demonstrated that rabbit and human antibody to PspA can enhance the deposition of C3 on pneumococci expressing either family 1 or 2 PspAs and either capsular types 2, 3, or 11. A vaccine candidate that can elicit immunity that neutralizes or compensates for S. pneumoniae's ability to thwart host immunity would be of value.     

Changes in serotype distribution and antibiotic resistance of nasopharyngeal isolates of Streptococcus pneumoniae from children in Korea, after optional use of the 7-valent conjugate vaccine

We investigated serotype distribution and antimicrobial resistance of pneumococcal carriage isolates from children after optional immunization with the 7-valent pneumococcal conjugate vaccine (PCV7) in Korea. From June 2009 to June 2010, 205 (16.5%) pneumococcal isolates were obtained from 1,243 nasopharyngeal aspirates of infants and children at Seoul National University Children's Hospital, Korea. Serotype was determined by Quellung reaction and antibiotic susceptibility was tested by E-test. The results were compared to previous studies done in the pre-PCV7 period. In this study, the most common serotypes were 6A (15.3%), 19A (14.7%), 19F (10.2%), 35B (7.3%), and 6D (5.6%). The proportion of PCV7 serotypes decreased from 61.9% to 23.8% (P < 0.001). The overall penicillin nonsusceptibility rate increased from 83.5% to 95.4% (P = 0.001). This study demonstrates the impact of optional PCV7 vaccination in Korea; the proportion of all PCV7 serotypes except 19F decreased while antimicrobial resistant serotypes 6A and 19A further increased.