Regulation of Streptococcus pneumoniae distribution by Toll-like receptor 2 in vivo

The phagocyte pattern recognition receptor Toll-like receptor 2 (TLR2) and the multi-receptor adaptor MyD88 contribute to the reduction of bacterial load in infections with intra- and extra-cellular Gram-positive bacteria. Their mechanism of antibacterial action is mostly unresolved but evident in vivo by an increased pathogen burden in infected TLR2-/- and MyD88-/- compared to C57BL/6 wild type (wt) mice. We had previously observed higher bacterial numbers in brains of TLR2-/- than of wt mice with meningitis. Here we study bacteria-phagocyte interaction by comparing S. pneumoniae distribution and localization in wt and TLR2-/- brain by confocal microscopy using a green fluorescent protein-transformed encapsulated S. pneumoniae (C5017). Colony-forming units were similarly distributed in TLR2-/- and wt mice and exclusively localized in meninges and ventricles. Bacteria were more abundant in ventricles, in and around TLR2-/- than wt GLT1v+ plexus choroideus epithelial cells. S. pneumoniae were also found in and around Gr-1+ granulocytes, but never in F4/80+ macrophages, Iba1+ microglia, GFAP+ astrocytes, Meca-31+ endothelial cells or Neun+ neurons of either mouse strain. The results indicate that TLR2 does not change bacterial distribution, but may contribute to antibacterial defense by modulating S. pneumoniae adherence and uptake in plexus epithelia.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Nitric oxide synthase in damaged sensory neuron

NADPH diaphorase colocalized with nitric oxide synthase was studied in damaged protoneurons of the nodose ganglion of rabbit vagus nerve. The number and activity of nitric oxide synthase-expressing neurons increased. Nitric oxide is involved in neuron remodeling. Translated fromByulleten' Eksperimental'noi biologii i Meditsiny, Vol. 128, No. 10, pp. 463–465, October, 1999     

Extract of the bristles of Dirphia sp. increases nitric oxide in a rat pleurisy model

Objectives In the present study we assessed the inflammatory potential of venom obtained from caterpillar genus Dirphia in an acute model of lung injury. Material and methods Injection of extract from the bristles of Dirphia sp. (EBD) into the pleural cavity of rats elicited an acute inflammation response characterized by fluid accumulation which contained a large number of polymorphonuclear neutrophils (PMNs). Results The results show that EBD induces an inflammatory response, with a significant increase in PMNs, exudate and nitric oxide within 4 h after a 0.04 mg/kg dose. The administration of anti-inflammatory drugs (fructose-1,6-bisphosphate, dexamethasone, rofecoxib, sodium diclofenac and pyrilamine) significantly reduced the inflammatory effect of EBD. Conclusions EBD causes an inflammatory reaction in the pleural cavity of rats involving a variety of inflammatory mediators, its action mechanism probably involving cellular injury and the exacerbated induction of cytokines and nitric oxide. Received 23 June 2005; returned for revision 24 August 2005; returned for final revision 2 October 2005; accepted by I. Ahnfelt-R?nne 13 October 2005     

Nitric oxide and pro-inflammatory cytokines correlate with pain intensity in chronic pain patients

Objective: Inflammatory cytokines as well as nitric oxide (NO) play a key role in the pathogenesis of persistent and exaggerated pain states. To document this, we investigated whether a range of cytokines and NO were detectable in the plasma of chronic pain patients and whether cytokine and NO levels correlated with pain severity. Methods: Plasma samples of 94 chronic pain patients and 6 healthy volunteers were obtained. Average pain intensity during the last 24h was assessed on a 11-point numeric rating scale and patients were distributed to three groups: light, moderate and severe pain. The concentrations of TNF-α, GM-CSF, interleukin (IL)-1β, IL-6, IL-8, interferon (IFN)-γ, IL-2, IL-4, IL-5, IL-10 and nitrate/nitrite were determined. Results: Patients with light pain demonstrated significantly increased levels of IL-6 compared to controls. In the severe pain group IL-6 and nitrate/nitrite were significantly increased. Serum concentrations of IL-1β, TNF-α, IL-2 and IL-4 were increased but as we adjusted the level of significance at p = 0.0045, most cytokine plasma levels failed to reach statistical significance. Conclusions: Pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IFN-γ, TNF-α) in the plasma correlate with increasing pain intensity. Chronic pain patients show a significant increase in plasma levels of NO in comparison to healthy controls. Received 30 May 2006; returned for revision 17 July 2006; accepted by G. Geisslinger 7 August 2006     

Nitric oxide synthase activity has limited influence on the control of Coccidioides infection in mice

The functions of inducible nitric oxide synthase (iNOS) activity in protection against microbial insults are still controversial. In this study, we explored the role of iNOS in protection against Coccidioides infection in mice. We observed that wild type (WT) and iNOS^−/− mice showed similar percent survival and fungal burden in their lungs at days 7 and 11 after intranasal challenge with Coccidioides. Vaccinated WT and iNOS^−/− mice revealed comparable fungal burden in their lungs and spleen at 7 and 11 days postchallenge. However, at 11 days the non-vaccinated, iNOS-deficient mice had significantly higher fungal burden in their spleen compared to WT mice. Additionally, higher numbers of lung-infiltrated neutrophils, macrophages and dendritic cells were observed in WT mice at day 11 postchallenge compared to iNOS^−/− mice. Moreover, no difference in numbers of T, B, NK or regulatory T cells, or concentrations of selected cytokines and chemokines were detected in lungs of both mouse strains at 7 and 11 days postchallenge. Although iNOS-derived NO production appears to influence the inflammatory response and dissemination of the fungal pathogen, our results suggest that iNOS activity does not play a significant role in the control of coccidioidal infection in mice and that other, still undefined mechanisms of host protection are involved.