Proteins SP-A Research Articles

Role of the Cell Wall Microenvironment in Expression of a Heterologous SpaP-S1 Fusion Protein by Streptococcus gordonii

The charge density in the cell wall microenvironment of Gram-positive bacteria is believed to influence the expression of heterologous proteins. To test this, the expression of a SpaP-S1 fusion protein, consisting of the surface protein SpaP of Streptococcus mutans and a pertussis toxin S1 fragment, was studied in the live vaccine candidate bacterium Streptococcus gordonii. Results showed that the parent strain PM14 expressed very low levels of SpaP-S1. By comparison, the dlt mutant strain, which has a mutation in the dlt operon preventing d-alanylation of the cell wall lipoteichoic acids, and another mutant strain, OB219(pPM14), which lacks the LPXTG major surface proteins SspA and SspB, expressed more SpaP-S1 than the parent. Both the dlt mutant and the OB219(pPM14) strain had a more negatively charged cell surface than PM14, suggesting that the negative charged cell wall played a role in the increase in SpaP-S1 production. Accordingly, the addition of Ca(2+), Mg(2+), and K(+), presumably increasing the positive charge of the cell wall, led to a reduction in SpaP-S1 production, while the addition of bicarbonate resulted in an increase in SpaP-S1 production. The level of SpaP-S1 production could be correlated with the level of PrsA, a peptidyl-prolyl cis/trans isomerase, in the cells. PrsA expression appears to be regulated by the cell envelope stress two-component regulatory system LiaSR. The results collectively indicate that the charge density of the cell wall microenvironment can modulate heterologous SpaP-S1 protein expression in S. gordonii and that this modulation is mediated by the level of PrsA, whose expression is regulated by the LiaSR two-component regulatory system.

Pulmonary surfactant in respiratory syncytial virus bronchiolitis: The role in pathogenesis and clinical implications

Respiratory syncytial virus (RSV) bronchiolitis is the leading cause of lower respiratory tract infection, and the most frequent reason for hospitalization among infants throughout the world. In addition to the acute consequences of the disease, RSV bronchiolitis in early childhood is related to further development of recurrent wheezing and asthma. Despite the medical and economic burden of the disease, therapeutic options are limited to supportive measures, and mechanical ventilation in severe cases. Growing evidence suggests an important role of changes in pulmonary surfactant content and composition in the pathogenesis of severe RSV bronchiolitis. Besides the well-known importance of pulmonary surfactant in maintenance of pulmonary homeostasis and lung mechanics, the surfactant proteins SP-A and SP-D are essential components of the pulmonary innate immune system. Deficiencies of such proteins, which develop in severe RSV bronchiolitis, may be related to impairment in viral clearance, and exacerbated inflammatory response. A comprehensive understanding of the role of the pulmonary surfactant in the pathogenesis of the disease may help the development of new treatment strategies. We conducted a review of the literature to analyze the evidences of pulmonary surfactant changes in the pathogenesis of severe RSV bronchiolitis, its relation to the inflammatory and immune response, and the possible role of pulmonary surfactant replacement in the treatment of the disease.

Mapping and use of seed protein loci for marker-assisted selection of growth habit and photoperiod response in Nuña bean (Phaseolus vulgaris L.)

The individual segregations of 14 seed protein loci named, SpA to SpM and Pha (phaseolin), were analyzed in a RIL population developed from the cross Xana × Cornell 49242. These seed protein loci were included in a genetic map previously developed in the same population. Protein loci, SpA, SpB, SpE, SpI, SpJ, and Pha, are organized in two different clusters, both located in linkage group (LG) 7; SpF, SpG, SpK, SpL, and SpM, form a single cluster in LG 4; SpC, is located in LG 3; and SpD, in LG 1. A close linkage was identified between the SpD seed protein locus, and the fin gene, controlling determinate growth habit. The usefulness of the SpD locus as a marker for the indirect selection of determinate growth habit and photoperiod insensitivity was checked in a F2 population derived from the cross G12587 (an indeterminate and photoperiod sensitive nuna bean) × Sanilac (determinate and photoperiod insensitive) and in a set of Mesoamerican and Andean genotypes. Results indicate that SpD protein locus was useful to detect individuals having determinate growth habit and photoperiod insensitivity in the cross G12587 × Salinac although some recombinants were found. However, the linkage between the SpD locus and the genes controlling growth habit and photoperiod sensitivity should be checked before using the SpD locus for the indirect selection of these traits in different backgrounds.

SpA, ClfA, and FnbA Genetic Variations Lead to Staphaurex Test-Negative Phenotypes in Bovine Mastitis Staphylococcus aureus Isolates

Staphylococcus aureus encodes many proteins that act as virulence factors, leading to a variety of diseases, including mastitis in cows. Among these virulence factors, SpA, ClfA, ClfB, FnbA, and FnbB are important for the ability of S. aureus to adhere to and invade host cells as well as to evade host immune responses. The interaction between these S. aureus surface proteins and human immunoglobulin G and fibrinogen that are coupled to latex particles is utilized to induce latex agglutination reactions, which are used widely in diagnostic kits for confirmation of presumptive S. aureus isolates. In this study, the Staphaurex latex agglutination test was performed on a collection of confirmed bovine mastitis S. aureus isolates. Notably, 54% (43/79 isolates) of these isolates exhibited latex agglutination-negative phenotypes (Staphaurex-negative result). To gain insights into the reasons for the high frequency of Staphaurex-negative bovine mastitis S. aureus isolates, the spa, clfA, clfB, fnbA, and fnbB genes were examined. Specific genetic changes in spa, clfA, and fnbA, as well as a loss of fnbB, which may impair SpA, ClfA, FnbA, and FnbB functions in latex agglutination reactions, were detected in Staphaurex-negative S. aureus isolates. The genetic changes included a premature stop codon in the spa gene, leading to a truncated SpA protein that is unable to participate in S. aureus cell-mediated agglutination of latex particles. In addition, clfA and fnbA genetic polymorphisms were detected that were linked to ClfA and FnbA amino acid changes that may significantly reduce fibrinogen-binding activity. The genetic variations in these S. aureus isolates might also have implications for their bovine mastitis virulence capacity.

Toll-like receptor 2-mediated sequential activation of MyD88 and MAPKs contributes to lipopolysaccharide-induced sp-a gene expression in human alveolar epithelial cells

Surfactant proteins (SPs) produced by pulmonary epithelial cells participate in the regulation of sepsis-induced acute lung injury. Our previous study has shown that lipopolysaccharide (LPS), a Gram-negative bacterial outer membrane component, can regulate sp-a gene expression in human lung carcinoma type II epithelial A549 cells. This study was further designed to evaluate the signal-transducing mechanisms of LPS-induced sp-a gene expression. Exposure of A549 cells to LPS induced SP-A mRNA and protein production in time-dependent manners. Application of toll-like receptor 2 (TLR2) siRNA into A549 cells decreased the levels of this receptor and simultaneously inhibited LPS-induced SP-A mRNA expression. Sequentially, LPS enhanced phosphorylation of mitogen-activated protein kinase (MEK) 4 and c-Jun NH 2 terminal kinase 1 (JNK1) in time-dependent manners. Application of TLR2 siRNA decreased LPS-enhanced phosphorylation of MEK4 and JNK1. After knocking-down the translation of MyD88 by RNA interference, the LPS-triggered MEK4 phosphorylation was attenuated. Consequently, LPS augmented the translocation of c-Jun from the cytoplasm to nuclei without affecting c-Fos. Pretreatment of A549 cells with SP600125, an inhibitor of JNK1, significantly lowered LPS-induced SP-A mRNA production. Analyses of an electrophoretic mobility shift assay and a reporter gene further showed that LPS increased the transactivation activity of AP-1 in A549 cells. Therefore, the present study demonstrates that LPS can induce sp-a gene expression in human type II epithelial A549 cells through TLR2-mediated sequential activation of MyD88–MEK4–JNK1–AP-1.

491 Recombinant Human Kgf/Palifermin® as An Alternative to Glucocorticoids- Effects on Pneumocyte Proliferation and Gene Expression in Neonatal Rats

Background: Surfactant decreases surface tension of pulmonary air:liquid interfaces. It mainly comprizes characteristic phosphatidylcholine (PC) species and proteins SP-A to -D. Betamethasone used to accelerate “lung maturation” is catabolic, while keratinocyte growth factor (KGF), expressed by fibroblasts, is non-catabolic, acts specifically on type-II-pneumocytes and correlates inversely with bronchopulmonary dysplasia incidence.Aims: To explore the potential of recombinant human KGF (rhKGF, Palifermin®) on neonatal lung development and surfactant metabolism. To contrast rhKGF with betamethasone effects in vivo.Methods: Postnatal rats (d1, d5, d19) were injected with rhKGF (2x5mg/kg), betamethasone (2x1mg/kg) or rhKGF+betamethasone over 48h. Pneumocyte proliferation was detected using bromodeoxyuridine incorporation. Expression of SP-A to -D, adipocyte triglyceride lipase (ATGL), lyso-PC-acyltransferase (LPCAT) and the ATGL activator CGI-58 were measured with PCR.Results: RhKGF, betamethasone and combination treatment increased SP-B expression throughout by +97-117%, +42-51% and +93-309% (P< 0.001), respectively. SP-C was increased by +35-48%, +30- 44% and +74-108% (P< 0.001) after the respective treatments, while SP-A&-D were unchanged in immature lungs. All treatments increased the expression of ATGL (+50-75%), LPCAT (+15-20%) und CGI-58 (+5-10%) (P< 0.05). However, whereas rhKGF effects were accompanied by increased pneumocyte proliferation, betamethasone blocked both basic (d7) and rhKGF-induced (d7+d21) proliferation.Conclusion: Short term medication of neonatal rats with rhKGF/Palifermin® increased the expression of genes relevant for surfactant function and metabolism. Actions were identical or superior to those of betamethasone, with combination treatment exerting maximal effects. However, while betamethasone effects were at the expense of lung anabolism/pneumocyte proliferation, rhKGF enhanced proliferation.

Myristate is selectively incorporated into surfactant and decreases dipalmitoylphosphatidylcholine without functional impairment

Lung surfactant mainly comprises phosphatidylcholines (PC), together with phosphatidylglycerols and surfactant proteins SP-A to SP-D. Dipalmitoyl-PC (PC16:0/16:0), palmitoylmyristoyl-PC (PC16:0/14:0), and palmitoylpalmitoleoyl-PC (PC16:0/16:1) together comprise 75-80% of surfactant PC. During alveolarization, which occurs postnatally in the rat, PC16:0/14:0 reversibly increases at the expense of PC16:0/16:0. As lipoproteins modify surfactant metabolism, we postulated an extrapulmonary origin of PC16:0/14:0 enrichment in surfactant. We, therefore, fed rats (d19-26) with trilaurin (C12:0(3)), trimyristin (C14:0(3)), tripalmitin (C16:0(3)), triolein (C18:1(3)) or trilinolein (C18:2(3)) vs. carbohydrate diet to assess their effects on surfactant PC composition and surface tension function using a captive bubble surfactometer. Metabolism was assessed with deuterated C12:0 (ω-d(3)-C12:0) and ω-d(3)-C14:0. C14:0(3) increased PC16:0/14:0 in surfactant from 12 ± 1 to 45 ± 3% and decreased PC16:0/16:0 from 47 ± 1 to 29 ± 2%, with no impairment of surface tension function. Combined phospholipase A(2) assay and mass spectrometry revealed that 50% of the PC16:0/14:0 peak comprised its isomer 1-myristoyl-2-palmitoyl-PC (PC14:0/16:0). While C12:0(3) was excluded from incorporation into PC, it increased PC16:0/14:0 as well. C16:0(3), C18:1(3), and C18:2(3) had no significant effect on PC16:0/16:0 or PC16:0/14:0. d(3)-C14:0 was enriched in lung PC, either via direct supply or via d(3)-C12:0 elongation. Enrichment of d(3)-C14:0 in surfactant PC contrasted its rapid turnover in plasma and liver PC, where its elongation product d(3)-C16:0 surmounted d(3)-C14:0. In summary, high surfactant PC16:0/14:0 during lung development correlates with C14:0 and C12:0 supply via specific C14:0 enrichment into lung PC. Surfactant that is high in PC16:0/14:0 but low in PC16:0/16:0 is compatible with normal respiration and surfactant function in vitro.

Cycloxygenase Inhibition Enhances the Effects of Surfactant Therapy in Endotoxin-Induced Rat Model of ARDS

The present study examines the relationships between inflammation and surfactant protein (SP) expression in a rodent model of acute respiratory distress syndrome (ARDS). Rats were intratracheally instilled with lipopolysaccharide (LPS) for 72 hours to induce ARDS and further treated with exogenous surfactant. Prostaglandin E(2) (PGE(2)) levels, cycloxygenase (COX) activity and alterations in SP-A apoprotein were measured. COX and SP-A expressions in lung tissue and SP-A-positive cells were determined by Western blot and immunofluorescence, respectively. PGE(2) levels and COX activity and its expression were increased with LPS exposure, whereas SP-A protein and percentage of SP-A-positive cells were decreased, which were subsequently reverted back by exogenous surfactant instillation. Because inhibition of COX-2 action is proposed to be useful in various inflammatory lung injuries, these results suggest that COX-2 expression and the possible beneficial effects of its inhibition on lung inflammation and dysfunction with LPS-ARDS corresponds closely with reduced SP-A expression.

Surfactant Replacement Therapy in Influenza A H1N1

To the Editors: A 22-month-old boy was admitted to the Clinic for Infective Diseases, University Clinical Center, Tuzla (Bosnia and Herzegovina), because of fever, cough, loss of appetite, and vomiting. An influenza virus type A H1N1 infection quick test (QuickVue, Influenza A+B—QuickVue Influenza A+B—bioMérieux) had a positive result, and 48 hours later infection with Influenza type A H1N1 was confirmed by polymerase chain reaction (multiplex real-time PCR, firma Roche Diagnostics, Laval, Quebec, Canada) at the University Clinical Center, Sarajevo. A chest radiograph showed bilateral pneumonia. During the first 4 days, he had attacks of hypoxemia, which finally became irremediable on oxygen therapy via nasal cannula. The boy was afebrile, unconscious, periodically agitated, cyanotic, dehydrated, pale, tachycardic and tachydyspnoic with clear signs of cardial decompensation, and worsening of the chest radiograph. He was transferred to the pediatric intensive care unit (PICU, Clinic for Pediatric Disease, University Clinic Center in Tuzla) for mechanical support. After 4 hours of mechanical ventilation with low lung compliance, it was decided to give bovine surfactant (Survanta, Abbott Laboratories) endotracheally. After 10 minutes, there was a significant improvement in lung compliance, oxygen saturation, and normalization of the heart rate. Therapy with surfactant was repeated every 8 hours. After 48 hours, auscultatory examination and chest radiograph of the boy were considerably improved. The role of surfactant in treating respiratory failure is doubtful in children, except in the newborn, even though surfactant can improve oxygenation and significantly decrease mortality in infants, children, and adolescents with acute lung injury and respiratory distress syndrome.1 That kind of therapy has been found ineffective in adults.2 Some investigations have shown that pulmonary surfactant proteins SP-A and SP-D can act as opsonins and neutralize influenza virus.3 In treatment of this child, we used medication that consisted of only surfactant proteins SP-B and SP-C. The medication led to significant improvement in lung function and the general condition, which was probably the result of lower alveolar surface tension and better lung compliance, but it is not possible to exclude a neutralizing effect of surfactant on influenza virus type A H1N1 by opsonization. Fahrija Skokić, MD, PhD Clinic of Pediatrics Igor Hudić, MD, PhD Clinic of Gynecology and Obstetrics Nešad Hotić, MD, PhD Belkisa Cǒlić, MD, PhD Clinic of Pediatrics Mirsada Prašo, MD, PhD Clinic of Anesthesiology and Reanimation Dubravka Bačaj, MD, PhD Clinic of Pediatrics University Clinical Center Tuzla, Bosnia and Herzegovina

Secreted surfactant protein A from fetal membranes induces stress fibers in cultured human myometrial cells

In the present study, we investigated the ability of human fetal membranes (amnion and choriodecidua) to regulate human maternal uterine cell functions through the secretion of surfactant protein (SP)-A and SP-D at the end of pregnancy. We detected the expression of both SP-A (SP-A1 and SP-A2) and SP-D by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed that human fetal membranes expressed both SP-A and SP-D. By Western blot analysis, we demonstrated that SP-A protein expression was predominant in choriodecidua, whereas the amnion predominantly expressed SP-D. Only the secretion of SP-A was evidenced in the culture supernatants of amnion and choriodecidua explants by immunodot blot and confirmed by Western blot. Exogenous human purified SP-A induced stress fiber formation in cultured human myometrial cells via a pathway involving Rho-kinase. Conditioned medium from choriodecidua and amnion explants mimicked the SP-A effect. Treatment of myometrial cells with SP-A-depleted conditioned medium from choriodecidua or amnion explants failed to change the actin dynamic. These data indicate that SP-A released by human fetal membranes is able to exert a paracrine regulation of F-actin filament organization in myometrial cells.

Effects of nicotine on pulmonary surfactant proteins A and D in ovine lung epithelia

Maternal smoking during pregnancy increases the incidence and severity of respiratory infections in neonates. Surfactant proteins A and D (SP-A and SP-D, respectively) are components of pulmonary innate immunity and have an important role in defense against inhaled pathogens. The purpose of this study was to determine if nicotine exposure during the third trimester of pregnancy alters the expression of SP-A and SP-D of fetal lung epithelia. Pregnant ewes were assigned to four groups; a nicotine-exposed full-term and pre-term group, and control full-term and pre-term group. Lung tissue was collected for Western blot and IHC analysis of SP-A level, Western blot analysis of SP-D level and qPCR analysis of SP-A and SP-D mRNA expression. Exposure to nicotine significantly decreased SP-A gene expression (P = 0.01) and SP-A protein level in pre-term lambs. This finding suggests that maternal nicotine exposure during the last trimester of pregnancy alters a key component of lung innate immunity in offspring.

Pulmonary surfactant and tuberculosis

Mycobacterium tuberculosis comes in contact with pulmonary surfactant, alveolar macrophages and type II epithelial cells. Alveolar type II epithelial cells secrete pulmonary surfactant, a complex mixture of phospholipids and proteins lining the alveolar surface, while alveolar macrophages are involved in surfactant catabolism. Surfactant proteins SP-A and SP-D modulate phagocytosis of M. tuberculosis by alveolar macrophages. We have reported that mice with decreased surfactant catabolism resulting from GM-CSF deficiency are highly susceptible to acute aerosol infection with 100 cfu of M. tuberculosis. Here, we evaluated the lungs of WT, GM-CSF-deficient, and GM-CSF-corrected mice surviving six months after sub-acute aerosol infection of 5-10 cfu M. tuberculosis. We show that GM-CSF-deficient mice develop intra-bronchial and intra-alveolar tuberculosis lesions with numerous mycobacteria, inflammatory cells, and extracellular proteinaceous material containing surfactant protein B (SP-B). In contrast, WT and GM-CSF-corrected mice develop typical epithelioid granulomas containing lymphocytes, SP-B positive cells, and M. tuberculosis bacilli inside macrophages. Our findings support the concept that whole pulmonary surfactant is an important component of host mycobacterial infection in the distal lung.

Identification of a cell wall-associated subtilisin-like serine protease involved in the pathogenesis of Streptococcus suis serotype 2

Streptococcus suis is an important swine and human pathogen, and also an emerging zoonotic agent. A surface-associated subtilisin-like serine protease (SspA) of S. suis was identified by screening a genomic expression library as fragments of this protein reacted most strongly with convalescent-phase pig sera. The sspA gene is present in 29 of 33 S. suis serotypes reference strains and is expressed on the surface of S. suis. Relative real-time quantitative PCR assay demonstrated that sspA mRNA expression in vivo was several thousand fold of that in vitro. A sspA − mutant was generated from a S. suis serotype 2 strain SC19 by allelic exchange. The mutant was not different from the wild type strain in subcellular structures and in hemolytic phenotype. However, the virulence of the sspA − mutant was markedly lower than the wild type in pigs as demonstrated in experimental infections. These data indicated that the surface-associated protein SspA is a conserved virulence factor of S. suis and is involved in the pathogenesis of S. suis.

Growth phase-dependent regulation of the global virulence regulator Rot in clinical isolates of Staphylococcus aureus

Current models for global virulence regulation in Sta phylococcus aureus are mainly based on studies performed with only a limited number of laboratory strains derived from NCTC8325. In these strains the small regulatory RNA, RNAIII, has a central role in virulence gene regulation. Recently, RNAIII was suggested to control transcription of target genes partly by inhibiting translation of the transcriptional regulator Rot. The present study was undertaken to examine if the model for RNAIII/Rot-dependent virulence regulation is conserved among clinical strains. To this end, we used Rot antibodies to directly assess the amount of Rot protein in 4 well-characterized S. aureus laboratory strains (8325-4, COL, Newman, and UAMS-1) and in 9 strains of clinical origin (encompassing USA300 and Mu50). Additionally, the cellular amount of RNAIII and rot mRNA was determined in all strains. The experiments revealed considerable variation in the Rot and RNAIII levels between strains. However, in the majority of strains the cellular amount of Rot was inversely correlated to the RNAIII level. As we demonstrate that Rot is a stable protein and that the level of rot transcript appeared similar in all strains, our data support that the model for RNAIII-mediated inhibition of rot mRNA translation is conserved among clinical strains. Assessment of Rot-dependent regulation of target genes revealed that Rot is a positive regulator of spa (protein A) transcription in all strains examined. In contrast, Rot repression of sspA (serine protease) and hlb (β-hemolysin) transcription was not conserved between strains. From this study, we conclude that while the paradigm for understanding RNAIII-dependent regulation of Rot is well-conserved, regulation of single genes is subject to considerable strain variation. We propose that variation in global regulatory networks contribute considerably to the phenotypic variation observed between S. aureus isolates.

Streptococcus gordonii Modulates Candida albicans Biofilm Formation through Intergeneric Communication

The fungus Candida albicans colonizes human oral cavity surfaces in conjunction with a complex microflora. C. albicans SC5314 formed biofilms on saliva-coated surfaces that in early stages of development consisted of approximately 30% hyphal forms. In mixed biofilms with the oral bacterium Streptococcus gordonii DL1, hyphal development by C. albicans was enhanced so that biofilms consisted of approximately 60% hyphal forms. Cell-cell contact between S. gordonii and C. albicans involved Streptococcus cell wall-anchored proteins SspA and SspB (antigen I/II family polypeptides). Repression of C. albicans hyphal filament and biofilm production by the quorum-sensing molecule farnesol was relieved by S. gordonii. The ability of a luxS mutant of S. gordonii deficient in production of autoinducer 2 to induce C. albicans hyphal formation was reduced, and this mutant suppressed farnesol inhibition of hyphal formation less effectively. Coincubation of the two microbial species led to activation of C. albicans mitogen-activated protein kinase Cek1p, inhibition of Mkc1p activation by H(2)O(2), and enhanced activation of Hog1p by farnesol, which were direct effects of streptococci on morphogenetic signaling. These results suggest that interactions between C. albicans and S. gordonii involve physical (adherence) and chemical (diffusible) signals that influence the development of biofilm communities. Thus, bacteria may play a significant role in modulating Candida carriage and infection processes in the oral cavity.

Effect of exogenous surfactant therapy on levels of pulmonary surfactant proteins A and D in preterm infants with respiratory distress syndrome

To examine whether exogenous pulmonary surfactants (exPS) substitute for or merely supplement endogenous pulmonary surfactants (enPS) by looking at sequential changes in the surfactant proteins (SP) SP-A and SP-D in alveolar pools. Fourteen preterm infants with RDS treated with an artificial surfactant were compared to five normal-term infants without RDS who were treated with artificial ventilation at birth. Immediately after birth, SP-A and SP-D were essentially absent in the alveolar pools of the RDS group, but were present at normal levels in the controls. Treatment with exPS apparently stimulated enPS production. In infants who responded well to exPS therapy, the SP concentration reached essentially normal levels within 48-72 h after birth.

Human parotid and submandibular glands express and secrete surfactant proteins A, B, C and D

The oral cavity and the salivary glands are open to the oral environment and are thus exposed to multiple microbiological, chemical and mechanical influences. The existence of an efficient defense system is essential to ensure healthy and physiological function of the oral cavity. Surfactant proteins play an important role in innate immunity and surface stability of fluids. This study aimed to evaluate the expression and presence of surfactant proteins (SP) A, B, C, and D in human salivary glands and saliva. The expression of mRNA for SP-A, -B, -C and -D was analyzed by RT-PCR in healthy parotid and submandibular glands. Deposition of all surfactant proteins was determined with monoclonal antibodies by means of Western blot analysis and immunohistochemistry in healthy tissues and saliva of volunteers. Our results show that all four surfactant proteins SP-A, SP-B, SP-C and SP-D are peptides of saliva and salivary glands. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D appear to be involved in immune defense inside the oral cavity. Furthermore, by lowering surface tension between saliva and the epithelial lining of excretory ducts, SP-B and SP-C may assist in drainage and outflow into the oral cavity. Further functions such as pellicle formation on teeth have yet to be determined.

Surfactant protein-A interacts with TLR4 and affects the phagocytic function of dendritic cells (135.41)

Abstract The surfactant protein (SP)-A and Toll-like receptor (TLR4) proteins are known as secretory and cellular pathogen pattern recognition receptors, respectively, and play an important role in the recognition of pathogens and host defense. In lung, the TLR4 is expressed by immune cells, and SP-A is synthesized by type II epithelial cells and secreted in alveoli. In this study, we investigated the interaction between purified baboon lung SP-A and recombinant TLR4 (extracellular domain)-MD2 proteins and effects of these proteins on phagocytic function of the human myelomonocytic cells (KG1) derived- and primary baboon lung-dendritic cells. The interaction between the two proteins was studied in vitro by a microwell based method. For measuring the changes in phagocytic ability of the dendritic cells, the cells were incubated with pHrodo-labeled, heat-killed Escherichia coli bioparticles in presence of native SP-A and/or recombinant TLR4-MD proteins and fluorescence released from ingested E. coli bioparticles inside the acidic phagosome was read on a fluorometer. We found that the SP-A protein binds to TLR4-MD2 protein. The exogenous addition of SP-A and TLR4-MD2 proteins was found to significantly increase the phagocytosis of E. coli bioparticles by dendritic cells. However, when the SP-A and TLR4-MD2 proteins were added together, the phagocytic ability of dendritic cells either remained unaltered or decreased from those treated with SP-A or TLR4-MD2 alone. These results suggest that the interaction between SP-A and TLR4 affects the bacterial phagocytosis by dendritic cells.

IRRAS Studies of the Host Defense Effect of Pulmonary Surfactants SP-A and SP-D

The pulmonary surfactant proteins SP-A and SP-D have been proven to play a role in host defense. SP-A binds phosphatidylcholine, galactosylceramide, and the lipid A portion of lipopolysaccharide (LPS), which is found in Gram-negative bacterial cell walls. SP-D binds phosphatidylinositol, glucosylceramide and LPS. Although X-ray crystallography and NMR spectroscopy have provided atomic level information about SP-A and SP-D structures, molecular level information about their binding to biological ligands is lacking. IR reflection-absorption spectroscopy (IRRAS) is used in the current work to study the properties of SP-A and SP-D at air/water interfaces and their interaction with biological ligands. The monolayer properties of recombinant rat neck + carbohydrate recognition domain (NCRD) SP-A and its point mutant D215A SP-A are compared. Both NCRD SP-A and D215A SP-A adsorb to the air/water interface, DPPC monolayers, and Lipid A monolayers, but D215A/PL SP-A displays higher affinity and greater stability. Measurements of surface pressure, Amide 1 intensity, and lipid acyl chain conformational ordering can help to elucidate the mechanism of interaction of SP-A and its ligands. Similar experiments have been carried out with SP-D.

Interactions of SP-B Based Peptide with Lipid and Protein Components of Lung Surfactant

Lung surfactant (LS) is a mixture of lipids and proteins that reduces the surface tension at the alveolar air-water interface and thus prevents lung collapse and enables normal breathing. Surfactant Protein B (SP-B) is an essential component of LS and is absolutely necessary for survival. SP-B is thought to function by facilitating large-scale rearrangements of lipids and stabilizing the structures at various stages of the breathing cycle. However, neither the structural basis for this ability nor the physiological ramifications of lipid rearrangements are yet understood, in part because a high-resolution structure of SP-B is not yet available. Mini-B is a peptide fragment of SP-B that has been shown in in vitro and in vivo studies to retain similar activity to the full-length protein. Previously, we determined the structure of Mini-B, first in organic solvent hexafluoroisopropanol (HFIP) and then in detergent micelles composed of sodiumdodecylsulfate (SDS) using solution NMR. In our present work, we have studied the interactions of Mini-B with dodecylphosphocholine (DPC) and SDS micelles. DPC and SDS micelles provide an interfacial environment, with lipid headgroups corresponding to the headgroups of the most abundant lipids in LS, phosphatidylcholine (PC) and phosphatidylglycerol (PG) and thus solution NMR studies of interactions between Mini-B and these micelles can provide insight into LS protein-lipid interactions. We have also investigated the interactions of Mini-B with the most abundant surfactant protein SP-A under similar conditions. These studies further the understanding of the mechanisms of SP-B interactions with other surfactant components in native lung environment.