Activity Of Normal Human Serum Research Articles

SARS-CoV-2 Nucleocapsid Protein Is Not Responsible for Over-Activation of Complement Lectin Pathway.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral structural protein that is abundant in the circulation of infected individuals. Previous published studies reported controversial data about the role of the N protein in the activation of the complement system. It was suggested that the N protein directly interacts with mannose-binding lectin-associated serine protease-2 (MASP-2) and stimulates lectin pathway overactivation/activity. In order to check these data and to reveal the mechanism of activation, we examined the effect of the N protein on lectin pathway activation. We found that the N protein does not bind to MASP-2 and MASP-1 and it does not stimulate lectin pathway activity in normal human serum. Furthermore, the N protein does not facilitate the activation of zymogen MASP-2, which is MASP-1 dependent. Moreover, the N protein does not boost the enzymatic activity of MASP-2 either on synthetic or on protein substrates. In some of our experiments, we observed that MASP-2 digests the N protein. However, it is questionable, whether this activity is biologically relevant. Although surface-bound N protein did not activate the lectin pathway, it did trigger the alternative pathway in 10% human serum. Additionally, we detected some classical pathway activation by the N protein. Nevertheless, we demonstrated that this activation was induced by the bound nucleic acid, rather than by the N protein itself.

Secreted Autotransporter Toxin (Sat) Mediates Innate Immune System Evasion.

Several strategies are used by Escherichia coli to evade the host innate immune system in the blood, such as the cleavage of complement system proteins by secreted proteases. Members of the Serine Proteases Autotransporters of Enterobacteriaceae (SPATE) family have been described as presenting proteolytic effects against complement proteins. Among the SPATE-encoding genes sat (secreted autotransporter toxin) has been detected in high frequencies among strains of E. coli isolated from bacteremia. Sat has been characterized for its cytotoxic action, but the possible immunomodulatory effects of Sat have not been investigated. Therefore, this study aimed to evaluate the proteolytic effects of Sat on complement proteins and the role in pathogenesis of BSI caused by extraintestinal E. coli (ExPEC). E. coli EC071 was selected as a Sat-producing ExPEC strain. Whole-genome sequencing showed that sat sequences of EC071 and uropathogenic E. coli CFT073 present 99% identity. EC071 was shown to be resistant to the bactericidal activity of normal human serum (NHS). Purified native Sat was used in proteolytic assays with proteins of the complement system and, except for C1q, all tested substrates were cleaved by Sat in a dose and time-dependent manner. Moreover, E. coli DH5α survived in NHS pre-incubated with Sat. EC071-derivative strains harboring sat knockout and in trans complementations producing either active or non-active Sat were tested in a murine sepsis model. Lethality was reduced by 50% when mice were inoculated with the sat mutant strain. The complemented strain producing active Sat partially restored the effect caused by the wild-type strain. The results presented in this study show that Sat presents immunomodulatory effects by cleaving several proteins of the three complement system pathways. Therefore, Sat plays an important role in the establishment of bloodstream infections and sepsis.

The Impact of Graphite Oxide Nanocomposites on the Antibacterial Activity of Serum.

Nanoparticles can interact with the complement system and modulate the inflammatory response. The effect of these interactions on the complement activity strongly depends on physicochemical properties of nanoparticles. The interactions of silver nanoparticles with serum proteins (particularly with the complement system components) have the potential to significantly affect the antibacterial activity of serum, with serious implications for human health. The aim of the study was to assess the influence of graphite oxide (GO) nanocomposites (GO, GO-PcZr(Lys)2-Ag, GO-Ag, GO-PcZr(Lys)2) on the antibacterial activity of normal human serum (NHS), serum activity against bacteria isolated from alveoli treated with nanocomposites, and nanocomposite sensitivity of bacteria exposed to serum in vitro (using normal human serum). Additionally, the in vivo cytotoxic effect of the GO compounds was determined with application of a Galleria mellonella larvae model. GO-PcZr(Lys)2, without IR irradiation enhance the antimicrobial efficacy of the human serum. IR irradiation enhances bactericidal activity of serum in the case of the GO-PcZr(Lys)2-Ag sample. Bacteria exposed to nanocomposites become more sensitive to the action of serum. Bacteria exposed to serum become more sensitive to the GO-Ag sample. None of the tested GO nanocomposites displayed a cytotoxicity towards larvae.

The role of Lsa23 to mediate the interaction of Leptospira interrogans with the terminal complement components pathway

Leptospirosis is a severe worldwide zoonotic disease caused by pathogenic Leptospira spp. It has been demonstrated that pathogenic leptospires are resistant to the bactericidal activity of normal human serum while saprophytic strains are susceptible. Pathogenic strains have the ability to bind soluble complement regulators and these activities are thought to contribute to bacterial immune evasion. One strategy used by some pathogens to evade the complement cascade, which is not well explored, is to block the terminal pathway. We have, thus, examined whether leptospires are able to interact with components of the terminal complement pathway. ELISA screening using anti-leptospires serum has shown that the pathogenic, virulent strain L. interrogans L1-130 can bind to immobilized human C8 (1 μg). However, virulent and saprophyte L. biflexa strains showed the ability to interact with C8 and C9, when these components were employed at physiological concentration (50 μg/mL), but the virulent strain seemed more competent. Lsa23, a putative leptospiral adhesin only present in pathogenic strains, interacts with C8 and C9 in a dose-dependent mode, suggesting that this protein could mediate the binding of virulent Leptospira with these components. To our knowledge, this is the first work reporting the binding of Leptospira to C8 and C9 terminal complement components, suggesting that the inhibition of this pathway is part of the strategy used by leptospires to evade the innate immunity.

Regulatory Protein OmpR Influences the Serum Resistance of Yersinia enterocolitica O:9 by Modifying the Structure of the Outer Membrane

The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS) compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC) and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.

Analysis of the SDS-PAGE patterns of outer membrane proteins from Escherichia coli strains that have lost the ability to form K1 antigen and varied in the susceptibility to normal human serum

We used SDS-polyacrylamide gel electrophoresis to investigate the outer membrane proteins (OMPs) band composition of 19 Escherichia coli K1 strains that have spontaneously lost the ability to form K1 polysaccharide capsule (E. coli K1−) and demonstrated different degrees of susceptibility to the bactericidal action of normal human serum. Presented results showed that there were differences between E. coli K1− strains in OMPs expressing capacity. The analysis performed on OMPs has not revealed a direct association between the different OMPs band composition and the susceptibility of these strains to the serum.

Identification of fibronectin as a major factor in human serum to recruit subchondral mesenchymal progenitor cells

Human serum has the potential for mesenchymal progenitor cell recruitment in repair of articular cartilage lesions. It is unclear which factor(s) in serum mediate this migratory effect. Our goal was to identify cell recruiting factors in human serum fractions obtained by ion exchange chromatography. The recruiting activity of serum fractions on human subchondral mesenchymal progenitor cells was analyzed using 96-well chemotaxis assays. Protein composition of recruiting serum fractions were analyzed by mass spectrometry and showed 58 potential candidates. Fibronectin, gelsolin, lumican, thrombospondin-1 and WNT-9a were identified as key candidates for progenitor cell recruitment. Only human plasma derived and recombinant fibronectin showed significant recruiting activity on progenitors reaching 50–90% of the recruiting activity of normal human serum. Presence of fibronectin in all human serum fractions with recruiting activity was verified by Western blot analysis. This study shows that fibronectin is a key factor in human serum to recruit mesenchymal progenitor cells and might be involved in subchondral mesenchymal progenitor cell migration into cartilage defects after microfracture.

Serine Protease PKF of Acinetobacter baumannii Results in Serum Resistance and Suppression of Biofilm Formation

Acinetobacter baumannii is an important nosocomial pathogen. Infections are often preceded by intubation or catheter use, promoting the formation of biofilm, and some strains are able to cause severe cases of bacteremia because of their ability to resist killing by complement. We identified a secreted serine protease, termed "PKF," that provided resistance to complement killing and suppressed biofilm formation. Serum resistance was abrogated in A. baumannii treated with protease inhibitors, as well as in a PKF-negative mutant. Serum resistance could be restored by recombinant PKF, which was shown to reduce the complement activity of normal human serum by almost 50%. PKF was shown to inhibit biofilm formation, because the PKF-negative mutant and wild-type A. baumannii treated with protease inhibitors produced biofilm that could be inhibited by addition of recombinant PKF. Our data indicate that PKF is required for serum resistance and that it suppresses biofilm formation in A. baumannii.

Clinical Evaluation of a New Creatine Kinase MB Activity Reagent Abrogating the Effect of Mitochondrial Creatine Kinase

Measuring creatine kinase (CK) MB activity using the immunoinhibition method remains useful in clinical laboratories. CK-MB activity is abnormally high when macro CK type 2 (mitochondrial creatine kinase, MtCK) is present in patient serum. In order to improve the accuracy of the CK-MB activity assay, we developed a new CK-MB activity method using highly specific anti-MtCK antibodies. We evaluated the clinical performance of the new method, which abrogates the effect of MtCK activity. Receiver operating characteristic analysis, CK-MB activity range, cut-off, and CK-MB to CK activity ratio were investigated. Mean CK-MB activity in normal human serum was 2.5 U/L by our method, in contrast to 12.0 U/L by the current method. Approximately 80% of CK-MB activity determined using the current kit corresponds to MtCK activity, and ubiquitous mitochondrial creatine kinase activity constitutes approximately 90% of MtCK activity. The cut-off and CK-MB activity ratio of our method were 12 U/L and 3 to 20%, respectively, in contrast to 22 U/L and 5 to 23%, respectively, using the current CK-MB method. The areas under the curve of our method, current CK-MB, electrophoresis, and CK-MB mass were 0.976, 0.928, 0.967, and 0.991, respectively. Our new method was superior to the electrophoresis and CK-MB mass as well as the conventional method due to jts promptness, simplification, and low cost. The new kit will improve the clinical diagnosis of acute myocardial infarction. CK-MB activity assay was considered as a suitable alternative to conventional cardiac markers due to its superior diagnostic validity.

Lipooligosaccharide Structure is an Important Determinant in the Resistance of Neisseria Gonorrhoeae to Antimicrobial Agents of Innate Host Defense.

The strict human pathogen Neisseria gonorrhoeae has caused the sexually transmitted infection termed gonorrhea for thousands of years. Over the millennia, the gonococcus has likely evolved mechanisms to evade host defense systems that operate on the genital mucosal surfaces in both males and females. Past research has shown that the presence or modification of certain cell envelope structures can significantly impact levels of gonococcal susceptibility to host-derived antimicrobial compounds that bathe genital mucosal surfaces and participate in innate host defense against invading pathogens. In order to facilitate the identification of gonococcal genes that are important in determining levels of bacterial susceptibility to mediators of innate host defense, we used the Himar I mariner in vitro mutagenesis system to construct a transposon insertion library in strain F62. As proof of principle that this strategy would be suitable for this purpose, we screened the library for mutants expressing decreased susceptibility to the bacteriolytic action of normal human serum (NHS). We found that a transposon insertion in the lgtD gene, which encodes an N-acetylgalactosamine transferase involved in the extension of the α-chain of lipooligosaccharide (LOS), could confer decreased susceptibility of strain F62 to complement-mediated killing by NHS. By complementation and chemical analyses, we demonstrated both linkage of the transposon insertion to the NHS-resistance phenotype and chemical changes in LOS structure that resulted from loss of LgtD production. Further truncation of the LOS α-chain or loss of phosphoethanolamine (PEA) from the lipid A region of LOS also impacted levels of NHS-resistance. PEA decoration of lipid A also increased gonococcal resistance to the model cationic antimicrobial polymyxin B. Taken together, we conclude that the Himar I mariner in vitro mutagenesis procedure can facilitate studies on structures involved in gonococcal pathogenesis.

Molecular Pathogenesis of Neisseria Gonorrhoeae

Molecular Pathogenesis of Neisseria Gonorrhoeae

Reptiles as a Source of Salmonella O48—Clinically Important Bacteria for Children: The Relationship Between Resistance to Normal Cord Serum and Outer Membrane Protein Patterns

Bacteria of the Salmonella O48 somatic antigen group are clinically important strains causing intestinal dysfunction and diarrhoea, especially in children. The susceptibility of Salmonella O48 strains containing sialic acid (N-acetylneuraminic acid (NeuAc)) in lipopolysaccharide (LPS) to the bactericidal action of normal cord serum (NCS) was determined. The authors' previous results published in Microbial Ecology in 2010 indicated that neither the presence of NeuAc in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of normal human serum (NHS), and that the presence of NeuAc in the LPS structure is not sufficient to block the activation of the alternative pathway of complement in NHS. The current results showed that the tested strains showed various sensitivities also to the bactericidal action of NCS. The authors postulate that the presence of certain outer membrane proteins (OMPs) are characteristic of the resistant and sensitive phenotypes of Salmonella O48 strains. To establish a possible relationship between resistance to NCS and OMPs band patterns, ten Salmonella O48 strains were studied as follows: susceptibility to the bactericidal effect of NCS, the mechanisms of NCS activation and OMP band patterns obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Opacity proteins increase Neisseria gonorrhoeae fitness in the female genital tract due to a factor under ovarian control.

The neisserial opacity (Opa) proteins are a family of antigenically distinct outer membrane proteins that undergo phase-variable expression. Opa(+) variants of Neisseria gonorrhoeae strain FA1090 are selected in a cyclical pattern from the lower genital tract of estradiol-treated mice. Here we show that cyclical recovery of Opa(+) gonococci does not occur in ovariectomized mice; therefore, the reproductive cycle plays a role in the selection kinetics in vivo. As predicted by the selection pattern shown by wild-type gonococci, we demonstrated that a constitutive Opa-expressing strain was more fit than an Opa-deficient mutant in the early and late phases of infection. We found no evidence that Opa-mediated colonization selects for Opa(+) variants during murine infection based on adherence assays with cultured murine epithelial cells. We also tested the hypothesis that complement selects for Opa protein expression during infection. Although some Opa(+) variants of a serum-sensitive derivative of strain FA1090 were more resistant to the bactericidal activity of normal human serum, selection for Opa expression was not abrogated in C3-depleted mice. Finally, as previously reported, Opa(+) gonococci were more sensitive to serine proteases. Thus, proteases or protease inhibitors may contribute to the observed in vivo selection pattern. We concluded that Opa proteins promote persistence of N. gonorrhoeae in the female genital tract and that opa gene phase variation allows gonococci to evade or capitalize upon unidentified host factors of the mammalian reproductive cycle. This work revealed an intimate interaction between pathogen and host and provides evidence that hormonally related factors shape bacterial adaptation.

Development and performance of an enzyme immunoassay to detect creatine kinase isoenzyme MB activity using anti-mitochondrial creatine kinase monoclonal antibodies

Objective: The MB fraction of creatine kinase (CK-MB) has long been used as a cardiac marker. It is known that the CK-MB immunoinhibition method lacks selectivity and accuracy, because the appearance of macro CK type 2, corresponding to mitochondrial creatine kinase (MtCK) in some patient serum may render CK-MB activity measured by conventional method abnormally high. Thus, to improve the specificity and accuracy of the CK-MB assay, we developed two types of monoclonal anti-MtCK antibodies against sarcomeric MtCK and ubiquitous MtCK, and present herein the performance of a new method using these antibodies. Material and methods: The performance of our test for detecting CK-MB activity was compared with other methods, and the range of CK-MB activities in normal human serum was investigated. Results: The two types of monoclonal antibodies developed by us were isoenzyme-specific to sMtCK or uMtCK. The correlation coefficients of our method and conventional method to electrophoresis were 0.973 and 0.873, respectively. The mean CK-MB activity in normal human serum by our method and the conventional method was 2.4 and 11.7 U/L, respectively. Thus, our data indicated that about 80% of CK-MB activity, determined using the conventional method, seems to correspond to the MtCK activity. Conclusion: Our method is novel in offering higher accuracy of measuring true CK-MB contents in human serum as compared to the conventional method. The possibility of accurately estimating CK-MB activity by our method which can inhibit MtCKs in healthy person and patient serum is likely to bring a break-through in clinical diagnostics.

Composition of the outer membrane proteins of Escherichia coli strains in relation to serum susceptibility after exposure to subinhibitory concentrations of amikacin and ciprofloxacin

The effect of subinhibitory concentrations of amikacin and ciprofloxacin on the susceptibility of Escherichia coli strains to the bactericidal action of normal human serum, outer membrane protein (OMP) expression and cell morphology was investigated. Sub-minimal inhibitory concentrations (sub-MICs) of both antibiotics were found to alter the expression of some OMPs of serum-resistant E. coli rods, the morphology of their cells and made them sensitive to the bactericidal activity of normal human serum.

Development and performance of an enzyme immunoassay to detect creatine kinase isoenzyme MB activity using anti-mitochondrial creatine kinase monoclonal antibodies

Objective: The MB fraction of creatine kinase (CK-MB) has long been used as a cardiac marker. It is known that the CK-MB immunoinhibition method lacks selectivity and accuracy, because the appearance of macro CK type 2, corresponding to mitochondrial creatine kinase (MtCK) in some patient serum may render CK-MB activity measured by conventional method abnormally high. Thus, to improve the specificity and accuracy of the CK-MB assay, we developed two types of monoclonal anti-MtCK antibodies against sarcomeric MtCK and ubiquitous MtCK, and present herein the performance of a new method using these antibodies. Material and methods: The performance of our test for detecting CK-MB activity was compared with other methods, and the range of CK-MB activities in normal human serum was investigated. Results: The two types of monoclonal antibodies developed by us were isoenzyme-specific to sMtCK or uMtCK. The correlation coefficients of our method and conventional method to electrophoresis were 0.973 and 0.873, respectively. The mean CK-MB activity in normal human serum by our method and the conventional method was 2.4 and 11.7 U/L, respectively. Thus, our data indicated that about 80% of CK-MB activity, determined using the conventional method, seems to correspond to the MtCK activity. Conclusion: Our method is novel in offering higher accuracy of measuring true CK-MB contents in human serum as compared to the conventional method. The possibility of accurately estimating CK-MB activity by our method which can inhibit MtCKs in healthy person and patient serum is likely to bring a break-through in clinical diagnostics.

Phosphoethanolamine Substitution of Lipid A and Resistance ofNeisseria gonorrhoeaeto Cationic Antimicrobial Peptides and Complement-Mediated Killing by Normal Human Serum

The capacity of Neisseria gonorrhoeae to cause disseminated gonococcal infection requires that such strains resist the bactericidal action of normal human serum. The bactericidal action of normal human serum against N. gonorrhoeae is mediated by the classical complement pathway through an antibody-dependent mechanism. The mechanism(s) by which certain strains of gonococci resist normal human serum is not fully understood, but alterations in lipooligosaccharide structure can affect such resistance. During an investigation of the biological significance of phosphoethanolamine extensions from lipooligosaccharide, we found that phosphoethanolamine substitutions from the heptose II group of the lipooligosaccharide beta-chain did not impact levels of gonococcal (strain FA19) resistance to normal human serum or polymyxin B. However, loss of phosphoethanolamine substitution from the lipid A component of lipooligosaccharide, due to insertional inactivation of lptA, resulted in increased gonococcal susceptibility to polymyxin B, as reported previously for Neisseria meningitidis. In contrast to previous reports with N. meningitidis, loss of phosphoethanolamine attached to lipid A rendered strain FA19 susceptible to complement killing. Serum killing of the lptA mutant occurred through the classical complement pathway. Both serum and polymyxin B resistance as well as phosphoethanolamine decoration of lipid A were restored in the lptA-null mutant by complementation with wild-type lptA. Our results support a role for lipid A phosphoethanolamine substitutions in resistance of this strict human pathogen to innate host defenses.

Identification of two late acyltransferase genes responsible for lipid A biosynthesis in Moraxella catarrhalis

Lipid A is a biological component of the lipo-oligosaccharide of a human pathogen, Moraxella catarrhalis. No other acyltransferases except for UDP-GlcNAc acyltransferase, responsible for lipid A biosynthesis in M. catarrhalis, have been identified. By bioinformatics, two late acyltransferase genes, lpxX and lpxL, responsible for lipid A biosynthesis were identified, and knockout mutants of each gene in M. catarrhalis strain O35E were constructed and named O35ElpxX and O35ElpxL. Structural analysis of lipid A from the parental strain and derived mutants showed that O35ElpxX lacked two decanoic acids (C10:0), whereas O35ElpxL lacked one dodecanoic (lauric) acid (C12:0), suggesting that lpxX encoded decanoyl transferase and lpxL encoded dodecanoyl transferase. Phenotypic analysis revealed that both mutants were similar to the parental strain in their toxicity in vitro. However, O35ElpxX was sensitive to the bactericidal activity of normal human serum and hydrophobic reagents. It had a reduced growth rate in broth and an accelerated bacterial clearance at 3 h (P < 0.01) or 6 h (P < 0.05) after an aerosol challenge in a murine model of bacterial pulmonary clearance. O35ElpxL presented similar patterns to those of the parental strain, except that it was slightly sensitive to the hydrophobic reagents. These results indicate that these two genes, particularly lpxX, encoding late acyltransferases responsible for incorporation of the acyloxyacyl-linked secondary acyl chains into lipid A, are important for the biological activities of M. catarrhalis.

Role of different moieties from the lipooligosaccharide molecule in biological activities of the Moraxella catarrhalis outer membrane

Lipooligosaccharide (LOS), a major component of the outer membrane of Moraxella catarrhalis, consists of two major moieties: a lipid A and a core oligosaccharide (OS). The core OS can be dissected into a linker and three OS chains. To gain an insight into the biological activities of the LOS molecules of M. catarrhalis, we used a random transposon mutagenesis approach with an LOS specific monoclonal antibody to construct a serotype A O35Elgt3 LOS mutant. MALDI-TOF-MS of de-O-acylated LOS from the mutant and glycosyl composition, linkage, and NMR analysis of its OS indicated that the LOS contained a truncated core OS and consisted of a Glc-Kdo(2) (linker)-lipid A structure. Phenotypic analysis revealed that the mutant was similar to the wild-type strain in its growth rate, toxicity and susceptibility to hydrophobic reagents. However, the mutant was sensitive to bactericidal activity of normal human serum and had a reduced adherence to human epithelial cells. These data, combined with our previous data obtained from mutants which contained only lipid A or lacked LOS, suggest that the complete OS chain moiety of the LOS is important for serum resistance and adherence to epithelial cells, whereas the linker moiety is critical for maintenance of the outer membrane integrity and stability to preserve normal cell growth. Both the lipid A and linker moieties contribute to the LOS toxicity.

The lysozyme and complement dependent bacteriolytic activity of normal human serum

The lysozyme and complement dependent bacteriolytic activity of normal human serum