NontypeableHaemophilus influenzaeInvasive Blood Isolates Are Mainly Phosphorylcholine Negative and Show Decreased Complement-Mediated Killing That Is Associated with Lower Binding of IgM and CRP in Comparison to Colonizing Isolates from the Oropharynx

ABSTRACTNontypeableHaemophilus influenzae(NTHi) bacteria express various molecules that contribute to their virulence. The presence of phosphocholine (PCho) on NTHi lipooligosaccharide increases adhesion to epithelial cells and is an advantage for the bacterium, enabling nasopharyngeal colonization, as measured in humans and animal models. However, when PCho is expressed on the lipooligosaccharide, it is also recognized by the acute-phase protein C-reactive protein (CRP) and PCho-specific antibodies, both of which are potent initiators of the classical pathway of complement activation. In this study, we show that blood isolates, which are exposed to CRP and PCho-specific antibodies in the bloodstream, have a higher survival in serum than oropharyngeal isolates, which was associated with a decreased presence of PCho. PCholowstrains showed decreased IgM, CRP, and complement C3 deposition, which was associated with increased survival in human serum. Consistent with the case for the PCholowstrains, removal of PCho expression bylicAgene deletion decreased IgM, CRP, and complement C3 deposition, which increased survival in human serum. Complement-mediated killing of PChohighstrains was mainly dependent on binding of IgM to the bacterial surface. These data support the hypothesis that a PCholowphenotype was selected in blood during invasive disease, which increased resistance to serum killing, mainly due to lowered IgM and CRP binding to the bacterial surface.

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Serum IgM and C-Reactive Protein Binding to Phosphorylcholine of NontypeableHaemophilus influenzaeIncreases Complement-Mediated Killing

Infection and Immunity ◽

10.1128/iai.00299-19 ◽

2019 ◽

Vol 87 (8) ◽

Cited By ~ 5

Author(s):

Jeroen D. Langereis ◽

Eva S. van der Pasch ◽

Marien I. de Jonge

Keyword(s):

Respiratory Tract ◽

Lower Respiratory Tract ◽

Classical Pathway ◽

Upper Respiratory Tract ◽

C Reactive Protein ◽

Surface Binding ◽

Specific Antibodies ◽

Bacterial Surface ◽

Reactive Protein ◽

Tract Infections

ABSTRACTNontypeableHaemophilus influenzae(NTHi) colonizes the human upper respiratory tract without causing disease symptoms, but it is also a major cause of upper and lower respiratory tract infections in children and elderly, respectively. NTHi synthesizes various molecules to decorate its lipooligosaccharide (LOS), which modulates the level of virulence. The presence of phosphorylcholine (PCho) on NTHi LOS increases adhesion to epithelial cells, which is an advantage for the bacterium enabling nasopharyngeal colonization. However, when PCho is incorporated on the LOS of NTHi, it is recognized by the acute-phase C-reactive protein (CRP) and PCho-specific antibodies, both potent initiators of the classical pathway of complement activation. We determined the presence of PCho and binding of IgG and IgM to the bacterial surface for 319 NTHi strains collected from the nasopharynx/oropharynx, middle ear, and lower respiratory tract. PCho detection was higher for NTHi strains collected from the nasopharynx/oropharynx, which was associated with increased binding of IgM and IgG to the bacterial surface. Binding of CRP and IgM to the bacterial surface of PChohighNTHi strains increased complement-mediated killing, which was largely dependent on PCho-specific IgM. The levels of PCho-specific IgM varied in sera from 12 healthy individuals, and higher PCho-specific IgM levels were associated with increased complement-mediated killing of a PChohighNTHi strain. In conclusion, incorporation of PCho on the LOS of NTHi marks the bacterium for binding of CRP and IgM, resulting in complement-mediated killing. Therefore, having a lower PCho might be beneficial in situations where sufficient PCho-specific antibodies and complement are present.

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Kingella kingaeSurface Polysaccharides Promote Resistance to Human Serum and Virulence in a Juvenile Rat Model

Infection and Immunity ◽

10.1128/iai.00100-18 ◽

2018 ◽

Vol 86 (6) ◽

Cited By ~ 7

Author(s):

Vanessa L. Muñoz ◽

Eric A. Porsch ◽

Joseph W. St. Geme

Keyword(s):

Human Serum ◽

Invasive Disease ◽

Complement Pathway ◽

Kingella Kingae ◽

Content Type ◽

Bacterial Surface ◽

Classical Complement Pathway ◽

Bactericidal Effects ◽

Pathogenic Process ◽

Classical Complement

ABSTRACTKingella kingaeis a Gram-negative coccobacillus that is increasingly being recognized as an important cause of invasive disease in young children. The pathogenesis ofK. kingaedisease begins with colonization of the oropharynx, followed by invasion of the bloodstream, survival in the intravascular space, and dissemination to distant sites. Recent studies have revealed thatK. kingaeproduces a number of surface factors that may contribute to the pathogenic process, including a polysaccharide capsule and an exopolysaccharide. In this study, we observed thatK. kingaewas highly resistant to the bactericidal effects of human serum complement. Using mutant strains deficient in expression of capsule, exopolysaccharide, or both in assays with human serum, we found that elimination of both capsule and exopolysaccharide was required for efficient binding of IgG, IgM, C4b, and C3b to the bacterial surface and for complement-mediated killing. Abrogation of the classical complement pathway using EGTA-treated human serum restored survival to wild-type levels by the mutant lacking both capsule and exopolysaccharide, demonstrating that capsule and exopolysaccharide promote resistance to the classical complement pathway. Consistent with these results, loss of both capsule and exopolysaccharide eliminated invasive disease in juvenile rats with an intact complement system but not in rats lacking complement. Based on these observations, we conclude that the capsule and the exopolysaccharide have important redundant roles in promoting survival ofK. kingaein human serum. Each of these surface factors is sufficient by itself to fully prevent serum opsonin deposition and complement-mediated killing ofK. kingae, ultimately facilitating intravascular survival and promotingK. kingaeinvasive disease.

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Resistance of Neisseria meningitidis to Human Serum Depends on T and B Cell Stimulating Protein B

Infection and Immunity ◽

10.1128/iai.03134-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1257-1264 ◽

Cited By ~ 7

Author(s):

Maike G. Müller ◽

Nina E. Moe ◽

Phillip Q. Richards ◽

Gregory R. Moe

Keyword(s):

B Cell ◽

Neisseria Meningitidis ◽

Human Serum ◽

Parent Strain ◽

Bacterial Pathogen ◽

Invasive Disease ◽

Binding Activity ◽

Classical Pathway ◽

Content Type ◽

Protein B

The ability of the human bacterial pathogenNeisseria meningitidisto cause invasive disease depends on survival in the bloodstream via mechanisms to suppress complement activation. In this study, we show that prophage genes coding for T and B cell stimulating protein B (TspB), which is an immunoglobulin-binding protein, are essential for survival ofN. meningitidisgroup B strain H44/76 in normal human serum (NHS). H44/76 carries three genes coding for TspB. Mutants having alltspBgenes inactivated did not survive in >5% NHS or IgG-depleted NHS. TspB appeared to inhibit IgM-mediated activation of the classical complement pathway, since survival of thetspBtriple knockout was the same as that of the parent strain or a complemented mutant when the classical pathway was inactivated by depleting NHS of C1q and was increased in IgM-depleted NHS. A mutant solely carryingtspBgenenmbh4476_0681was as resistant as the parent strain, while mutants carrying onlynmbh4476_0598ornmbh4476_1698were killed in ≥5% NHS. The phenotype associated with TspB is formation of a matrix containing TspB, IgG, and DNA that envelopes aggregates of bacteria. Recombinant proteins corresponding to particular subdomains of TspB were found to have human IgG Fcγ- and/or DNA-binding activity, but only TspB derivatives containing both domains formed large, biofilm-like aggregates when combined with purified IgG and DNA. Recognizing the role of TspB in serum resistance may lead to a better understanding of why strains that carrytspBgenes are associated with invasive meningococcal disease.

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Antibody Binding and Complement-Mediated Killing of Invasive Haemophilus influenzae Isolates from Spain, Portugal, and the Netherlands

Infection and Immunity ◽

10.1128/iai.00454-20 ◽

2020 ◽

Vol 88 (10) ◽

Author(s):

Elena Dudukina ◽

Laura de Smit ◽

Giel J. A. Verhagen ◽

Arie van de Ende ◽

José María Marimón ◽

...

Keyword(s):

The Netherlands ◽

Haemophilus Influenzae ◽

Human Serum ◽

Opportunistic Pathogen ◽

Invasive Disease ◽

Chronic Obstructive ◽

Bacterial Survival ◽

Upper Respiratory Tract ◽

Content Type ◽

Bacterial Surface

ABSTRACT Haemophilus influenzae is a Gram-negative bacterium that can be classified into typeable (types a through f) and nontypeable (NTHi) groups. This opportunistic pathogen asymptomatically colonizes the mucosal epithelium of the upper respiratory tract, from where it spreads to other neighboring regions, potentially leading to disease. Infection with NTHi can cause otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and pneumonia, but it is increasingly causing invasive disease, including bacteremia and meningitis. Invasive NTHi strains are more resistant to complement-mediated killing. However, the mechanisms of complement resistance have never been studied in large numbers of invasive NTHi strains. In this study, we determined the relationship between binding of IgG or IgM and the bacterial survival in normal human serum for 267 invasive H. influenzae strains from Spain, Portugal, and the Netherlands, of which the majority (200 [75%]) were NTHi. NTHi bacteria opsonized with high levels of IgM had the lowest survival in human serum. IgM binding to the bacterial surface, but not IgG binding, was shown to be associated with complement-mediated killing of NTHi strains. We conclude that evasion of IgM binding by NTHi strains increases survival in blood, thereby potentially contributing to their ability to cause severe invasive diseases.

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Role of sapA and yfgA in Susceptibility to Antibody-Mediated Complement-Dependent Killing and Virulence of Salmonella enterica Serovar Typhimurium

Infection and Immunity ◽

10.1128/iai.00419-17 ◽

2017 ◽

Vol 85 (9) ◽

Cited By ~ 1

Author(s):

Edna M. Ondari ◽

Jennifer N. Heath ◽

Elizabeth J. Klemm ◽

Gemma Langridge ◽

Lars Barquist ◽

...

Keyword(s):

Salmonella Enterica ◽

Protein Translocation ◽

Invasive Disease ◽

Immune Serum ◽

Insertion Mutant ◽

Content Type ◽

Serovar Typhimurium ◽

Serum Killing ◽

Increased Sensitivity

ABSTRACT The ST313 pathovar of Salmonella enterica serovar Typhimurium contributes to a high burden of invasive disease among African infants and HIV-infected adults. It is characterized by genome degradation (loss of coding capacity) and has increased resistance to antibody-dependent complement-mediated killing compared with enterocolitis-causing strains of S. Typhimurium. Vaccination is an attractive disease-prevention strategy, and leading candidates focus on the induction of bactericidal antibodies. Antibody-resistant strains arising through further gene deletion could compromise such a strategy. Exposing a saturating transposon insertion mutant library of S. Typhimurium to immune serum identified a repertoire of S. Typhimurium genes that, when interrupted, result in increased resistance to serum killing. These genes included several involved in bacterial envelope biogenesis, protein translocation, and metabolism. We generated defined mutant derivatives using S. Typhimurium SL1344 as the host. Based on their initial levels of enhanced resistance to killing, yfgA and sapA mutants were selected for further characterization. The S. Typhimurium yfgA mutant lost the characteristic Salmonella rod-shaped appearance, exhibited increased sensitivity to osmotic and detergent stress, lacked very long lipopolysaccharide, was unable to invade enterocytes, and demonstrated decreased ability to infect mice. In contrast, the S. Typhimurium sapA mutants had similar sensitivity to osmotic and detergent stress and lipopolysaccharide profile and an increased ability to infect enterocytes compared with the wild type, but it had no increased ability to cause in vivo infection. These findings indicate that increased resistance to antibody-dependent complement-mediated killing secondary to genetic deletion is not necessarily accompanied by increased virulence and suggest the presence of different mechanisms of antibody resistance.

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The Streptococcuspneumoniae Capsule Inhibits Complement Activity and Neutrophil Phagocytosis by Multiple Mechanisms

Infection and Immunity ◽

10.1128/iai.00881-09 ◽

2009 ◽

Vol 78 (2) ◽

pp. 704-715 ◽

Cited By ~ 206

Author(s):

Catherine Hyams ◽

Emilie Camberlein ◽

Jonathan M. Cohen ◽

Katie Bax ◽

Jeremy S. Brown

Keyword(s):

Normal Serum ◽

Classical Pathway ◽

Limited Data ◽

C Reactive Protein ◽

Fcγ Receptors ◽

Complement Activity ◽

Bacterial Surface ◽

Antibody Recognition ◽

Reactive Protein ◽

Classical Complement

ABSTRACT The Streptococcus pneumoniae capsule is vital for virulence and may inhibit complement activity and phagocytosis. However, there are only limited data on the mechanisms by which the capsule affects complement and the consequences for S. pneumoniae interactions with phagocytes. Using unencapsulated serotype 2 and 4 S. pneumoniae mutants, we have confirmed that the capsule has several effects on complement activity. The capsule impaired bacterial opsonization with C3b/iC3b by both the alternative and classical complement pathways and also inhibited conversion of C3b bound to the bacterial surface to iC3b. There was increased binding of the classical pathway mediators immunoglobulin G (IgG) and C-reactive protein (CRP) to unencapsulated S. pneumoniae, indicating that the capsule could inhibit classical pathway complement activity by masking antibody recognition of subcapsular antigens, as well as by inhibiting CRP binding. Cleavage of serum IgG by the enzyme IdeS reduced C3b/iC3b deposition on all of the strains, but there were still marked increases in C3b/iC3b deposition on unencapsulated TIGR4 and D39 strains compared to encapsulated strains, suggesting that the capsule inhibits both IgG-mediated and IgG-independent complement activity against S. pneumoniae. Unencapsulated strains were more susceptible to neutrophil phagocytosis after incubation in normal serum, normal serum treated with IdeS, complement-deficient serum, and complement-deficient serum treated with IdeS or in buffer alone, suggesting that the capsule inhibits phagocytosis mediated by Fcγ receptors, complement receptors, and nonopsonic receptors. Overall, these data show that the S. pneumoniae capsule affects multiple aspects of complement- and neutrophil-mediated immunity, resulting in a profound inhibition of opsonophagocytosis.

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Pleiotropic Effects of Cell Wall Amidase LytA on Streptococcus pneumoniae Sensitivity to the Host Immune Response

Infection and Immunity ◽

10.1128/iai.02811-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 591-603 ◽

Cited By ~ 25

Author(s):

Elisa Ramos-Sevillano ◽

Ana Urzainqui ◽

Susana Campuzano ◽

Miriam Moscoso ◽

Fernando González-Camacho ◽

...

Keyword(s):

Immune Response ◽

Cell Wall ◽

Streptococcus Pneumoniae ◽

Complement Activation ◽

Host Immune Response ◽

Factor H ◽

Pleiotropic Effects ◽

Complement C3 ◽

Classical Pathway ◽

Content Type

The complement system is a key component of the host immune response for the recognition and clearance ofStreptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein toS. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for thelytA plydouble mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia.

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Streptococcus pneumoniae TIGR4 Phase-Locked Opacity Variants Differ in Virulence Phenotypes

mSphere ◽

10.1128/msphere.00386-17 ◽

2017 ◽

Vol 2 (6) ◽

Cited By ~ 17

Author(s):

Melissa B. Oliver ◽

Ankita Basu Roy ◽

Ranjit Kumar ◽

Elliot J. Lefkowitz ◽

W. Edward Swords

Keyword(s):

Gene Expression ◽

Recent Work ◽

Bacterial Species ◽

Phase Variation ◽

Nasopharyngeal Colonization ◽

Content Type ◽

Bacterial Surface ◽

Variable Expression ◽

Dna Methylase ◽

Single Allele

ABSTRACT A growing number of bacterial species undergo epigenetic phase variation due to variable expression or specificity of DNA-modifying enzymes. For pneumococci, this phase variation has long been appreciated as being revealed by changes in colony opacity, which are reflected in changes in expression or accessibility of factors on the bacterial surface. Recent work showed that recombination-generated variation in alleles of the HsdS DNA methylase specificity subunit mediated pneumococcal phase variation. We generated phase-locked populations of S. pneumoniae TIGR4 expressing a single nonvariant hsdS allele and observed significant differences in gene expression and virulence. These results highlight the importance of focused pathogenesis studies within specific phase types. Moreover, the generation of single-allele hsdS constructs will greatly facilitate such studies. Streptococcus pneumoniae (pneumococcus) is a leading human pathogen that can cause serious localized and invasive diseases. Pneumococci can undergo a spontaneous and reversible phase variation that is reflected in colony opacity and which allows the population to adapt to different host environments. Generally, transparent variants are adapted for nasopharyngeal colonization, whereas opaque variants are associated with invasive disease. In recent work, colony phase variation was shown to occur by means of recombination events to generate multiple alleles of the hsdS targeting domain of a DNA methylase complex, which mediates epigenetic changes in gene expression. A panel of isogenic strains were created in the well-studied S. pneumoniae TIGR4 background that are “locked” in the transparent (n = 4) or opaque (n = 2) colony phenotype. The strains had significant differences in colony size which were stable over multiple passages in vitro and in vivo. While there were no significant differences in adherence for the phase-locked mutant strains to immortalized epithelial cells, biofilm formation and viability were reduced for the opaque variants in static assays. Nasopharyngeal colonization was stable for all strains, but the mortality rates differed between them. Transcript profiling by transcriptome sequencing (RNA-seq) analyses revealed that the expression levels of certain virulence factors were increased in a phase-specific manner. As epigenetic regulation of phase variation (often referred to as "phasevarion") is emerging as a common theme for mucosal pathogens, these results serve as a model for future studies of host-pathogen interactions. IMPORTANCE A growing number of bacterial species undergo epigenetic phase variation due to variable expression or specificity of DNA-modifying enzymes. For pneumococci, this phase variation has long been appreciated as being revealed by changes in colony opacity, which are reflected in changes in expression or accessibility of factors on the bacterial surface. Recent work showed that recombination-generated variation in alleles of the HsdS DNA methylase specificity subunit mediated pneumococcal phase variation. We generated phase-locked populations of S. pneumoniae TIGR4 expressing a single nonvariant hsdS allele and observed significant differences in gene expression and virulence. These results highlight the importance of focused pathogenesis studies within specific phase types. Moreover, the generation of single-allele hsdS constructs will greatly facilitate such studies.

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The Capsular Serotype of Streptococcus pneumoniae Is More Important than the Genetic Background for Resistance to Complement

Infection and Immunity ◽

10.1128/iai.00740-10 ◽

2010 ◽

Vol 78 (12) ◽

pp. 5262-5270 ◽

Cited By ~ 36

Author(s):

Merit Melin ◽

Krzysztof Trzciński ◽

Seppo Meri ◽

Helena Käyhty ◽

Merja Väkeväinen

Keyword(s):

Streptococcus Pneumoniae ◽

Invasive Disease ◽

Relative Effect ◽

Clinical Isolates ◽

Complement C3 ◽

Bacterial Surface ◽

Complement Resistance ◽

Opsonophagocytic Killing ◽

Complement Deposition ◽

Phagocytic Killing

ABSTRACT The polysaccharide capsule of Streptococcus pneumoniae inhibits phagocytic killing by innate immune mechanisms. Certain serotypes are associated with invasive disease while others with a nasopharyngeal carriage. The invasiveness of serotypes may partly be explained by ability to resist deposition of complement (C3) on the bacterial surface and consequent opsonophagocytic killing. In our previous studies, we observed that clinical isolates of serotypes 1 and 5, which are rarely detected in asymptomatic carriage, were resistant to complement deposition and opsonophagocytosis, whereas serotypes 6B and 23F, both common in carriage, were more sensitive to deposition of C3 and opsonophagocytic killing. However, presence of significant variation in C3 deposition between isolates of the same serotype indicated that factors other than the capsule also affect complement resistance. To distinguish the relative effect of the capsular serotype and other virulence factors on C3 deposition, we compared capsule-switched mutants prepared in genetic backgrounds of pneumococcal strains TIGR4, 603, and 618. Clinical isolates which had the same multilocus sequence type but expressed different serotypes were also compared. We found that the serotype had a significant impact on complement resistance and that the more resistant the strain was to complement, the higher was the concentration of polysaccharide-specific antibodies required for opsonophagocytic killing. Comparison of strains expressing the same capsular polysaccharides in the different genetic backgrounds and various capsular mutants of the same strain suggests that while the genotype affects complement resistance, the serotype is the most important determinant. Differences between serotypes were more significant than the differences between strains.

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Phase-Variable Changes in the Position of O-Methyl Phosphoramidate Modifications on the Polysaccharide Capsule of Campylobacter jejuni Modulate Serum Resistance

Journal of Bacteriology ◽

10.1128/jb.00027-17 ◽

2017 ◽

Vol 199 (14) ◽

Cited By ~ 14

Author(s):

Brittany Pequegnat ◽

Renee M. Laird ◽

Cheryl P. Ewing ◽

Christina L. Hill ◽

Eman Omari ◽

...

Keyword(s):

Human Serum ◽

Campylobacter Jejuni ◽

Phase Variation ◽

Normal Human Serum ◽

Phase Variable ◽

Content Type ◽

Enhanced Sensitivity ◽

Serum Killing ◽

Human Sera ◽

Normal Human

ABSTRACT Campylobacter jejuni polysaccharide capsules (CPS) are characterized by the presence of nonstoichiometric O-methyl phosphoramidate (MeOPN) modifications. The lack of stoichiometry is due to phase variation at homopolymeric tracts within the MeOPN transferase genes. C. jejuni strain 81-176 contains two MeOPN transferase genes and has been shown previously to contain MeOPN modifications at the 2 and 6 positions of the galactose (Gal) moiety in the CPS. We demonstrate here that one of the two MeOPN transferases, encoded by CJJ81176_1435, is bifunctional and is responsible for the addition of MeOPN to both the 2 and the 6 positions of Gal. A new MeOPN at the 4 position of Gal was observed in a mutant lacking the CJJ81176_1435 transferase and this was encoded by the CJJ81176_1420 transferase. During routine growth of 81-176, the CJJ81176_1420 transferase was predominantly in an off configuration, while the CJJ81176_1435 transferase was primarily on. However, exposure to normal human serum selected for cells expressing the CJJ81176_1420 transferase. MeOPN modifications appear to block binding of naturally occurring antibodies to the 81-176 CPS. The absence of MeOPN-4-Gal resulted in enhanced sensitivity to serum killing, whereas the loss of MeOPN-2-Gal and MeOPN-6-Gal resulted in enhanced resistance to serum killing, perhaps by allowing more MeOPN to be put onto the 4 position of Gal. IMPORTANCE Campylobacter jejuni undergoes phase variation in genes encoding surface antigens, leading to the concept that a strain of this organism consists of multiple genotypes that are selected for fitness in various environments. Methyl phosphoramidate modifications on the capsule of C. jejuni block access of preexisting antibodies in normal human sera to the polysaccharide chain, thus preventing activation of the classical arm of the complement cascade. We show that the capsule of strain 81-176 contains more sites of MeOPN modifications than previously recognized and that one site, on the 4 position of galactose, is more critical to complement resistance than the others. Exposure to normal human serum selects for variants in the population expressing this MeOPN modification.

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