Mediates Serum Resistance Research Articles

Outer membrane vesicle-mediated serum protection in Aggregatibacter actinomycetemcomitans

ABSTRACT Aggregatibacter actinomycetemcomitans belongs to the HACEK group of fastidious Gram-negative organisms, a recognized cause of infective endocarditis. A. actinomycetemcomitans is also implicated in periodontitis, with rapid progress in adolescents. We recently demonstrated that the major outer membrane protein, OmpA1 was critical for serum survival of the A. actinomycetemcomitans serotype a model strain, D7SS, and that the paralogue, OmpA2 could operate as a functional homologue to OmpA1 in mediating serum resistance. In the present work, an essentially serum-sensitive ompA1 ompA2 double mutant A. actinomycetemcomitans strain derivative was exploited to elucidate if A. actinomycetemcomitans OMVs can contribute to bacterial serum resistance. Indeed, supplementation of OMVs resulted in a dose-dependent increase of the survival of the serum-sensitive strain in incubations in 50% normal human serum (NHS). Whereas neither OmpA1 nor OmpA2 was required for the OMV-mediated serum protection, OMVs and LPS from an A. actinomycetemcomitans strain lacking the LPS O-antigen polysaccharide part were significantly impaired in protecting D7SS ompA1 ompA2. Our results using a complement system screen assay support a model where A. actinomycetemcomitans OMVs can act as a decoy, which can trigger complement activation in an LPS-dependent manner, and consume complement components to protect serum-susceptible bacterial cells.

Expression of Rickettsia Adr2 protein in E. coli is sufficient to promote resistance to complement-mediated killing, but not adherence to mammalian cells.

Bacteria exposed to host serum are subject to the antibacterial effects to the complement system. However, pathogenic microorganisms have evolved mechanisms of evading this immune attack. We have previously demonstrated that at least two R. conorii antigens, RC1281/Adr1 and OmpB β-peptide, contribute to the evasion of complement-mediated killing by binding the complement regulatory proteins vitronectin and factor H. RC1282/Adr2, a protein related to Adr1, is predicted to share similar structural features, suggesting that this protein may also contribute to evasion of complement-mediated killing. Interestingly, the R. prowazekii Adr1 and Adr2(RP828) proteins were originally found to interact with host cell surface proteins, suggesting their putative roles as adhesins in this pathogenic rickettsial species. In this study, we expressed both R. conorii and R. prowazekii Adr2 on the surface of a non-adherent, serum-sensitive strain of E. coli to examine the potential role of this protein to mediate evasion of complement-mediated killing and adherence to host cells. We demonstrate that, similar to R. conorii Adr1, R. conorii and R. prowazekii Adr2 are sufficient to mediate serum resistance and to promote interaction with the host complement regulator vitronectin. Furthermore, we demonstrate that expression of Adr2 in a non-adherent strain of E. coli is insufficient to mediate adherence to cultured mammalian endothelial cells. Together, our data demonstrate that the R. conorii and R. prowazekii Adr2 protein does not participate in the interactions with mammalian cells, but rather, participates in the evasion of killing by complement.

Yersinia enterocolitica YadA Mediates Complement Evasion by Recruitment and Inactivation of C3 Products

Yersinia adhesin A (YadA) is a major virulence factor of Yersinia enterocolitica. YadA mediates host cell binding and autoaggregation and protects the pathogen from killing by the complement system. Previous studies demonstrated that YadA is the most important single factor mediating serum resistance of Y. enterocolitica, presumably by binding C4b binding protein (C4BP) and factor H, which are both complement inhibitors. Factor H acts as a cofactor for factor I-mediated cleavage of C3b into the inactive form iC3b and thus prevents formation of inflammatory effector compounds and the terminal complement complex. In this study, we challenged the current direct binding model of factor H to YadA and show that Y. enterocolitica YadA recruits C3b and iC3b directly, without the need of an active complement cascade or additional serum factors. Enhanced binding of C3b does not decrease survival of YadA-expressing Yersiniae because C3b becomes readily inactivated by factor H and factor I. Binding of factor H to YadA is greatly reduced in the absence of C3. Experiments using Yersinia lacking YadA or expressing YadA with reduced trimeric stability clearly demonstrate that both the presence and full trimeric stability of YadA are essential for complement resistance. A novel mechanism of factor H binding is presented in which YadA exploits recruitment of C3b or iC3b to attract large amounts of factor H. As a consequence, formation of the terminal complement complex is limited and bacterial survival is enhanced. These findings add a new aspect of how Y. enterocolitica effectively evades the host complement system.

The Rickettsial OmpB β-Peptide of Rickettsia conorii Is Sufficient To Facilitate Factor H-Mediated Serum Resistance

Pathogenic species of the spotted fever group Rickettsia are subjected to repeated exposures to the host complement system through cyclic infections of mammalian and tick hosts. The serum complement machinery is a formidable obstacle for bacteria to overcome if they endeavor to endure this endozoonotic cycle. We have previously demonstrated that that the etiologic agent of Mediterranean spotted fever, Rickettsia conorii, is susceptible to complement-mediated killing only in the presence of specific monoclonal antibodies. We have also shown that in the absence of particular neutralizing antibody, R. conorii is resistant to the effects of serum complement. We therefore hypothesized that the interactions between fluid-phase complement regulators and conserved rickettsial outer membrane-associated proteins are critical to mediate serum resistance. We demonstrate here that R. conorii specifically interacts with the soluble host complement inhibitor, factor H. Depletion of factor H from normal human serum renders R. conorii more susceptible to C3 and membrane attack complex deposition and to complement-mediated killing. We identified the autotransporter protein rickettsial OmpB (rOmpB) as a factor H ligand and further demonstrate that the rOmpB β-peptide is sufficient to mediate resistance to the bactericidal properties of human serum. Taken together, these data reveal an additional function for the highly conserved rickettsial surface cell antigen, rOmpB, and suggest that the ability to evade complement-mediated clearance from the hematogenous circulation is a novel virulence attribute for this class of pathogens.

Functional Recruitment of the Human Complement Inhibitor C4BP to Yersinia pseudotuberculosis Outer Membrane Protein Ail

Ail is a 17-kDa chromosomally encoded outer membrane protein that mediates serum resistance (complement resistance) in the pathogenic Yersiniae (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis). In this article, we demonstrate that Y. pseudotuberculosis Ail from strains PB1, 2812/79, and YPIII/pIB1 (serotypes O:1a, O:1b, and O:3, respectively) can bind the inhibitor of the classical and lectin pathways of complement, C4b-binding protein (C4BP). Binding was observed irrespective of serotype tested and independently of YadA, which is the primary C4BP receptor of Y. enterocolitica. Disruption of the ail gene in Y. pseudotuberculosis resulted in loss of C4BP binding. Cofactor assays revealed that bound C4BP is functional, because bound C4BP in the presence of factor I cleaved C4b. In the absence of YadA, Ail conferred serum resistance to strains PB1 and YPIII, whereas serum resistance was observed in strain 2812/79 in the absence of both YadA and Ail, suggesting additional serum resistance factors. Ail from strain YPIII/pIB1 alone can mediate serum resistance and C4BP binding, because its expression in a serum-sensitive laboratory strain of Escherichia coli conferred both of these phenotypes. Using a panel of C4BP mutants, each deficient in a single complement control protein domain, we observed that complement control protein domains 6-8 are important for binding to Ail. Binding of C4BP was unaffected by increasing heparin or salt concentrations, suggesting primarily nonionic interactions. These results indicate that Y. pseudotuberculosis Ail recruits C4BP in a functional manner, facilitating resistance to attack from complement.

Characterization of Diverse Subvariants of the Meningococcal Factor H (fH) Binding Protein for Their Ability To Bind fH, To Mediate Serum Resistance, and To Induce Bactericidal Antibodies

Neisseria meningitidis is a commensal of the human nasopharynx but is also a major cause of septicemia and meningitis. The meningococcal factor H binding protein (fHbp) binds human factor H (fH), enabling downregulation of complement activation on the bacterial surface. fHbp is a component of two serogroup B meningococcal vaccines currently in clinical development. Here we characterize 12 fHbp subvariants for their level of surface exposure and ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies. Flow cytometry and Western analysis revealed that all strains examined expressed fHbp on their surface to different extents and bound fH in an fHbp-dependent manner. However, differences in fH binding did not always correlate with the level of fHbp expression, indicating that this is not the only factor affecting the amount of fH bound. To overcome the issue of strain variability in fHbp expression, the MC58ΔfHbp strain was genetically engineered to express different subvariants from a constitutive heterologous promoter. These recombinant strains were characterized for fH binding, and the data confirmed that each subvariant binds different levels of fH. Surface plasmon resonance revealed differences in the stability of the fHbp-fH complexes that ranged over 2 orders of magnitude, indicating that differences in residues between and within variant groups can influence fH binding. Interestingly, the level of survival in human sera of recombinant MC58 strains expressing diverse subvariants did not correlate with the level of fH binding, suggesting that the interaction of fHbp with fH is not the only function of fHbp that influences serum resistance. Furthermore, cross-reactive bactericidal activity was seen within each variant group, although the degree of activity varied, suggesting that amino acid differences within each variant group influence the bactericidal antibody response.

Crystallographic characterization of the passenger domain of theBordetellaautotransporter BrkA

Autotransporters (ATs) are proteins that deliver effectors (the passenger domain) to the surface of Gram-negative bacteria by the type V secretion pathway. The passenger domain of BrkA, a Bordetella pertussis autotransporter mediating serum resistance and adherence, was cloned in a pET expression system and overexpressed in Escherichia coli. The gene product was correctly refolded, purified to homogeneity and crystallized. The crystals diffracted to 2.8 A resolution. The space group was assumed to be P4(1)2(1)2, with unit-cell parameters a = b = 108.19, c = 115.35 A.

CspA-Mediated Binding of Human Factor H Inhibits Complement Deposition and Confers Serum Resistance in Borrelia burgdorferi

Borrelia burgdorferi has developed efficient mechanisms for evading the innate immune response during mammalian infection and has been shown to be resistant to the complement-mediated bactericidal activity of human serum. It is well recognized that B. burgdorferi expresses multiple lipoproteins on its surface that bind the human complement inhibitors factor H and factor H-like protein 1 (FH/FHL-1). The binding of FH/FHL-1 on the surface of B. burgdorferi is thought to enhance its ability to evade serum-mediated killing during the acute phase of infection. One of the key B. burgdorferi FH/FHL-1 binding proteins identified thus far was designated CspA. While it is known that CspA binds FH/FHL-1, it is unclear how the interaction between CspA and FH/FHL-1 specifically enhances serum resistance. To better understand how CspA mediates serum resistance in B. burgdorferi, we inactivated cspA in a virulent strain of B. burgdorferi. An affinity ligand blot immunoassay and indirect immunofluorescence revealed that the CspA mutant does not efficiently bind human FH to its surface. Consistent with the lack of FH binding, the CspA mutant was also highly sensitive to killing by human serum. Additionally, the deposition of complement components C3, C6, and C5b-9 was enhanced on the surface of the CspA mutant compared to that of the wild-type strain. The combined data lead us to conclude that the CspA-mediated binding of human FH confers serum resistance by directly inhibiting complement deposition on the surface of B. burgdorferi.

Contribution of interactions between complement inhibitor C4b-binding protein and pathogens to their ability to establish infection with particular emphasis on Neisseria gonorrhoeae

Complement activation and resulting opsonisation with C3b form key arms of the innate immune defense against infections. However, a wide variety of pathogens subvert complement attack by binding host complement inhibitors, which results in diminished opsonophagocytosis and killing of bacteria by lysis. Human C4b-binding protein (C4BP) binds Neisseria gonorrhoeae and Streptococcus pyogenes, both uniquely human pathogens. This binding specificity is circumvented by other bacterial species, which bind C4BP from numerous mammalian hosts that they infect. Binding of C4BP to Neisseria is mediated by outer membrane porin proteins and appears to be one of the main factors mediating serum resistance. Targeting C4BP binding sites on bacterial surfaces with vaccine-induced antibodies may block binding of C4BP and enhance a common vaccine design strategy that depends on the generation of complement-dependent bactericidal and opsonophagocytic antibody activities.

Signature-Tagged Mutagenesis of Klebsiella pneumoniae To Identify Genes That Influence Biofilm Formation on Extracellular Matrix Material

Klebsiella pneumoniae causes urinary tract infections, respiratory tract infections, and septicemia in susceptible individuals. Strains of Klebsiella frequently produce extended-spectrum beta-lactamases, and infections with these strains can lead to relatively high mortality rates (approximately 15%). Other virulence factors include production of an antiphagocytic capsule and outer membrane lipopolysaccharide (LPS), which mediates serum resistance, as well as fimbriae on the surface of the bacteria. Type 1 fimbriae mediate adherence to many types of epithelial cells and may facilitate adherence of the bacteria to the bladder epithelium. Type 3 fimbriae can bind in vitro to the extracellular matrix of urinary and respiratory tissues, suggesting that they mediate binding to damaged epithelial surfaces. In addition, type 3 fimbriae are required for biofilm formation by Klebsiella pneumoniae on plastics and human extracellular matrix; thus, they may facilitate the formation of treatment-resistant biofilm on indwelling plastic devices, such as catheters and endotracheal tubing. The presence of these devices may cause tissue damage, allowing Klebsiella to grow as a biofilm on exposed tissue basement membrane components. Though in vivo biofilm growth may be an important step in the infection process, little is known about the genetic factors required for biofilm formation by Klebsiella pneumoniae. Thus, we performed signature-tagged mutagenesis to identify factors produced by K. pneumoniae strain 43816 that are required for biofilm formation. We identified mutations in the cps capsule gene cluster, previously unidentified transcriptional regulators, fimbrial, and sugar phosphotransferase homologues, as well as genetic loci of unknown function, that affect biofilm formation.

A novel interaction of outer membrane protein A with C4b binding protein mediates serum resistance of Escherichia coli K1.

Escherichia coli is an important pathogen that causes meningitis in neonates. The development of bacteremia preceding the traversal across the blood-brain barrier is a prerequisite for this pathogen that obviously must survive the bactericidal activity of serum. Here we report that outer membrane protein A (OmpA) of Escherichia coli contributes to serum resistance by binding to C4b binding protein (C4bp), a complement fluid phase regulator. C4bp contains seven identical alpha-chains and one beta-chain linked together with disulfide bridges. We found that OmpA binds the alpha-chain of C4bp, which is composed of eight homologous complement control protein (CCP) modules. Binding studies using mutants of recombinant C4bp that lack one CCP at a time suggest that CCP3 is the major site of interaction with OmpA. Furthermore, we demonstrate that the N terminus of OmpA interacts with C4bp. Binding of C4bp to OmpA is not significantly inhibited in the presence of either C4b or heparin and is not salt sensitive, implying that it is hydrophobic in nature, suggesting a novel interaction between OmpA and C4bp. A compelling observation in this study is that synthetic peptides corresponding to CCP3 sequences block the binding of C4bp to OmpA and also significantly enhance serum bactericidal activity.

Antibodies to BrkA augment killing of Bordetella pertussis

BrkA is a Bvg-regulated Bordetella pertussis protein that mediates serum resistance and adherence. It shares sequence identity with another B. pertussis virulence factor called pertactin, and it is a member of the diverse group of proteins found in Gram-negative bacteria that are secreted by an autotransporter mechanism. Sera, either from individuals who have been vaccinated with acellular pertussis vaccines, or from individuals who have no re-collection of recent infection with B. pertussis fail to kill wild-type B. pertussis, but kill brkA mutant strains very well. We examined whether BrkA could be neutralised in serum fitting this profile. BrkA is synthesised as a 103 kDa precursor that is processed into a surface-associated N-terminal 73 kDa passenger domain, and an outer-membrane embedded C-terminal 30 kDa transporter moiety. Polyclonal antibodies were raised to a recombinant, re-folded histidine-tagged fusion protein representing the 73 kDa passenger region. These anti-BrkA antibodies were shown to boost the existing bactericidal capacity of human serum against B. pertussis by neutralising BrkA.

A novel sialic acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae.

Factor H (fH), a key alternative complement pathway regulator, is a cofactor for factor I-mediated cleavage of C3b. fH consists of 20 short consensus repeat (SCR) domains. Sialic acid binding domains have previously been localized to fH SCRs 6-10 and 13. To examine fH binding on a sialylated microbial surface, we grew Neisseria gonorrhoeae in the presence of 5'-cytidinemonophospho-N-acetylneuraminic acid, which sialylates lipooligosaccharide and converts to serum resistance gonococci previously sensitive to nonimmune serum killing. fH domains necessary for binding sialylated gonococci were determined by incubating organisms with recombinant human fH (rH) and nine mutant rH molecules (deletions spanning the entire fH molecule). rH and all mutant rH molecules that contained SCRs 16-20 bound to the sialylated strain; no mutant molecule bound to serum-sensitive nonsialylated organisms. Sialic acid was demonstrated to be the fH target by flow cytometry that showed a fourfold increase in fH binding that was reversed by neuraminidase-mediated cleavage of sialic acid off gonococci. Functional specificity of fH was confirmed by decreased total C3 binding and almost complete conversion to iC3b on sialylated gonococci. Sialic acid can therefore bind fH uniquely through SCRs 16-20. This blocks complement pathway activation for N. gonorrhoeae at the level of C3.

Surface exclusion specificity of the TraT lipoprotein is determined by single alterations in a five-amino-acid region of the protein.

The TraT protein is a highly cell-surface-exposed lipoprotein specified by F-like plasmids that confers serum resistance and blocks the conjugative transfer of plasmids to cells bearing identical or closely related plasmids, a process known as surface exclusion. The TraT protein specified by the antibiotic-resistance plasmid R6-5 was purified to apparent homogeneity. When added to mating mixtures, TraT blocked the transfer of plasmids belonging to Surface Exclusion Group IV (Sfx IV) but had no significant effect on the transfer of plasmids belonging to other groups. Additionally, the purified protein has a protective effect on bacterial cells incubated in serum, indicating that it does not have to be located on the cell surface to mediate serum resistance. To localize regions of the protein that were responsible for surface exclusion specificity, the amino acid sequence of the TraT protein specified by CoIB2-K98 (Sfx II) was determined by cloning and sequencing of the corresponding gene. Comparison of the derived sequence with those of the F and R100-1 proteins indicated that surface exclusion specificity of TraT is determined by single alterations in a five-amino-acid region (residues 116-120). This was confirmed by segment swapping experiments in which the specificity of the R6-5 TraT protein (Sfx IV) was switched to that of the CoIB2-K98 protein (Sfx II). Our results suggest that the region defined by residues 116-120 is located on the external face of the outer membrane and interacts specifically with the donor cell in surface exclusion.

The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure–function relationships in the TraT protein

The TraT protein is a surface-exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion. The structure and function of the TraT protein determined by plasmid R6-5 was probed by genetic insertion of a foreign antigenic determinant, the C3 epitope of polio virus, at residues 61, 125, 180, 200 or 216 of the protein. The chimaeric proteins were transported to the outer membrane and, in three cases, immunoassays with an anti-C3 monoclonal antibody indicated that the C3 epitope was exposed on the cell surface. Three of the hybrids, with insertions at residues 125, 180 and 200, assembled into the trypsin-resistant oligomeric form characteristic of the wild-type protein, which suggested that these regions are not involved in TraT subunit:subunit interactions. Additionally, the hybrid protein carrying the C3 epitope at position 180 functioned in a genetic suppression assay and retained partial surface-exclusion activity. Thus, its localization, folding and organization does not appear to be grossly altered from that of the wild-type protein. Applications of the protein for the transport of foreign antigenic determinants to the cell surface are discussed.

Adaptive alterations in the outer membrane of gram-negative bacteria during human infection.

The outer membrane of gram-negative bacteria is a dynamic structure that is capable of altering its ultrastructure and chemistry in order to adapt to changes in its environment. In human infections, outer-membrane alterations are known to play a role in mediating serum resistance, iron uptake, adaptation by Pseudomonas aeruginosa to colonization of the lungs of cystic fibrosis patients, and adaptive resistance to the polymyxin and aminoglycoside antibiotics. This adaptive antibiotic resistance is due to alterations in the cation binding sites within the outer membrane so that these cationic antibiotics can no longer penetrate through the membrane effectively. Adaptive resistance is not stable but is maintained only in the continued presence of the antibiotic. Hence, the role that this type of resistance to cationic antibiotics plays in clinical treatment of human infections remains inadequately assessed.

Mediation of serum resistance in Salmonella typhimurium by an 11-kilodalton polypeptide encoded by the cryptic plasmid.

A cosmid bank of the DNA (including cryptic plasmid DNA) of a virulent strain of Salmonella typhimurium was prepared in Escherichia coli K12, and clones that contained cryptic plasmid DNA were detected by probing. Two such clones expressed a new outer membrane protein of 11 kilodaltons (kDa) and were serum resistant (E. coli K12 is serum sensitive). The gene encoding the 11-kDa protein was subcloned in a 2.1-kilobase fragment and shown to mediate serum resistance in both E. coli K12 and a cryptic plasmid-free (serum-sensitive) strain of S. typhimurium. The cryptic plasmid-free S. typhimurium strain did not express normal lipopolysaccharide, but introduction of the 11-kDa protein gene into the strain rendered the strain serum resistant without restoration of normal lipopolysaccharide synthesis. The 11-kDa protein gene was not sufficient to restore either macrophage resistance or virulence to a cryptic plasmid-free strain of S. typhimurium.

Contributions oftraTandissgenes to the serum resistance phenotype of plasmid ColV2-K94

A genetic determinant for serum resistance, designated iss, has been found previously on the colicinogenic plasmid ColV2-K94. In this work we have identified a second serum resistance gene, traT, on ColV2-K94. The serum resistance mediated by derivatives of ColV2-K94 was due to presence of one or both of the iss and traT genes. Plasmid pWS12 (TraT+ Iss+) contained the kanamycin (Km) resistance transposon Tn903 inserted near the origin of replication of ColV2-K94, and plasmids pWS15 (TraT+), pWS16 (TraT+) and pWS18 (TraT+ Iss+) were deletion derivatives of pWS12 constructed in vitro and in vivo. pWS12 and pWS18 conferred a 20-fold increase in relative resistance to 20% guinea pig serum when introduced into the serum-susceptible, genetically defined recA strain of Escherichia coli K-12, AB2463. Plasmids pWS15 and pWS16, from which iss had been deleted, still conferred 5-fold increases in relative resistance on AB2463. The level of resistance conferred on this strain by the antibiotic resistance plasmid R100–1 (which expresses the traT serum resistance gene) was comparable to that of plasmids pWS15 and pWS16. The 25-kDa traT gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the outer membrane proteins of strain AB2463 carrying ColV2-K94. This protein cross-reacted immunologically with the traT protein expressed by F or R100–1. Our results indicated that both traT and iss are capable of mediating serum resistance in ColV2-K94.