Complement Resistance Research Articles

Analysis of Moraxella catarrhalis by DNA typing: evidence for a distinct subpopulation associated with virulence traits.

Two DNA typing methods, probe-generated restriction fragment length polymorphism analysis and single-adapter amplified fragment length polymorphism analysis, were used to study the genetic relationships among 90 Moraxella catarrhalis strains. Both methods were found to be highly concordant, generating a dendrogram with 2 main branches. The division of the M. catarrhalis population into 2 subspecies was supported by analysis of the 16S rRNA sequences. Both beta-lactamase-positive and beta-lactamase-negative strains were found in all main branches, suggesting horizontal transfer of the beta-lactamase gene. In contrast, 2 virulence traits, complement resistance and adherence to epithelial cells, were strongly associated with 1 of the 2 subspecies. The branch depth suggested that complement-resistant adherent strains diverged from a common ancestor more recently than did complement-sensitive nonadherent strains. These findings suggest the existence of subpopulations of M. catarrhalis that differ in virulence, and they may have implications for vaccine development.

Surface Antigen Expression and Complement Susceptibility of Differentiated Neuroblastoma Clones

Human neuroblastoma cell lines typically consist of heterogenous subpopulations of cells that are morphologically and biochemically distinct. The cell types are characterized as neuroblastic (N-type), substrate-adherent Schwann-like (S-type), or intermediate (I). These cell types can undergo spontaneous or induced transdifferentiation in vitro. We investigated the complement sensitivity of different neuroblastoma cell lines and of matched sets of cloned N- and S-type neuroblastoma cell lines. Human neuroblastoma cell lines that consisted predominantly of a neuroblastic phenotype were shown to be significantly more susceptible to human complement-mediated lysis than cell lines of other cancer types. Complement sensitivity of neuroblastoma cell lines was correlated with low levels of CD59, decay-accelerating factor, and membrane cofactor protein expression. We found that cloned S-type neuroblastoma cells were much more resistant to complement-mediated lysis than cloned N-type cells. The increased complement resistance of S-type cells was shown to be due to increased expression of membrane-bound complement inhibitors. CD59 was the single most important protein in providing S-type cells with protection from complement lysis. S-type cells were also found to express lower levels of GD2, a target antigen for a complement activating monoclonal antibody currently in clinical trials for neuroblastoma immunotherapy. The ability of S-type cells to evade complement, and the ability of S-type cells to differentiate into the more tumorigenic N-type cells, may represent a mechanism of tumor survival and regrowth, a phenomenon often observed with this cancer.

Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates.

We have previously demonstrated the existence of Klebsiella pneumoniae clinical isolates deficient in the lipopolysaccharide O side chain, the major factor for resistance to complement-mediated killing in this bacterial species. These isolates are complement resistant, and their mechanisms to resist complement were investigated by selecting transposon-generated complement-sensitive mutants. One mutant with a drastically reduced capacity to grow in nonimmune human serum carried the transposon inserted in an open reading frame of a gene cluster involved in capsule synthesis. This mutant produced less capsule, bound more molecules of the complement component C3, and was more sensitive to complement-mediated and opsonophagocytic killings than was the parent strain. Four additional clinical isolates representing four different K serotypes were studied, and results showed that capsular polysaccharide is a major complement resistance factor in these O side chain-deficient isolates.

Molecular Aspects of Complement-mediated Bacterial Killing: PERIPLASMIC CONVERSION OF C9 FROM A PROTOXIN TO A TOXIN

As part of the membrane attack complex complement protein C9 is responsible for direct killing of bacteria. Here we show that in the periplasmic space of an Escherichia coli cell C9 is converted from a protoxin to a toxin by periplasmic conditions missing in spheroplasts. This conversion is independent of the pathway by which C9 enters the periplasm. Both, C9 shocked into the periplasm and plasmid-expressed C9 targeted to the periplasm via a signal sequence are toxic. Toxicity requires disulfide-linked C9 because export into the periplasm of cells defective in disulfide bond synthesis (dsbA and dsbB mutants) is not toxic unless N-acetylcysteine is added externally to promote cystines. A N-terminal fragment, C9[1-144], is not toxic nor is cytoplasmically expressed C9, even in trxB mutants that are able to form disulfide bonds in the cytoplasm. Importantly, expression of full-length C9 in complement-resistant cells has no effect on their viability. Expression and translocation into the periplasm may provide a novel model to identify molecular mechanisms of other bactericidal disulfide-linked proteins and to investigate the nature of bacterial complement resistance.

Complement Resistance-Related Traits among Escherichia coli Isolates from Apparently Healthy Birds and Birds with Colibacillosis

In this study, 294 Escherichia coli isolates from birds with colibacillosis were collected from disease outbreaks throughout the United States and were compared with 75 fecal E. coli isolates of apparently healthy chickens by their possession of several purported virulence genes, resistance to rough-lipopolysaccharide-specific bacteriophages (rLPSr), and elaboration of capsule. Traits were selected for study on the basis of their association with complement resistance. The genes targeted in this study included those encoding colicin V (cvaC) and the outer membrane proteins TraT (traT), OmpA (ompA), and Iss (iss). No significant differences were found between the two groups of isolates in the occurrence of cvaC-, traT-, or ompA-homologous sequences or in rLPSr. Only a few isolates were encapsulated, and the isolates of healthy birds were significantly more likely to be encapsulated than were the isolates of sick birds. However, iss, whether detected through hybridization or amplification, was found in more of the disease-associated isolates than in those of healthy birds. This difference was highly significant. Further, iss sequences were widely distributed among isolates of different serotypes from various avian host species and sites within these hosts. Such results suggest that possession of the iss sequence by an avian E. coli isolate may be a good indicator of that isolate's potential to cause disease. This association warrants further study because iss and the protein it encodes may be useful targets of future colibacillosis control efforts.

Protein kinase activation and protein phosphorylation in Naegleria fowleri amebae in response to normal human serum.

Activation of signal transduction pathways in response to serum complement in Naegleria fowleri amebae was investigated. We examined the activation of protein kinases and changes in the phosphorylation state of proteins in N. fowleri stimulated by normal human serum (NHS). To determine differences in phosphorylation of proteins when amebae were exposed to NHS or heat inactivated serum (HIS) lacking complement, amebae were labeled with [32P] orthophosphate. An increase in phosphorylation of relatively low molecular weight proteins was noted in N. fowleri incubated in NHS with a concomitant decrease in phosphorylation of high molecular mass polypeptides. To investigate whether serine/threonine or tyrosine kinases were stimulated by NHS, amebae were treated with protein kinase inhibitors H7, staurosporine or genistein, prior to serum exposure and examined for susceptibility to complement. Treatment with each of these inhibitors resulted in increased complement lysis. Incubation of N. fowleri with genistein specifically inhibited tyrosine phosphorylation of proteins stimulated by NHS. A tyrosine kinase activity assay using exogenous polyGlu-Tyr substrate demonstrated differential activation of tyrosine kinases in amebae treated with NHS when compared to treatment with HIS. The results suggest that activation of protein kinases and subsequent protein phosphorylation are important in mediating complement resistance in N. fowleri.

Complement resistance of tumor cells: basal and induced mechanisms

Clinical and experimental studies have suggested that complement may play a role in tumor cytotoxicity. However, the efficiency of complement-mediated tumor cell lysis is hampered by various protective mechanisms, which may be divided into two categories: basal and induced mechanisms. The basal mechanisms are spontaneously expressed in cells without a need for prior activation, whereas the induced mechanisms develop in cells subjected to stimulation with cytokines, hormones, drugs or with sublytic doses of complement and other pore-formers. Membrane-associated complement regulatory proteins, such as CD55 (DAF, Decay-Accelerating Factor), CD46 (MCP, Membrane Cofactor Protein), CD35 (CR1, Complement Receptor type 1) and CD59, which serve as an important mechanism of self protection and render autologous cells insensitive to the action of complement, appear to be over-expressed on certain tumors. Furthermore, tumor cells secrete several soluble complement inhibitors. Tumor cells may also express proteases that degrade complement proteins, such as C3, or ecto-protein kinases which can phosphorylate complement components, such as C9. Besides this basal resistance, nucleated cells resist, to some extent, complement damage by removing the membrane attack complexes (MAC) from their surface. Several biochemical pathways, including protein phosphorylation, activation of G-proteins and turnover of phosphoinositides have been implicated in resistance to complement. Calcium ion influx and activation of protein kinase C (PKC) and of mitogen-activated protein kinase (MAPK) have also been demonstrated to be associated with the complement-induced enhanced resistance to lysis. The complete elucidation of the molecular mechanisms involved in basal and induced tumor cell resistance will enable the development of strategies for interfering with these evasion mechanisms and the use of the cytotoxic complement system against tumor cells.

Complement resistance inBorrelia burgdorferistrain 297: outer membrane proteins prevent MAC formation at lysis susceptible sites

Two variants ofBorrelia burgdorferistrain 297, complement-resistant wild-type (WT297) and complement-sensitive mutant (MUT297), were used as a model to study the mechanism of resistance to the alternative complement pathway in this organism. No difference in the quantity of membrane attack complex (MAC) deposition on WT297 and MUT297 was observed after 2 h incubation with normal human serum (NHS), at which time 4% of WT297 and 95% of MUT297 were killed. The polymerization of C9 bound to WT297 and MUT297 was demonstrated by immunoblotting using an anti-C9 polyclonal antibody. Immunofluorescence and thin-section immunoelectron microscopy showed MAC to be diffusely distributed on the outer membrane of both variants. Furthermore, MAC appeared to be tightly bound to the surface of both variants as demonstrated by elution studies. Protease treatment rendered WT297 susceptible to killing by NHS, suggesting that outer membrane proteins may be associated with complement resistance of WT297. One- and two-dimensional gel electrophoreses showed that proteins of 20 and 30 kDa, and 66 kDa were present in WT297 but were absent or sparse in trypsin-treated WT297 and MUT297. Interestingly, immunoblotting using a polyclonal antibody against C3 showed that C3 fragments appeared to bind different acceptors on WT297 than on trypsin-treated WT297, or MUT297. Therefore, the binding of C3 fragments to acceptors on WT297, in contrast to MUT297, may not direct the formation of the MAC to lysis-susceptible sites on the surface of the bacterium, resulting in the complement resistance of WT297.

Phenotypic changes in the lipopolysaccharide of Pseudomonas aeruginosa and Escherichia coli grown in milk-based enteral nutrition solutions.

Previous studies have shown enteral nutritional solutions (ENS) contaminated with large numbers of microorganisms from the environment or gastrointestinal (GI) tract of patients have caused respiratory infections, acute and chronic enteritis, and septicemia. The introduction of "closed" enteral feeding systems has been used to prevent contaminating organisms from entering enteral feeding systems in large numbers. However, there is some discussion as to whether this has been an effective measure in reducing ENS-related infections because there is anecdotal evidence to suggest that disease processes resulting from enteral feeding are still commonplace in the hospital and home. This is because there is very little information about the growth of microorganisms in ENS and whether growth in ENS may affect the virulence and pathogenicity of microorganisms. This study shows that Escherichia coli and Pseudomonas aeruginosa may grow at 25 degrees C from either high or low initial numbers to up to 9.2 log colony-forming units per mL in a range of milk-based ENS. However, these organisms did not grow in the fruit-based ENS. The effect on the lipopolysaccharide (LPS) of culturing E. coli and P. aeruginosa in milk-based ENS as opposed to standard laboratory media was examined using polyacrylamide gel electrophoresis. We found that there were significant qualitative changes in the phenotype of O-polysaccharide side chains of the LPS from these organisms. O-polysaccharide is known to mediate in the complement, antibiotic and bile resistance, and affect adherence. Therefore, changes in the virulence and pathogenicity of these microorganisms when cultured in ENS may be indicated. Thus, the study provides further evidence for reevaluating the microbiologic and immunologic effects of enteral feeding, especially on the microbial flora of the GI tract.

Characterization of BrkA expression in Bordetella bronchiseptica.

BrkA confers resistance to killing by complement in Bordetella pertussis. Complement resistance in Bordetella bronchiseptica was examined. Four B. bronchiseptica strains possessed the brkA gene; however, only three expressed the protein. Only the strain lacking BrkA was susceptible to complement. Introduction of the B. pertussis brkA gene restored BrkA expression to this strain but did not confer resistance. brkA was mutated in the strains that naturally expressed BrkA, and loss of BrkA did not confer sensitivity to complement. As a species, B. bronchiseptica is more resistant to complement than B. pertussis, and BrkA does not mediate resistance.

Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope.

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

Up-regulated expression of decay-accelerating factor (CD55) confers increased complement resistance to sprouting neural cells.

We studied gene expression in relation to induced neural differentiation in a human neural crest-derived cell line, Paju. Messenger RNA isolated before and after treatment with phorbol 12-myristate 13-acetate was analyzed by differential display reverse transcription PCR. A strongly up-regulated expression of decay-accelerating factor (DAF, CD55) was found to parallel the induced neural sprouting while the expression of two other complement regulatory proteins (CD59/protectin, CD46/membrane cofactor protein) remained unaltered during neural differentiation. The increased membrane expression of DAF, which was also seen on neural processes and growth cones, conferred elevated resistance to complement-mediated lysis. Our findings suggest that in sprouting neurons DAF expression is up-regulated to provide additional complement resistance to pathfinding axons/dendrites invading new environment. It is also suggested that membrane expression of DAF may constitute a marker of growing and regenerating neurons.

Up-regulated expression of decay-accelerating factor (CD55) confers increased complement resistance to sprouting neural cells

Up-regulated expression of decay-accelerating factor (CD55) confers increased complement resistance to sprouting neural cells

Immunohistochemical assessment of 17-1A and complement resistance factors CD55 and CD59 in liver metastases of colon cancer

Immunohistochemical assessment of 17-1A and complement resistance factors CD55 and CD59 in liver metastases of colon cancer

Immunohistochemical assessment of 17-1A and complement resistance factors CD55 and CD59 in esophageal cancer

Immunohistochemical assessment of 17-1A and complement resistance factors CD55 and CD59 in esophageal cancer

Resistance of Acanthamoeba Species to Complement Lysis

Acanthamoeba species were evaluated for susceptibility to complement lysis as determined by release of radiolabeled uridine. The 3 Acanthamoeba species tested, A. culbertsoni (ATCC 30171), A. castellanii (ATCC 30010), and A. polyphaga (ATCC 30461), depleted hemolytic complement activity from normal human serum (NHS), yet were resistant to its lytic effects. Examination of microtiter plates containing amoebae incubated in NHS demonstrated formation of a pellet in the wells. Pellet formation was not observed when amoebae were incubated in human cord serum, heat-inactivated serum, or C1q-deficient serum. Ultrastructural examination of serum-treated amoebae revealed the presence of a finely granular substance that surrounded the amoebae. Treatment of amoebae with enzymes or metabolic inhibitors prior to incubation in NHS was performed to investigate the mechanism of complement resistance. Cycloheximide or cytochalasin D pretreatment increased the susceptibility of A. culbertsoni and A. castellanii to complement lysis. Cytochalasin D treatment also increased the susceptibility of A. polyphaga to complement lysis. Inhibition of serine protease activity by phenylmethylsulfonylfluoride increased complement susceptibility of all 3 species of Acanthamoeba. Enzymatic removal of surface components from A. polyphaga or A. castellanii, with trypsin, neuraminidase, or phosphatidylinositol-specific phospholipase C (PIPLC), did not affect serum resistance. In contrast, PIPLC treatment of A. culbertsoni significantly increased lysis by complement. The ability of Acanthamoeba species to activate the alternative complement pathway yet resist complement-mediated cellular lysis can be attributed to both the release of a transport-dependent extracellular matrix as well as the presence of complement inhibitory surface proteins.

Differential sensitivity of New World Leishmania spp. promastigotes to complement-mediated lysis: Correlation with the expression of three parasite polypeptides

American tegumentary leishmaniasis is caused by Leishmania of the subgenera Leishmania and Viannia. In this paper, we demonstrate that promastigotes of these two subgenera display distinct characteristic patterns of complement sensitivity during growth in vitro. Using fresh normal human serum in lytic assays, we show that while promastigotes of two species of the subgenus Leishmania differentiate into forms that are more resistant to the lytic action of complement, promastigotes of three species of the subgenus Viannia remain sensitive to complement mediated lysis during all stages of their growth in vitro. Complement resistance of the subgenus Leishmania is temporary, reaching its peak at the beginning of the stationary phase of growth, and decreasing thereafter. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we detected in L. amazonensis (subgenus Leishmania), but not in L. guyanensis (subgenus Viannia), three polypeptides whose expression parallels the resistance of promastigotes to complement-mediated lysis.

Human immunodeficiency virus type 1 incorporates both glycosyl phosphatidylinositol-anchored CD55 and CD59 and integral membrane CD46 at levels that protect from complement-mediated destruction.

Human immunodeficiency virus type 1 (HIV-1) can be either resistant or sensitive to complement-mediated destruction depending on the host cells. Incorporation of different levels of host cell CD46, CD55 and CD59 may account for this differential sensitivity to complement. However, it has not been determined whether CD46, CD55 and CD59 can all be incorporated at levels which protect virions. To determine whether each of these proteins can protect HIV-1, virions were derived from CHO cells expressing either human CD46, CD55 or CD59. Virions were shown to incorporate both glycosyl phosphatidylinositol (GPI)-anchored CD55 and CD59 as well as transmembrane CD46. Importantly, all three virus preparations were significantly more resistant to complement lysis than control virus. This study demonstrates that HIV-1 incorporates both transmembrane and GPI-anchored complement control proteins from host cells and that both types of protein increase complement resistance of virus.

Intrastrain variation of lipopolysaccharide of Pasteurella multocida in turkeys

Abstract Objective To document intrastrain variation of lipopolysaccharide (LPS) in Pasteurella multocida and correlate these changes with changes in determinants associated with virulence. Animals 25 broad-breasted white turkeys. Procedure Phenotypic bacterial variants were identified by lectin affinity and were assayed for adherence to epithelial cells and complement resistance in vitro. The LPS purified from these variants was subjected to polyacrylamide gel electrophoresis and lectin affinity analysis. Turkeys were challenge exposed, then observed for 1 week. At first sign of disease, or at the end of the study, turkeys were euthanatized, necropsied, and inspected for gross lesions. Results The LPS variant designated as Ricinus communis agglutinin-positive had greater adherence to epithelial cells, complement resistance, and virulence in turkeys than did the variant designated as R communis agglutinin-negative. Conclusions Intrastrain variation of LPS exists in P multocida, and changes in LPS are correlated with changes in virulence. (Am J Vet Res 1997;58:755–759)

Complement resistance of capsulated strains ofAeromonas salmonicida

The complement resistance ofAeromonas salmonicidastrains grown under conditions promoting capsule formation was investigated using well characterized strains and their isogenic mutants. Complement resistance was previously studied using the same strains growing under noncapsulating conditions. The serum resistant strains were found to activate complement, but rapidly degrade C3b preventing productive formation of the lytic complex C5b-9. Isogenic lipopolysaccharide rough mutants grown under non-capsulating conditions were serum sensitive, binding a large amount of C3b and leading to productive formation of C5b-9. When grown under conditions promoting capsule formation, these mutants were partially resistant to complement because less C3b is bound to them and also partially degraded, with a concomitant reduction in lytic C5b-9.