Integrin binding by Borrelia burgdorferi P66 facilitates dissemination but is not required for infectivity

P66, a Borrelia burgdorferi surface protein with porin and integrin‐binding activities, is essential for murine infection. The role of P66 integrin‐binding activity in B. burgdorferi infection was investigated and found to affect transendothelial migration. The role of integrin binding, specifically, was tested by mutation of two amino acids (D205A,D207A) or deletion of seven amino acids (Del202–208). Neither change affected surface localization or channel‐forming activity of P66, but both significantly reduced binding to α_vβ₃. Integrin‐binding deficient B. burgdorferi strains caused disseminated infection in mice at 4 weeks post‐subcutaneous inoculation, but bacterial burdens were significantly reduced in some tissues. Following intravenous inoculation, the Del202–208 bacteria were below the limit of detection in all tissues assessed at 2 weeks post‐inoculation, but bacterial burdens recovered to wild‐type levels at 4 weeks post‐inoculation. The delay in tissue colonization correlated with reduced migration of the Del202–208 strains across microvascular endothelial cells, similar to Δp66 bacteria. These results indicate that integrin binding by P66 is important to efficient dissemination of B. burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle.     

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.     

The lipid raft proteome of Borrelia burgdorferi

Eukaryotic lipid rafts are membrane microdomains that have significant amounts of cholesterol and a selective set of proteins that have been associated with multiple biological functions. The Lyme disease agent, Borrelia burgdorferi, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured B. burgdorferi as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft‐associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC‐MS/MS or MudPIT. Identified proteins were analyzed in silico for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of Borrelia, other pathogenic bacteria, as well as for the evolution of these structures. All MS data have been deposited in the ProteomeXchange with identifier PXD002365 ( http://proteomecentral.proteomexchange.org/dataset/PXD002365 ).     

The salt‐sensitive structure and zinc inhibition of Borrelia burgdorferi protease BbHtrA

HtrA serine proteases are highly conserved and essential ATP‐independent proteases with chaperone activity. Bacteria express a variable number of HtrA homologues that contribute to the virulence and pathogenicity of bacterial pathogens. Lyme disease spirochetes possess a single HtrA protease homologue, Borrelia burgdorferi HtrA (BbHtrA). Previous studies established that, like the human orthologue HtrA1, BbHtrA is proteolytically active against numerous extracellular proteins in vitro. In this study, we utilized size exclusion chromatography and blue native polyacrylamide gel electrophoresis (BN‐PAGE) to demonstrate BbHtrA oligomeric structures that were substrate independent and salt sensitive. Examination of the influence of transition metals on the activity of BbHtrA revealed that this protease is inhibited by Zn²⁺ > Cu²⁺ > Mn²⁺. Extending this analysis to two other HtrA proteases, E. coli DegP and HtrA1, revealed that all three HtrA proteases were reversibly inhibited by ZnCl₂ at all micro molar concentrations examined. Commercial inhibitors for HtrA proteases are not available and physiologic HtrA inhibitors are unknown. Our observation of conserved zinc inhibition of HtrA proteases will facilitate structural and functional studies of additional members of this important class of proteases.     

The HtrA protease of Borrelia burgdorferi degrades outer membrane protein BmpD and chemotaxis phosphatase CheX

Borrelia burgdorferi, the spirochaetal agent of Lyme disease, codes for a single HtrA protein, HtrABb (BB0104) that is homologous to DegP of Escherichia coli (41% amino acid identity). HtrABb shows physical and biochemical similarities to DegP in that it has the trimer as its fundamental unit and can degrade casein via its catalytic serine. Recombinant HtrABb exhibits proteolytic activity in vitro, while a mutant (HtrABbS198A) does not. However, HtrABb and DegP have some important differences as well. Native HtrABb occurs in both membrane‐bound and soluble forms. Despite its homology to DegP, HtrABb could not complement an E. coli DegP deletion mutant. Late stage Lyme disease patients, as well as infected mice and rabbits developed a robust antibody response to HtrABb, indicating that it is a B‐cell antigen. In co‐immunoprecipitation studies, a number of potential binding partners for HtrABb were identified, as well as two specific proteolytic substrates, basic membrane protein D (BmpD/BB0385) and chemotaxis signal transduction phosphatase CheX (BB0671). HtrABb may function in regulating outer membrane lipoproteins and in modulating the chemotactic response of B. burgdorferi.     

The β(3) -integrin ligand of Borrelia burgdorferi is critical for infection of mice but not ticks

P66 is a Borrelia burgdorferi surface protein with β₃ integrin binding and channel forming activities. In this study, the role of P66 in mammalian and tick infection was examined. B. burgdorferiΔp66 strains were not infectious in wild‐type, TLR2^?/?‐ or MyD88^?/?‐deficient mice. Strains with p66 restored to the chromosome restored near wild‐type infectivity, while complementation with p66 on a shuttle vector did not restore infectivity. Δp66 mutants are cleared quickly from the site of inoculation, but analyses of cytokine expression and cellular infiltrates at the site of inoculation did not reveal a specific mechanism of clearance. The defect in these mutants cannot be attributed to nutrient limitation or an inability to adapt to the host environment in vivo as Δp66 bacteria were able to survive as well as wild type in dialysis membrane chambers in the rat peritoneum. Δp66 bacteria were able to survive in ticks through the larva to nymph moult, but were non‐infectious in mice when delivered by tick bite. Independent lines of evidence do not support any increased susceptibility of the Δp66 strains to factors in mammalian blood. This study is the first to define a B. burgdorferi adhesin as essential for mammalian, but not tick infection.     

Borrelia burgdorferi locus BB0795 encodes a BamA orthologue required for growth and efficient localization of outer membrane proteins

The outer membrane (OM) of the pathogenic diderm spirochete, Borrelia burgdorferi, contains integral β‐barrel outer membrane proteins (OMPs) in addition to its numerous outer surface lipoproteins. Very few OMPs have been identified in B. burgdorferi, and the protein machinery required for OMP assembly and OM localization is currently unknown. Essential OM BamA proteins have recently been characterized in Gram‐negative bacteria that are central components of an OM β‐barrel assembly machine and are required for proper localization and insertion of bacterial OMPs. In the present study, we characterized a putative B. burgdorferi BamA orthologue encoded by open reading frame bb0795. Structural model predictions and cellular localization data indicate that the B. burgdorferi BB0795 protein contains an N‐terminal periplasmic domain and a C‐terminal, surface‐exposed β‐barrel domain. Additionally, assays with an IPTG‐regulatable bb0795 mutant revealed that BB0795 is required for B. burgdorferi growth. Furthermore, depletion of BB0795 results in decreased amounts of detectable OMPs in the B. burgdorferi OM. Interestingly, a decrease in the levels of surface‐exposed lipoproteins was also observed in the mutant OMs. Collectively, our structural, cellular localization and functional data are consistent with the characteristics of other BamA proteins, indicating that BB0795 is a B. burgdorferi BamA orthologue.     

CD14 Targets Complement Receptor 3 to Lipid Rafts during Phagocytosis of Borrelia burgdorferi

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is mediated partly by the interaction of the spirochete with Complement Receptor (CR) 3. CR3 requires the GPI-anchored protein, CD14, in order to efficiently internalize CR3-B. burgdorferi complexes. GPI-anchored proteins reside in cholesterol-rich membrane microdomains, and through its interaction with partner proteins, help initiate signaling cascades. Here, we investigated the role of CD14 on the internalization of B. burgdorferi mediated by CR3. We show that CR3 partly colocalizes with CD14 in lipid rafts. The use of the cholesterol-sequestering compound methyl-β-cyclodextran completely prevents the internalization of the spirochete in CHO cells that co-express CD14 and CR3, while no effect was observed in CD11b-deficient macrophages. These results show that lipid rafts are required for CR3-dependent, but not independent, phagocytosis of B. burgdorferi. Our results also suggest that CD14 interacts with the C-lectin domain of CR3, favoring the formation of multi-complexes that allow their internalization, and the use of β-glucan, a known ligand for the C-lectin domain of CR3, can compensate for the lack of CD14 in CHO cells that express CR3. These results provide evidence to understand the mechanisms that govern the interaction between CR3 and CD14 during the phagocytosis of B. burgdorferi.     

The BBA33 lipoprotein binds collagen and impacts Borrelia burgdorferi pathogenesis

Borrelia burgdorferi, the etiologic agent of Lyme disease, adapts to the mammalian hosts by differentially expressing several genes in the BosR and Rrp2‐RpoN‐RpoS dependent pathways, resulting in a distinct protein profile relative to that seen for survival in the Ixodes spp. tick. Previous studies indicate that a putative lipoprotein, BBA33, is produced in an RpoS‐dependent manner under conditions that mimic the mammalian component of the borrelial lifecycle. However, the significance and function for BBA33 is not known. Given its linkage to the BosR/Rrp2‐RpoN‐RpoS regulatory cascade, we hypothesized that BBA33 facilitates B. burgdorferi infection in the mammalian host. The deletion of bba33 eliminated B. burgdorferi infectivity in C3H mice, which was rescued by genetic complementation with intact bba33. With regard to function, a combinatorial peptide approach, coupled with subsequent in vitro binding assays, indicated that BBA33 binds to collagen type VI and, to a lesser extent, collagen type IV. Whole cell binding assays demonstrated BBA33‐dependent binding to human collagen type VI. Taken together, these results suggest that BBA33 interacts with collagenous structures and may function as an adhesin in a process that is required to prevent bacterial clearance.     

The Borrelia burgdorferi CheY3 response regulator is essential for chemotaxis and completion of its natural infection cycle

Borrelia burgdorferi possesses a sophisticated and complex chemotaxis system, but how the organism utilizes this system in its natural enzootic life cycle is poorly understood. Of the three CheY chemotaxis response regulators in B. burgdorferi, we found that only deletion of cheY3 resulted in an altered motility and significantly reduced chemotaxis phenotype. Although ΔcheY3 maintained normal densities in unfed ticks, their numbers were significantly reduced in fed ticks compared with the parental or cheY3‐complemented spirochetes. Importantly, mice fed upon by the ΔcheY3‐infected ticks did not develop a persistent infection. Intravital confocal microscopy analyses discovered that the ΔcheY3 spirochetes were motile within skin, but appeared unable to reverse direction and perform the characteristic backward–forward motility displayed by the parental strain. Subsequently, the ΔcheY3 became ‘trapped’ in the skin matrix within days of inoculation, were cleared from the skin needle‐inoculation site within 96 h post‐injection and did not disseminate to distant tissues. Interestingly, although ΔcheY3 cells were cleared within 96 h post‐injection, this attenuated infection elicited significant levels of B. burgdorferi‐specific IgM and IgG. Taken together, these data demonstrate that cheY3‐mediated chemotaxis is crucial for motility, dissemination and viability of the spirochete both within and between mice and ticks.     

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook‐associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non‐motile mutant cells that are unable to assemble flagellar filaments and pentagon‐shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella‐deficient mutants (i.e., in the hook, rod, or MS‐ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.     

Interferon‐gamma production in Lyme arthritis synovial tissue promotes differentiation of fibroblast‐like synoviocytes into immune effector cells

Lyme arthritis (LA), a late disease manifestation of Borrelia burgdorferi infection, usually resolves with antibiotic therapy. However, some patients develop proliferative synovitis lasting months to several years after spirochetal killing, called postinfectious LA. In this study, we phenotyped haematopoietic and stromal cell populations in the synovial lesion ex vivo and used these findings to generate an in vitro model of LA using patient‐derived fibroblast‐like synoviocytes (FLS). Ex vivo analysis of synovial tissue revealed high abundance of IFNγ‐producing T cells and NK cells. Similar to marked IFNγ responses in tissue, postinfectious LA synovial fluid also had high levels of IFNγ. HLA‐DR‐positive FLS were present throughout the synovial lesion, particularly in areas of inflammation. FLS stimulated in vitro with B. burgdorferi, which were similar to conditions during infection, expressed 68 genes associated primarily with innate immune activation and neutrophil recruitment. In contrast, FLS stimulated with IFNγ, which were similar to conditions in the postinfectious phase, expressed >2,000 genes associated with pathogen sensing, inflammation, and MHC Class II antigen presentation, similar to the expression profile in postinfectious synovial tissue. Furthermore, costimulation of FLS with B. burgdorferi and IFNγ induced greater expression of IL‐6 and other innate immune response proteins and genes than with IFNγ stimulation alone. These results suggest that B. burgdorferi infection, in combination with IFNγ, initiates the differentiation of FLS into a highly inflammatory phenotype. We hypothesise that overexpression of IFNγ by lymphocytes within synovia perpetuates these responses in the postinfectious period, causing proliferative synovitis and stalling appropriate repair of damaged tissue.     

BGA66 and BGA71 facilitate complement resistance of Borrelia bavariensis by inhibiting assembly of the membrane attack complex

Borrelia (B.) bavariensis exhibits a marked tropism for nervous tissues and frequently causes neurological manifestations in humans. The molecular mechanism by which B. bavariensis overcomes innate immunity, in particular, complement remains elusive. In contrast to other serum‐resistant spirochetes, none of the B. bavariensis isolates investigated bound complement regulators of the alternative (AP) and classical pathway (CP) or proteolytically inactivated complement components. Focusing on outer surface proteins BGA66 and BGA71, we demonstrated that both molecules either inhibit AP, CP and terminal pathway (TP) activation, or block activation of the CP and TP respectively. Both molecules bind complement components C7, C8 and C9, and thereby prevent assembly of the terminal complement complex. This inhibitory activity was confirmed by the introduction of the BGA66 and BGA71 encoding genes into a serum‐sensitive B. garinii strain. Transformed spirochetes producing either BGA66 or BGA71 overcome complement‐mediated killing, thus indicating that both proteins independently facilitate serum resistance of B. bavariensis. The generation of C‐terminally truncated proteins as well as a chimeric BGA71 protein lead to the localization of the complement‐interacting binding site within the N‐terminus. Collectively, our data reveal a novel immune evasion strategy of B. bavariensis that is directed against the activation of the TP.     

Compartmentalized DCC signalling is distinct from DCC localized to lipid rafts

Background information. Netrin‐1 is a bi‐functional cue that attracts or repels different classes of neurons during development. The netrin‐1 receptor DCC (deleted in colorectal cancer) acts as a tyrosine kinase‐associated receptor to mediate the attractive response towards netrin‐1. The lipid raft‐localized Src family kinase Fyn is required for DCC‐mediated axon guidance. DCC functions are also dependent on lipid rafts, membrane microdomains corresponding to a low‐density, detergent‐resistant membrane fraction. However, it remains unclear how the association of DCC with lipid rafts controls netrin‐1 signalling. Results. DCC targeted to lipid rafts represented a minor proportion of total DCC inside the cell, but predominated on the cell surface of both IMR‐32 human neuroblastoma cells and embryonic cortical neurons. Netrin‐1 accumulated in lipid rafts, but had no effect on the targeting of DCC to that compartment, with DCC remaining on the cell surface in lipid rafts through 60 min post‐treatment. However, DCC was able to interact with Fyn, both in the lipid rafts and soluble compartments isolated from embryonic E19 rat brains, whereas early downstream signalling components such as Nck‐1, and total and active focal adhesion kinase were mainly localized to the non‐lipid raft compartment. Conclusions. Together, these results suggest that DCC can be found in raft and non‐raft portions of the plasma membrane, with early signalling events propagated by non‐raft associated DCC.     

MARCKS regulates lamellipodia formation induced by IGF‐I via association with PIP2 and β‐actin at membrane microdomains

Myristoylated alanine‐rich C kinase substrate (MARCKS) is considered to participate in formation of F‐actin‐based lamellipodia, which represents the first stage of neurite formation. However, the mechanism of how MARCKS is involved in lamellipodia formation is not precisely unknown. Using SH‐SY5Y cells, we demonstrated here that MARCKS was translocated from cytosol to detergent‐resistant membrane microdomains, known as lipid rafts, within 30 min after insulin‐like growth factor‐I (IGF‐I) stimulation, which was accompanied by MARCKS dephosphorylation, β‐actin accumulation in lipid rafts, and lamellipodia formation. The protein kinase C inhibitor, Ro‐31‐8220, and Rho‐kinase inhibitors, HA1077 and Y27632, themselves decreased basal phosphorylation levels of MARCKS and coincidently elicited translocation of MARCKS to lipid rafts. On the other hand, the phosphoinositide 3‐kinase inhibitor, LY294002, abolished IGF‐I‐induced dephosphorylation, translocation of MARCKS to lipid rafts, and lamellipodia formation. Treatment of cells with neomycin, a PIP₂‐masking reagent, attenuated the translocation of MARCKS to lipid rafts and the lamellipodia formation induced by IGF‐I, although dephosphorylation of MARCKS was not affected. Immunocytochemical and immunoprecipitation analysis indicated that IGF‐I stimulation induced the translocation of MARCKS to lipid rafts in the edge of lamellipodia and formation of the complex with PIP₂. Moreover, we demonstrated that knockdown of endogenous MARCKS resulted in significant attenuation of IGF‐I‐induced β‐actin accumulation in the lipid rafts and lamellipodia formation. These results suggest a novel role for MARCKS in lamellipodia formation induced by IGF‐I via the translocation of MARCKS, association with PIP₂, and accumulation of β‐actin in the membrane microdomains. J. Cell. Physiol. 220: 748–755, 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of the high‐affinity choline transporter activity and trafficking by its association with cholesterol‐rich lipid rafts

The sodium‐coupled, hemicholinium‐3‐sensitive, high‐affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol‐rich lipid rafts in both SH‐SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH‐SY5Y cells expressing rat CHT with filipin, methyl‐β‐cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol‐saturated MβC. Kinetic analysis of binding of [³H]hemicholinium‐3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (K_D) and maximum number of binding sites (B_max); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol‐rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.

     

Evaluation of Borrelia burgdorferi BbHtrA Protease as a Vaccine Candidate for Lyme Borreliosis in Mice

Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA) which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrA^S226A). Although inoculation with either BbHtrA or BbHtrA^S226A produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.     

P66 porins are present in both Lyme disease and relapsing fever spirochetes: A comparison of the biophysical properties of P66 porins from six Borrelia species

The genus Borrelia is the cause of the two human diseases: Lyme disease (LD) and relapsing fever (RF). Both LD and RF Borrelia species are obligate parasites and are dependent on nutrients provided by their hosts. The first step of nutrient uptake across the outer membrane of these Gram-negative bacteria is accomplished by water-filled channels, so-called porins. The knowledge of the porin composition in the outer membranes of the different pathogenic Borrelia species is limited. Only one porin has been described in relapsing fever spirochetes to date, whereas four porins are known to be present in Lyme disease agents. From these, the Borrelia burgdorferi outer membrane channel P66 is known to act as an adhesin and was well studied as a porin. To investigate if P66 porins are expressed and similarly capable of pore formation in other Borrelia causing Lyme disease or relapsing fever three LD species (B. burgdorferi, B. afzelii, B. garinii) and three RF species (B. duttonii, B. recurrentis and B. hermsii) were investigated for outer membrane proteins homologous to P66. A search in current published RF genomes, comprising the ones of B. duttonii, B. recurrentis and B. hermsii, indicated that they all contained P66 homologues. The P66 homologues of the six Borrelia species were purified to homogeneity and their pore-forming abilities as well as the biophysical properties of the pores were analyzed using the black lipid bilayer assay.     

The apicomplexan parasite Babesia divergens internalizes band 3, glycophorin A and spectrin during invasion of human red blood cells

Plasmodium falciparum invades human red blood cells (RBC), while Babesia divergens infects bovine and, occasionally, human RBC. The mammalian RBC is normally unable to endocytose or phagocytose and the events leading to invasion are incompletely understood. Initially, both parasites are surrounded by the RBC plasma membrane‐derived parasitophorous vacuolar membrane (PVM) that is formed during invasion. In P. falciparum‐infected RBC, the PVM persists at least until parasite replication is completed whereas it has been proposed that the B. divergens PVM is disintegrated soon upon invasion. Here, we have used a B. divergens strain adapted to human RBC to investigate the formation and fate of the PVM. Using ultrastructural analysis and whole‐mount or on‐section immunofluorescence and immunogold labelling, we demonstrate that the initial vacuolar membrane is formed from protein and lipid components of the RBC plasma membrane. Integral membrane proteins band 3 and glycophorin A and the cytoskeletal protein spectrin are associated with the PVM of the B. divergens, but are absent from the PVM of P. falciparum at the ring or the trophozoite stage. Our results provide evidence that the biophysical properties of the RBC cytoskeleton per se do not preclude the internalization of cytoskeletal proteins by invading parasites.