Dawn R. Clifton

Delineating the Requirement for the Borrelia burgdorferi Virulence Factor OspC in the Mammalian Host

ABSTRACT We previously demonstrated that outer surface protein C (OspC) of Borrelia burgdorferi is essential for establishing mammalian infection. However, the role of OspC in mammalian infection is unknown. Here, we report experiments designed to distinguish between two models of OspC function in the mammalian host: (i) OspC fulfills an essential physiological role for growth and host adaptation or (ii) OspC provides a protective role for evasion of components of the innate immune response. We found that a B. burgdorferi ospC mutant, previously demonstrated to be noninfectious in both immunocompetent and SCID mice, could survive in the relatively immune-privileged environment of dialysis membrane chambers implanted within the peritoneum of a rat. The ospC mutant also adapts to the mammalian environment, as determined by the protein profiles of the chamber-cultivated spirochetes. Therefore, OspC does not appear to provide a physiological function for the survival of B. burgdorferi within the mammalian host. The second model, evasion of the innate immune system, was tested by assessing the infectivity of the ospC mutant in mice deficient for myeloid differentiation protein 88 (MyD88). Recent studies have shown that B. burgdorferi is prevented from reaching high cell numbers in the mammalian host by MyD88-dependent signaling pathways. The ospC mutant was incapable of infecting MyD88-deficient mice, suggesting that the role of OspC cannot be related solely to evasion of MyD88-mediated innate immunity. These results reiterate the importance of OspC in mammalian infection and eliminate simple models of function for this enigmatic protein.

Tyrosine Phosphorylation of the Chlamydial Effector Protein Tarp Is Species Specific and Not Required for Recruitment of Actin

ABSTRACT Chlamydiae are obligate intracellular pathogens that efficiently induce their endocytosis by susceptible eukaryotic host cells. Recently, a Chlamydia trachomatis type III secreted effector protein, Tarp, was found to be translocated and tyrosine phosphorylated at the site of entry and associated with the recruitment of actin that coincides with endocytosis. C. trachomatis Tarp possesses up to six direct repeats of approximately 50 amino acids each. The majority of the tyrosine residues are found within this repeat region. Here we have ectopically expressed distinct domains of Tarp in HeLa 229 cells and demonstrated that tyrosine phosphorylation occurs primarily within the repeat region, while recruitment of actin is mediated by the C-terminal domain of the protein. A comparison of other sequenced chlamydial genomes revealed that each contains an ortholog of Tarp, although Chlamydia muridarum, Chlamydophila caviae, and Chlamydophila pneumoniae Tarp lack the large repeat region. Immunofluorescence and immunoblotting using an antiphosphotyrosine antibody show no evidence of phosphotyrosine at the site of entry of C. muridarum, C. caviae, and C. pneumoniae, although each species similarly recruits actin. Ectopic expression of full-length C. trachomatis and C. caviae Tarp confirmed that both recruit actin but only C. trachomatis Tarp is tyrosine phosphorylated. The data indicate that the C-terminal domain of Tarp is essential for actin recruitment and that tyrosine phosphorylation may not be an absolute requirement for actin recruitment. The results further suggest the potential for additional, unknown signal transduction pathways associated specifically with C. trachomatis.

Temporal Expression Analysis of the Borrelia burgdorferi Paralogous Gene Family 54 Genes BBA64, BBA65, and BBA66 during Persistent Infection in Mice

ABSTRACT Members of the Borrelia burgdorferi paralogous gene family 54 (pgf 54) are regulated by conditions simulating mammalian infection and are thought to be instrumental in borrelial host survival and pathogenesis. To explore the activities of these genes in vivo, a comprehensive analysis of pgf 54 genes BBA64, BBA65, and BBA66 was performed to assess the genetic stability, host antibody responses, and kinetics of gene expression in the murine model of persistent infection. DNA sequencing of pgf 54 genes obtained from reisolates at 1 year postinfection demonstrated that all genes of this family are stable and do not undergo recombination to generate variant antigens during persistent infection. Antibodies against BBA64 and BBA66 appeared soon after infection and were detectable throughout the infection, suggesting that there was gene expression during infection. However, quantitative reverse transcription-PCR revealed that BBA64 gene expression was considerably decreased in Borrelia residing in the mouse ear tissue compared to the expression in cultured spirochetes by 20 days postinfection and that the levels of expression remained low throughout the infection. Conversely, transcription of the BBA65 and BBA66 genes was increased, and both of these genes were continuously expressed until 100 days postinfection; this was followed by periods of differential expression late in infection. The expression profile of the BBA64 gene suggests that this gene has an important role during tick-to-host transmission and early infection, whereas the expression profile of the BBA65 and BBA66 genes suggests that these genes have a role in persistent infection. The differential regulation of pgf 54 genes observed during infection may help confer a survival advantage during persistent infection, influencing mechanisms for B. burgdorferi dissemination, tissue tropism, or evasion of the adaptive immune response.

The bba64 gene of Borrelia burgdorferi, the Lyme disease agent, is critical for mammalian infection via tick bite transmission

The spirochetal agent of Lyme disease, Borrelia burgdorferi, is transmitted by bites of Ixodes ticks to mammalian reservoir hosts and humans. The mechanism(s) by which the organism is trafficked from vector to host is poorly understood. In this study, we demonstrate that a B. burgdorferi mutant strain deficient in the synthesis of the bba64 gene product was incapable of infecting mice via tick bite even though the mutant was (i) infectious in mice when introduced by needle inoculation, (ii) acquired by larval ticks feeding on infected mice, and (iii) able to persist through tick molting stages. This finding of a B. burgdorferi gene required for pathogen transfer and/or survival from the tick to the susceptible host represents an important breakthrough toward understanding transmission mechanisms involved for the Lyme disease agent.

Borrelia burgdorferi Surface-Localized Proteins Expressed during Persistent Murine Infection Are Conserved among Diverse Borrelia spp.

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease in the United States, regulates numerous genes encoding lipoproteins on linear plasmid 54 in response to environmental cues. We analyzed a subset of these genes/proteins that were historically categorized as paralogous gene family 54 (BBA64, BBA65, BBA66, BBA68, BBA69, BBA70, BBA71, and BBA73) and found that the expression of several genes was influenced by the σN-σS regulatory cascade at the level of transcription and protein synthesis. Moreover, we established in this and a previous study that BBA65, BBA66, BBA69, BBA71, and BBA73 are temporally expressed during persistent infection of immunocompetent mice, as determined by quantitative real time-PCR of ear tissue, by enzyme-linked immunosorbent assay, and by immunoblotting. Correspondingly, BBA65, BBA66, BBA71, and BBA73 proteins were detectable in infectious B. burgdorferi B31 isolates but undetectable in noninfectious isolates. BBA65, BBA66, BBA71, and BBA73 proteins were also found to partition into the Triton X-114 detergent phase and were sensitive to protease treatment of intact cells, indicating that they are membrane associated and surface localized. Lastly, Southern blotting and PCR with specific gene primer/probes for BBA64, BBA65, BBA66, BBA71, and BBA73 suggest that many of these genes are conserved among the B. burgdorferi sensu lato isolates and the relapsing-fever Borrelia species. Together, the data presented suggest that these genes may play a part in Borrelia infection and/or pathogenicity that could extend beyond the sensu lato group.

NF-κB Activation during Rickettsia rickettsii Infection of Endothelial Cells Involves the Activation of Catalytic IκB Kinases IKKα and IKKβ and Phosphorylation-Proteolysis of the Inhibitor Protein IκBα

ABSTRACT Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-κB (NF-κB) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-κB subunits, p65 and p50, and IκB proteins, IκBα and IκBβ. The transcript and protein levels of p50, p65, and IκBβ remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IκBα at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IκBα mRNA. The level of IκBα mRNA gradually returned toward baseline, whereas that of total IκBα protein remained lower than the corresponding controls. The activities of IKKα and IKKβ, the catalytic subunits of IκB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-κB response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKKα and IKKβ, leading to attenuation of nuclear translocation of NF-κB. Also, increased activity of IKKα was evident later during the infection, coinciding with the late phase of NF-κB activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-κB activation. Since NF-κB is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.

Regulation and expression of bba66 encoding an immunogenic infection‐associated lipoprotein in Borrelia burgdorferi

When Borrelia burgdorferi (Bb) is transmitted from a tick vector to a mammalian host the spirochaete alters gene expression, allowing for adaptation to the new host. We evaluated the regulation of paralogous gene family (pgf) 54 members in response to environmental cues and focused our efforts on determining the molecular mechanisms influencing bba66 expression. By qRT‐PCR, bba65, bba66, bba71 and bba73 displayed regulation similar to ospC under mammalian‐like conditions. Of the pgf 54 members, bba66 demonstrated the greatest and second greatest change in expression in response to pH or temperature shift respectively. Furthermore, Bb‐infected mice and patients with early disseminated Lyme disease produced detectable antibodies to BBA66. A protein(s) active in Bb at pH 7 was able to interact with the bba66 upstream region and was specific as bba64 and ospC promoters were unable to out‐compete for binding. bba66 promoter mapping revealed putative σ70 and σS consensus sequences, enabling us to narrow the protein binding site to a region within an imperfect inverted repeat upstream of the −35 region. Moreover, BBA66 production is associated with an infectious phenotype, and loss of either σN or σS resulted in loss of BBA66. Promoter‐GFP fusion analysis indicated that the σ70 and/or σS consensus sequences alone were not sufficient to initiate transcription and a portion of the upstream inverted repeat was required. These results suggest a primary role for BBA66 in Bb transmission and infection.

Borrelia burgdorferi bba66 Gene Inactivation Results in Attenuated Mouse Infection by Tick Transmission

ABSTRACT The impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tick-mouse cycle. The BBA66-deficient mutant isolate, BbΔA66, remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired BbΔA66 following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of BbΔA66-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of BbΔA66 with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.