Adam S. Coleman

A Chromosomally Encoded Virulence Factor Protects the Lyme Disease Pathogen against Host-Adaptive Immunity

Borrelia burgdorferi, the bacterial pathogen of Lyme borreliosis, differentially expresses select genes in vivo, likely contributing to microbial persistence and disease. Expression analysis of spirochete genes encoding potential membrane proteins showed that surface-located membrane protein 1 (lmp1) transcripts were expressed at high levels in the infected murine heart, especially during early stages of infection. Mice and humans with diagnosed Lyme borreliosis also developed antibodies against Lmp1. Deletion of lmp1 severely impaired the pathogens ability to persist in diverse murine tissues including the heart, and to induce disease, which was restored upon chromosomal complementation of the mutant with the lmp1 gene. Lmp1 performs an immune-related rather than a metabolic function, as its deletion did not affect microbial persistence in immunodeficient mice, but significantly decreased spirochete resistance to the borreliacidal effects of anti-B. burgdorferi sera in a complement-independent manner. These data demonstrate the existence of a virulence factor that helps the pathogen evade host-acquired immune defense and establish persistent infection in mammals.

BBA52 Facilitates Borrelia burgdorferi Transmission from Feeding Ticks to Murine Hosts

Borrelia burgdorferi, the pathogen of Lyme borreliosis, persists in nature through a tick-rodent transmission cycle. A selective assessment of the microbial transcriptome, limited to gene-encoding putative membrane proteins, reveals that bba52 transcription in vivo is strictly confined to the vector-specific portion of the microbial life cycle, with the highest levels of expression noted in feeding ticks and with swift down-regulation noted in mice. bba52 deletion did not affect murine disease as assessed by the genesis of arthritis and carditis or long-term persistence of pathogens in mice or ticks. However, bba52 deficiency did impair microbial transitions between hosts and vector, defects that could be fully rescued when bba52 expression was genetically restored to the original genomic locus. These studies establish that BBA52 facilitates vector-host transitions by the pathogen and therefore is a potential antigenic target for interference with transmission of B. burgdorferi from ticks to mammalian hosts.

Borrelia burgdorferi complement regulator-acquiring surface protein 2 does not contribute to complement resistance or host infectivity.

Borrelia burgdorferi, the pathogen of Lyme disease, cycles in nature through Ixodes ticks and mammalian hosts. At least five Complement Regulator-Acquiring Surface Proteins (BbCRASPs) are produced by B. burgdorferi, which are thought to assist spirochetes in host immune evasion. Recent studies established that BbCRASP-2 is preferentially expressed in mammals, and elicits robust antibody response in infected hosts, including humans. We show that BbCRASP-2 is ubiquitously expressed in diverse murine tissues, but not in ticks, reinforcing a role of BbCRASP-2 in conferring B. burgdorferi defense against persistent host immune threats, such as complement. BbCRASP-2 immunization, however, fails to protect mice from B. burgdorferi infection and does not modify disease, as reflected by the development of arthritis. An infectious BbCRASP-2 mutant was generated, therefore, to examine the precise role of the gene product in spirochete infectivity. Similar to wild type B. burgdorferi, BbCRASP-2 mutants remain insensitive to complement-mediated killing in vitro, retain full murine infectivity and induce arthritis. Quantitative RT-PCR assessment indicates that survivability of BbCRASP-2-deficient B. burgdorferi is not due to altered expression of other BbCRASPs. Together, these results suggest that the function of a selectively expressed B. burgdorferi gene, BbCRASP-2, is not essential for complement resistance or infectivity in the murine host.

The BarA-UvrY Two-Component System Regulates Virulence in Avian Pathogenic Escherichia coli O78:K80:H9

ABSTRACT The BarA-UvrY two-component system (TCS) in Escherichia coli is known to regulate a number of phenotypic traits. Both in vitro and in vivo assays, including the chicken embryo lethality assay, showed that this TCS regulates virulence in avian pathogenic E. coli (APEC) serotype O78:K80:H9. A number of virulence determinants, such as the abilities to adhere, invade, persist within tissues, survive within macrophages, and resist bactericidal effects of serum complement, were compromised in mutants lacking either the barA or uvrY gene. The reduced virulence was attributed to down regulation of type 1 and Pap fimbriae, reduced exopolysaccharide production, and increased susceptibility to oxidative stress. Our results indicate that BarA-UvrY regulates virulence properties in APEC and that the chicken embryo lethality assay can be used as a surrogate model to determine virulence determinants and their regulation in APEC strains.

Methods for rapid transfer and localization of lyme disease pathogens within the tick gut.

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle. A tick-borne murine model has been developed to study Lyme disease in the laboratory. While naíve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

BBK07 Immunodominant Peptides as Serodiagnostic Markers of Lyme Disease

ABSTRACT Lyme disease (LD) is a tick-borne infection caused by the bacterial pathogen Borrelia burgdorferi. Current diagnostic tests mostly use borrelial lysates or select antigens to detect serum antibodies against B. burgdorferi. These immunoassays are not entirely effective, especially for detection of early infection. We have recently characterized an in vivo-induced antigen, BBK07, as a serodiagnostic marker for LD. We now report that in a line blot assay, recombinant BBK07 protein-based detection is 90% sensitive and nearly 100% specific against B. burgdorferi infection in humans. Using an overlapping peptide library of 23 peptides encompassing full-length BBK07, we identified the immunodominant epitopes of BBK07 during human infection. We show that a select combination of amino-terminal peptides significantly enhanced BBK07-based diagnostic accuracy compared to that with the full-length protein. Although in enzyme-linked immunosorbent assay (ELISA) studies BBK07 peptides had overall lower sensitivity than established serodiagnostic peptides, such as the VlsE peptide C6 and OspC peptide pepC10, for the detection of early human LD, a subset of serum samples that failed to recognize either VlsE or OspC peptides were preferentially reactive to BBK07 peptides. These results highlight the fact that BBK07 peptides could be useful to complement the efficacy of VlsE and OspC peptide-based serodiagnostic assays. Finally, using a panel of canine sera, we show that BBK07 peptide is also effective for LD diagnosis in infected dogs. Together, our data show that peptides from the B. burgdorferi surface protein BBK07 are highly specific and sensitive serodiagnostic markers, and we suggest their future use in LD diagnostic assays.

Borrelia burgdorferi lipoprotein BmpA activates pro-inflammatory responses in human synovial cells through a protein moiety

Borrelia burgdorferi invasion of mammalian joints results in genesis of Lyme arthritis. Other than spirochete lipids, existence of protein antigens, which are abundant in joints and participate in B. burgdorferi-induced host inflammatory response, is unknown. Here, we report that major products of the B. burgdorferi basic membrane protein (bmp) A/B operon that are induced in murine and human joints, possess inflammatory properties. Compared to the wild type B. burgdorferi, an isogenic bmpA/B mutant induced significantly lower levels of pro-inflammatory cytokines TNF-alpha and IL-1beta in cultured human synovial cells, which could be restored using bmpA/B-complemented mutants, and more directly, upon addition of recombinant BmpA, but not BmpB or control spirochete proteins. Non-lipidated and lipidated versions of BmpA induced similar levels of cytokines, and remained unaffected by treatment with lipopolysaccharide inhibitor, polymyxin B. The bmpA/B mutant was also impaired in the induction of NF-kappaB and p38 MAP kinase signaling pathways in synovial cells, which were activated by non-lipidated BmpA. These results show that a protein moiety of BmpA can induce cytokine responses in synovial cells via activation of the NF-kappaB and p38 MAP kinase pathways and thus, could potentially contribute to the genesis of Lyme arthritis.

BBK07, a Dominant In Vivo Antigen of Borrelia burgdorferi, Is a Potential Marker for Serodiagnosis of Lyme Disease

ABSTRACT One of the recently identified Borrelia burgdorferi immunogens, BBK07, is characterized for its expression in the spirochete infection cycle and evaluated for its potential use as a serodiagnostic marker for Lyme disease. We show that the BBK07 gene is expressed at extremely low levels in vitro and in ticks but is dramatically induced by spirochetes once introduced into the host and is highly expressed throughout mammalian infection. In contrast, the expression of BBK12, a paralog of BBK07 with 87% amino acid identity, although expressed in vitro, remained undetectable in vivo throughout murine infection and in ticks. BBK07 is localized in the outer membrane, and the amino-terminal domain of the antigen is exposed on the microbial surface. A truncated BBK07 protein representing the amino-terminal domain is able to effectively detect antibodies to B. burgdorferi, both in experimentally infected mice and in humans. Further characterization of the immunodominant antigens of B. burgdorferi, such as BBK07, could contribute to the development of novel serodiagnostic markers for detection of Lyme disease.

Antibody Profiling of Borrelia burgdorferi Infection in Horses

ABSTRACT Infection with Borrelia burgdorferi is common in horses and ponies from the New England and mid-Atlantic regions of the United States. Here, we evaluated luciferase immunoprecipitation systems (LIPS) for profiling antibody responses against three different antigenic targets for the diagnosis of equine B. burgdorferi infection. LIPS testing of horse serum samples suspected of Lyme infection revealed that approximately 75% of the horse samples (114/159) were seropositive against the synthetic VOVO antigen, comprising repeated immunodominant C6 epitopes as well as OspC immunodominant epitopes. A comparison of VOVO and immunofluorescence assays (IFA) showed that 51% of the samples were positive in both assays (VOVO+/IFA+), 13% were VOVO−/IFA+, 21% were VOVO+/IFA−, and 15% were negative in both. To further understand humoral responses to B. burgdorferi and reconcile the diagnostic differences between IFA and VOVO, two additional B. burgdorferi LIPS tests were performed with DbpA and DbpB. Robust seropositive antibody responses against DbpA and/or DbpB were detected in 98% (79/81) of the VOVO+/IFA+ and 93% (50/54) of the discrepant samples. Additionally, some of the samples negative by both VOVO and IFA showed immunoreactivity against DbpA and/or DbpB. Overall, 94% of the suspected horse samples were seropositive by LIPS, and heat map analysis revealed that seropositive samples often were immunoreactive with at least two of the three antigens. These results suggest that LIPS tests employing multiple recombinant antigens offer a promising approach for the evaluation of antibody responses in Lyme disease.

Integration of AI-2 Based Cell-Cell Signaling with Metabolic Cues in Escherichia coli.

The quorum sensing molecule Autoinducer-2 (AI-2) is generated as a byproduct of activated methyl cycle by the action of LuxS in Escherichia coli. AI-2 is synthesized, released and later internalized in a cell-density dependent manner. Here, by mutational analysis of the genes, uvrY and csrA, we describe a regulatory circuit of accumulation and uptake of AI-2. We constructed a single-copy chromosomal luxS-lacZ fusion in a luxS + merodiploid strain and evaluated its relative expression in uvrY and csrA mutants. At the entry of stationary phase, the expression of the fusion and AI-2 accumulation was positively regulated by uvrY and negatively regulated by csrA respectively. A deletion of csrA altered message stability of the luxS transcript and CsrA protein exhibited weak binding to 5’ luxS regulatory region. DNA protein interaction and chromatin immunoprecipitation analysis confirmed direct interaction of UvrY with the luxS promoter. Additionally, reduced expression of the fusion in hfq deletion mutant suggested involvement of small RNA interactions in luxS regulation. In contrast, the expression of lsrA operon involved in AI-2 uptake, is negatively regulated by uvrY and positively by csrA in a cell-density dependent manner. The dual role of csrA in AI-2 synthesis and uptake suggested a regulatory crosstalk of cell signaling with carbon regulation in Escherichia coli. We found that the cAMP-CRP mediated catabolite repression of luxS expression was uvrY dependent. This study suggests that luxS expression is complex and regulated at the level of transcription and translation. The multifactorial regulation supports the notion that cell-cell communication requires interaction and integration of multiple metabolic signals.