In Vivo Imaging of Bioluminescent Mycobacterium ulcerans: A Tool to Refine the Murine Buruli Ulcer Tail Model.

Buruli ulcer (BU) is a neglected tropical disease caused by infection with Mycobacterium ulcerans. Unclear transmission, no available vaccine, and suboptimal treatment regimens hamper the control of this disease. Carefully designed preclinical research is needed to address these shortcomings. In vivo imaging (IVIS®, Perkin Elmer, Waltham, MA) of infection is an emerging tool that permits monitoring of disease progression and reduces the need to using large numbers of mice at different time-points during the experiment, as individual mice can be imaged at multiple time-points. We aimed to further describe the use of in vivo imaging (IVIS) in BU. We studied the detection of M. ulcerans in experimentally infected BALB/c mouse tails and the subsequent histopathology and immune response in this pilot study. IVIS-monitoring was performed weekly in ten infected BALB/c mice to measure light emitted as a proxy for bacterial load. Nine of 10 (90%) BALB/c mice infected subcutaneously with 3.3 × 105 M. ulcerans JKD8049 (containing pMV306 hsp16+luxG13) exhibited light emission from the site of infection, indicating M. ulcerans growth in vivo, whereas only five of 10 (50%) animals developed clinical signs of the disease. Specific antibody titers were detected within 2 weeks of the infection. Interferon (IFN)-γ and interleukin (IL)-10 were elevated in animals with pathology. Histopathology revealed clusters of acid-fast bacilli in the subcutaneous tissue, with macrophage infiltration and granuloma formation resembling human BU. Our study successfully showed the utility of M. ulcerans IVIS monitoring and lays a foundation for further research.

Expression of the Tick-Associated Vtp Protein of Borrelia hermsii in a Murine Model of Relapsing Fever.

Borrelia hermsii, a spirochete and cause of relapsing fever, is notable for its immune evasion by multiphasic antigenic variation within its vertebrate host. This is based on a diverse repertoire of surface antigen genes, only one of which is expressed at a time. Another major surface protein, the Variable Tick Protein (Vtp), is expressed in the tick vector and is invariable at its genetic locus. Given the limited immune systems of ticks, the finding of considerable diversity among the Vtp proteins of different strains of B. hermsii was unexpected. We investigated one explanation for this diversity of Vtp proteins, namely expression of the protein in mammals and a consequent elicitation of a specific immune response. Mice were infected with B. hermsii of either the HS1 or CC1 strain, which have antigenically distinctive Vtp proteins but otherwise have similar repertoires of the variable surface antigens. Subsequently collected sera were examined for antibody reactivities against Vtp and other antigens using Western blot analysis, dot blot, and protein microarray. Week-6 sera of infected mice contained antibodies that were largely specific for the Vtp of the infecting strain and were not attributable to antibody cross-reactivities. The antibody responses of the mice infected with different strains were otherwise similar. Further evidence of in vivo expression of the vtp gene was from enumeration of cDNA sequence reads that mapped to a set of selected B. hermsii genes. This measure of transcription of the infecting strain's vtp gene was ~10% of that for the abundantly-expressed, serotype-defining variable antigen gene but similar to that of genes known for in vivo expression. The findings of Vtp expression in a vertebrate host and elicitation of a specific anti-Vtp antibody response support the view that balancing selection by host adaptive immunity accounts in part for the observed diversity of Vtp proteins.

Pathogen and Host Response Dynamics in a Mouse Model of Borrelia hermsii Relapsing Fever.

Most Borrelia species that cause tick-borne relapsing fever utilize rodents as their natural reservoirs, and for decades laboratory-bred rodents have served as informative experimental models for the disease. However, while there has much progress in understanding the pathogenetic mechanisms, including antigenic variation, of the pathogen, the host side of the equation has been neglected. Using different approaches, we studied, in immunocompetent inbred mice, the dynamics of infection with and host responses to North American relapsing fever agent B. hermsii. The spirochete's generation time in blood of infected mice was between 4-5 h and, after a delay, was matched in rate by the increase of specific agglutinating antibodies in response to the infection. After initiating serotype cells were cleared by antibodies, the surviving spirochetes were a different serotype and, as a population, grew more slowly. The retardation was attributable to the host response and not an inherently slower growth rate. The innate responses at infection peak and immediate aftermath were characterized by elevations of both pro-inflammatory and anti-inflammatory cytokines and chemokines. Immunodeficient mice had higher spirochete burdens and severe anemia, which was accounted for by aggregation of erythrocytes by spirochetes and their partially reversible sequestration in greatly enlarged spleens and elsewhere.

Fibronectin-binding protein of Borrelia hermsii expressed in the blood of mice with relapsing fever.

To identify and characterize surface proteins expressed by the relapsing fever (RF) agent Borrelia hermsii in the blood of infected mice, we used a cell-free filtrate of their blood to immunize congenic naive mice. The resultant antiserum was used for Western blotting of cell lysates, and gel slices corresponding to reactive bands were subjected to liquid chromatography-tandem mass spectrometry, followed by a search of the proteome database with the peptides. One of the immunogens was identified as the BHA007 protein, which is encoded by a 174-kb linear plasmid. BHA007 had sequence features of lipoproteins, was surface exposed by the criteria of in situ protease susceptibility and agglutination of Vtp(-) cells by anti-BHA007 antibodies, and was not essential for in vitro growth. BHA007 elicited antibodies during experimental infection of mice, but immunization with recombinant protein did not confer protection against needle-delivered infection. Open reading frames (ORFs) orthologous to BHA007 were found on large plasmids of other RF species, including the coding sequences for the CihC proteins of Borrelia duttonii and B. recurrentis, but not in Lyme disease Borrelia species. Recombinant BHA007 bound both human and bovine fibronectin with Kd (dissociation constant) values of 22 and 33 nM, respectively, and bound to C4-binding protein with less affinity. The distant homology of BHA007 and its orthologs to BBK32 proteins of Lyme disease species, as well as to previously described BBK32-like proteins in relapsing fever species, indicates that BHA007 is a member of a large family of multifunctional proteins in Borrelia species that bind to fibronectin as well as other host proteins.

Use of a high-throughput screen to identify Leptospira mutants unable to colonize the carrier host or cause disease in the acute model of infection.

The molecular basis for leptospirosis infection and colonization remains poorly understood, with no efficient methods available for screening libraries of mutants for attenuation. We analysed the attenuation of leptospiral transposon mutants in vivo using a high-throughput method by infecting animals with pooled sets of transposon mutants. A total of 95 mutants was analysed by this method in the hamster model of acute infection, and one mutant was identified as attenuated (M1233, lb058 mutant). All virulence factors identified in Leptospira to date have been characterized in the acute model of infection, neglecting the carrier host. To address this, a BALB/c mouse colonization model was established. The lb058 mutant and two mutants defective in LPS synthesis were colonization deficient in the mouse model. By applying the high-throughput screening method, a further five colonization-deficient mutants were identified for the mouse model; these included two mutants in genes encoding proteins with a predicted role in iron uptake (LB191/HbpA and LB194). Two attenuated mutants had transposon insertions in either la0589 or la2786 (encoding proteins of unknown function). The final attenuated mutant had an unexpected deletion of genes la0969-la0975 at the point of transposon insertion. This is the first description of defined, colonization-deficient mutants in a carrier host for Leptospira. These mutants were either not attenuated or only weakly attenuated in the hamster model of acute leptospirosis, thus illustrating that different factors that may be required in the carrier and acute models of leptospiral infection. High-throughput screening can reduce the number of animals used in virulence studies and increase the capacity to screen mutants for attenuation, thereby enhancing the likelihood of detecting unique virulence factors. A comparison of virulence factors required in the carrier and acute models of infection will help to unravel colonization and dissemination mechanisms of leptospirosis.

Alp, an arthropod-associated outer membrane protein of Borrelia species that cause relapsing fever.

Borrelia hermsii and other relapsing fever (RF) species are noted for their highly polymorphic surface antigens, the variable major proteins (VMP). Less is known about other surface proteins of these pathogens in either their vertebrate reservoirs or arthropod vectors. To further characterize these proteins, we elicited antibodies against VMP-less cells, noted antibody reactions against whole cells and cell components, and then subjected selected antigens to mass spectroscopy for amino acid sequencing for comparison against a B. hermsii genome database. One of the derived monoclonal antibodies, H0120, agglutinated spirochetes, and in Western blot analyses, it bound to a 14-kDa protein of whole cells and their membrane fractions but not after protease treatment. A search of open reading frames of the B. hermsii genome with extracted peptides identified the 14-kDa protein with bha128, a 453-nucleotide gene of the 175-kb linear plasmid. The bha128 gene was synthesized and expressed in Escherichia coli. The protein product was bound by antibody H0120. Genes homologous to bha128 occur in the RF species Borrelia turicatae, B. duttonii, and B. recurrentis but not in Lyme disease Borrelia species or other organisms. The following findings indicated an association of BHA128, renamed Alp, with the tick environment: (i) Alp was produced at higher levels at 23°C than at 34 °C; (ii) almost all spirochetes in tick salivary glands were bound by the H0120 antibody, but only ~1% of spirochetes in the blood of infected mice were bound; and (iii) infected mice produced antibodies to several B. hermsii antigens but not detectably to native or recombinant Alp.

Cyclic and acyclic defensins inhibit human immunodeficiency virus type-1 replication by different mechanisms.

Defensins are antimicrobial peptides expressed by plants and animals. In mammals there are three subfamilies of defensins, distinguished by structural features: alpha, beta and theta. Alpha and beta-defensins are linear peptides with broad anti-microbial activity that are expressed by many mammals including humans. In contrast, theta-defensins are cyclic anti-microbial peptides made by several non-human primates but not humans. All three defensin types have anti-HIV-1 activity, but their mechanisms of action differ. We studied the anti-HIV-1 activity of one defensin from each group, HNP-1 (alpha), HBD-2 (beta) and RTD-1 (theta). We examined how each defensin affected HIV-1 infection and demonstrated that the cyclic defensin RTD-1 inhibited HIV-1 entry, while acyclic HNP-1 and HBD-2 inhibited HIV-1 replication even when added 12 hours post-infection and blocked viral replication after HIV-1 cDNA formation. We further found that all three defensins downmodulated CXCR4. Moreover, RTD-1 inactivated X4 HIV-1, while HNP-1 and HBD-2 inactivated both X4 and R5 HIV-1. The data presented here show that acyclic and cyclic defensins block HIV-1 replication by shared and diverse mechanisms. Moreover, we found that HNP-1 and RTD-1 directly inhibited firefly luciferase enzymatic activity, which may affect the interpretation of previously published data.