Human TLR1 Deficiency Is Associated with Impaired Mycobacterial Signaling and Protection from Leprosy Reversal Reaction

Toll-like receptors (TLRs) are important regulators of the innate immune response to pathogens, including Mycobacterium leprae, which is recognized by TLR1/2 heterodimers. We previously identified a transmembrane domain polymorphism, TLR1_T1805G, that encodes an isoleucine to serine substitution and is associated with impaired signaling. We hypothesized that this TLR1 SNP regulates the innate immune response and susceptibility to leprosy. In HEK293 cells transfected with the 1805T or 1805G variant and stimulated with extracts of M. leprae, NF-κB activity was impaired in cells with the 1805G polymorphism. We next stimulated PBMCs from individuals with different genotypes for this SNP and found that 1805GG individuals had significantly reduced cytokine responses to both whole irradiated M. leprae and cell wall extracts. To investigate whether TLR1 variation is associated with clinical presentations of leprosy or leprosy immune reactions, we examined 933 Nepalese leprosy patients, including 238 with reversal reaction (RR), an immune reaction characterized by a Th1 T cell cytokine response. We found that the 1805G allele was associated with protection from RR with an odds ratio (OR) of 0.51 (95% CI 0.29–0.87, p = 0.01). Individuals with 1805 genotypes GG or TG also had a reduced risk of RR in comparison to genotype TT with an OR of 0.55 (95% CI 0.31–0.97, p = 0.04). To our knowledge, this is the first association of TLR1 with a Th1-mediated immune response. Our findings suggest that TLR1 deficiency influences adaptive immunity during leprosy infection to affect clinical manifestations such as nerve damage and disability

Genes for the Major Structural Components of Thermotogales Species’ Togas Revealed by Proteomic and Evolutionary Analyses of OmpA and OmpB Homologs

The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompα) and the porin OmpB (or Ompβ). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant β-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had β-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath

Deciphering the Outer Envelope of Thermotoga maritima

Thermotoga maritima is the representative member of the order Thermotogales, which is comprised of extremely thermophilic anaerobic bacteria. All members of this order are characterized by the presence of a unique outer envelope, the toga. The outer envelope appears as polar cellular distensions that sometimes are as large as the cytoplasm. The outer envelope increases in size while the cytoplasm remains the same leading to an outer envelope distension. The outer envelope distension development and increase in its size most likely occurs for storage of nutrients. Initial observations suggest that cells create larger distensions at the poles as they enter stationary phase. Preliminary data showed an increase in the number of cells with larger outer envelope distensions from the mid-log to stationary phase. Comparative measurements of cells were taken in their respective growth phases to determine cytoplasm sizes. There was a significant (p=4.44E-31) increase in the ratio of the whole cell to the cytoplasm between the mid-log and stationary phases. There was a significant (p=1.25E-20) increase in the area of the outer envelope and an insignificant (p=0.02) increase in cytoplasm area between mid-log and stationary phase. This indicates a 1.69-fold increase in the outer envelope size while the cytoplasmic aspect of the cells remained the same. Furthermore, expression analysis was conducted to confirm growth phase specific gene expression. There was an increase in the expression of the (Outer membrane protein encoding genes) ompA1, ompB, ompA2 and ompA3 by 7.9 and 5.2, 3 and 2.2 fold respectively from mid-log to stationary phase. The beta barrel assembly machinery protein gene (bamA) showed only a 1.2 fold while the csaB was up regulated 4.4 fold between phases

Genes for the Major Structural Components of Thermotogales Species’ Togas Revealed by Proteomic and Evolutionary Analyses of OmpA and OmpB Homologs

The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompα) and the porin OmpB (or Ompβ). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant β-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had β-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath

TLR1 Polymorphism Frequency in Different Leprosy Types.

1<p>P value for comparison of allele frequencies.</p>2<p>P value for comparison of genotype frequencies.</p>3<p><b><i>A</i></b> denotes common allele, <b><i>a</i></b> denotes minor allele.</p>4<p>Tuberculoid includes TT and BT. Lepromatous includes LL, BL, and BB.</p

Baseline characteristics of leprosy subjects.

1<p>Percentages of individuals in some categories do not sum to 100 due to missing data. Not shown among all leprosy are 8 subjects with peripheral neuropathy (PN), and one with leprosy of indeterminate type (IN).</p>2<p>The four most frequent ethnic groups (from > 8 ethnic groups) are tabulated.</p>3<p>P represents exact <i>P</i> values for overall distribution of gender and age groups within the different ethnic groups and for the overall distribution of leprosy type and immune reactions among the 4 different ethnic groups.</p

IL-β and TNF-α production in human mononuclear cells following stimulation with <i>M. leprae</i>.

<p>Peripheral blood mononuclear cells were stimulated for 18 hours and supernatants were assayed for cytokine production by luminex multiplex bead assay. PBMCs were obtained from 11 individuals with the genotype 1805TT or 1805TG (TT/TG: dark circles) and 10 individuals with the 1805GG genotypes (GG: open circles). PBMCs stimulated with media, PAM2 at 75 ng/mL (P2), PAM3 at 75 ng/mL (P3), or LPS at 10 ng/ml (LPS), whole irradiated ML at 20 µg/ml (ML) or MLcw at 10 µg/ml (MLcw). IL-1β production following stimulation is shown in (A), and TNF-α production in (B). The mean level and standard error of the mean are depicted and were derived from averaging the responses of individual within each genotype group. The median level is depicted by a bar; *p≤0.05, **p≤0.001 by Mann–Whitney U-test.</p

Frequency of TLR1 haplotypes in reversal reaction.

1<p>Order for 6 SNP haplotypes, left to right: Trs5743563C, Ars5743565G, Trs5743592C, Trs5743595C, G743A, T1805G.</p>2<p>Order for 2 SNP haplotypes: Trs5743592C, T1805G.</p>3<p>P value represents comparison of a given haplotype with the reference haplotype TATTAT.</p

TLR1 Polymorphism Frequency in Reversal Reaction.

1<p>P value for comparison of allele frequencies by Chi-square.</p>2<p>P value for comparison of genotype frequencies calculated by Chi-square unless otherwise indicated.</p>3<p><b><i>A</i></b> denotes common allele, <b><i>a</i></b> denotes minor allele.</p>4<p>P value calculated by Fisher's Exact test due to small cell number.</p

IL-6 production by human primary cells following stimulation with <i>M. leprae.</i>

<p>Peripheral blood mononuclear cells were stimulated for 18 hours and supernatants were assayed for cytokine production by ELISA. PBMCs were derived from whole blood taken from 15 individuals with the genotype 1805TTor 1805TG (TT/TG: dark circles) and 13 individuals with the 1805GG genotypes (GG: open circles). (A): PBMCs stimulated in triplicate with media, PAM2 at 75 ng/ml (P2 75), PAM3 at 75 ng/ml (P3 75), or LPS at 10 ng/ml (LPS 10). (B): PBMCs stimulated in triplicate with whole irradiated ML at 20 or 100 µg/ml (ML20 or ML100) or MLcw at 2 or 10 µg/ml (MLcw 2 or MLcw10). The mean level and standard error of the mean are depicted and were derived from averaging the responses of individuals stimulated in triplicate. The median level is depicted by a bar. *P≤0.01, **P≤0.001 by Mann–Whitney U-test.</p