The role of neutrophils in tuberculosis (TB) resistance and pathology is poorly understood. Neutrophil reactions are meant to target the offending pathogen but may lead to destruction of the host lung tissue, making the defending cells an enemy. Here, we show that mice of the I/St strain which are genetically susceptible to TB show an unusually high and prolonged neutrophil accumulation in their lungs after intratracheal infection. Compared to neutrophils from more resistant A/Sn mice, I/St neutrophils display an increased mobility and tissue influx, prolonged lifespan, low expression of the CD95 (Fas) apoptotic receptor, relative resistance to apoptosis, and an increased phagocytic capacity for mycobacteria. Segregation genetic analysis in (I/St ؋ A/Sn)F 2 hybrids indicates that the alleles of I/St origin at the chromosome 3 and 17 quantitative trait loci which are involved in the control of TB severity also determine a high level of neutrophil influx. These features, along with the poor ability of neutrophils to restrict mycobacterial growth compared to that of lung macrophages, indicate that the prevalence of neutrophils in TB inflammation contributes to the development of pathology, rather than protection of the host, and that neutrophils may play the role of a "Trojan horse" for mycobacteria.

ABSTRACT

The traditional classification of nucleic acid polymerases as either DNA or RNA polymerases is based, in large part, on their fundamental preference for the incorporation of either deoxyribonucleotides or ribonucleotides during chain elongation. The refined structure determination of Moloney murine leukemia virus reverse transcriptase, a strict DNA polymerase, recently allowed the prediction that a single amino acid residue at the active site might be responsible for the discrimination against the 2OH group of an incoming ribonucleotide. Mutation of this residue resulted in a variant enzyme now capable of acting as an RNA polymerase. In marked contrast to the wild-type enzyme, the K m of the mutant enzyme for ribonucleotides was comparable to that for deoxyribonucleotides. The results are consistent with proposals of a common evolutionary origin for both classes of enzymes and support models of a common mechanism of nucleic acid synthesis underlying catalysis by all such polymerases.A key characteristic of nucleic acid polymerases is their traditional classification as either DNA or RNA polymerases, which is determined by a given enzyme's ability to selectively use either deoxyribonucleotides (dNTPs) or ribonucleotides (rNTPs) as substrates for incorporation into a growing chain (1, 2). This classification, however, may not be as fundamental as originally thought (3)(4)(5). Crystallographic studies have demonstrated that DNA and RNA polymerases have remarkable structural similarities (refs. 6-15; reviewed in ref. 16), even though they lack extensive primary sequence homology. Both have a characteristic protein fold forming a nucleic acid binding cleft and a trio of carboxylic acid residues thought to participate directly in catalysis through two bound divalent metal ions. Steady-state analyses further support the notion of a common stepwise polymerization mechanism (17, 18). These observations suggest that it might be possible to convert a DNA polymerase into an RNA polymerase by relatively minor alterations in its structure.Reverse transcriptases (RTs), encoded by all retroviruses, play a defining role in the retroviral life cycle (refs. 19 and 20; for reviews see ref. 21). The enzyme is responsible for the synthesis of a double-stranded linear DNA copy of the RNA genome, which is subsequently inserted into the host genome to form the integrated proviral DNA.The reverse transcription reaction is complex, requiring RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and an associated RNase activity specific for RNA in RNA:DNA hybrid form (22). Although the enzyme can copy either RNA or DNA templates, RT, like all DNA polymerases, can only use deoxyribonucleotides, and not ribonucleotides, as substrates. Studies of the HIV-1 RT have permitted modeling of the position of the incoming nucleotide at the active site (23, 24), with ␣-helices C and E and ␤-sheet strands 6 and 9-11, setting the major topology of the dNTP binding site. A recently determined crystal structure of a catal...

Transcription elongation factors stimulate the activity of DNA-dependent RNA polymerases by increasing the overall elongation rate and the completion of RNA chains. One group of such factors, which includes Escherichia coli GreA, GreB and eukaryotic SII (TFIIS), acts by inducing hydrolytic cleavage of the transcript within the RNA polymerase, followed by release of the 3'-terminal fragment. Here we report the crystal structure of GreA at 2.2 A resolution. The structure contains an amino-terminal domain consisting of an antiparallel alpha-helical coiled-coil dimer which extends into solution, reminiscent of the coiled coil in seryl-tRNA synthetases. A site near the tip of the coiled-coil 'finger' plays a direct role in the transcript cleavage reaction by contacting the 3'-end of the transcript. The structure exhibits an unusual asymmetric charge distribution which indicates the manner in which GreA interacts with the RNA polymerase elongation complex.

Host genetics has an important role in leprosy, and variants in the shared promoter region of PARK2 and PACRG were the first major susceptibility factors identified by positional cloning. Here we report the linkage disequilibrium mapping of the second linkage peak of our previous genome-wide scan, located close to the HLA complex. In both a Vietnamese familial sample and an Indian case-control sample, the low-producing lymphotoxin-alpha (LTA)+80 A allele was significantly associated with an increase in leprosy risk (P = 0.007 and P = 0.01, respectively). Analysis of an additional case-control sample from Brazil and an additional familial sample from Vietnam showed that the LTA+80 effect was much stronger in young individuals. In the combined sample of 298 Vietnamese familial trios, the odds ratio of leprosy for LTA+80 AA/AC versus CC subjects was 2.11 (P = 0.000024), which increased to 5.63 (P = 0.0000004) in the subsample of 121 trios of affected individuals diagnosed before 16 years of age. In addition to identifying LTA as a major gene associated with early-onset leprosy, our study highlights the critical role of case- and population-specific factors in the dissection of susceptibility variants in complex diseases.

No abstract

The GreA and GreB proteins of Escherichia coli induce cleavage of the nascent transcript in ternary elongation complexes of RNA polymerase. Gre factors are presumed to have two biologically important and evolutionarily conserved functions: the suppression of elongation arrest and the enhancement of transcription fidelity. A three-dimensional structure of GreB was generated by homology modeling on the basis of the known crystal structure of GreA. Both factors display similar overall architecture and surface charge distribution, with characteristic C-terminal globular and Nterminal coiled-coil domains. One major difference between the two factors is the "basic patch" on the surface of the coiled-coil domain, which is much larger in GreB than in GreA. In both proteins, a site near the basic patch cross-links to the 3 terminus of RNA in the ternary transcription complex. GreA/GreB hybrid molecules were constructed by genetic engineering in which the N-terminal domain of one protein was fused to the C-terminal domain of the other. In the hybrid molecules, both the coiled-coil and the globular domains contribute to specific binding of Gre factors to RNA polymerase, whereas the antiarrest activity and the GreA or GreB specificity of transcript cleavage is determined by the N-terminal domain. These results implicate the basic patch of the N-terminal coiled-coil domain as an important functional element responsible for the interactions with nascent transcript and determining the size of the RNA fragment to be excised during the course of the cleavage reaction.Two closely related Escherichia coli proteins, GreA and GreB, participate in RNA polymerase (RNAP) 1 transcription elongation by preventing and/or suppressing the condition of elongation arrest (1, 2). In addition, both factors have been shown to facilitate the transition of RNAP from the stage of abortive initiation to productive elongation (3). GreA and GreB may also have a proofreading role in transcription (4). It is thought that the Gre activity is accomplished by endonucleolytic cleavage of RNAs within the ternary elongation complexes (TECs) (2). The cleavage is followed by dissociation of the 3Ј-terminal fragment and restart of elongation from a newly generated 3Ј-OH terminus (2). Similar reactions are induced in eukaryotic TECs of RNA polymerase II by transcription elongation factor TFIIS (5-8), which performs the same functions as GreA and GreB but lacks any sequence similarity.GreA and GreB have almost the same molecular mass and share substantial amino acid sequence homology (2). However, there are several differences in their functional and biochemical properties in vitro. First, in all studied TECs that are susceptible for cleavage reactions, GreA induces hydrolysis of short RNAs 2 or 3 nucleotides long from the RNA 3Ј terminus ("type A" cleavage activity), whereas GreB stimulates hydrolysis of RNAs that are 2-18 nucleotides long, depending on the stage of transcription elongation ("type B" cleavage activity) (2, 9). Second, GreA can only prevent the ...

BackgroundDepending on the epidemiological setting, a variable proportion of leprosy patients will suffer from excessive pro-inflammatory responses, termed type-1 reactions (T1R). The LRRK2 gene encodes a multi-functional protein that has been shown to modulate pro-inflammatory responses. Variants near the LRRK2 gene have been associated with leprosy in some but not in other studies. We hypothesized that LRRK2 was a T1R susceptibility gene and that inconsistent association results might reflect different proportions of patients with T1R in the different sample settings. Hence, we evaluated the association of LRRK2 variants with T1R susceptibility.MethodologyAn association scan of the LRRK2 locus was performed using 156 single-nucleotide polymorphisms (SNPs). Evidence of association was evaluated in two family-based samples: A set of T1R-affected and a second set of T1R-free families. Only SNPs significant for T1R-affected families with significant evidence of heterogeneity relative to T1R-free families were considered T1R-specific. An expression quantitative trait locus (eQTL) analysis was applied to evaluate the impact of T1R-specific SNPs on LRRK2 gene transcriptional levels.Principal FindingsA total of 18 T1R-specific variants organized in four bins were detected. The core SNP capturing the T1R association was the LRRK2 missense variant M2397T (rs3761863) that affects LRRK2 protein turnover. Additionally, a bin of nine SNPs associated with T1R were eQTLs for LRRK2 in unstimulated whole blood cells but not after exposure to Mycobacterium leprae antigen.SignificanceThe results support a preferential association of LRRK2 variants with T1R. LRRK2 involvement in T1R is likely due to a pathological pro-inflammatory loop modulated by LRRK2 availability. Interestingly, the M2397T variant was reported in association with Crohn’s disease with the same risk allele as in T1R suggesting common inflammatory mechanism in these two distinct diseases.