Strain-Specific Differences in the Genetic Control of Two Closely Related Mycobacteria

The host response to mycobacterial infection depends on host and pathogen genetic factors. Recent studies in human populations suggest a strain specific genetic control of tuberculosis. To test for mycobacterial-strain specific genetic control of susceptibility to infection under highly controlled experimental conditions, we performed a comparative genetic analysis using the A/J- and C57BL/6J-derived recombinant congenic (RC) mouse panel infected with the Russia and Pasteur strains of Mycobacterium bovis Bacille Calmette Guérin (BCG). Bacillary counts in the lung and spleen at weeks 1 and 6 post infection were used as a measure of susceptibility. By performing genome-wide linkage analyses of loci that impact on tissue-specific bacillary burden, we were able to show the importance of correcting for strain background effects in the RC panel. When linkage analysis was adjusted on strain background, we detected a single locus on chromosome 11 that impacted on pulmonary counts of BCG Russia but not Pasteur. The same locus also controlled the splenic counts of BCG Russia but not Pasteur. By contrast, a locus on chromosome 1 which was indistinguishable from Nramp1 impacted on splenic bacillary counts of both BCG Russia and Pasteur. Additionally, dependent upon BCG strain, tissue and time post infection, we detected 9 distinct loci associated with bacillary counts. Hence, the ensemble of genetic loci impacting on BCG infection revealed a highly dynamic picture of genetic control that reflected both the course of infection and the infecting strain. This high degree of adaptation of host genetics to strain-specific pathogenesis is expected to provide a suitable framework for the selection of specific host-mycobacteria combinations during co-evolution of mycobacteria with humans

The genetic dissection of mycobacterial infection

The host response to mycobacterial infection is highly variable. A role for hostand bacterial factors in this variability is well established, although it is not knownwhether these factors act independently of each other or whether infectionoutcome results from a joint effect of host and pathogen. To address thisquestion, the present thesis concurrently examined the effect of geneticallycontrolled host and bacterial factors using a mouse model of infection. A panel ofrecombinant congenic (RC) mouse strains and their A/J and C57BL/6J inbredprogenitors were infected with virulent Mycobacterium tuberculosis or theattenuated M. bovis Bacille Calmette Guérin (BCG) Russia and Pasteur strains. Ajoint effect of host and pathogen on the course of mycobacterial infection wasobserved at both the phenotypic and genetic level. In the A/J and C57BL/6Jmouse strains, pathogen-associated factors had a major impact on biologicalphenotypes (pulmonary replication, lung histopathology) as well as mechanisticphenotypes (pulmonary transcription of Ifng, Il12b, Il4 and chemokine genes)with the host genetic background modulating the magnitude of these responses.At the genetic level, a comparative analysis of the pulmonary and splenic countsof BCG Russia and BCG Pasteur in the RC strains revealed that mycobacterialinfection is under the control of both generic and strain-specific genetic effects. Alocus on chromosome 1 indistinguishable from Nramp1 controlled early BCGPasteur and early and late BCG Russia infection in a spleen-specific manner.Loci impacting on the counts of BCG Russia but not BCG Pasteur were identifiedon chromosome 13 for the spleen and on chromosome 11 for the lung and spleenat the late phase of infection. M. tuberculosis infection was also under distinctgenetic control in the RC strains, further demonstrating that genetic control ofmycobacterial infection is adapted to the infecting mycobacterial strain. A stronggenetic effect detected on chromosome 10 was linked with early death followingM. tuberculosis infection. Analysis conditional on this locus identified a set ofgenetic control elements on chromosomes 2, 4, and 13. Together, these studiesprovide compelling evidence for strong specificity of the host response to theinfecting pathogen.La réponse de l'hôte aux infections par des mycobactéries est hautement variable.Des facteurs de l'hôte et de la bactérie ont un rôle à jouer dans cette variabilitébien qu'on ne sache pas si ces facteurs agissent indépendamment ou si le résultatde l'infection découle d'un effet combiné de l'hôte et du pathogène. Pour répondreà cette question, la présente thèse a évalué l'impact de facteurs de l'hôte et dupathogène sous contrôle génétique à l'aide d'un modèle murin d'infection. Unpanel de souches de souris congéniques recombinantes (CR) et leurs progéniteursconsanguins A/J et C57BL/6J furent infectés avec du Mycobacterium tuberculosisvirulent ou les souches atténuées M. bovis Bacille Calmette Guérin (BCG) Russieet Pasteur. Un effet combiné de l'hôte et du pathogène sur le déroulement del'infection mycobactérienne fut observé autant aux niveaux phénotypiques quegénétiques. Chez les souches A/J et C57BL/6J, des facteurs associés aupathogène ont eu un impact majeur sur les phénotypes biologiques (réplication auniveau du poumon, histopathologie pulmonaire) de même que sur les phénotypesmécanistes (transcription de Ifng, Il12b, Il4 et de gènes de chimiokines dans lepoumon), alors que le fond génétique modulait la magnitude de ces réponses. Auniveau génétique, une analyse comparative des comptes pulmonaires et spléniquesde BCG Russie et BCG Pasteur chez les souches CR a révélé que l'infection pardes mycobactéries est sous le contrôle d'effets génétiques génériques de mêmeque spécifiques à la souche. Dans la rate, un locus sur le chromosome 1 qui nepouvait être discriminé de Nramp1 a contrôlé l'infection précoce par BCG Pasteuret l'infection précoce et tardive par BCG Russie. Dans la phase tardived'infection, des locus influençant les comptes de BCG Russie mais non de BCGPasteur ont été identifiés sur le chromosome 13 pour la rate et sur le chromosome11 pour la rate et le poumon. L'infection par M. tuberculosis était également souscontrôle génétique distinct chez les souches CR, ce qui démontre à nouveau que lecontrôle génétique d'infections mycobactériennes est adapté à la souche demycobactérie qui infecte. Un important effet génétique détecté sur lechromosome 10 fut lié à une mortalité précoce suite à une infection par M.tuberculosis. Une analyse conditionnelle à ce locus a identifié un ensemble d'éléments de contrôle génétique sur les chromosomes 2, 4 et 13. Globalement,ces études relèvent la forte spécificité de la réponse de l'hôte au pathogène quiinfecte

Impact of Methoxymycolic Acid Production by Mycobacterium bovis BCG Vaccines

BCG vaccines are a family of closely related daughter strains of an attenuated isolate of Mycobacterium bovis derived by in vitro passage from 1908 to 1921. During subsequent laboratory propagation of the vaccine strain until its lyophilization in 1961, BCG Pasteur underwent at least seven further genomic mutations. The impact of these mutations on the properties of the vaccine is currently unknown. One mutation, a glycine-to-aspartic acid substitution in the mmaA3 gene, occurred between 1927 and 1931 and impairs methoxymycolic acid synthesis in BCG strains obtained from the Pasteur Institute after this period. Mycolic acids of the cell wall are classified into three functional groups (alpha-, methoxy-, and ketomycolic acids), and together these lipids form a highly specialized permeability barrier around the bacterium. To explore the impact of methoxymycolic acid production by BCG strains, we complemented the functional gene of mmaA3 into BCG Denmark and tested a number of in vitro and in vivo phenotypes. Surprisingly, restoration of methoxymycolic acids alone had no effect on cell wall permeability, resistance to antibiotics, or growth in cultured macrophages and C57BL/6 mice. Our results demonstrate that the loss of methoxymycolic acid production did not apparently affect the virulence of BCG strains

Specific Dysregulation of IFNγ Production by Natural Killer Cells Confers Susceptibility to Viral Infection

<div><p>Natural Killer (NK) cells contribute to the control of viral infection by directly killing target cells and mediating cytokine release. In C57BL/6 mice, the Ly49H activating NK cell receptor plays a key role in early resistance to mouse cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. Here we show that transgenic expression of Ly49H failed to provide protection against MCMV infection in the naturally susceptible A/J mouse strain. Characterization of Ly49H⁺ NK cells from <i>Ly49h</i>-A transgenic animals showed that they were able to mount a robust cytotoxic response and proliferate to high numbers during the course of infection. However, compared to NK cells from C57BL/6 mice, we observed an intrinsic defect in their ability to produce IFNγ when challenged by either m157-expressing target cells, exogenous cytokines or chemical stimulants. This effect was limited to NK cells as T cells from C57BL/6 and <i>Ly49h</i>-A mice produced comparable cytokine levels. Using a panel of recombinant congenic strains derived from A/J and C57BL/6 progenitors, we mapped the genetic basis of defective IFNγ production to a single 6.6 Mb genetic interval overlapping the <i>Ifng</i> gene on chromosome 10. Inspection of the genetic interval failed to reveal molecular differences between A/J and several mouse strains showing normal IFNγ production. The chromosome 10 locus is independent of MAPK signalling or decreased mRNA stability and linked to MCMV susceptibility. This study highlights the existence of a previously uncovered NK cell-specific <i>cis</i>-regulatory mechanism of <i>Ifnγ</i> transcript expression potentially relevant to NK cell function in health and disease.</p></div

Chromosome 10 controls both IFNγ production and MCMV resistance.

<p>(A, B) Freshly isolated splenocytes from B6, Css10 and BcA9 mice were stimulated with P/I for 4 h. The plots are gaited (A) on CD3⁻DX5⁺ NK cells (B) CD3⁺DX5⁻ T cells and the percentage of cells expressing intracellular IFNγ is shown. (C) Splenocytes from indicated strains were incubated for 4 h with RMAS-m157 and the percentage of Ly49H⁺ NK cells expressing intracellular IFNγ is shown. Data were analyzed using one way ANOVA with Bonferoni post-test and presented as mean ± SEM. **<i>P</i><0.01, ***<i>P</i><0.001. (D) Indicated mice were infected with MCMV and viral load was quantified from the spleen at day 3 p.i. Data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM. ***<i>P</i><0.001. Similar results were obtained in another independent experiment.</p

Decreased IFNγ production by Ly49H⁺ NK cells in A/J mice.

<p>Splenocytes from B6 and A-<i>Ly49h</i> mice were harvested and stimulated with RMAs-m157, BAF-m157, IL12/IL18 or PMA and ionomycin (P/I) for 3–5 h. (A) Representative dot plots demonstrating IFNγ production following stimulation and gaited on CD3⁻DX5⁺ Ly49H⁺ NK cells. The numbers represent the percentage of Ly49H⁺ producing IFNγ. (B) Graphical representation of IFNγ production by CD3⁻DX5⁺ Ly49H⁺ NK cells following stimulation. (C) Representative dot plots showing IFNγ production following stimulation and gaited on CD3⁺ or CD3⁻ cells. Numbers represent the percentage of cells producing IFNγ. (D) Graphical representation of IFNγ production by CD3⁻ DX5⁺ (T cells) or CD3⁺ DX5⁻ (NK cells) after P/I stimulation. Data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM and <i>P</i> values of significant results between groups are indicated. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Mapping of IFNγ production by NK cells reveals a single locus on chromosome 10.

<p>(A) Genome-wide linkage analysis was done using mice from the 33 RCS strains outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004511#ppat-1004511-g004" target="_blank">Figure 4A</a>. IFNγ production by NK cells following P/I treatment was used as the mapping trait. The negative log genome-wide <i>p</i> values are shown. (B) Chr 10 negative log genome-wide <i>p</i> values of IFNγ production by NK cells upon P/I treatment. (C) Map of the 6.6 Mbp relevant interval in chr 10 harboring 45 genes in black rectangles (adapted from UCSC mouse genome browser, mm9).</p

NK cells from A/J mice can proliferate and produce Granzyme B and Perforin following MCMV inoculums.

<p>Splenocytes were harvested from naïve B6 and A-<i>Ly49h</i> mice (day 0) or mice infected with 2500 PFU MCMV (n = 3 mice/time point). Time points post-infection are indicated in the figure. (A) BrdU incorporation was analyzed on CD3⁻DX5⁺ Ly49H⁺ NK cells by flow cytometry. (B) Spleen viral titers were determined by PA. Intracellular (C) Granzyme (D) Perforin expression was analyzed by flow cytometry on CD3-DX5+ Ly49H+ NK cells. Representative plots from individual mice are shown. The percent of Ly49H+ NK cells positive for Gzmb, and Prf1 are summarized for one experiment. Data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM and significant <i>P</i> values are indicated. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. These data are representative of 2–3 independent experiments.</p