An attenuated quadruple gene mutant of Mycobacterium tuberculosis imparts protection against tuberculosis in guinea pigs

Previously we had developed a triple gene mutant of Mycobacterium tuberculosis (MtbΔmms) harboring disruption in three genes, namely mptpA, mptpB and sapM. Though vaccination with MtbΔmms strain induced protection in the lungs of guinea pigs, the mutant strain failed to control the hematogenous spread of the challenge strain to the spleen. Additionally, inoculation with MtbΔmms resulted in some pathological damage to the spleens in the early phase of infection. In order to generate a strain that overcomes the pathology caused by MtbΔmms in spleen of guinea pigs and controls dissemination of the challenge strain, MtbΔmms was genetically modified by disrupting bioA gene to generate MtbΔmmsb strain. Further, in vivo attenuation of MtbΔmmsb was evaluated and its protective efficacy was assessed against virulent M. tuberculosis challenge in guinea pigs. MtbΔmmsb mutant strain was highly attenuated for growth and virulence in guinea pigs. Vaccination with MtbΔmmsb mutant generated significant protection in comparison to sham-immunized animals at 4 and 12 weeks post-infection in lungs and spleen of infected animals. However, the protection imparted by MtbΔmmsb was significantly less in comparison to BCG immunized animals. This study indicates the importance of attenuated multiple gene deletion mutants of M. tuberculosis for generating protection against tuberculosis

Histopathological analysis of guinea pig lungs post intradermal administration of <i>M</i>. <i>tuberculosis</i>, MtbΔ<i>bioA</i> or BCG.

<p>The figure depicts representative 40x magnification photomicrographs of formalin fixed, paraffin embedded and haematoxylin-eosin (H&E) stained 5 μm sections of lung tissue of guinea pigs intradermally inoculated with <i>M</i>. <i>tuberculosis</i>, MtbΔ<i>bioA</i> or BCG and euthanized at (a) 1 week, (b) 3 weeks and (c) 6 weeks post inoculation. AS denotes alveolar spaces and G denotes granuloma. The graphical representation of the total granuloma score and granuloma fraction are shown alongside by box plot of 5 animals per group (the mean is represented by ‘+’, median value is denoted by horizontal line, box represents the inter quartile range and the minimum and maximum value is denoted by whiskers). While animals inoculated with <i>M</i>. <i>tuberculosis</i> showed increasing pathological damage, the animals infected with MtbΔ<i>bioA</i> or BCG continued to exhibit no granulomatous pathology. The scale bars depict 200 μm. *<i>p</i> <0.05 (Mann-Whitney test, two-tailed).</p

Evaluation of protective efficacy of MtbΔ<i>bioA</i> in guinea pig model of experimental tuberculosis.

<p>(a) Experimental protocol for assessing the protective efficacy of MtbΔ<i>bioA</i> against infection with virulent <i>M</i>. <i>tuberculosis</i> in guinea pigs. Guinea pigs (n = 6) were vaccinated with BCG or a single dose of MtbΔ<i>bioA</i> or two doses of MtbΔ<i>bioA</i> administered at 6-week interval (Δ<i>bioA/</i> Δ<i>bioA</i>). Vaccinated guinea pigs were challenged via the aerosol route with ~50 bacilli of virulent <i>M</i>. <i>tuberculosis</i> at 12 weeks from primary immunization and euthanized at 4 weeks post challenge. Sham-immunized animals were taken as control. (b, c) Enumeration of bacillary load in the lungs and spleen of vaccinated guinea pigs at 4 weeks post challenge. In both lungs and spleen, MtbΔ<i>bioA</i> imparted significant protection over sham-immunized controls. Booster dose with MtbΔ<i>bioA</i> failed to confer any improvement in protective efficacy imparted by single dose of MtbΔ<i>bioA</i>. Each data point represents the log₁₀ CFU/ organ for an individual animal and the bar depicts mean (±SE) for each group. **<i>p</i> <0.01 and ***<i>p</i> <0.001 (one-way ANOVA followed by Tukey’s multiple comparison test).</p

Influence of disruption of the <i>bioA</i> gene on the attenuation of <i>M</i>. <i>tuberculosis</i> in guinea pigs.

<p>(a) Experimental protocol for evaluating the influence of disruption of the <i>bioA</i> gene on the pathogenicity of <i>M</i>. <i>tuberculosis</i> in guinea pigs infected via aerosol. The figure shows the bacillary load in the (b) lungs and (c) spleen of guinea pigs at 3, 6 and 12 weeks post infection with <i>M</i>. <i>tuberculosis</i> (○), MtbΔ<i>bioA</i> (□) or MtbΔ<i>bioA</i>.comp (△) strains. Three animals per group were euthanized to determine the retained dose in the lungs following aerosol infection. Each data point at 3, 6, and 12 weeks represents the mean (±SE) of the log₁₀ CFU/organ obtained with six guinea pigs per experimental group. Dotted line depicts the limit of detection for pulmonary bacillary load, meaning thereby that at 6 and 12 weeks post infection no bacilli were obtained from the total homogenate of half lung of MtbΔ<i>bioA</i> infected animals. No bacilli were obtained from the complete spleen homogenates of MtbΔ<i>bioA</i> infected animals at 12 weeks post infection. *** <i>p</i> <0.001 [unpaired <i>t</i>-test (two-tailed)] when compared with MtbΔ<i>bioA</i> infected animals.</p

Histopathological analysis of vaccinated animals at 4 weeks post challenge.

<p>(a) The figure depicts representative 40x magnification photomicrographs of formalin fixed, paraffin embedded and haematoxylin-eosin (H&E) stained 5 μm sections of lung tissue of guinea pigs (n = 6) vaccinated with BCG, MtbΔ<i>bioA</i>, two doses of MtbΔ<i>bioA</i> (Δ<i>bioA</i>/Δ<i>bioA</i>) or saline and euthanized at 4 weeks post challenge. The scale bars depict 200 μm. AS and G denote alveolar spaces and granuloma, respectively. (b) The graphical representation of the total granuloma score of the lung sections is shown by box plot of 6 animals per group (the mean is represented by ‘+’, median value is denoted by horizontal line, box represents the inter quartile range and the minimum and maximum value is denoted by whiskers). *<i>p</i> <0.05 and **<i>p</i> <0.01 (Mann-Whitney test, two tailed). (c) The figure depicts the graphical representation of the number of granulomas that were scored in lung section from each animal in a group. The black region depicts the number of necrotic granulomas out of the total number of granulomas seen in lung section from each animal. (d) The graphical representation of the total granuloma score of the liver sections of 6 animals per group is shown by box plot (the mean is represented by ‘+’, median value is denoted by horizontal line, box represents the inter quartile range and the minimum and maximum value is denoted by whiskers). **<i>p</i> <0.01 (Mann-Whitney test, two tailed).</p

<i>bioA</i> mutant of <i>Mycobacterium tuberculosis</i> shows severe growth defect and imparts protection against tuberculosis in guinea pigs

<div><p>Owing to the devastation caused by tuberculosis along with the unsatisfactory performance of the Bacillus Calmette–Guérin (BCG) vaccine, a more efficient vaccine than BCG is required for the global control of tuberculosis. A number of studies have demonstrated an essential role of biotin biosynthesis in the growth and survival of several microorganisms, including mycobacteria, through deletion of the genes involved in <i>de novo</i> biotin biosynthesis. In this study, we demonstrate that a <i>bioA</i> mutant of <i>Mycobacterium tuberculosis</i> (MtbΔ<i>bioA</i>) is highly attenuated in the guinea pig model of tuberculosis when administered aerogenically as well as intradermally. Immunization with MtbΔ<i>bioA</i> conferred significant protection in guinea pigs against an aerosol challenge with virulent <i>M</i>. <i>tuberculosis</i>, when compared with the unvaccinated animals. Booster immunization with MtbΔ<i>bioA</i> offered no advantage over a single immunization. These experiments demonstrate the vaccinogenic potential of the attenuated <i>M</i>. <i>tuberculosis bioA</i> mutant against tuberculosis.</p></div

Histopathology of guinea pig tissues post aerosol infection with <i>M</i>. <i>tuberculosis</i>, MtbΔ<i>bioA</i> or MtbΔ<i>bioA</i>.comp strains.

<p>The figure depicts representative 40x magnification photomicrographs of formalin fixed, paraffin embedded and haematoxylin-eosin (H&E) stained 5 μm sections of lung tissue of guinea pigs aerogenically infected with <i>M</i>. <i>tuberculosis</i>, MtbΔ<i>bioA</i> or MtbΔ<i>bioA</i>.comp and euthanized at (a) 3 weeks, (b) 6 weeks and (c) 12 weeks post infection. AS, G and N denote alveolar spaces, granuloma and necrosis, respectively. The graphical representation of the total granuloma score and granuloma fraction are shown alongside by box plot of all six animals per group (the mean is represented by ‘+’, median value is denoted by horizontal line, box represents the inter quartile range and the minimum and maximum value is denoted by whiskers). (d) Total granuloma score for liver sections of infected animals (n = 6 animals per group) at 3, 6 and 12 weeks post infection. MtbΔ<i>bioA</i> infected animals showed negligible pulmonary as well as hepatic granulomatous pathological damage. <i>M</i>. <i>tuberculosis</i> and MtbΔ<i>bioA</i>.comp infected animals displayed increasing number of granulomas in the lungs as well as liver. The scale bars depict 200 μm. *<i>p</i> <0.05 and **<i>p</i> <0.01 (Mann-Whitney test, two-tailed).</p

Evaluation of the gross pathological damage of the organs of vaccinated guinea pigs at 4 weeks post challenge.

<p>(a) The figure depicts representative photographs of lungs, spleen and liver of guinea pigs belonging to different groups of vaccination at 4 weeks post challenge. Graphical representation of gross pathological scores assigned to the lungs, spleen and liver of guinea pigs euthanized at 4 weeks post challenge is represented alongside. Each data point represents the score assigned to an individual animal. The bar depicts median (± interquartile range) for each group. * <i>p</i> <0.05 and **<i>p</i> <0.01 (Kruskal-Wallis test followed by the Dunn's multiple comparison test). (b) The graphical representation of organ (lungs and spleen) weights of guinea pigs at 4 weeks post challenge. The bar depicts mean (± SD) for each group. * <i>p</i> <0.05, **<i>p</i> <0.01 and ***<i>p</i> <0.001 [unpaired (two-tailed) <i>t</i>-test].</p

Bacterial strains, plasmids and primers employed in this study.

<p>Bacterial strains, plasmids and primers employed in this study.</p

Confirmation of disruption of the <i>bioA</i> gene in <i>M</i>. <i>tuberculosis</i>.

<p>(a) Diagrammatic representation of the <i>bioA</i> locus in <i>M</i>. <i>tuberculosis</i> and MtbΔ<i>bioA</i>. The <i>bioA</i> gene is the first gene of an operon consisting of three downstream genes <i>bioF1</i>, <i>bioD</i> and Rv1571. The figure depicts the disruption of the <i>bioA</i> gene in MtbΔ<i>bioA</i> by homologous recombination. Arrows show the location of the <i>bioA</i> gene specific primers F-<i>bioA</i> and R-<i>bioA</i> employed for the confirmation of disruption of the <i>bioA</i> gene by PCR. (b) PCR based confirmation of disruption of <i>bioA</i> gene in MtbΔ<i>bioA</i>. A 1.3 kb PCR amplification product was obtained with the <i>M</i>. <i>tuberculosis</i> genomic DNA as template (lane 1) and 2.4 kb PCR amplification product was obtained with MtbΔ<i>bioA</i> genomic DNA as template (lane 2). λ<i>Hind</i>III DNA molecular mass marker and 100 bp ladder were loaded in lanes 3 and 4, respectively. (c) Confirmation of disruption of <i>bioA</i> in MtbΔ<i>bioA</i> by immunoblot analysis. 10 μg of cell lysate of <i>M</i>. <i>tuberculosis</i> (lane 1), MtbΔ<i>bioA</i> (lane 2) and MtbΔ<i>bioA</i>.comp (lane 3) strains were separated on a 12.5% polyacrylamide gel and immunoblot analysis was carried out with anti-BioA polyclonal antiserum. BioA (~48 kDa band) was detected in the cell lysate of the <i>M</i>. <i>tuberculosis</i> and MtbΔ<i>bioA</i>.comp. The disruption of <i>bioA</i> in MtbΔ<i>bioA</i> was confirmed by the absence of the ~48 kDa band in lane 2. Protein molecular weight marker and 150 ng of purified BioA were loaded in lanes 4 and 5, respectively. (d, e, f) Influence of disruption of the <i>bioA</i> gene on the growth of <i>M</i>. <i>tuberculosis</i> in culture media. <i>M</i>. <i>tuberculosis</i>, MtbΔ<i>bioA</i> and MtbΔ<i>bioA</i>.comp were cultured separately in (d) MB7H9 medium, (e) modified 7H9 medium (prepared without biotin) and (f) modified 7H9 medium supplemented with 1 mg/L of biotin. Growth at 37°C/200 rpm was monitored for 14 days by measuring the absorbance at 600 nm. MtbΔ<i>bioA</i> failed to grow in modified 7H9 medium and the growth was restored to wild type levels when modified 7H9 was supplemented with 1 mg/L or 0.5 mg/L of biotin. Two independent growth analysis experiments were carried out in duplicates and the values of absorbance are represented as the mean (±SE).</p