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. 2012 Jan 10:2:266.
doi: 10.3389/fmicb.2011.00266. eCollection 2011.

Mycobacterium tuberculosis RNA Expression Patterns in Sputum Bacteria Indicate Secreted Esx Factors Contributing to Growth are Highly Expressed in Active Disease

Archana Bukka  1 , Christopher T D Price, Douglas S Kernodle, James E Graham
Affiliations

Mycobacterium tuberculosis RNA Expression Patterns in Sputum Bacteria Indicate Secreted Esx Factors Contributing to Growth are Highly Expressed in Active Disease

Archana Bukka et al. Front Microbiol. .

Abstract

To identify factors contributing to the ability of tubercle bacilli to grow in the lung during active infection, we analyzed RNA expression patterns in bacteria present in patient sputum. Prominent among bacterial transcripts identified were those encoding secreted peptides of the Esat-6 subfamily that includes EsxK and EsxL (Rv1197 and Rv1198). H37Rv esxKL and esxJI transcripts were differentially expressed under different growth conditions, and disruption of these genes altered growth phase kinetics in typical laboratory batch broth cultures. These growth defects, including the reduced intracellular growth of an ΔesxKL mutant in primary human macrophages, were reversed by either low multiplicity co-infection or co-culture with wild-type bacteria, demonstrating the ability of the secreted factors to rescue isogenic mutants. Complementing either only esxL or esxI alone (Rv1198 or Rv1037c) also reduced observed growth defects, indicating these genes encode factors capable of contributing to growth. Our studies indicate that the Mycobacterium tuberculosis Mtb9.9 family secreted factors EsxL and EsxI can act in trans to modulate growth of intracellular bacteria, and are highly expressed during active human lung infection.

Keywords: ESX-5; QILLS-paired secreted factors; WGX100; esxN; esxO; esxV.

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Figures

Figure 1
Figure 1
Mycobacterium tuberculosis genomic array hybridized with radiolabeled cDNAs obtained by SCOTS from sputum bacteria. Array patterns obtained by hybridizing cDNAs obtained by three rounds of SCOTS from bacteria in five un-induced sputum specimens as described in Section “Materials and Methods” are shown. Array features showing the highest levels of hybridization for patient 2 (circled) were readily detected for all specimens, and correspond to ORFs Rv1197 (esxK) and Rv1198 (esxL) on the left, and Rv2346c (esxP), Rv2347c (esxO), and Rv1047 on the right.
Figure 2
Figure 2
ΔesxKL mutant is defective in intracellular growth. (A) THP-1 cells were infected with wild-type (open squares) or ΔesxKL mutant (open triangles) at a MOI of 1: l. Data points indicate the mean number of CFUs for three independently infected wells and error bars indicate SD from means. Intracellular growth of the ΔesxKL mutant was significantly reduced in three independent experiments (P < 0.005)* at 96 h as determined by unpaired t-test. (B) THP-1 cells were infected at MOI of 1:1 and a growth index as CFU at 96 h relative to CFU at 24 h was determined for wild-type (open bar), ΔesxKL (light gray bar), esxK complemented (dark gray bar), and esxL complemented (black bar) strains. Reduced intracellular growth of ΔesxKL (P < 0.005)* was reversed by restoring only esxL. Data shown is a representative of two independent experiments. (C) The ΔesxKL mutant showed this growth defect in similar experiments with a primary single-donor PBMC infection model, and restoring esxL allowed normal intracellular growth.
Figure 3
Figure 3
Co-infection with wild-type allows intracellular growth of the ΔesxKL mutant population. (A) THP-1 monolayers were infected with wild-type or ΔesxKL mutant or 1:1 ratio of wild-type and ΔesxKL mutant strains at a MOI of 1:1. CFUs were determined at 24 and 96 h post-infection. Growth indices are shown for wild-type (open bar), ΔesxKL (gray bar), and ΔesxKL sub-population in co-infection (black bar). Reduced intracellular growth of ΔesxKL mutant (P < 0.005)* was rescued by co-infection with wild-type bacteria. A representative of three independent experiments is shown (B) THP-1 monolayers were co-infected with fixed number of ΔesxKL and varying concentration of wild-type H37Rv. Growth index at 96 h post-infection for wild-type (open bar) and ΔesxKL (gray bar) in each co-infection well was determined. Data from a single experiment is shown.
Figure 4
Figure 4
esxKL mutant and wild-type bacteria do not inhabit the same cells during low MOI co-infection. A representative confocal microscopy image of THP-1 cells simultaneously infected with equal proportion of fluorescently labeled wild-type (red) and ΔesxKL mutant (green) at MOI of 1:1 is shown. Cells were infected and fixed at (A) 24 h, and (B) 72 h post-infection as described in Section “Materials and Methods.”
Figure 5
Figure 5
Secondary structure prediction for M. tuberculosis EsxL (Rv1198) and EsxI (Rv1037c). The figures depicts the difference in the alpha-helical secondary structures of EsxL and EsxI based on analysis with nnpredict (Kneller et al., 1990).
Figure 6
Figure 6
esxJIi mutation reduces growth in cultures. (A) Cultures were grown in 7H9 broth supplemented with 0.05% Tween and OADC and optical density measurements taken at 600 nm. Growth of wild-type is indicated with squares, esxJIi mutant with circles, and esxI complemented strain with triangles. The inset shows wet weight measurements of wild-type (bar), esxJIi (gray bar), and esxI complemented strain (black bar) at 15 days of growth. A representative of three independent experiments is shown. (B) Co-culturing with wild-type bacteria rescues growth of esxJIi mutant. Growth of H37Rv (squares), the esxJIi mutant (triangles), and the esxJIi mutant in co-culture (closed circles) were determined by enumerating CFUs. Plate counts were in triplicate and averaged. Inset shows growth indices for wild-type (bar), esxJIi mutant (gray bar), and esxJIi mutant in co-culture (black bar) calculated as CFU at 9 days relative day 0. Data shown is a representative of three independent experiments where the wild-type and mutant were grown both independently and in co-culture, and showed this difference in growth of the mutant.
Figure 7
Figure 7
Reduced in vitro growth of ΔesxKL mutant. Cultures were grown in M7H9 broth supplemented with 0.05% Tween and OADC and optical density measurements taken at 600 nm. Circles indicate optical density for wild-type, squares for ΔesxKL mutant, and triangles for esxL complemented mutant strain. The reduced growth rate of the mutant was restored to wild-type levels by complementing with esxL. A representative of three experiments is shown.
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