Transcriptional Profile of <i>Mycobacterium tuberculosis</i> Replicating in Type II Alveolar Epithelial Cells

<div><p><i>Mycobacterium tuberculosis</i> (<i>M</i>. <i>tb</i>) infection is initiated by the few bacilli inhaled into the alveolus. Studies in lungs of aerosol-infected mice provided evidence for extensive replication of <i>M</i>. <i>tb</i> in non-migrating, non-antigen-presenting cells in the alveoli during the first 2–3 weeks post-infection. Alveoli are lined by type II and type I alveolar epithelial cells (AEC) which outnumber alveolar macrophages by several hundred-fold. <i>M</i>. <i>tb</i> DNA and viable <i>M</i>. <i>tb</i> have been demonstrated in AEC and other non-macrophage cells of the kidney, liver, and spleen in autopsied tissues from latently-infected subjects from TB-endemic regions indicating systemic bacterial dissemination during primary infection. <i>M</i>. <i>tb</i> have also been demonstrated to replicate rapidly in A549 cells (type II AEC line) and acquire increased invasiveness for endothelial cells. Together, these results suggest that AEC could provide an important niche for bacterial expansion and development of a phenotype that promotes dissemination during primary infection. In the current studies, we have compared the transcriptional profile of <i>M</i>. <i>tb</i> replicating intracellularly in A549 cells to that of <i>M</i>. <i>tb</i> replicating in laboratory broth, by microarray analysis. Genes significantly upregulated during intracellular residence were consistent with an active, replicative, metabolic, and aerobic state, as were genes for tryptophan synthesis and for increased virulence (ESAT-6, and ESAT-6-like genes, <i>esxH</i>, <i>esxJ</i>, <i>esxK</i>, <i>esxP</i>, and <i>esxW</i>). In contrast, significant downregulation of the DevR (DosR) regulon and several hypoxia-induced genes was observed. Stress response genes were either not differentially expressed or were downregulated with the exception of the heat shock response and those induced by low pH. The intra-type II AEC <i>M</i>. <i>tb</i> transcriptome strongly suggests that AEC could provide a safe haven in which <i>M</i>. <i>tb</i> can expand dramatically and disseminate from the lung prior to the elicitation of adaptive immune responses.</p></div

Numbers of <i>M</i>. <i>tb</i> genes differentially expressed in A549 cells arranged by functional category.

<p>^a Functional categories as designated in the Tuberculist website (<a href="http://tuberculist.epfl.ch/index.html" target="_blank">http://tuberculist.epfl.ch/index.html</a>).</p><p>^b Numbers in parentheses below Functional Category indicate total number of genes in the category in the <i>M</i>. <i>tb</i> H37Rv genome.</p><p>^c Percentages in parentheses indicate the percentage of genes differentially expressed within the indicated category.</p><p>Numbers of <i>M</i>. <i>tb</i> genes differentially expressed in A549 cells arranged by functional category.</p

quantitative RT-PCR verification of select genes.

<p>^aFold change indicates the ratio of the normalized number of transcripts for the select gene during <i>M</i>. <i>tb</i> infection of A549 cells to the normalized number of transcripts of the same gene during mid-log growth of <i>M</i>. <i>tb</i> in 7H9 media with standard deviation in parentheses.</p><p>*P-value indicates the statistical comparison of fold change of the select gene to fold change of the negative control (23S RNA) by unpaired two-tail t test. P-values ≤ 0.5 are statistically significant and are highlighted in bold.</p><p>quantitative RT-PCR verification of select genes.</p

Differentially expressed functional categories indicative of <i>M</i>. <i>tb</i> active state in type II AEC.

<p>^a Number within parentheses indicates total number of genes in the <i>M</i>. <i>tb</i> genome within this functional group</p><p>^b Bold number within parentheses indicates statistical significance, i.e., P value ≤ 0.05</p><p>Differentially expressed functional categories indicative of <i>M</i>. <i>tb</i> active state in type II AEC.</p

Differentially expressed functional categories indicative of <i>M</i>. <i>tb</i> replication in type II AEC.

<p>^a Number within parentheses indicates total number of genes in the <i>M</i>. <i>tb</i> genome within this functional group</p><p>^b Bold number within parentheses indicates statistical significance, i.e., P value ≤ 0.05</p><p>Differentially expressed functional categories indicative of <i>M</i>. <i>tb</i> replication in type II AEC.</p

Genes encoding ESAT-6-like proteins differentially expressed by <i>M</i>. <i>tb</i> in type II AEC.

<p>^a Number within parentheses indicates total number of genes in the <i>M</i>. <i>tb</i> genome within this functional group</p><p>^b Bold number within parentheses indicates statistical significance, i.e., P value ≤ 0.05</p><p>Genes encoding ESAT-6-like proteins differentially expressed by <i>M</i>. <i>tb</i> in type II AEC.</p

Differentially expressed functional categories indicative of <i>M</i>. <i>tb</i> response to stress in type II AEC.

<p>^a Number within parentheses indicates total number of genes in the <i>M</i>. <i>tb</i> genome within this functional group</p><p>^b Bold number within parentheses indicates statistical significance, i.e., P value ≤ 0.05</p><p>^c (Sherman et al., 2001) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123745#pone.0123745.ref056" target="_blank">56</a>]</p><p>^d (O’Toole and Williams, 2004) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123745#pone.0123745.ref061" target="_blank">61</a>]</p><p>^e (Fisher et al., 2002) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123745#pone.0123745.ref058" target="_blank">58</a>]</p><p>Differentially expressed functional categories indicative of <i>M</i>. <i>tb</i> response to stress in type II AEC.</p

Differentially expressed functional categories indicative of <i>M</i>. <i>tb</i> respiratory and metabolic state in type II AEC.

<p>^a Number within parentheses indicates total number of genes in the <i>M</i>. <i>tb</i> genome within this functional group</p><p>^b Bold number within parentheses indicates statistical significance, i.e., P value ≤ 0.05</p><p>Differentially expressed functional categories indicative of <i>M</i>. <i>tb</i> respiratory and metabolic state in type II AEC.</p

Differentially expressed functional categories indicative of <i>M</i>. <i>tb</i> access to iron and nutrients in type II AEC.

<p>^a Number within parentheses indicates total number of genes in the <i>M</i>. <i>tb</i> genome within this functional group</p><p>^b Bold number within parentheses indicates statistical significance, i.e., P value ≤ 0.05</p><p>Differentially expressed functional categories indicative of <i>M</i>. <i>tb</i> access to iron and nutrients in type II AEC.</p

The National Spatial Strategy: Lessons for implementing a National Planning Framework

The Irish Government is in the process of developing a National Planning Framework (NPF). This will replace the National Spatial Strategy for Ireland 2002-2020 (NSS). The NSS is generally considered to have been unsuccessful, mainly due to a lack of implementation driven by shortcomings in governance. This paper explores these shortcomings, and suggests ways to prevent similar difficulties with the NPF. The paper concludes that the political process needs to be at the heart of the preparation and adoption of the NPF. There is the danger that the NPF will fail if the political environment remains embedded in traditional approaches to planning across the state