Differential fadE28 expression associated with phenotypic virulence of Mycobacterium tuberculosis

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Abstract

Ability to persist in human macrophages is central to the virulence of Mycobacterium tuberculosis and is not invariable among various strains. Differential gene expression that is associated with phenotypic virulence may provide additional information of virulent genes involved in the pathogenesis of M. tuberculosis, which is not fully elucidated. Three hypervirulent strains of M. tuberculosis isolated from patients suffering with tuberculous meningitis were shown to grow more rapidly inside human macrophages in a previous study. In the current investigation, expression of 7 mycobacterial genes (fadE28, mce1A, mymA, acr, sigA, sugC, and Rv3723) of these strains during ex vivo macrophage challenge and in vitro acid shock was quantified by real-time PCR. Using rrs gene as a normalisation gene, fadE28 gene exhibited differential gene expression that is associated with phenotypic virulence, whereas the other 6 genes showed indistinguishable expression patterns. Up-regulation of fadE28 gene in the hypervirulent strains may account for virulence by increasing the efficiency of beta-oxidation, which is important for the persistence in macrophages as M. tuberculosis uses fatty acids preferably inside phagosome of macrophages. The fadE28 gene, together with its adjacent genes may also be critical in the process of lipid modification that could facilitate parasitism in human macrophages.

Introduction

Tuberculosis remains to be a major health issue worldwide. The etiological agent of tuberculosis, Mycobacterium tuberculosis, causes more than 8 million morbidity and 2 million mortality annually [1]. The health issue caused by tuberculosis is worsened by the fatal combination of infection with human immunodeficiency virus (HIV) and extensive drug-resistant (XDR) M. tuberculosis [2]. To overcome the threat of tuberculosis, development of more effective drugs and vaccines, which requires a thorough understanding of the pathogenesis of M. tuberculosis, is imperative.

Although humans are equipped by an advanced immune system, M. tuberculosis has evolved to be capable of persistence in humans distinctively. The intracellular parasitism of M. tuberculosis in alveolar macrophages is apparent [3]. However, understanding of the mechanisms behind, especially at the genetic level, has been rudimentary after decades of slow progress. Roles of a few virulent genes, such as katG [4], [5], mce1A [6], [7], [8], icl [9], and pks1-15 [10], were clearly illustrated. Inactivation of katG, which encodes catalase–peroxidase–peroxynitritase, weakens the ability of M. tuberculosis to counteract reactive oxygen intermediates (ROIs) as catalase–peroxidase–peroxynitritase is capable of neutralising ROIs [5]. mce1A encodes an MCE-family protein that facilitates the entry of M. tuberculosis into mammalian cell [6], [7]. Escherichia coli expressing mce1A gene and latex micropheres coated with Mce1A protein were shown to be capable of entering Henrietta Lacks' (HeLa) cells, which are non-phagocytic [6], [7]. The active domain of Mce1A was subsequently confined to 22 highly basic amino acids located in the core region of protein, termed Inv3, which is able to induce the cytoskeletal rearrangement of HeLa cells [8]. icl has been believed to be vital for the persistent infection of M. tuberculosis [9]. The possible role of icl on persistence may be explained by the enzyme encoded, isocitrate lyase, which can further metabolise the breakdown products of beta-oxidation (β-oxidation) such as acetyl-coenzyme A, and thereby produce energy efficiently when using fatty acid as a sole carbon source [9]. pks1-15 encodes a polyketide synthase that is essential for the production of immunosuppressive phenolic glycolipid (PGL) [10]. Failure to produce PGL in certain strains, like H37Rv, was probably owing to deletion of 7 nucleotides leading to a shift of reading frame in the pks1-15 gene [10]. Nevertheless, virulent role of numerous virulent gene candidates, like mymA [11], [12], [13], acr [14], [15], [16], sigA [17], [18], [19], fadE28 [20], [21], [22], sugC [20], [21], [23], and Rv3723 [20], [21] remains to be verified, despite putative functions have been proposed for these candidates. mymA encodes a probable mycobacterial monooxygenase that belongs to an acid-induced operon (Rv3083Rv3089). This mymA operon has been speculated to be involved in alternative synthesis of mycolic acids, which are subject to be damaged by macrophages [11], [12], [13]. acr encodes a homologue of alpha-crystallin, which is a stress-induced protein induced in many stressful conditions but not in favourable conditions [14], [15]. Intriguingly, expression of acr is inversely proportional to the growth rate of M. tuberculosis [14], [15]. These findings lead to a hypothesis that acr may be involved in the deceleration of mycobacterial multiplication in order to balance the survival of M. tuberculosis and host [16]. sigA encodes a principal sigma factor that regulates the transcription of housekeeping genes [17]. Direct proportion between growth rate and sigA expression inside macrophages indicates sigA may regulates expression of not only housekeeping genes but also unknown virulent genes [18], [19]. Genes fadE28, sugC, and Rv3723 are responsible for diverse functions: fadE28 encodes acyl-coenzyme A (acyl-CoA) dehydrogenase, which participates the first step of β-oxidation [22]; SugC protein may be a member of ATP-binding cassette (ABC) transport system, which is involved in active import of sugar across the bacterial membrane [23]; Rv3723 encodes a probable transmembrane protein that has an unknown function [24]. These 3 candidates may have certain roles in the pathogenesis of M. tuberculosis since inactivation of fadE28, sugC, or Rv3723 was shown to attenuate the survival of M. tuberculosis inside both macrophages and mice [20], [21].

As individual strains of M. tuberculosis were demonstrated to be more virulent than the others as determined by the models of macrophage [25], [26], [27] and mouse [28], [29], intrinsic difference must be present and thereby contributes to the heterogeneous phenotypic virulence. Three hypervirulent strains of M. tuberculosis were previously isolated from patients suffering tuberculous meningitis [27]. These 3 hypervirulent strains exhibited intracellular multiplication inside human macrophages after 10 days of infection, which grew similarly with or faster than the virulent strain H37Rv [27]. In contrast, 122 hypovirulent clinical isolates exhibited no intracellular survival on day 10, like the avirulent strain H37Ra [27]. Differential gene expression present in these strains under the challenge of human macrophages may provide a clue to identify the virulent genes of M. tuberculosis.

In the current study, mycobacterial gene expression of 7 genes, which included mce1A, mymA, acr, sigA, fadE28, sugC, and Rv3723, during ex vivo macrophage challenge and in vitro acid shock was measured by means of relative quantification in an attempt to identify possible differential gene expression that is associated with the phenotypic virulence of M. tuberculosis.

Section snippets

Differential gene expression during ex vivo macrophage challenge

Using rrs as a normalisation gene, differential gene expression that is associated with the phenotypic virulence of M. tuberculosis was observed in the fadE28 gene, whereas the other 6 genes investigated (mce1A, mymA, acr, sigA, sugC, and Rv3723) exhibited indistinguishable expression patterns during 48 h of ex vivo macrophage challenge (Fig. 1). In general, the fadE28 gene was up-regulated in 3 hypervirulent strains (H107, H108, and H112) and the H37Rv, but was down-regulated in 3 hypovirulent

M. tuberculosis strains

The 3 hypervirulent strains (H107, H108, and H112) and the 3 hypovirulent strains (H9, H54, and H111) were characterised in our previous study [27]. Standard virulent strain H37Rv (ATCC 27294) was purchased from American type culture collection (ATCC). All M. tuberculosis strains were cultured in Bactec Myco/F lytic culture vials (Becton Dickinson) at 37 °C with continuous shaking at 130 rpm in a tilted position until mid-logarithmatic phase, which was monitored spectrophotometrically at 600 nm (OD

Acknowledgements

We would like to thank The Hong Kong Red Cross and Blood Transfusion Services for providing buffy coat from normal human blood donors.

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