Strategic Excellence: MAP Gene Expression Programmes Following Host Cell Entry

Sun Tzu states ‘What is of supreme importance in war is to attack the enemy's strategy' (Tzu, 19 71, p. 77). The host relies on phagocytosis and phagosome maturation for infection clear­ance, but as noted above, MAP undermines these fundamental mechanisms through stress resistance and interference with host signalling.

Several sigma factors (e.g. sigH and sigE) in MAP were differentially co-regulated with a large number of genes, depending on the type of stressor applied (Wu et al., 2007b). This was further explored by Ghosh et al. (2013), clari­fying SigH as a stress-response sigma factor for early (rv3671c yielded bacterial clear­ance of up to a 3.6-log reduction by 98 days, in contrast with no reduction at 1year post­infection for wild-type strains. Gross pathology showed marked reduction in pulmonary lesions by the disrupted strain consistent with attenu­ation. This serine protease was labelled myco­bacterial acid resistance protease (MarP), and homologues are present across Mycobacterium. Further research showed ∆marP attenuation in zebrafish larvae for M. marinum (Levitte et al., 2016), as well as further exploring its potential targets (Small et al., 2013; Botella et al., 2017). MAP possesses a homologue of this target (MAP0403c or MAP_RS02055), and investiga­tion uncovered rapid MAP marP gene expression in bovine MDMs as well as bovine mammary epi­thelial (MAC-T) cells. This expression was closely tied to phagosome acidification, and if host cells were pre-treated with Bafilomycin A1, an in­hibitor of phagosomal acidification, marP gene expression was not observed (Kugadas et al., 2016). This pattern was not observed for non- pathogenic M. smegmatis’ more distant putative marP homologue, supporting a conserved mech­anism responsible for intracellular survival and persistence of pathogenic mycobacteria. Botella et al. (2017) provided evidence for peptidoglycan hydrolase RipA being a target of MarP-digestion, which ties back again to the importance of cell wall maintenance for acid stress-survival.

Unlike other mycobacteria, MAP has spe­cial iron requirements that enhance its sur­vival while inside the host. For optimal growth in vitro, MAP requires supplementation of the siderophore mycobactin J. Whole genome se­quencing of MAP K-10 provided one explana­tion for this dependency, revealing a truncation of the mbtA gene. It has been suggested that this truncation impairs the production of myco­bactin from the mbtA- J operon (Li et al., 2005; Wang et al., 2014b). Despite this truncation, Zhu et al. (2008) showed that MAP is still able to transcribe mycobactin synthesis genes inside macrophages. To corroborate these findings, Janagama et al. (2010) described the upregu­lation of several genes responsible for iron ac­quisition in infected tissues, including genes responsible for mycobactin biosynthesis.

Iron is vital to fundamental biological processes; however, high intracellular concen­trations of free iron are toxic to bacteria. As such, cells have developed tightly regulated processes for intracellular metal homeostasis (Eckelt et al., 2014). Bacteria control metal ho­meostasis by activating a set of genes regulated by metal-sensing transcription factors known as metalloregulatory proteins (Chandrangsu et al., 2017). In prokaryotes, there are two ma­jor families of metalloregulators: diphtheria toxin (DtxR) and ferric uptake regulator (FUR) (Hantke, 1981). In 2009, Janagama and others identified and characterized MAP282 7, an iron­dependent regulator (IdeR) in MAP. A member of the DtxR protein family, IdeR is involved in regulatory mechanisms to acquire, store or pre­vent excess accumulation of iron.

In addition to IdeR, the MAP genome also encodes three putative FUR boxes, which are iron- regulated transcriptional control motifs present in a MAP-specific genomic island (Stratmann et al., 2004). To date, there is no information about the potential roles of FUR in MAP. In 2009, Alexander and others confirmed that the MAP genome also contains another putative metal transport operon, LSP^p 15, which includes a FUR-like transcriptional regulator (Alexander et al., 2009). Additionally, using transposon mutagenesis, Wang etal. (2016) demonstrated that this specific operon provides an alternative iron uptake system. However, a deeper investigation is necessary into how FUR in MAP behaves in an organism where IdeR is the protago­nist. As a key virulence determinant, iron regula­tion in MAP and its role in pathogen survival and infection are important areas of research that may lead to advances in the ability to culture MAP and improve diagnostic techniques.

During MAP replication within the mac­rophage phagosome, host cell resources and space become limited. Therefore, MAP egresses from the cell by inducing apoptosis and is able to infect neighbouring macrophages within the subepithe- lial dome. Infected macrophages migrate into local lymphatics concurrent with disease progression, resulting in bacterial spread to regional lymph nodes, including mesenteric lymph nodes (Ayele et al., 2004; Sivakumar et al., 2005; Tiwari et al., 2006; Zhu et al., 2008). MAP is able to replicate within regional lymph nodes. Macroscopic lesions develop in the intestine and mesenteric lymph nodes, causing the intestinal wall to thicken and become corrugated (Sivakumar et al., 2005). Infection may spread to the supramammary lymph node and mammary gland and therefore cause MAP contamination in colostrum and milk (Sweeney et al., 1992a, b). Several studies have shown MAP can survive the commercial pasteuri­zation and processing and therefore poses chal­lenges to containment by presence in commercial calf milk replacer (Grant et al., 2017), and with respect to the potential zoonotic nature of MAP, may pose a significant public health concern for humans susceptible to Crohn's disease (Sweeney et al., 1992b; Grant, 1998).

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