Mycobacterium avium-triggered diseases: pathogenomics

Microreview

Mycobacterium avium-triggered diseases:pathogenomics

Dmitriy Ignatov,1 Elena Kondratieva,2

Tatyana Azhikina1 and Alexander Apt2*1Shemyakin and Ovchinnikov Institute of BioorganicChemistry, Moscow, Russia.2Central Institute for Tuberculosis, Moscow, Russia.

Summary

The species Mycobacterium avium includesseveral subspecies representing highly special-ized avian and mammalian pathogens, non-obligatory pathogens of immune compromisedhumans and saprophitic organisms. Recentlyobtained information concerning the diversity ofM. avium genomic structures not only clarifiedphylogenic relationships within this species, butbegan to shed light on the question of how suchclosely related microorganisms adapt to the occu-pation of distinct ecological niches. In this reviewwe discuss specific features of M. avium geneticcomposition, as well as genetic and molecularaspects of M. avium hominissuis (MAH)-triggereddisease pathogenesis, including virulence, pen-etration, immune response manipulation and hostgenetic control.

Introduction

According to current taxonomy, the genus Mycobacteriumincludes more than 60 species and more than 100 sub-species (http://www.dsmz.de). Three species whose overtpathogenicity for humans was recognized long ago,Mycobacterium tuberculosis, M. leprae and M. bovis,have been characterized far better than non-pathogenicand conditionally pathogenic so-called non-tuberculousmycobacteria (NTM), predominantly populating soil andwater. However, among the latter, about 20 species causehuman and animal diseases (Wallace et al., 1997;

Heifets, 2004), and thus receive constantly growing inter-est from researchers.

The epidemiology of conditions caused by NTM and thegeographic distribution of these species are not welldefined. In the USA, the general frequency of NTM-caused diseases was reported lower than 1.8 cases per100 000 individuals, and varied substantially betweenstates, peaking at 11 cases per 100 000 in Massachusetts(Wallace et al., 1997). Apparently, the highest frequencyon the globe – 100–150 per 100 000 – was reported forGhana (Amofah et al., 2002), demonstrating both highNTM burden in an endemic country and their importancefor the general epidemic structure of mycobacterial dis-eases in such a country.

Among NTM, the species Mycobacterium aviumattracts serious attention. This species includes four sub-species: M. avium avium (MAA), M. avium hominissuis(MAH), M. avium silvaticum (MAS) and M. avium paratu-berculosis (MAP) (Thorel et al., 1990; Turenne et al.,2008). MAA and MAS are specific avian pathogenscausing a tuberculosis (TB)-like disease in birds (McDi-armid, 1948; Dvorska et al., 2003). MAP is a well-knownpathogen causing Johne’s disease, i.e. chronic enteritis ofruminants (Harris and Barletta, 2001), and may have arole in human Crohne’s disease (Feller et al., 2007).However, the most prominent impact on public health isexerted by MAH, a dangerous intracellular human patho-gen in the absence of normal T cell immunity. Thesemycobacteria are present in approximately 70% ofpatients with advanced untreated AIDS and are consid-ered the major killer in this cohort (Horsburgh, 1991;Nightingale et al., 1992; Inderlied et al., 1993; Falkinham,2003). On the background of less severely impairedimmunity, e.g. in older persons and in children, M. aviummay cause chronic lung diseases (Benson, 1994; Griffith,1997; Nolt et al., 2003).

General characteristics of infection caused by MAH

MAH invades the human host via either the respiratorytract causing lung infection which slowly disseminates toother organs, or the intestinal epithelium causing rapidlydisseminating infections (Damsker and Bottone, 1985;

Received 25 November, 2011; revised 3 February, 2012; accepted 8February, 2012. *For correspondence. E-mail [email protected]; Tel. (+7)499 785 9072; Fax (+7) 499 785 1961.

Cellular Microbiology (2012) doi:10.1111/j.1462-5822.2012.01776.x

© 2012 Blackwell Publishing Ltd

cellular microbiology

Falkinham, 1996). In experimental models, intravenousinfection leading to a human-like disseminated pathologyis commonly used (Flórido et al., 2002). To model chroniclung infection, intratracheal, intranasal and aerogenicroutes are applied (Appelberg and Sarmento, 1990;Saunders et al., 1998; Gilbertson et al., 2004; Appelberg,2006).

After successfully infecting the host, the lifestyle ofM. avium is similar to that of other pathogenic mycobac-teria: it resides and multiplies within macrophages, relyingon the capacity to either inhibit macrophage activation,or resist bactericidal mechanisms, or both (Frehel et al.,1991). To enter the macrophage, M. avium interacts withvarious surface receptors, such as complement receptors(except, probably, CR3 and CR4 (Bermudez et al., 1999;Bohlson et al., 2001), mannose receptors and fibronectin(Bermudez et al., 1991; Polotsky et al., 1997), type Ascavenger receptors and, in conjunction with antibodies,Fc-g receptors (Fenton et al., 2005). Data are accumulat-ing that recognition of M. avium via Toll-like receptors(TLR) is also important. Thus, TLR-2 knock-out micedisplay enhanced susceptibility to M. avium comparedwith their wild-type counterparts (Feng et al., 2003;Gomes et al., 2004; 2008). Given that mice with theswitched-off MyD88 adapter molecule are even more sus-ceptible than TLR-2-/- mice (Feng et al., 2003), it wassuggested that intracellular TLR-9 may also play a role inM. avium infection (Appelberg, 2006), as it was shown forM. tuberculosis (Bafica et al., 2005).

After entering the macrophage, M. avium resides withinphagosomes, specific cytoplasmic vacuoles encapsulat-ing the engulfed bacteria. Similar to other pathogenicmycobacteria, a major mechanism of intracellular survivalof M. avium is their capacity to inhibit phagosome matu-ration, which prevents acidification of its internal compart-ment below pH = 6.4, and to block fusion of phagosomeswith extremely acidic (pH = 4.8) lysosomes (Oh andStraubinger, 1996; Sturgill-Koszycki et al., 1996; Guerinand de Chastellier, 2000). After recognition and phagocy-tosis of M. avium, macrophages instantly start to produceand secrete numerous chemokines, cytokines and theirligands, e.g. TNF-a, LT-a/b; IL-1, -6, -12, -18; G-CSF andGM-CSF; CXCL-1, -2, -3, -8; CCL-2, -3, -4, -5, -20 (Furneyet al., 1992; Shiratsuchi et al., 1993; Fattorini et al., 1994;Sarmento and Appelberg, 1996; Shiratsuchi and Ellner,2001; Blumenthal et al., 2005). This powerful ‘cytokinestorm’ marks the starting phase of two tightly interdepen-dent processes: the immune response and inflammationof infected tissue. Since many aspects of these complexreactions were the subject of excellent reviews by Appel-berg (2006) and Stabel (2006), the corresponding datawill not be discussed here. However, it is worth mention-ing that studies in mice with genetically disrupted genesoperating along different axes of the immune response

clearly demonstrated that during adaptive immunity, likein other mycobacterial diseases, protective responsesagainst M. avium largely depend upon activation ofmacrophage oxidative/bactericidal functions by type 1pro-inflammatory cytokines provided by immune T lym-phocytes and other cell types (Ehlers et al., 2000; Floridoet al., 2005; Petrofsky and Bermudez, 2005).

Host genetic control

While the general picture of immune responses againstM. avium and M. tuberculosis looks similar, the hostgenetic control of susceptibility to and severity of thediseases caused by these two related pathogens is strik-ingly different (Kondratieva et al., 2010). TB infectioncontrol, in both mice and humans, involves numerousinteracting and independent genes, which makes thewhole picture extremely complicated (for a review seeFortin et al., 2007; Schurr and Kramnik, 2008; Apt, 2011).On the other hand, the progress and outcome of infectionwith M. avium is largely dependent upon a single gene –Nramp1 (or Slc11a1). Since clinical forms of M. aviuminfection are observed exclusively in individuals withimmune deficiency, and since a totally non-functionalhuman NRAMP1 allele analogous to murine Nramp1S isnot known, there are no reports of case–control associa-tion studies of susceptibility to M. avium in humans.However, in mice, strong dependence of the infectionseverity upon the presence of functional versus dysfunc-tional Nramp1 allele has been clearly demonstrated inassociative (Orme et al., 1986; Stokes et al., 1986; Appel-berg and Sarmento, 1990), congenic (Nakamura, 1992;De Chastellier et al., 1993) and segregation (Kondratievaet al., 2007) genetic studies. The most likely explanationfor this apparent difference between M. avium andM. tuberculosis in genetic control of the two infectionsis that the gene Nramp1 is particularly important whenthe pathogen is effectively contained within macrophagephagosomes.

A suggested functional role of this gene in sequesteringiron and, possibly, other divalent cations from the endo-somal system (Forbes and Gros, 2001; Kuhn et al., 2001)(see Fortin et al., 2007 for review), raises the questionabout the difference between M. avium and M. tuberculo-sis in intraendosomal, 2+-cation-dependent metabolism.Studies in the in vitro macrophage-infection system dem-onstrated a similar ability of the two species to accumulateand retain iron in the phagosome using transferrin recep-tor and siderophore production (Wagner et al., 2005). Inthe absence of direct evidence, it is tempting to speculatethat a substantially stronger dependence of M. avium onthe Slc11a1 function may be due to the differences indynamic integrity of M. avium- and M. tuberculosis-infected phagosomes. Evidence is accumulating that

2 D. Ignatov, E. Kondratieva, T. Azhikina and A. Apt

© 2012 Blackwell Publishing Ltd, Cellular Microbiology

M. tuberculosis and M. leprae are able to escape fromphagosomes into the cytosol (Majlessi et al., 2005; vander Wel et al., 2007), which suggests that these uncondi-tionally pathogenic mycobacteria acquire access to cyto-solic sources of iron after breaking the phagosomemembrane. On the other hand, M. avium-containing pha-gosomes showed no fusion with endoplasmic reticulum(Touret et al., 2005). In line with this, there are indicationsthat M. avium stay longer within integrated phagosomessurrounded by an intact membrane compared with morevirulent M. tuberculosis (U. Schaible, pers. comm.). Thus,the fully functional r allele of Slc11a1 may depriveM. avium of the host sources of iron much more efficientlythan M. tuberculosis, which provides an explanation for amore prominent role of this gene in the former infection.

Comparative genomics

Genome plasticity

Selective pressure due to environmental diversity leads tostructural genomic changes, such as gene duplication,deletion and horizontal transmission, as well as tochanges in gene expression (Mira et al., 2002; Vicenteand Mingorance, 2008). Since the subspecies ofM. avium represent highly specialized avian (MAA), rumi-nant (MAP) and conditional human (MAH) pathogens,comparative studies of genome plasticity and geneexpression may provide new knowledge about mecha-nisms allowing evolutionarily similar organisms to occupydifferent niches.

The genomic sequences of two M. avium subspecies,namely, MAP strain K-10 and MAH strain 104, have beenannotated, the genome sequence of MAA strain ATCC25291 is close to completion, and the genomes of another21 strains are being sequenced (http://www.ncbi.nlm.nih.gov). MAH appears to contain a somewhat longergenome compared with MAP and MAA which is in agree-ment with the general trend for taxonomically similar non-pathogenic/conditionally pathogenic versus obligatorypathogenic organisms (Cases et al., 2003). Thus, theMAH genome contains 846-kb-long 24 long sequencepolymorphisms (LSP) lacking in MAP (Wu et al., 2006).

Within a single subspecies, the highest level of genomicheterogeneity is observed in MAH which almost certainlyrepresents the evolutional prototype that gave rise to theMAP and MAA-MAS branches (Wu et al., 2006; Turenneet al., 2008). Analysis of the LSP distribution within apanel of M. avium strains suggested that the origin ofMAP from a putative MAH-like progenitor strain had beenlikely a two-step event. First, the progenitor strain hadacquired by horizontal transmission 6 MAP-specific LSPhomologous to those of environmental Actinomyces, withthe total length of ~ 125 kb and containing 82 open

reading frames (ORF). Second, some genetic materialhas been lost: one mutual deletion is observed in all MAPstrains, and some deletions are specific for the cattle-affecting and some for the sheep-affecting strains (Alex-ander et al., 2009).

Two of six MAP-specific LSP are putative prophages,while the other four contain genes encoding proteins pre-dominantly involved in inorganic ion transport andmetabolism. Other genetic elements potentially importantin pathogenesis include MAP2189–2194 encoding anuncommon mammalian cell entry (mce) operon, andMAP3740–3746 and MAP3726–3729, involved, respec-tively, in siderophore synthesis and capture (Alexanderet al., 2009).

Cell wall and secretion system 7

Generally speaking, M. avium cell wall structure is similarto that of M. tuberculosis, relying on an arabinogalactan-peptidoglycan mycolic acid core. However, some promi-nent differences are obvious. For example, unlike highlypathogenic M. tuberculosis and M. leprae, M. aviumdoes not synthesize phtiocerol dimycocerosate (PDIM)(Onwueme et al., 2005). An important role of this myco-bacteria outer cell wall component in TB pathogenesiswas first demonstrated in experiments with knockoutmutant strains of M. tuberculosis: compared with their wildtype counterparts, mutants lacking PDIM were consider-ably attenuated in a mouse TB model and failed to preventacidification and maturation of phagosomes in murinemacrophages (Camacho et al., 1999; Pethe et al., 2004).Later it was suggested that PDIM is involved in reorgani-zation of the macrophage membrane during M. tubercu-losis engulfment changing its physical characteristics andaltering signalling pathways downstream of receptorsinvolved in phagocytosis (Astarie-Dequeker et al., 2009).In the context of this review, it is important to note thatseveral genes participating in PDIM synthesis and trans-portation are lacking in M. avium genome. Those include:the ppsABCDE operon encoding the type I modularpolyketide synthase responsible for phtiocerole and phe-nolphtiocerole synthesis; the mas gene involved in myco-cerosic acid synthesis; the papA5 gene encodingphtiocerole and phtiodiolone diestherases; and themmpL7 gene involved in a correct orientation of PDIM inthe cell wall (Li et al., 2005; Banerjee et al., 2011). Theseimportant deletions clearly indicate that M. avium andM. tuberculosis substantially differ regarding their interac-tions with macrophages of the host.

Another important difference between M. avium andM. tuberculosis is the presence of glycopeptidolipids(GPL) in the cell wall of the former species. GPL areproduced by a few NTM representing both saprophytes(Mycobacterium smegmatis) and parasites (Mycobacte-

Mycobacterium avium pathogenomics 3


rium porcinum, M. senegalense, M. avium), and theirstructure, biosynthesis and the role in pathogenesis ofM. avium-triggered disease are well defined (see Schoreyand Sweet, 2008 for the review). Interestingly, the capac-ity to produce GPL is thought to be involved in biofilmdevelopment (Yamazaki et al., 2006) which, in turn, maybe important for MAH survival and proliferation in watersupply systems (Freeman et al., 2006). The fact that MAHis capable of colonizing water supply systems by formingbiofilms was recently established, and its possible role ininfection of individuals with immune deficiencies was sug-gested (Johansen et al., 2009). Besides MAH, capacity toform biofilms was shown for MAP. MAP mutants with thedisrupted pstA gene encoding the enzyme involved inearly stage of GPL synthesis demonstrated a decreasedability to form biofilms, accompanied by altered cell mor-phology (Wu et al., 2009).

The use of the type-7 secretion systems (T7SS) is acharacteristic feature of many mycobacterial species (seeAbdallah et al., 2007 for the review). M. tuberculosis pos-sesses five gene clusters (Esx1–5), each encoding aunique T7SS. In the M. avium genome, the cluster Esx1 isdeleted (Gey van Pittius et al., 2001). This may explainthe capacity of macrophages from immune competenthumans to inhibit the growth of M. avium much moreeffectively than M. tuberculosis: the ESAT-6 secretedprotein encoded by Esx1 and thus present in M. tubercu-losis but not in M. avium is an important virulence factorinterfering with the host immune responses (Wang et al.,2009). In addition, Esx1 (and Esx5) are involved incaspase-independent cell death of M. tuberculosis-infected macrophages and might play a role in the trans-location of M. tuberculosis from phagosomes tocytoplasm (van der Wel et al., 2007).

Transcription regulation

The main bacterial transcription regulation systems in-clude one-component signal-transduction systems (OCSS),two-component signal-transduction systems (TCSS),serine-threonine protein kinases (STPKs) and extra-cytoplasmic function (ECF) sigma-factors (Ulrich et al.,2005; Bretl et al., 2011). The M. avium genome contains 14genetically linked TCSS, 11 of which are present inthe M. tuberculosis genome (Ulrich and Zhulin, 2010).Comparative genomic analysis demonstrated that of12 genetically linked M. tuberculosis TCSS, only Rv0600-0601-tcrA is absent in the M. avium genome (Bretl et al.,2011). Nothing is known about the stimuli recognizedor the genes regulated by these TCSS, as well as bythe three genetically linked TCSS of M. avium whichare absent in M. tuberculosis: MAV_3282-MAV_3283,MAV_4112-MAV_4114, MAV_5281-MAV_5280 (http://mistdb.com/). Using bioinformatic approaches, a set of

M. avium genes possibly activated by the DosR-DosS-DosT system (so-called ‘DosR regulon’) was recently pre-dicted (Gerasimova et al., 2011). The authors suggestedthat the core of the DosR regulon may be similar inM. avium and M. tuberculosis and contain genes encodinguniversal stress proteins, nitroreductases, diacylglycerolacyltransferases, heat shock proteins and ferredoxins.TCSS MtrA–MtrB is present in all mycobacterial genomesanalysed to date (Bretl et al., 2011). In M. avium, this TCSSparticipates in shifting the cell wall phenotype resulting inacquisition of antibiotic resistance (Cangelosi et al., 2006).It was suggested that recognition of the signal provided byMtrA–MtrB depends upon interaction of MtrA with LpqBlipoprotein (Nguyen et al., 2010).

Mce operons

The M. tuberculosis genome possesses four mceoperons (mce1–4) containing 8–13 genes each. The keyfunctional elements are six mce genes (mceA–F) andtwo yrbE (yrbEA and yrbEB) genes. Mce proteinsdemonstrate homology with SBR components of ABCtransporters, whereas YrbE demonstrate homology withpermeases (Zhang and Xie, 2011). Mce1A and Mce3Amolecules are important for penetration into non-phagocytic cells, e.g. epitheliocytes (El-Shazly et al.,2007). The mce4 operon encodes a cholesterol transpor-tation system allowing M. tuberculosis to consume thiscomponent of the host cell membrane during the para-site’s persistence within macrophages, even after activa-tion with IFN-g (Pandey and Sassetti, 2008). M. aviumcontains more mce operons compared with M. tuberculo-sis: nine in MAV and eight in MAP, including homologuesof all four M. tuberculosis mce operons (Li et al., 2005).Although mce4-like operons are remarkably conservedthroughout mycobacterial species (Zhang and Xie, 2011),MAP carries unique stop codons in the first and thirdgenes of the mce4 operon leading to the expression oftruncated proteins, as well as a long deletion of the firstfive genes in mce3 (Kumar et al., 2005). The possiblephysiological role of these mutations remains unknown.

PE/PPE families

The function of numerous mycobacterial proteins belong-ing to these two families remains unknown despite theconstant research effort since the genome of M. tubercu-losis was sequenced (Cole et al., 1998). Only recently itwas shown that PE/PPE proteins are located on thesurface of the mycobacterial cell wall (Newton et al.,2009; Sani et al., 2010; Sampson, 2011). Physiologicaldata are scarce, but a few studies indicate that theseproteins play a role in the pathogenesis of M. avium-triggered disease. Thus, in a panel of MAH mutants



obtained by random transposon mutagenesis, a strainbearing the mutated ppe gene MAV_2928 failed to inhibitmaturation and acidification of phagosomes and demon-strated decreased virulence in infected mice (Li et al.,2004). Later it was shown that infection of macrophageswith MAV_2928 wild type and mutant strains resulted indifferent protein expression profiles within phagosomes.In addition, phagosomes in macrophages infected withthe mutant strain displayed lower levels of Ca++ and Zn++cations, suggesting better activation of protective mecha-nisms of these cells and possible involvement ofMAV_2928 in endosome remodelling (Jha et al., 2010).Similar results were published for a gene belonging to thePE family: the MAH strain bearing the mutated MAV_1346gene displayed a decreased virulence in both macroph-ages and infected mice (Li et al., 2010). More work isneeded to clarify the biology of these mycobacteria-specific molecules.

Within the M. avium species proteins of the PE/PPEfamily demonstrate a relatively high level of conservation,although a few genes encoding PPE proteins appeared tobe unique for MAH and MAP. In addition, some pe/ppegenes, being reasonably conserved in their nucleotidesequences in the two subspecies, encoded highly diver-gent AA sequences due to the shifts in reading frames(Mackenzie et al., 2009). It remains to be determinedwhether or not these differences underlie the host speci-ficity of the two subspecies.

The MAP genome contains 10 pe and 37 ppe genes,which is substantially fewer than in M. tuberculosis (61and 68 respectively); the subgroup PE_PGRS is totallylacking in MAP and only a single MAP3939c gene repre-sents the PPE_MPTR subgroup (Li et al., 2005). Obvi-ously, acquisition and expansion of these geneticelements in bacteria belonging to the M. tuberculosiscomplex occurred after separation of the correspondinglimb from the mutual phylogenetic branch (Gey van Pittiuset al., 2006). A substantially lower number and diversity ofPE/PPE in M. avium compared with M. tuberculosis mayform the basis for different virulence, genetic control andpathological features of the corresponding infections.

Functional genomics

Host cell invasion

Several M. avium genes operating along the invasionpathway have been identified. In particular, the capacity toinvade epithelial cells was shown to depend uponMAV_3616, a homologue of fadE20 which in M. tubercu-losis encodes acyl-CoA dehydrogenase; MAV_0383encoding cyclopropane-fatty-acyl-phospholipid synthase;and a homologue of fadD2 encoding fatty acyl synthase.Thus, the enzymes involved in biosynthesis, degradation

and transportation of fatty acids were important for pen-etration (Miltner et al., 2005; Dam et al., 2006). In addi-tion, a few membrane proteins with unknown functions,e.g. homologues of M. tuberculosis proteins encoded byRv2724c, Rv3802c and Rv3723, as well as by MAV_2054and its MAP homologue, were involved in epithelial cellinvasion (Miltner et al., 2005).

More recently it was demonstrated that interaction ofFadD2 with the epithelial cell surface upregulates theexpression of several mycobacterial genes, includingMAV_5138 and MAV_3679, which encode regulators oftranscription. Overexpression of these factors increasesthe invasion of epithelial cells by M. avium and results inupregulation of MAV_1190-, MAV_4139-encoded mol-ecules and, importantly, the CipA protein. The latter isthought to be located on the surface of M. avium during itscontact with the host epithelial cell where it interacts withthe Cdc42 molecule of the host (Harriff et al., 2009).

Studies in cell cultures and in mouse models demon-strated that MAH invades intestinal mucosa via entero-cytes (Sangari et al., 2001). The mechanism of enterocyteinvasion is not completely understood; it is generallybelieved that during its early contact with host cellsM. avium somehow upregulates the activity of small RhoGTPase Cdc42, which interacts with and phosphorylatesN-WASP protein. The latter interacts with the Arp2/3complex which acquires capacity to directly stimulatepolymerization and remodelling of actin required for trans-membrane invasion (Rohatgi et al., 1999; Dam et al.,2006; Harriff et al., 2009). Taken together, these datasuggest the following provisional model of M. avium inter-action with epithelial cells. Host cell binds ‘molecules ofinvasion’ located on the surface of M. avium, e.g. FadD2,which leads to activation of transcription regulators, e.g.MAV_5138 and MAV_3679, via presently unknown signal-ling pathways. This, in turn, upregulates the expression ofseveral proteins, such as CipA, which may alter signallingpathways of the host cell (Harriff et al., 2009).

As mentioned above, infection of host macrophages isa key feature of M. avium-triggered disease pathogen-esis, and macrophage receptors involved in phagocytosisare relatively well characterized. Some information is alsoavailable concerning M. avium genes required for mac-rophage invasion. Thus, it was reported that a few flankinggenes, including the one involved in GPL synthesis, forma pathogenicity island (PI) which is important for macroph-age penetration by MAH (Danelishvili et al., 2007). This PIis lacking in MAP and M. tuberculosis and has low GCcontent, which, in the opinion of the authors, suggests itsintroduction in MAH by horizontal transmission. Theyspeculate that corresponding products are expressed onthe MAH cell wall and may alter macrophage signallingpathways activated during phagocytosis. Such an activitywas better documented for strain-specific GPL (ssGPL),



the class of molecules residing on the M. avium surface,interacting with macrophage receptors and altering theprofile of cytokine secretion (Schorey and Sweet, 2008;Rocco and Irani, 2011). MAH mutants lacking ssGPL elic-ited higher secretion of pro-inflammatory TNF-a, IL-6,IL-12 and RANTES molecules compared with the wildtype strain (Irani and Maslow, 2005; Krzywinska et al.,2005). Furthermore, it was demonstrated that ssGPLinteract with TLR2 and activate antibacterial functions ofmacrophages via the MyD88 adapter molecule (Sweetand Schorey, 2006).

Survival in macrophage

Similar to M. tuberculosis, inside the macrophage MAHinhibits phagosome maturation and phagosome–lysosome fusion preventing decrease in pH levels withinthe vacuole (Oh and Straubinger, 1996). Apparently, themembrane of the MAH-containing phagosome undergoesa certain type of remodelling: the content of some cellsurface-derived glyco-conjugates gradually decreases,presumably due to not well-defined cholesterol-dependent interactions between the membrane and theentire MAH surface, requiring their tight contact (De Chas-tellier and Thilo, 2002; 2006; Pietersen et al., 2004; DeChastellier et al., 2009). Although there are huge gaps inour understanding of how MAH genetically controls itspersistence within macrophages, some essential geneswere described in a few studies.

Thus, the expression of the icl gene which encodes oneof key glyoxalate shunt enzymes, isocitrate lyase, wasobserved in MAH populating macrophages but lacking inbacteria from culture medium (Sturgill-Koszycki et al.,1997; Honer Zu Bentrup et al., 1999). Similarly, dozens ofMAH genes upregulate their expression within macroph-ages, e.g. mbtE and mbtF involved in mycobactin, andpks10/pks11 involved in polyketide biosynthesis (Houet al., 2002).

Indirect information concerning the intracellular genom-ics of MAH was obtained by screening attenuatedmutants in a mouse model of infection after applyingsignature-tagged mutagenesis, and a few genes requiredfor inhibition of phagosome oxidation and successfulestablishment of infection were identified. Those includeMAV_2450 (pks12), MAV_4292 (hypothetical protein),MAV_4012 (conserved hypothetical protein), MAV_4264(hypothetical protein, homologous with a bacterial regula-tory protein TetR domain). Interestingly, strains withmutated MAV_4292 appeared to be sensitive to ROI,those with mutated MAV_4264 were sensitive to RNO,and MAV_4012 mutants were sensitive to both oxidativepathways, suggesting a key role of putative MAV_4012-encoded protein for MAH protection within macrophages(Li et al., 2010).

Gene expression in vivo

So far, only two whole-genome gene expression studiesof M. avium obtained from infected host tissues were pub-lished. Janagama et al. applied whole-genome oligo-nucleotide microarray to a MAP strain extracted from themesenterial lymph nodes and intestine of a cow sufferingfrom Johne’s disease, and from macrophages infectedwith this strain. In ex vivo samples, there was a remark-able increase in the expression of transcripts involved incell wall biosynthesis and, presumably, latency transfer. Inmacrophages, genes involved in intracellular traffickingand secretion, as well as those from the PI responsible foracquisition of iron, were upregulated (Janagama et al.,2010).

For identification of MAA genes that are differentiallyexpressed during pulmonary infection of mice of geneti-cally susceptible Nramp1s and resistant Nramp1r strains,we applied the cDNA pyrosequencing technique usingtissue samples highly enriched for bacterial cDNA(Azhikina et al., 2009; Ignatov et al., 2010). It was foundthat multiplication in the susceptible host is accompaniedwith upregulation of several genes operating in the Krebs’cycle, oxidative phosphorylation, NO reduction, fatty acidsbiosynthesis, replication and translation. Characteristicfeatures of infection in the resistant host were upregula-tion in expression of genes involved in anaerobic oxida-tion, fatty acid degradation and biosynthesis of theirpolycyclic derivatives, and biosynthesis of mycobactinand other polyketides. These findings may suggest thatduring well-controlled chronic infection in a geneticallyresistant host, M. avium acquires a ‘latent phenotype’ byswitching to anaerobic oxidation, fatty acid degradation asthe main source of energy, and altered cell wall structure.

Conclusion

Information concerning the genome structure of, andphylogenic relations between, different subspecies ofM. avium is rapidly accumulating. Briefly, the peculiaritiesof M. avium pathogenomics are summarized in Table 1.However, despite successful applications of transposonmutagenesis and the initial achievements in gene expres-sion analysis, molecular pathogenic mechanisms ofinfections caused by these mycobacteria are poorlyunderstood. Clearly, these mechanisms differ substan-tially from those operating during M. tuberculosis infec-tion: genes determining PDIM synthesis are lacking andPE/PPE genes are scarce in M. avium, while the abun-dance of ssGPL on its cell wall surface provides a uniquetool for host response modulation. More data are neededto permit a complete understanding of how M. aviuminhibits protective responses of the host. In this regard,application of short non-coding RNA techniques and



direct comparisons of gene expression profiles in intra-and extracellular bacteria appear to be promisingapproaches.

Acknowledgements

We are grateful to Prof. D. McMurray for critically reading themanuscript and valuable suggestions. The work was supportedby grants from the ‘Leading Scientific Schools of Russia’ (projectNSh 5638.2010.4), ‘Molecular and Cellular Biology’ programmeof the Presidium of the Russian Academy of Sciences, by RBRFGrant 11-04-01325 (to T.A.) and NIH Grant R01-AI078864(to A.A.).

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Table 1. M. avium and M. tuberculosis pathogenomic factors.

M. avium M. tuberculosis

Genome features The length of M. avium genome varies between 4.8 Mb(MAP K10) and 5.5 Mb (MAH 104)

The length of M. tuberculosis genome is about 4.4 Mb inall sequenced strains. Obligatory pathogens often haveshorter genomes than their non-pathogenic oropportunistic relatives

PE/PPE genes: Low number and diversity comparedwith MTb.

PE/PPE genes occupy up to 10% of the genome.PE-GPRS and PPE_MPTR genes are widespread

Lack of the widespread PE-GPRS and PPE_MPTRgenes

Lack of several enzymes involved in synthesis andcorrect orientation of PDIM in the cell wall

PDIM is an essential cell wall component involved inMTb virulence

Cell wall Synthesis of GPLs, glycolipids involved inimmunomodulation and biofilm formation. Presence ofssGPLs is a specific feature of M. avium complex

Lack of GPL biosynthesis

Host cell invasion MAH often invades the host through intestinal mucosavia enterocytes

The primary way of invasion is via alveolar macrophagesafter inhaling bacterial aerosols. NormallyM. tuberculosis does not invade intestinal mucosa

Both pathogens use similar receptors to enter macrophages, but peculiarities of M. avium genome suggest specificways of interaction with the phagocytes. Apparently, ssGPLs can modulate macrophage signalling pathways.Furthermore, the MAH-specific pathogenicity island encodes proteins expressed on the MAH cell wall which altermacrophage signalling pathways activated during phagocytosis

Host genetic control Largely depends on Slc11a1 in mouse models. Thisprotein deprives intraphagosomal M. avium of divalentcations. The mode of human genetic control is notknown

Depends on numerous interacting genes in both miceand humans



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