Autophagy and Burkholderia - Nature

Immunology and Cell Biology (2015) 93, 18–24 & 2015 Australasian Society for Immunology Inc. All rights reserved 0818-9641/15 www.nature.com/icb

REVIEW

Autophagy and Burkholderia Rodney J Devenish and Shu-chin Lai Autophagy has become increasingly viewed as an important component of the eukaryotic innate immune system. The elimination of intracellular pathogens by autophagy in mammalian cells (xenophagy) results not only in the degradation of invading bacteria, viruses, fungi and parasites, but also liberation of metabolites that may have been utilized during pathogen infection, thus promoting cell survival. After gaining entry into the cell, intracellular bacterial pathogens attempt to escape from phagosomes (or endosomes) into the cytosol where they endeavour to continue the infection cycle unhindered by host cell protective mechanisms. Bacterial recognition resulting from either their cytosolic location, the secretion of bacterial products, or phagosomal membrane damage, can induce autophagy. In this context, induction of autophagy results in the clearance of some bacterial pathogens, whereas other bacteria are able to manipulate autophagy for their own benefit and appear to effectively replicate within autophagosome-like vesicles. Some bacteria are seemingly able to evade autophagy and Burkholderia pseudomallei is one of them. This review will discuss the autophagic processes that may be activated by host cells to provide protection against infection by this bacterial pathogen. Immunology and Cell Biology (2015) 93, 18–24; doi:10.1038/icb.2014.87; published online 21 October 2014

AN OVERVIEW OF AUTOPHAGIC PROCESSES THAT INTERFACE WITH INTRACELLULAR BACTERIA Autophagy is a multifunctional, intracellular process that has an important role in protecting eukaryotic cells and maintaining intracellular homoeostasis. Autophagic processes have a crucial role in eliminating intracellular bacteria (xenophagy). However, some bacterial pathogens have evolved strategies to avoid, or manipulate, autophagic processes to facilitate their own survival.1,2 It is pertinent here to distinguish between two different autophagic processes in relation to bacterial infection of mammalian cells. The first process is macroautophagy (hereafter referred to as autophagy) that involves recognition of intracellular bacteria and their sequestration in double-membrane vesicles—autophagosomes. The cellular machinery for autophagosome formation has been recently comprehensively reviewed.3 Briefly, autophagosome formation is initiated at the isolation membrane. The coordinated action of ATG (autophagyrelated) proteins elicits expansion of this membrane with protein and lipid recruitment resulting in the formation of the autophagosome. A key component and signature marker of the autophagosomal membrane is the protein LC3, which must be conjugated to phosphatidylethanolamine in order to be incorporated into the isolation membrane. During the process of isolation membrane expansion, the cargo is sequestered (in this context, bacteria) and eventually enclosed within the completed autophagosome. Autophagosomes subsequently fuse with lysosomes, to become autolysosomes, and the contents within are degraded. The products of degradation are transported to the cytosol for reuse by the cell. The second process by which autophagy participates in bacterial infection is LC3-asociated phagocytosis (LAP). LAP involves LC3

being directly recruited to single-membrane phagosomes.4 LAP is initiated following infection by several bacterial pathogens including Escherichia coli,5 Salmonella typhimurium,6 Mycobacterium marinum,7 Burkholderia pseudomallei8 and Listeria monocytogenes.9 LAP requires some of the molecular machinery used for autophagosome formation, but the initiating ULK1 complex is dispensable.10 The role of LAP, in the context of bacterial infection, is proposed to be the rapid clearance of bacterial pathogens by means of an increased level of phagocytosis and bacterial killing as first demonstrated for E. coli.5 Notably, both autophagosomes and phagosomes that are undergoing LAP are marked by the presence of LC3 in the membrane. A standard measure of autophagy is to determine co-localization of bacteria with LC3, the latter almost invariably being detected as GFP-LC3 puncta. However, there is no readily applicable, facile technique available to differentiate between those puncta which identify phagosomes from those that identify autophagosomes. Therefore, the role of LAP may be underappreciated in bacterial infection. Electron microscopy is the method of choice for distinguishing the ultrastructural difference of a single-membrane (phagosome) compared with a double-membrane (autophagosome) compartment,11 but it is not always used by investigators. B. PSEUDOMALLEI AS THE CAUSATIVE AGENT OF MELIOIDOSIS B. pseudomallei is a Gram-negative bacterial pathogen, designated a Category B select agent by the US Centers for Disease Control. This pathogen is the causative agent of melioidosis, a serious invasive disease, with a mortality rate of up to 40% and which is endemic in Southeast Asia and Northern Australia.12 It is an environmental

Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, VIC, Australia Correspondence: Professor RJ Devenish, Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia. E-mail: [email protected] Received 29 July 2014; revised 11 September 2014; accepted 16 September 2014; published online 21 October 2014

Autophagy and Burkholderia RJ Devenish and S-c Lai 19

organism found in soil and water and infections are generally contracted through contact with contaminated soil or water,13 with increased infection rates following severe weather events such as cyclones and flooding.14 All strains of B. pseudomallei are intrinsically resistant to many antibiotics, thereby limiting the antibiotics that can be used for therapy and although such therapy can be effective, further optimization is required.15 Relapse is common even after apparently appropriate antibiotic therapy and recovery from acute infection.16 B. pseudomallei vaccine development has focused on a wide variety of candidates,17 but a high efficacy vaccine remains a goal of current research. AUTOPHAGIC PROCESSES AND B. PSEUDOMALLEI Our laboratory performed the first analyses of B. pseudomallei interacting with host cell autophagic processes. Through extensive analysis of electron microscopy images of B. pseudomallei-infected RAW264.7 murine macrophage-like cells, we demonstrated that intracellular bacteria are either free in the cytosol or sequestered in single-membrane phagosomes (Figure 1), but only very rarely contained in double-membrane autophagosomes.8 Given that a subset of intracellular bacteria are co-localized with GFP-LC3,18 we concluded that LC3 is actively recruited to B. pseudomallei-containing phagosomes8 via LAP. Furthermore, B. pseudomallei mutants defective in phagosome escape show increased co-localization with LC3 and the lysosome marker LAMP-1. This is accompanied by decreased intracellular survival,8 suggesting that LC3 recruitment is associated with enhanced levels of phagolysosome maturation and subsequent bacterial degradation as had previously been observed for E. coli infection.5 More recently we have extended our analysis of B. pseudomallei infection of RAW macrophages to examining rapamycin or starvation treatment on the consequences for LAP in the presence of BECN1 knockdown (by siRNA). Although we observed enhanced LAP of B. pseudomallei in both cases, rapamycin-elicited Beclin 1-independent

LAP whereas the starvation response was Beclin 1 dependent.19 The significance of this finding is currently under further investigation. Nevertheless, following infection of macrophages most bacteria can escape from phagosomes and once free in the cytoplasm are rarely targeted by autophagosomes (Figure 1).8 Critically, the mechanism(s) by which cytoplasmic B. pseudomallei evade autophagy remains unknown and is a focus of current studies. Some intracellular bacterial pathogens can avoid killing by host autophagic processes Bacteria may be able to escape LAP as exemplified by B. pseudomallei (see above). Escape from phagosomes (whether LC3-associated or not) into the cytosol is a direct means by which bacteria avoid lysosomal proteolysis. Alternatively, remodelling of the phagosomal compartment to block phagosomal maturation and fusion with lysosomes, as exemplified by L. monocytogenes, provides another means of LAP subversion. L moncytogenes remains viable inside LC3-associated phagosomes that are also associated with LAMP-1 (that usually denotes maturation into a lytic compartment). In this case the modified compartment is used as a niche for intracellular replication.9 As indicated above, B. pseudomallei is able to escape LAP and evade autophagy to exist ‘free’ in the cytosol. Evasion of autophagy by B. pseudomallei is contrasted with the fate of two other bacterial species, L. monocytogenes and S. flexneri, which having escaped from phagosomes into the cytosol, are targeted by autophagy. L. monocytogenes escapes from phagosomes through the action of the pore-forming toxin listeriolysin O and two phospholipase C enzymes. Once within the cytosol, bacteria mediate actin polymerization and intracellular motility. This occurs through recruitment of host actin nucleation complex ARP2/3 by the bacterial protein ActA. Evasion of uptake by autophagosomes occurs via a mechanism dependent on the actinbased motility of bacteria as well as their ‘camouflage’ derived from

Figure 1 Comparison of the fates of intracellular B. pseudomallei (Bp) and B. cenocepacia (Bc). Bp (top) is subject to LAP but most bacteria escape from phagosomes, gain an actin tail and replicate ‘free’ in the cytoplasm. The sequestration of bacteria in autophagsosomes is blocked, seemingly by a failure of autophagosome initiation. Bc either escapes from endosomes and is free in the cytoplasm, or resides in a damaged endosomal compartment. In either case, despite the apparent recruitment of components of the autophagic machinery, autophagosomes are not completed. Immunology and Cell Biology


recruitment of ARP2/3 and the host major vault protein to the bacterial surface.20,21 S. flexneri also induces membrane damage to mediate escape from phagosomes. The bacterial protein IcsA mediates actin polymerisation which occurs at one pole of the bacteria facilitating their spread into neighbouring cells. These bacteria secrete the T3SS effector protein, IcsB, which by binding to IcsA competitively inhibits its interaction with the core autophagy protein Atg5 thus abrogating the induction of autophagy.20,22 Although no details of the molecular interaction between IcsB and IcsA have been reported; effectively this interaction masks the recognition of bacteria by the autophagy machinery. The inhibition of binding of Atg5 to IcsA through the action of IcsB occurs at 4–6 h after infection.22 A recent study suggests that IcsB has a distinctly different role, much earlier after infection (40 min) by S. flexneri.23 Here IcsB recruits the F-BAR domain containing protein, Toca-1, to the vicinity of intracellular bacteria and together they modulate LC3 recruitment. This IcsAindependent process potentially serves to restrict LAP and/or LC3 recruitment to membrane remnants that are elicited by phagosomal escape. However this process is not ‘classical’ LAP given that bacteria are not sequestered within intact phagosomes, but are associated with phagosomal membrane fragments.23 B. pseudomallei evades autophagy by a mechanism different from that utilised by S. flexneri B. pseudomallei can invade and survive within phagocytic and nonphagocytic cells.24 It possesses a genetic locus with similarity to the Salmonella and Shigella type III secretion systems (T3SS), designated T3SS3,25 which is required for endosomal escape,25,26 but not for invasion.26 Thus, B. pseudomallei mutants with insertions in either bsaZ (predicted to encode a structural component of the T3SS3 secretion apparatus) or bipD (a T3SS3 needle tip protein) were unable to escape from phagosomes, form actin tails or replicate within macrophage cell lines.25 More recently we have shown that loss of the secreted T3SS3 effector BopA leads to a significant delay in phagosome escape.8 Normally, within 15 min of internalization, B. pseudomallei lyses phagosome membranes and escapes into the cytoplasm where its survival is mediated, at least in part, by its resistance to antimicrobial peptides27 and its inhibition of inducible nitric oxide synthase (iNOS) synthesis.28 Once within the cytoplasm, B. pseudomallei stimulates actin polymerisation and the formation of an actin ‘tail’ at one pole of the bacterial cell, facilitating intracellular motility.29 This suggests that an autophagy evasion mechanism similar to that used by Shigella, where the B. pseudomallei proteins BimA and BopA serve as the functional homologues of IcsA and IcsB, respectively. The actin-based motility of B. pseudomallei is mediated by BimA,29 a T3SS3 effector protein, that has 11% amino acid identity to S. flexneri IcsA, but lacks the IcsA-binding domain targeted by Atg5.22 Moreover, although the B. pseudomallei BopA protein shows 23% amino acid identity to S. flexneri IcsB, our data (Cullinane M, Allwood E, Prescott M, Devenish RJ, Adler B and Boyce JD, unpublished) suggest that the B. pseudomallei proteins, BimA and BopA, do not interact with each other in a manner comparable to their S. flexneri counterparts. Therefore, B. pseudomallei evades autophagy by an alternative mechanism to that of S. flexneri. Furthermore, neither a B. pseudomallei bimA mutant (Cullinane M, Allwood E, Prescott M, Devenish RJ, Adler B and Boyce JD, unpublished) nor a bopA mutant27 exhibits increased susceptibility to autophagy.18 Nevertheless, BopA is involved in modulating the host cell response to infection, as bopA mutant bacteria show delayed escape from phagosomes and reduced intracellular survival.8,18 Immunology and Cell Biology

Bioinformatic analysis of the B. pseudomallei genome sequence identified bpsl1631, a largely uncharacterised ORF that encodes an 1124 amino acid putative outer membrane protein, as having 19% identity to S. flexneri IcsA. BPSS1631 and its B. mallei homologue had been identified independently as a putative autotransporter protein, likely to function as a surface or secreted protein and therefore with the potential to modulate interactions with the intracellular environment of the host.30,31 In addition, we identified three regions within BPSL1631 that have 27% amino acid identity with IcsA when the Atg5 binding-site region with IcsA was used as the reference sequence. The presence of this sequence homology suggested that BPSL1631 may have the capacity to bind Atg5. However, mutant bacteria lacking a functional bpsl1631 gene have the capacity to escape from phagosomes and replicate efficiently within host cells. Furthermore, loss of bpsl1631 did not affect virulence in a mouse model of infection (Lai S, Boyce JD, Adler B and Devenish RJ, unpublished data). Therefore, it is unlikely that this protein has a role subverting antibacterial autophagy. CONJUGATION TO UBIQUITIN (Ub) AS A SIGNAL FOR THE AUTOPHAGIC TARGETING OF BACTERIA A recently recognized signal for autophagic targeting of different intracellular bacteria is conjugation to Ub.32 This process requires initial binding of Ub to cargo designated for degradation and the subsequent recognition by adaptor proteins (including p62/SQSTM1, NDP52/CALCOCO2, NBR1 and optineurin) that facilitate ubiquitinated cargo engulfment by autophagosomes.33,34 The nature of the Ub chains decorating cytosolic bacteria is generally undefined. A sizeable spectrum of configurations is possible. A single Ub molecule can be added at a single site (mono-Ub), single Ub molecules can be added to multiple sites (multi-Ub), or multiple Ub molecules can be added to either single or multiple sites (poly-Ub). For poly-Ub successive Ub molecules are assembled by ubiquitination of the Ub molecule itself. Such poly-Ub chains can be generated by linking of the free carboxy terminal of distal Ub to any of the seven lysine residues (at positions 6, 11, 27, 29, 33, 48 and 63) on the surface of Ub as well as its N-terminal methionine.32 The limited data available indicate Salmonella is decorated with linear and K63 Ub chains,35 whereas M. marinum in macrophages is conjugated with both K48 and K63 Ub chains.36 It is not yet established whether different adaptor proteins have different affinities for different types of Ub chain. Posttranslational modification of adaptor proteins in the proximity of Ub-binding domains can affect their binding preferences.37 The conjugation of cargo with Ub requires the activity of three enzymes: an activating enzyme (E1), a conjugating enzyme (E2) and finally a Ub ligase (E3). The last step is highly regulated and occurs in a substratespecific manner catalysed by E3 Ub ligases that interact directly with the target proteins. E3 ligases are of particular interest because of their role in substrate selection. Few of the host E3 ligases responsible for catalysing Ub chains that target intracellular bacteria have been identified. LRSAM1 (leucine-rich repeat and sterile α-motif containing 1), is responsible for addition of Ub to intracellular S. typhimurium prior to its elimination by autophagy.38 Parkin, although first characterised for its function in recruiting Ub to damaged mitochondria,39 also catalyzes the Ub chains that target intracellular M. tuberculosis.40 Ub conjugation can be reversed by the action of deubiquitinating enzymes. Deubiquitinating enzymes hydrolyse the bond between the carboxy-terminus of a Ub molecule or Ub chain and the substrate to which it is conjugated.32 The deubiquitinase activity of the S. typhimurium virulence protein, SseL, reduces autophagic flux in infected cells facilitating avoidance of autophagy and favouring bacterial replication.41 As indicated above, a small


number of adaptor proteins directly mediate an interaction between Ub on ubiquitinated targets and LC3 located in the phagophore membrane (and eventually the completed autophagosome). These LC3/adaptor interactions are themselves mediated through LC3interaction regions42 that facilitate the recruitment and organization of LC3-expressing membranes at the target site. The specificity of particular adaptor proteins for particular bacterial species is presently poorly defined, but it is known that NBR1 targets Francisella tularenis,43 whereas p62, NDP52 and optineurin are all involved in autophagy of S. typhimurium.44–46 Does B. pseudomallei infection modulate host Ub responses? At present there is limited information concerning Ub and the host response in B. pseudomallei infection, and how its modulation might lead to evasion of host autophagy. There is, however, strong evidence for at least two Ub-processing enzymes in B. pseudomallei that may modulate host ubiquitination (Figure 2). The first is CHBP, a Cif

Figure 2 Two Ub-modulating activities that B. pseudomallei might use to mediate avoidance of autophagy. (A) CHBP, which deamidates Ub (and NEDD8); and (B) BPSS1512, which acts as a deubiquitinase. A bacterium having escaped from a phagosome is not delivered to an autophagosome. The lack of ‘proper’ ubiquitination (of unidentified substrates) might result in the bacterium now being recognized by host autophagy.

(cycle inhibiting factor) homologue, which is an inhibitor of ubiquitination that specifically deamidates Gln40 of Ub. Deamidated Ub does not support Ub ligase-catalysed Ub-chain synthesis.47 Subsequently, it has been shown that deamidation of Ub inhibits its interaction with p62/SQSTM1, which suggests that CHBP activity may, in part, relate to interference with the association between Ub and Ub-binding proteins.48 CHBP protein also functions as a glutamine deamidase able to target NEDD8, a Ub-like protein, thereby interfering with its function to trigger macrophage-specific apoptosis.49 Killing of macrophages in this manner would serve as a potential virulence mechanism for B. pseudomallei by which it could counteract macrophage-mediated host defence. CHBP is recognised by melioidosis patient sera50 indicating that it is expressed in vivo and may have a role in pathogenesis. Intriguingly, in a recent survey not all B. pseudomallei genome sequences surveyed (33/43) were shown to include the bpss1385 gene encoding CHBP.51 Moreover, western blot analysis using an antiserum raised against a synthetic peptide only detected CHBP in 7/15 clinical isolates.51 CHBP is localized within the cytoplasm of B. pseudomallei-infected U937 cells and secretion is dependent on T3SS3 function. Although a B. pseudomallei bpss1385 insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared with its wild-type (WT) parent strain, no defect in actin-based motility or multinucleated giant cell formation was observed.51 It remains unclear how CHBP secretion mediates the observed virulence-associated phenotypes. The second protein, a Ub-processing enzyme, is encoded by the bpss1512 gene (also designated tssM) and that has almost 100% identity with a B. mallei protein,52 previously shown to display deubiquitinating enzyme activity in vitro.53 TssM acts to inhibit NF‑κB activation and type I IFN activation by deubiquitinase activity targeting TRAF3, TRAF6 and IκBα.52 Previous studies have failed to identify the mechanism of secretion of TssM. Although TssM has been described as a T3SS effector,32 recent evidence shows TssM to possess an atypical signal sequence with its secretion mediated by the single B. pseudomallei T2SS.54 Our laboratory has constructed deletion mutants for both bpss1385 and bpss1512 and have examined the importance of these two genes in allowing B. pseudomallei to avoid autophagic degradation following phagosomal escape. No significant alteration in colocalization of bacteria with LC3 or with Ub was observed (Lai S, Gong L, Boyce JD, Adler B and Devenish RJ, unpublished data). Thus, there is no prima facie evidence for a change in susceptibility of these mutant bacteria to LAP and/or canonical autophagy. Furthermore, the loss of either gene did not affect virulence in a mouse model of infection (Lai S, Treerat P, Gong L, Boyce JD, Adler B and Devenish RJ, unpublished data). Although co-localization and microscopy studies of genetically defined mutants will provide direct evidence for the importance of specific genes in modulating either the extent of ubiquitination or susceptibility to LAP/autophagy, they will not provide for characterization of the target proteins or the sites of Ub additions. A more comprehensive and definitive analysis of the changes in ubiquitination that arise following infection requires application of mass spectrometry-based proteomics. Such analyses are facilitated by recent improvements in instrument sensitivity, mass accuracy, peptide fragmentation and database searching,55,56 and are currently underway in our laboratory. It is possible that following escape of bacteria from phagosomes to the cytosol the phagosome membrane remnants (rather than escaped bacteria) are ubiquitinated such that they will be targeted by autophagy. A precedent exists in S. flexneri infection where proteins including TRAF6 (involved in NF-kB signalling), are ubiquinated, recruit the autophagy marker LC3 and the adaptor SQSTM1/p62. Immunology and Cell Biology


The presence of both SQSTM1/p62 and other IKK components trapped in the membrane remnants led to the suggestion that the autophagy pathway is used to regulate the activation of inflammatory responses at early time points in infection.57 As indicated above, following infection by B. pseudomallei most bacteria escape from phagosomes, however, whether detectable ubiquitination of phagosome membrane fragments occurs has not been reported. Although one could predict that B. pseudomallei expresses proteins that directly modulate host ubiquitination of intracellular bacteria (that is, E3 Ub ligases and deubiquitinating enzymes; see above) it is also possible that B. pseudomallei expresses proteins that act as decoy components of the host response, thereby allowing the bacterium to evade autophagy. There is a precedent for use of this strategy by viral pathogens that encode proteins which mediate antagonism of host autophagy,58 for example a Bcl-2 homologue (vBcl-2).59 In the bacterial pathogen context a decoy protein might act to 'induce nonbactericidal autophagy' by serving as an attachment site for Ub, or by the binding and sequestration of adaptor proteins (bridging between Ub and LC3), or LC3. There is potential for any such bacterial ubiquitinated proteins to be identified by mass spectrometryproteomics approaches. A bacterial inducer of host autophagy B. pseudomallei, contains a large genome (7.2 Mb) with many genes of putative or unknown function. Interactions with potential hosts and environmental factors may induce rapid adaptations in these genes. Tan and colleagues have undertaken an evolutionary analysis of multiple B. pseudomallei genomes leading to the identification of several previously uncharacterized genes bearing genetic signatures of rapid adaptation (positive selection).60 Diverse cellular phenotypes were observed when these genes were expressed individually, including induction of autophagy. Subsequently, more detailed characterisation of one particular gene bpss0180, a T6SS cluster 4-associated gene, has revealed that the encoded protein is capable of inducing autophagy in both phagocytic and nonphagocytic mammalian cells.61 Following infection of macrophages, a mutant disrupted in bpss0180 exhibited significantly decreased co-localization with LC3 and impaired intracellular survival; these phenotypes were rescued by complementation with an intact bpss0180 gene. On this basis it has been proposed that BPSS0180 acts as an autophagy inducer targeted to host components.61 This would be to the advantage of bacteria through increased production of nutrient resources for their utilization in intracellular replication. However, detailed characterisation of the function of BPSS0180 in infected cells has yet to be undertaken. Intriguingly there are putative homologues of BPSS0180 in the B. pseudomallei genome, which also warrant further investigation as potential inducers of autophagy. Lessons learnt from another Burkholderia species? B. cenocepacia is a virulent pathogen that causes significant morbidity and mortality in patients with cystic fibrosis (CF), and like B. pseudomallei it is difficult to treat because it is resistant to nearly all available antibiotics.62 B. cenocepacia is first localized to early endosomes, but survives intracellularly by subverting the fusion of endosomes with lysosomes.63 An interesting feature of the studies on B. cenocepacia is that ‘opposing’ autophagy outcomes can be observed in WT compared with CF macrophages. CF macrophages in mice (and humans) have compromised macroautophagic activity64 resulting from impaired Beclin 1 activity. This arises following its cross-linking mediated by tissue transglutaminase resulting in its sequestration as a component of the phosphatidylinositol 3-kinase complex III within aggregsomes.64 Analysis of transmission electron microscopy images Immunology and Cell Biology

shows that in WT macrophages, a fraction of the intracellular bacteria resides in compartments classified as autophagosomes, whereas in CF macrophages most bacteria remain inside single-membrane vacuoles that do not fuse with lysosomes. Moreover, in WT macrophages, a fraction of the intracellular bacterial compartments are LC3-labelled, but this is not the case in CF macrophages.65 The availability of the adaptor SQSTM1/p62 dictates the fate of infection.66 In WT macrophages, SQSTM1/p62 depletion or overexpression leads to increased or decreased bacterial intracellular survival, respectively. By contrast, depletion of SQSTM1/p62 in CF macrophages results in decreased bacterial survival, whereas overexpression leads to increased intracellular growth. In CF macrophages the depletion of SQSTM1/p62 results in the release of Beclin 1 from aggregates and allows its redistribution throughout the cell. Subsequently, it can be recruited to bacteria containing vacuoles, mediating the recruitment of LC3 thereby leading to bacterial clearance by autophagy. A final interesting point relates to the observation that following treatment of infected CF macrophages with rapamycin, enhanced co-localization of bacteria with LC3 was observed.65 However, the nature of the compartment was not reported and it remains to be determined whether bacteria undergo LAP or canonical autophagy following treatment of CF macrophages with rapamycin. Recently, the intracellular life cycle of B. cenocepacia strain J2315 and its interaction with autophagy in human cells was described.67 Escape to the cytosol from the endocytic pathway triggers the recruitment of Ub, Ub-binding adaptors SQSTM1/p62 and NDP52 together with LC3, to the vicinity of bacteria. Despite the recruitment of these key effectors, as noted above, B. cenocepacia blocks autophagosome completion and continues to replicate in the host cytosol (Figure 1). Notably, however, induction of host cell autophagy prior to bacterial infection was demonstrated to significantly inhibit B. cenocepacia replication. A parallel study has demonstrated that IFN-γ promotes autophagy-mediated clearance of B. cenocepacia in human CF macrophages.68 IFN-γ-treated CF macrophages showed increased co-localization of bacteria with SQSTM1/p62 and increased trafficking to lysosomes compared with untreated CF macrophages. Electron microscopy analysis confirmed that autophagy was promoted by IFN-γ as double-membrane compartments were observed around bacteria in CF-treated cells, whereas only single-membrane compartments were found in untreated CF cells. In summary, the evidence to date suggests that by comparison with B. pseudomallei, during infection of macrophages by B. cenocepacia there are conditions under which antibacterial autophagy can be effective. Further ‘comparative’ studies should prove useful in uncovering mechanisms by which both of these species interact with host autophagy processes. Comparative metabolic systems analysis of B. cenocepacia and B. multivorans (which exhibit important differences in pathogenesis) identified functional pathways indicating B. cenocepacia produces a wider array of virulence factors. This supports the clinical observation that B. cenocepacia is more virulent.69 Extension of this type of analysis to B. pseudomallei (as well as other members of the Burkholderia genus) might also provide clues as to the basis of the differences in host autophagic response to infection between the two closely related bacterial strains. Concluding remarks In summary, B. pseudomallei can be cleared from cells by LAP, but most bacteria are able to escape from phagosomes and replicate in the cytosol. These bacteria evade autophagy by a mechanism that remains to be defined. Evidence suggests that B. pseudomallei blocks the initiation of autophagosome formation, unlike B. cenocepacia where


autophagy stalls after commencement of isolation membrane synthesis. Defining the molecular mechanisms by which B. pseudomallei evades autophagy will potentially identify specific bacterial targets that might underpin the foundations of future disease alleviation strategies. Significantly, recent advances in pharmacological manipulation of autophagic pathways suggest the feasibility of such intervention as therapeutic strategy for a variety of pathological conditions.70 This approach could include infection by bacterial pathogens. The use of autophagy-stimulating drugs for treatment of melioidosis, or the use of IFN-γ, will require further study to build on the limited reports to date71 and to fully establish efficacy outside of cell culture or animal models. Additionally the potential for development of new drugs targeting the Ub system has been recognized,72,73 and could be exploited in future treatment regimes if evasion of autophagy by B. pseudomallei is clearly demonstrated to be linked to Ub signalling in host cells. Finally, a recent study suggests that the role of the large cryptic secondary metabolome encoded within the B. pseudomallei genome warrants further investigation for its contributions to virulence.74 Whether or not the host autophagic response to infection is modulated by products of secondary metabolism remains to be determined. If this were to be the case then inhibition of the relevant small molecule biosynthetic pathway(s) might prove to be an effective strategy for developing novel melioidosis-specific therapeutics. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS Work from the authors’ laboratory was supported by grants from the Australian Research Council and the National Health and Medical Research Council, Australia.

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