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Survival and persistence of opportunistic Burkholderia species in host cells
Survival and persistence of opportunistic Burkholderia species in host cells Miguel A Valvano, Karen E Keith and Silvia T Cardona Burkholderia are microorganisms that have a unique ability to adapt and survive in many different environments. They can also serve as biopesticides and be used for the biodegradation of organic compounds. Usually harmless while living in the soil, these bacteria are opportunistic pathogens of plants and immunocompromised patients, and occasionally infect healthy individuals. Some of the species in this genus can also be utilised as biological weapons. They all possess very large genomes and have two or more circular chromosomes. Their survival and persistence, not only in the environment but also in host cells, offers a remarkable example of bacterial adaptation. Addresses Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada Corresponding author: Valvano, Miguel A ([email protected])
Current Opinion in Microbiology 2005, 8:99–105 This review comes from a themed issue on Host–microbe interactions: bacteria Edited by Pascale Cossart and Jorge Gala´n Available online 6th January 2005 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2004.12.002
Introduction The Burkholderia genus contains over 32 species that occupy very diverse ecological niches, ranging from contaminated soils to the respiratory tract of humans [1]. Most members of the genus are known to be plant pathogens and soil bacteria. B. mallei and B. pseudomallei are well-recognised as pathogenic for humans and animals, but virtually all Burkholderia can be opportunistic pathogens in immunocompromised individuals. B. pseudomallei is the causative agent of melioidosis (see Glossary), a septicemic disease associated with bacterial dissemination to several organs, which is endemic in Southeast Asia and Northern Australia. B. mallei is the causative agent of the zoonotic disease glanders, and is a host-adapted pathogen that does not appear to persist in nature outside its equine host. Both glanders and melioidosis can be acquired by aerosol, exhibit high mortality rates without antibiotic treatment and can be potentially difficult to diagnose at an early stage. Therefore, B. pseudomallei and B. mallei are a great concern as www.sciencedirect.com
biological weapons and are listed by the Centers for Disease Control and Prevention as category B agents. B. cepacia, initially described as the causative agent of soft rot in onions, has emerged as a multi-drug resistant nosocomial pathogen in immunocompromised patients, particularly in those with chronic granulomatous diseases and cystic fibrosis (CF). Nearly all CF patients suffer from chronic infections of the major airways. The infection exacerbates the progressive pulmonary deterioration and correlates with substantial morbidity and mortality. B. cepacia is not a single microorganism but rather a collection of related species or genomovars collectively referred to as the B. cepacia complex (Bcc) [1]. Of these, B. cenocepacia (formerly genomovar III) and B. multivorans (formerly genomovar II) are the most prevalent species isolated from patients with CF [2]. Pseudomonas aeruginosa and Bcc commonly infect patients with CF. However, Bcc infections are more serious for three main reasons: first, infected patients can deteriorate more rapidly and also develop a life-threatening pneumonia termed cepacia syndrome; second, treatment of these infections is usually difficult because of the intrinsic resistance of Bcc to most clinically useful antibiotics [3]; and third, unlike P. aeruginosa, some Bcc isolates are transmissible from patient to patient [2]. A feature common to B. mallei, B. pseudomallei and Bcc infections is the localisation of the infection to the lungs and airways. Furthermore, persistence of the bacterium without causing overt disease has also been documented for B. pseudomallei and Bcc. In this review, we discuss the ability of B. pseudomallei, B. mallei and Bcc to survive and persist in host cells.
Virulence factors in Burkholderia A number of potential virulence factors have been described and characterised in Bcc isolates. They include cable pili [4,5], flagella [6], a type III secretion system (TTSS) [7], surface exopolysaccharide [8], the production of melanin [9], catalase and superoxide dismutase [10], iron-scavenging siderophores [11], proteases [12] and other secreted enzymes [13], quorum sensing systems [14,15] and the ability to form biofilms [16]. Not all Bcc species produce each of the proposed virulence factors and, to date, only a few of the individual factors have been clearly demonstrated to contribute significantly to lung disease. Resistance to antibiotics commonly used for genetic selection makes it difficult to conduct genetic studies Current Opinion in Microbiology 2005, 8:99–105
100 Host–microbe interactions: bacteria
Glossary Category B agents: Second highest priority agents as defined by the Centers for Disease Control and Prevention (CDC) because they are moderately easy to disseminate, result in moderate morbidity rates and low mortality rates, and require specific enhancements of CDC’s diagnostic capacity and enhanced disease surveillance. Chronic granulomatous disease: A group of rare inherited disorders that result in the inability of phagocytes to undergo the respiratory burst necessary to kill certain types of bacteria and fungi. This leads to recurrent life-threatening bacterial and fungal infections. Cystic fibrosis: A genetic disease that affects approximately 1 in 2000 individuals. Cftr encodes a protein called the cystic fibrosis transmembrane conductance regulator. This is a chloride ion transporter. A defective Cftr gene causes a dysfunction of secretory epithelial cells in the respiratory, digestive and reproductive systems. Glanders: An infectious disease that is caused by the bacterium Burkholderia mallei, which primarily affects horses, donkeys and mules but that can be naturally contracted by goats, dogs and cats. Glanders can be transmitted to humans by direct contact with infected animals. The types of infection include localised, pus-forming cutaneous infections, pulmonary infections, bloodstream infections and chronic suppurative infection of the skin. Melioidosis: This is also known as Whitmore’s disease. It is clinically and pathologically similar to glanders, but is redominately found in tropical climates, especially in Southeast Asia where it is endemic. The bacteria causing melioidosis (Pseudomonas pseudomallei) are found in contaminated water and soil, and are spread to humans and animals through direct contact with the contaminated source. Melioidosis can be categorised as an acute or localised infection, acute pulmonary infection, acute bloodstream infection and chronic suppurative infection. Inapparent infections are also possible. Type III secretion system (TTSS): Specialised multi-protein secretion systems found in various Gram-negative bacteria for the export of virulence factors delivered directly to host cells. These factors subvert normal host cell functions in ways that seem beneficial to invading bacteria. The genes encoding several TTSSs reside on pathogenicity islands, which are inserted DNA segments within the chromosome that confer a variety of virulence traits upon the host bacterium, such as the ability to acquire iron and to adhere to or to enter host cells.
in Bcc strains; however, new genetic tools have been developed that allow for their gene expression [17]. Recently, a modified signature-tagged mutagenesis (STM) screen using a widely disseminated strain of B. cenocepacia resulted in the identification of 102 transposon mutants that were attenuated for survival in a rat model of chronic lung infection [18]. Despite the elimination of auxotrophic mutants before the in vivo selection step, an abundance of mutants with insertions in genes encoding functions involved in cellular metabolism were isolated from the STM screen. This suggests that bacterial metabolic adjustments are necessary for survival in the rat lung environment. In addition, genes required for resistance to cationic peptides (pmrF), magnesium transport (mgtC), lipopolysaccharide synthesis and capsule biosynthesis were identified [18]. A study using subtractive hybridisation has also identified a capsular polysaccharidebiosynthesis gene cluster as a major virulence determinant for B. pseudomallei [19]. The complete genome sequences of B. pseudomallei and B. mallei have recently been reported [20,21]. Each Current Opinion in Microbiology 2005, 8:99–105
organism possesses two circular chromosomes and a total of 5855 and 5435 coding sequences, respectively. In both cases, the large chromosome encodes many of the core functions associated with central metabolism and cell growth, whereas the small chromosome encodes accessory functions probably associated with adaptation and survival in various niches. The genome of a clinical isolate of B. cenocepacia has also been sequenced, revealing three chromosomes and one large plasmid totalling 8 Mb (http://www.sanger.ac.uk/Projects/B_cenocepacia). Analysis of the genome sequence of B. pseudomallei revealed several genes encoding survival and virulence functions, including three TTSSs. The only virulence factors clearly demonstrated in B. mallei to be essential for pathogenicity are an exopolysaccharide capsule [22] and a Salmonella enterica-like TTSS [23]. Analysis of the B. mallei genome identified several additional candidate virulence factors, the function of which is supported by comparative genome hybridisation and expression profiling of the bacterium in hamster liver in vivo. This identified secreted hydrolases, a toxin biosynthesis protein, and phosphorous and iron acquisition proteins [21], but the determination of their precise role awaits the appropriate genetic experiments using specific mutants in an animal infection model.
Burkholderia species as intracellular pathogens Bcc isolates are able to survive intracellularly in vitro within macrophages [24,25], respiratory epithelial cells [26,27] and amoebae [28,29]. It is not clear, however, if intracellular survival occurs in vivo, although the capacity of strains to penetrate epithelial cells has been correlated with mouse infectivity [30,31]. Bcc can survive intracellularly with minimal or no replication and establish residence in an acidic membrane-bound vacuole that does not fuse with lysosomes [29]. This suggests that the intracellular Bcc redirects maturation of the phagosome and, as a result, Bcc resides in a specialised compartment that we designated the B. cepacia-containing vacuole [29]. In macrophages, intracellular Bcc are localised within spacious vacuoles (Figure 1) that are characterised by low pH, rapid accumulation of the late endosomal glycoprotein markers LAMP1 (lysosome-associated membrane protein 1) and LAMP2, exclusion of fluid phase endocytic markers that accumulate in lysosomes and impaired fusion with lysosomes (Table 1; J Lamothe and MA Valvano, unpublished; [29]). Remarkably, B. cepacia-containing vacuoles have many features in common with S. enterica-containing vacuoles [32,33]; although, unlike Salmonella, intracellular Bcc isolates do not appear to replicate. Heat-inactivated bacteria are targeted to the lysosome where they are degraded. Therefore, it is probable that live intracellular bacteria produce molecules that interfere with the normal maturation pathway of the phagosome (see below). www.sciencedirect.com
Survival and persistence of opportunistic Burkholderia species in host cells Valvano, Keith and Cardona 101
Figure 1
Phase contrast micrographs of RAW 264.7 macrophages infected with a B. dolosa (Bcc genomovar VI) strain. Photographs of two macrophage cells taken at 2 h post infection show spacious vacuoles that contain bacteria.
B. pseudomallei has also been shown to survive within a variety of phagocytic and nonphagocytic cells. In contrast to the Bcc, when B. pseudomallei is taken up by macrophage-like cell lines it is capable of escaping from endocytic vacuoles and resides freely in the cytosol, where it can replicate. The internalisation of B. pseudomallei by RAW 264.7 macrophages also induces cell–cell fusion, resulting in multi-nucleated giant cell formation [34]. This phenomenon might facilitate the spread of B. pseudomallei from one cell to another, and has not been observed in Bcc infections. Eventually, intracellular B. pseudomallei escape the macrophage cell, but the mechanism by which this happens is not yet understood. A rhamnolipid, comprising two molecules of rhamnose and two molecules of b-hydroxytetradecanoic acid, has been detected in the supernatant of B. pseudomallei cultures [35]. At high concentrations this rhamnolipid induced morphological alteration of the mammalian cells and exhibited a time- and dose-dependent cytotoxicity against nonphagocytic and phagocytic cell lines, probably due to its detergent-like properties. Cellular functions such as phagocytosis were affected in rhamnolipid-
treated cells and cell cycle progression was impaired. These alterations were thought to be caused by the reorganisation of the cytoskeleton, characterised by loss of central stress fibres [35]. It was therefore suggested that the B. pseudomallei rhamnolipid could be targeting small GTPases, activating Rac and inactivating Rho, thus causing a loss of stress fibres and pronounced peripheral actin staining [35]. B. cenocepacia-induced actin cytoskeleton rearrangements in well-differentiated human airway epithelial cells have also been observed [36]. Similar patterns of actin filament network disruption were observed in cell cultures exposed to B. multivorans or treated with cytochalasin D, a potent inhibitor of actin filament formation. This experiment suggests that B. multivorans promotes disruption of the actin filament network during epithelial invasion. It was also demonstrated that B. multivorans uses actin-independent entry mechanisms. Indeed, single-cell entry and translocation by way of the paracellular shunt are facilitated in the presence of actin disruption [37]. For many years, the description of actin-based intracellular motility of intracellular pathogens has been limited
Table 1 Similarities and differences between the intracellular survival of B. cenocepacia and S. enterica. Property
B. cenocepacia (live)
B. cenocepacia (heat-killed)
S. enterica (live)
Bacteria in vacuoles Intracellular bacteria have an intact morphology Acidic vacuole as determined by Lysosensor Co-localisation with Lysotracker Accumulation of fluorescent dextrans Co-localisation with LAMP-1 Co-localisation with LAMP-2 Live bacteria Bacterial replication Defect in phosphoinositide targeting Defect in NADPH oxidase trafficking
+ + (for up to 5 days) +
+ (after 2 h) + + + + +
+ + +
+ + + UI UI
UI UI
+ + + + + +
UI, under investigation.
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to members of the genera Shigella, Listeria and Rickettsia. The intracellular motility of these organisms is strictly coupled to a polarised actin polymerisation process at one pole of the bacterium, commonly known as an actin comet or rocket tail. The actin rearrangement is initiated at one pole of B. pseudomallei leading to actin tail formation and actin-associated peripheral membrane protrusions [34]. As it has been shown for the actin-based motility of Listeria, Shigella and vaccinia virus, B. pseudomallei uses the Arp2/3 complex to induce actin polymerisation, albeit by a different activation mechanism [38].
Type III secretion systems Three TTSSs have been identified in B. pseudomallei: two plant-type systems and a third TTSS, termed Bsa, which is similar to the Inv/Spa/Prg system of S. enterica serovar Typhimurium and the Ipa/Mxi/Spa TTSS of Shigella flexneri [39–41]. This bsa locus is also conserved in B. mallei and B. cenocepacia [42]. Mutagenesis experiments demonstrated that a functional TTSS is required for the full pathogenicity of B. mallei in the BALB/c mouse and Syrian hamster models of infection [23]. A mutant in the B. cenocepacia bscN gene, which encodes a putative ATPase, elicited reduced inflammation in the lungs of a mouse infection model [7]. Also, a mutation in the B. cenocepacia TTSS causes mistargeting of intracellular bacteria to a degradative vacuole, effectively reducing intracellular survival (J Lamothe and MA Valvano, unpublished). These results implicate the TTSS as an important determinant in the pathogenesis of B. cenocepacia. Characterisation of several B. pseudomallei transposon mutants in the structural gene bsaZ, the translocator gene bipD and the effector genes bopA, bopB and bopE, has shed light on the putative function of these proteins encoded by the Bsa TTSS. A B. pseudomallei bipD mutant was drastically reduced in its ability to invade epithelial cells, replicate in murine macrophage-like cell lines, escape from endocytic vesicles and form an actin tail [40]. Furthermore, a B. pseudomallei bopE mutant showed diminished ability to invade HeLa cells. BopE exhibits guanine nucleotide exchange factor activity acting on Cdc42 and Rac1, promoting membrane ruffling [43]. Further characterisation of bsa mutants in a murine model revealed that a bipD-deficient strain exhibited a 2160-fold reduction in the mean lethal dose for BALB/c mice. Reductions in the replication rate of B. pseudomallei bipD mutants were combined with a reduction in both splenomegaly and abscess formation in the spleen and liver [44]. This reduction in replication rate mirrors that of a B. mallei bipD mutant [23]. However, the B. mallei bipD mutant was less attenuated than mutants in either capsular polysaccharide- or branched chain amino acidsynthesis genes [45,46]. It was also shown that immunisation of mice with purified BipD before B. pseudomallei challenge provided no significant protection against infection. No significant attenuation was observed when Current Opinion in Microbiology 2005, 8:99–105
a bopE mutant was administered intraperitoneally or intranasally in BALB/c mice, whereas bopA and bopB mutants exhibited a statistically significant increase in the median time to death when administered to BALB/c mice by the intraperitoneal route [44]. Like for Salmonella, this suggests that B. pseudomallei effector proteins work together in concert to promote invasion of eukaryotic host cells.
Evasion of host reactive oxygen and nitrogen intermediates The resistance of Bcc strains to cationic antimicrobial peptides renders them resistant to non-oxidative killing by phagocytes [2]. In vivo, host defense against B. cepacia is critically dependent on reactive oxygen intermediates [47]. For instance, patients with chronic granulomatous disease, whose polymorphonuclear leukocytes (PMNs) fail to mount an effective oxidative burst, are often infected with Bcc [2]. These observations contrast with the apparent tolerance of the Bcc to the oxidative environment in the CF lung, where the inflammatory response is dominated by PMNs [48]. It has been reported that Bcc strains isolated from CF patients, especially B. cenocepacia, are strong catalase and superoxide dismutase producers [10]. Recent evidence, which shows that catalase-defective mutants of B. cenocepacia have low survival rates in amoebae and macrophages, suggests that intracellular Bcc strains/isolates escape oxidative damage in macrophages (KE Keith et al., unpublished). Furthermore, intracellular survival of Bcc isolates in murine macrophages occurs despite induction of an oxidative burst and is associated with reduced nitric oxide production [24]. Amoebae and macrophages are far less competent than PMNs at generating toxic oxygen radicals, which might explain the persistence of Bcc within macrophages, perhaps enhancing their capacity for intracellular parasitism. Conditions in which reactive oxygen intermediates are quenched may exist in the CF lung, which would suggest that Bcc isolates are better suited to cause infection than P. aeruginosa, which are more susceptible to non-oxidative killing. B. pseudomallei can invade macrophages without the activation of inducible nitric oxide synthase (NOS), an essential enzyme needed for the generation of reactive nitrogen intermediates (RNIs), which regulates survival and multiplication of intracellular bacteria [49,50]. Failure to induce NOS expression in macrophages might be related to the fact that B. pseudomallei possess lipopolysaccharide (LPS) that is different from other Gramnegative bacteria and is a poor activator of macrophage cell responses. In the presence of IFN-g, inducible NOS expression was upregulated resulting in enhanced B. pseudomallei killing.
Infection models One of the difficulties in investigating the pathogenesis of Burkholderia is the lack of suitable animal models of www.sciencedirect.com
Survival and persistence of opportunistic Burkholderia species in host cells Valvano, Keith and Cardona 103
Table 2 Infection models to investigate the pathogenesis of Burkholderia species. Model
Route
Strains
Effects
Reference
BALB/c mice
Intranasal
B. cenocepacia
[58]
C57BL/6 mice
Intranasal Agar bead Agar bead Intranasal Oral Oral
Clearance of pathogen; induction of a strong inflammatory response in the lung; high levels of IL-1b Association with pulmonary macrophages More resistant to infection than BALB/c mice Reduced mortality with an fliCII mutant (major flagellin subunit) Reduced inflammation with a TTSS mutant Chronic severe bronchopneumonia Reduced killing with cep quorum-sensing mutant Intestinal infection; slow and fast killing
Reproduces naturally-acquired glanders Necrosis; lack of root hair and chlorosis (varies according to strain) Survival (varies according to strain) Survival
[51] [55] [28] [54]
Cftr / mice C. elegans
Horse Alfalfa Acanthamoeba
Intratracheally
B. multivorans B. pseudomallei B. cenocepacia B. cenocepacia B. cenocepacia B. cenocepacia B. thailandensis B. pseudomallei B. mallei B. mallei Bcc Bcc B. pseudomallei
infection. Except in the case of B. mallei, where a horse model that reproduces the natural disease has recently been established [51], there is a general lack of infection models that mimic human infection, especially those for chronic infection and long-term bacterial persistence (Table 2). The agar-bead model of lung infection provides the opportunity for a long-term chronic infection with the Bcc (PA Sokol, personal communication) but might not completely mimic CF disease. In contrast, a murine model of chronic pneumonia using Cftr / mice reflects, in part, the situation in human patients and might help to elucidate the mechanisms leading to defective host defense and infection in CF [52]. An interesting way to circumvent some of the problems with animal models is to use ex vivo models involving welldifferentiated cultures established from airway epithelia of CF patients. These models exhibit features resembling the physiopathology of the airways, illustrating differences in the way that epithelia of CF patients and normal subjects handle B. cenocepacia infection. In this model, invading bacteria were free within the cytoplasm and surrounded by intermediate filaments as well as between cells. These results suggest that differences might exist between epithelial and phagocytic cells in the characteristics of the intracellular infection by B. cenocepacia [53]. It is possible that the ability of Burkholderia to survive in host cells reflects the adaptability of these bacteria to many different environments. For instance, the ability of B. pseudomallei and many Bcc isolates to persist within amoeba [29,54] raises the question of the role of protozoa as potential environmental reservoirs for these opportunistic pathogens. Furthermore, nonmammalian infection models using plants [55], amoebae and nematodes such as Caenorhabditis elegans [56,57] might aid to better understand the physiopathology of Burkholderia. www.sciencedirect.com
[58] [59] [60] [7] [52] [56] [57]
Conclusions Burkholderia species are important opportunistic pathogens that have the ability to survive in many different environments, including susceptible human hosts. These bacteria possess large genomes and carry an enormous genetic potential for adaptation to many different environments. The availability of host models for infection, together with the exploitation of genomic sequence data and the development of new genetic tools will afford the possibility to better understand the biology of these opportunistic pathogens.
Acknowledgements We apologise to all researchers whose work has not been cited owing to the lack of space. This work was supported by grants from the Canadian Cystic Fibrosis Foundation and the Canadian Institutes of Health Research to MAV, and STC has been supported by a Fellowship from the Canadian Cystic Fibrosis Foundation. MAV holds a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis.
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29. Lamothe J, Thyssen S, Valvano MA: Burkholderia cepacia complex isolates survive intracellularly without replication within acidic vacuoles of Acanthamoeba polyphaga. Cell Microbiol 2004, 12:1127-1138. This paper demonstrates that Bcc survival occurs in a late phagosomal compartment that cannot fuse with lysosomes, providing the first demonstration that live, intracellular Bcc strains alter the process of endosomal maturation. 30. Cieri MV, Mayer-Hamblett N, Griffith A, Burns JL: Correlation between an in vitro invasion assay and a murine model of Burkholderia cepacia lung infection. Infect Immun 2002, 70:1081-1086. 31. Chiu CH, Ostry A, Speert DP: Invasion of murine respiratory epithelial cells in vivo by Burkholderia cepacia. J Med Microbiol 2001, 50:594-601. 32. Linehan SA, Holden DW: The interplay between Salmonella typhimurium and its macrophage host — what can it teach us about innate immunity? Immunol Lett 2003, 85:183-192. 33. Drecktrah D, Knodler LA, Steele-Mortimer O: Modulation and utilization of host cell phosphoinositides by Salmonella spp. Infect Immun 2004, 72:4331-4335. 34. Kespichayawattana W, Rattanachetkul S, Wanun T, Utaisincharoen P, Sirisinha S: Burkholderia pseudomallei induces cell fusion and actin-associated membrane protrusion: a possible mechanism for cell-to-cell spreading. Infect Immun 2000, 68:5377-5384. 35. Ha¨ ußler S, Rohde M, von Neuhoff N, Nimtz M, Steinmetz I: Structural and functional cellular changes induced by Burkholderia pseudomallei rhamnolipid. Infect Immun 2003, 71:2970-2975. 36. Schwab U, Leigh M, Ribeiro C, Yankaskas J, Burns K, Gilligan P, Sokol P, Boucher R: Patterns of epithelial cell invasion by different species of the Burkholderia cepacia complex in well-differentiated human airway epithelia. Infect Immun 2002, 70:4547-4555. 37. Schwab UE, Ribeiro CM, Neubauer H, Boucher RC: Role of actin filament network in Burkholderia multivorans invasion in www.sciencedirect.com
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well-differentiated human airway epithelia. Infect Immun 2003, 71:6607-6609. 38. Breitbach K, Rottner K, Klocke S, Rohde M, Jenzora A, Wehland J, Steinmetz I: Actin-based motility of Burkholderia pseudomallei involves the Arp 2/3 complex, but not N-WASP and Ena/VASP proteins. Cell Microbiol 2003, 5:385-393. This study describes that the mechanism of actin-based motility in B. pseudomallei is different from that described for Listeria and Rickettsia. 39. Attree O, Attree I: A second type III secretion system in Burkholderia pseudomallei: who is the real culprit? Microbiology 2001, 147:3197-3199. 40. Stevens MP, Wood MW, Taylor LA, Monaghan P, Hawes P, Jones PW, Wallis TS, Galyov EE: An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen. Mol Microbiol 2002, 46:649-659. 41. Rainbow L, Hart CA, Winstanley C: Distribution of type III secretion gene clusters in Burkholderia pseudomallei, B. thailandensis and B. mallei. J Med Microbiol 2002, 51:374-384. 42. Glendinning KJ, Parsons YN, Duangsonk K, Hales BA, Humphreys D, Hart CA, Winstanley C: Sequence divergence in type III secretion gene clusters of the Burkholderia cepacia complex. FEMS Microbiol Lett 2004, 235:229-235. 43. Stevens MP, Friebel A, Taylor LA, Wood MW, Brown PJ, Hardt WD, Galyov EE: A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity. J Bacteriol 2003, 185:4992-4996. 44. Stevens MP, Haque A, Atkins T, Hill J, Wood MW, Easton A, Nelson M, Underwood-Fowler C, Titball RW, Bancroft GJ et al.: Attenuated virulence and protective efficacy of a Burkholderia pseudomallei Bsa type III secretion mutant in murine models of melioidosis. Microbiology 2004, 150:2669-2676. 45. Atkins T, Prior R, Mack K, Russell P, Nelson M, Prior J, Ellis J, Oyston PC, Dougan G, Titball RW: Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis. J Med Microbiol 2002, 51:539-547. 46. Atkins T, Prior RG, Mack K, Russell P, Nelson M, Oyston PC, Dougan G, Titball RW: A mutant of Burkholderia pseudomallei, auxotrophic in the branched chain amino acid biosynthetic pathway, is attenuated and protective in a murine model of melioidosis. Infect Immun 2002, 70:5290-5294. 47. Segal BH, Ding L, Holland SM: Phagocyte NADPH oxidase, but not inducible nitric oxide synthase, is essential for early control of Burkholderia cepacia and Chromobacterium violaceum infection in mice. Infect Immun 2003, 71:205-210. 48. Bals R, Weiner DJ, Wilson JM: The innate immune system in cystic fibrosis lung disease. J Clin Invest 1999, 103:303-307. 49. Utaisincharoen P, Anuntagool N, Arjcharoen S, Limposuwan K, Chaisuriya P, Sirisinha S: Induction of iNOS expression and
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antimicrobial activity by interferon (IFN)-b is distinct from IFN-g in Burkholderia pseudomallei-infected mouse macrophages. Clin Exp Immunol 2004, 136:277-283. 50. Utaisincharoen P, Tangthawornchaikul N, Kespichayawattana W, Anuntagool N, Chaisuriya P, Sirisinha S: Kinetic studies of the production of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) in macrophages stimulated with Burkholderia pseudomallei endotoxin. Clin Exp Immunol 2000, 122:324-329. 51. Lopez J, Copps J, Wilhelmsen C, Moore R, Kubay J, St-Jacques M, Halayko S, Kranendonk C, Toback S, DeShazer D et al.: Characterization of experimental equine glanders. Microbes Infect 2003, 5:1125-1131. This is an important study describing a disease model that mimics natural glanders. It will make it possible to directly test vaccines and other preventative strategies to combat glanders. 52. Sajjan U, Thanassoulis G, Cherapanov V, Lu A, Sjolin C, Steer B, Wu YJ, Rotstein OD, Kent G, McKerlie C et al.: Enhanced susceptibility to pulmonary infection with Burkholderia cepacia in CftrS/S mice. Infect Immun 2001, 69:5138-5150. 53. Sajjan U, Keshavjee S, Forstner J: Responses of welldifferentiated airway epithelial cell cultures from healthy donors and patients with cystic fibrosis to Burkholderia cenocepacia infection. Infect Immun 2004, 72:4188-4199. 54. Inglis TJ, Robertson T, Woods DE, Dutton NS, Chang BJ: Flagellum-mediated adhesion by Burkholderia pseudomallei precedes invasion of Acanthamoeba astronyxis. Infect Immun 2003, 71:2280-2282. 55. Bernier SP, Silo-Suh L, Woods DE, Ohman DE, Sokol PA: Comparative analysis of plant and animal models for characterization of Burkholderia cepacia virulence. Infect Immun 2003, 71:5306-5313. 56. Ko¨ the M, Antl M, Huber B, Stoecker K, Ebrecht D, Steinmetz I, Eberl L: Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system. Cell Microbiol 2003, 5:343-351. 57. Gan YH, Chua KL, Chua HH, Liu B, Hii CS, Chong HL, Tan P: Characterization of Burkholderia pseudomallei infection and identification of novel virulence factors using a Caenorhabditis elegans host system. Mol Microbiol 2002, 44:1185-1197. 58. Chu KK, MacDonald KL, Davidson DJ, Speert DP: Persistence of Burkholderia multivorans within the pulmonary macrophage in the murine lung. Infect Immun 2004, 72:6142-6147. 59. Liu B, Koo GC, Yap EH, Chua KL, Gan YH: Model of differential susceptibility to mucosal Burkholderia pseudomallei infection. Infect Immun 2002, 70:504-511. 60. Urban TA, Griffith A, Torok AM, Smolkin ME, Burns JL, Goldberg JB: Contribution of Burkholderia cenocepacia flagella to infectivity and inflammation. Infect Immun 2004, 72:5126-5134.
Current Opinion in Microbiology 2005, 8:99–105
Current Opinion in Microbiology 2005, 8:99–105 This review comes from a themed issue on Host–microbe interactions: bacteria Edited by Pascale Cossart and Jorge Gala´n Available online 6th January 2005 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2004.12.002
Introduction The Burkholderia genus contains over 32 species that occupy very diverse ecological niches, ranging from contaminated soils to the respiratory tract of humans [1]. Most members of the genus are known to be plant pathogens and soil bacteria. B. mallei and B. pseudomallei are well-recognised as pathogenic for humans and animals, but virtually all Burkholderia can be opportunistic pathogens in immunocompromised individuals. B. pseudomallei is the causative agent of melioidosis (see Glossary), a septicemic disease associated with bacterial dissemination to several organs, which is endemic in Southeast Asia and Northern Australia. B. mallei is the causative agent of the zoonotic disease glanders, and is a host-adapted pathogen that does not appear to persist in nature outside its equine host. Both glanders and melioidosis can be acquired by aerosol, exhibit high mortality rates without antibiotic treatment and can be potentially difficult to diagnose at an early stage. Therefore, B. pseudomallei and B. mallei are a great concern as www.sciencedirect.com
biological weapons and are listed by the Centers for Disease Control and Prevention as category B agents. B. cepacia, initially described as the causative agent of soft rot in onions, has emerged as a multi-drug resistant nosocomial pathogen in immunocompromised patients, particularly in those with chronic granulomatous diseases and cystic fibrosis (CF). Nearly all CF patients suffer from chronic infections of the major airways. The infection exacerbates the progressive pulmonary deterioration and correlates with substantial morbidity and mortality. B. cepacia is not a single microorganism but rather a collection of related species or genomovars collectively referred to as the B. cepacia complex (Bcc) [1]. Of these, B. cenocepacia (formerly genomovar III) and B. multivorans (formerly genomovar II) are the most prevalent species isolated from patients with CF [2]. Pseudomonas aeruginosa and Bcc commonly infect patients with CF. However, Bcc infections are more serious for three main reasons: first, infected patients can deteriorate more rapidly and also develop a life-threatening pneumonia termed cepacia syndrome; second, treatment of these infections is usually difficult because of the intrinsic resistance of Bcc to most clinically useful antibiotics [3]; and third, unlike P. aeruginosa, some Bcc isolates are transmissible from patient to patient [2]. A feature common to B. mallei, B. pseudomallei and Bcc infections is the localisation of the infection to the lungs and airways. Furthermore, persistence of the bacterium without causing overt disease has also been documented for B. pseudomallei and Bcc. In this review, we discuss the ability of B. pseudomallei, B. mallei and Bcc to survive and persist in host cells.
Virulence factors in Burkholderia A number of potential virulence factors have been described and characterised in Bcc isolates. They include cable pili [4,5], flagella [6], a type III secretion system (TTSS) [7], surface exopolysaccharide [8], the production of melanin [9], catalase and superoxide dismutase [10], iron-scavenging siderophores [11], proteases [12] and other secreted enzymes [13], quorum sensing systems [14,15] and the ability to form biofilms [16]. Not all Bcc species produce each of the proposed virulence factors and, to date, only a few of the individual factors have been clearly demonstrated to contribute significantly to lung disease. Resistance to antibiotics commonly used for genetic selection makes it difficult to conduct genetic studies Current Opinion in Microbiology 2005, 8:99–105
100 Host–microbe interactions: bacteria
Glossary Category B agents: Second highest priority agents as defined by the Centers for Disease Control and Prevention (CDC) because they are moderately easy to disseminate, result in moderate morbidity rates and low mortality rates, and require specific enhancements of CDC’s diagnostic capacity and enhanced disease surveillance. Chronic granulomatous disease: A group of rare inherited disorders that result in the inability of phagocytes to undergo the respiratory burst necessary to kill certain types of bacteria and fungi. This leads to recurrent life-threatening bacterial and fungal infections. Cystic fibrosis: A genetic disease that affects approximately 1 in 2000 individuals. Cftr encodes a protein called the cystic fibrosis transmembrane conductance regulator. This is a chloride ion transporter. A defective Cftr gene causes a dysfunction of secretory epithelial cells in the respiratory, digestive and reproductive systems. Glanders: An infectious disease that is caused by the bacterium Burkholderia mallei, which primarily affects horses, donkeys and mules but that can be naturally contracted by goats, dogs and cats. Glanders can be transmitted to humans by direct contact with infected animals. The types of infection include localised, pus-forming cutaneous infections, pulmonary infections, bloodstream infections and chronic suppurative infection of the skin. Melioidosis: This is also known as Whitmore’s disease. It is clinically and pathologically similar to glanders, but is redominately found in tropical climates, especially in Southeast Asia where it is endemic. The bacteria causing melioidosis (Pseudomonas pseudomallei) are found in contaminated water and soil, and are spread to humans and animals through direct contact with the contaminated source. Melioidosis can be categorised as an acute or localised infection, acute pulmonary infection, acute bloodstream infection and chronic suppurative infection. Inapparent infections are also possible. Type III secretion system (TTSS): Specialised multi-protein secretion systems found in various Gram-negative bacteria for the export of virulence factors delivered directly to host cells. These factors subvert normal host cell functions in ways that seem beneficial to invading bacteria. The genes encoding several TTSSs reside on pathogenicity islands, which are inserted DNA segments within the chromosome that confer a variety of virulence traits upon the host bacterium, such as the ability to acquire iron and to adhere to or to enter host cells.
in Bcc strains; however, new genetic tools have been developed that allow for their gene expression [17]. Recently, a modified signature-tagged mutagenesis (STM) screen using a widely disseminated strain of B. cenocepacia resulted in the identification of 102 transposon mutants that were attenuated for survival in a rat model of chronic lung infection [18]. Despite the elimination of auxotrophic mutants before the in vivo selection step, an abundance of mutants with insertions in genes encoding functions involved in cellular metabolism were isolated from the STM screen. This suggests that bacterial metabolic adjustments are necessary for survival in the rat lung environment. In addition, genes required for resistance to cationic peptides (pmrF), magnesium transport (mgtC), lipopolysaccharide synthesis and capsule biosynthesis were identified [18]. A study using subtractive hybridisation has also identified a capsular polysaccharidebiosynthesis gene cluster as a major virulence determinant for B. pseudomallei [19]. The complete genome sequences of B. pseudomallei and B. mallei have recently been reported [20,21]. Each Current Opinion in Microbiology 2005, 8:99–105
organism possesses two circular chromosomes and a total of 5855 and 5435 coding sequences, respectively. In both cases, the large chromosome encodes many of the core functions associated with central metabolism and cell growth, whereas the small chromosome encodes accessory functions probably associated with adaptation and survival in various niches. The genome of a clinical isolate of B. cenocepacia has also been sequenced, revealing three chromosomes and one large plasmid totalling 8 Mb (http://www.sanger.ac.uk/Projects/B_cenocepacia). Analysis of the genome sequence of B. pseudomallei revealed several genes encoding survival and virulence functions, including three TTSSs. The only virulence factors clearly demonstrated in B. mallei to be essential for pathogenicity are an exopolysaccharide capsule [22] and a Salmonella enterica-like TTSS [23]. Analysis of the B. mallei genome identified several additional candidate virulence factors, the function of which is supported by comparative genome hybridisation and expression profiling of the bacterium in hamster liver in vivo. This identified secreted hydrolases, a toxin biosynthesis protein, and phosphorous and iron acquisition proteins [21], but the determination of their precise role awaits the appropriate genetic experiments using specific mutants in an animal infection model.
Burkholderia species as intracellular pathogens Bcc isolates are able to survive intracellularly in vitro within macrophages [24,25], respiratory epithelial cells [26,27] and amoebae [28,29]. It is not clear, however, if intracellular survival occurs in vivo, although the capacity of strains to penetrate epithelial cells has been correlated with mouse infectivity [30,31]. Bcc can survive intracellularly with minimal or no replication and establish residence in an acidic membrane-bound vacuole that does not fuse with lysosomes [29]. This suggests that the intracellular Bcc redirects maturation of the phagosome and, as a result, Bcc resides in a specialised compartment that we designated the B. cepacia-containing vacuole [29]. In macrophages, intracellular Bcc are localised within spacious vacuoles (Figure 1) that are characterised by low pH, rapid accumulation of the late endosomal glycoprotein markers LAMP1 (lysosome-associated membrane protein 1) and LAMP2, exclusion of fluid phase endocytic markers that accumulate in lysosomes and impaired fusion with lysosomes (Table 1; J Lamothe and MA Valvano, unpublished; [29]). Remarkably, B. cepacia-containing vacuoles have many features in common with S. enterica-containing vacuoles [32,33]; although, unlike Salmonella, intracellular Bcc isolates do not appear to replicate. Heat-inactivated bacteria are targeted to the lysosome where they are degraded. Therefore, it is probable that live intracellular bacteria produce molecules that interfere with the normal maturation pathway of the phagosome (see below). www.sciencedirect.com
Survival and persistence of opportunistic Burkholderia species in host cells Valvano, Keith and Cardona 101
Figure 1
Phase contrast micrographs of RAW 264.7 macrophages infected with a B. dolosa (Bcc genomovar VI) strain. Photographs of two macrophage cells taken at 2 h post infection show spacious vacuoles that contain bacteria.
B. pseudomallei has also been shown to survive within a variety of phagocytic and nonphagocytic cells. In contrast to the Bcc, when B. pseudomallei is taken up by macrophage-like cell lines it is capable of escaping from endocytic vacuoles and resides freely in the cytosol, where it can replicate. The internalisation of B. pseudomallei by RAW 264.7 macrophages also induces cell–cell fusion, resulting in multi-nucleated giant cell formation [34]. This phenomenon might facilitate the spread of B. pseudomallei from one cell to another, and has not been observed in Bcc infections. Eventually, intracellular B. pseudomallei escape the macrophage cell, but the mechanism by which this happens is not yet understood. A rhamnolipid, comprising two molecules of rhamnose and two molecules of b-hydroxytetradecanoic acid, has been detected in the supernatant of B. pseudomallei cultures [35]. At high concentrations this rhamnolipid induced morphological alteration of the mammalian cells and exhibited a time- and dose-dependent cytotoxicity against nonphagocytic and phagocytic cell lines, probably due to its detergent-like properties. Cellular functions such as phagocytosis were affected in rhamnolipid-
treated cells and cell cycle progression was impaired. These alterations were thought to be caused by the reorganisation of the cytoskeleton, characterised by loss of central stress fibres [35]. It was therefore suggested that the B. pseudomallei rhamnolipid could be targeting small GTPases, activating Rac and inactivating Rho, thus causing a loss of stress fibres and pronounced peripheral actin staining [35]. B. cenocepacia-induced actin cytoskeleton rearrangements in well-differentiated human airway epithelial cells have also been observed [36]. Similar patterns of actin filament network disruption were observed in cell cultures exposed to B. multivorans or treated with cytochalasin D, a potent inhibitor of actin filament formation. This experiment suggests that B. multivorans promotes disruption of the actin filament network during epithelial invasion. It was also demonstrated that B. multivorans uses actin-independent entry mechanisms. Indeed, single-cell entry and translocation by way of the paracellular shunt are facilitated in the presence of actin disruption [37]. For many years, the description of actin-based intracellular motility of intracellular pathogens has been limited
Table 1 Similarities and differences between the intracellular survival of B. cenocepacia and S. enterica. Property
B. cenocepacia (live)
B. cenocepacia (heat-killed)
S. enterica (live)
Bacteria in vacuoles Intracellular bacteria have an intact morphology Acidic vacuole as determined by Lysosensor Co-localisation with Lysotracker Accumulation of fluorescent dextrans Co-localisation with LAMP-1 Co-localisation with LAMP-2 Live bacteria Bacterial replication Defect in phosphoinositide targeting Defect in NADPH oxidase trafficking
+ + (for up to 5 days) +
+ (after 2 h) + + + + +
+ + +
+ + + UI UI
UI UI
+ + + + + +
UI, under investigation.
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to members of the genera Shigella, Listeria and Rickettsia. The intracellular motility of these organisms is strictly coupled to a polarised actin polymerisation process at one pole of the bacterium, commonly known as an actin comet or rocket tail. The actin rearrangement is initiated at one pole of B. pseudomallei leading to actin tail formation and actin-associated peripheral membrane protrusions [34]. As it has been shown for the actin-based motility of Listeria, Shigella and vaccinia virus, B. pseudomallei uses the Arp2/3 complex to induce actin polymerisation, albeit by a different activation mechanism [38].
Type III secretion systems Three TTSSs have been identified in B. pseudomallei: two plant-type systems and a third TTSS, termed Bsa, which is similar to the Inv/Spa/Prg system of S. enterica serovar Typhimurium and the Ipa/Mxi/Spa TTSS of Shigella flexneri [39–41]. This bsa locus is also conserved in B. mallei and B. cenocepacia [42]. Mutagenesis experiments demonstrated that a functional TTSS is required for the full pathogenicity of B. mallei in the BALB/c mouse and Syrian hamster models of infection [23]. A mutant in the B. cenocepacia bscN gene, which encodes a putative ATPase, elicited reduced inflammation in the lungs of a mouse infection model [7]. Also, a mutation in the B. cenocepacia TTSS causes mistargeting of intracellular bacteria to a degradative vacuole, effectively reducing intracellular survival (J Lamothe and MA Valvano, unpublished). These results implicate the TTSS as an important determinant in the pathogenesis of B. cenocepacia. Characterisation of several B. pseudomallei transposon mutants in the structural gene bsaZ, the translocator gene bipD and the effector genes bopA, bopB and bopE, has shed light on the putative function of these proteins encoded by the Bsa TTSS. A B. pseudomallei bipD mutant was drastically reduced in its ability to invade epithelial cells, replicate in murine macrophage-like cell lines, escape from endocytic vesicles and form an actin tail [40]. Furthermore, a B. pseudomallei bopE mutant showed diminished ability to invade HeLa cells. BopE exhibits guanine nucleotide exchange factor activity acting on Cdc42 and Rac1, promoting membrane ruffling [43]. Further characterisation of bsa mutants in a murine model revealed that a bipD-deficient strain exhibited a 2160-fold reduction in the mean lethal dose for BALB/c mice. Reductions in the replication rate of B. pseudomallei bipD mutants were combined with a reduction in both splenomegaly and abscess formation in the spleen and liver [44]. This reduction in replication rate mirrors that of a B. mallei bipD mutant [23]. However, the B. mallei bipD mutant was less attenuated than mutants in either capsular polysaccharide- or branched chain amino acidsynthesis genes [45,46]. It was also shown that immunisation of mice with purified BipD before B. pseudomallei challenge provided no significant protection against infection. No significant attenuation was observed when Current Opinion in Microbiology 2005, 8:99–105
a bopE mutant was administered intraperitoneally or intranasally in BALB/c mice, whereas bopA and bopB mutants exhibited a statistically significant increase in the median time to death when administered to BALB/c mice by the intraperitoneal route [44]. Like for Salmonella, this suggests that B. pseudomallei effector proteins work together in concert to promote invasion of eukaryotic host cells.
Evasion of host reactive oxygen and nitrogen intermediates The resistance of Bcc strains to cationic antimicrobial peptides renders them resistant to non-oxidative killing by phagocytes [2]. In vivo, host defense against B. cepacia is critically dependent on reactive oxygen intermediates [47]. For instance, patients with chronic granulomatous disease, whose polymorphonuclear leukocytes (PMNs) fail to mount an effective oxidative burst, are often infected with Bcc [2]. These observations contrast with the apparent tolerance of the Bcc to the oxidative environment in the CF lung, where the inflammatory response is dominated by PMNs [48]. It has been reported that Bcc strains isolated from CF patients, especially B. cenocepacia, are strong catalase and superoxide dismutase producers [10]. Recent evidence, which shows that catalase-defective mutants of B. cenocepacia have low survival rates in amoebae and macrophages, suggests that intracellular Bcc strains/isolates escape oxidative damage in macrophages (KE Keith et al., unpublished). Furthermore, intracellular survival of Bcc isolates in murine macrophages occurs despite induction of an oxidative burst and is associated with reduced nitric oxide production [24]. Amoebae and macrophages are far less competent than PMNs at generating toxic oxygen radicals, which might explain the persistence of Bcc within macrophages, perhaps enhancing their capacity for intracellular parasitism. Conditions in which reactive oxygen intermediates are quenched may exist in the CF lung, which would suggest that Bcc isolates are better suited to cause infection than P. aeruginosa, which are more susceptible to non-oxidative killing. B. pseudomallei can invade macrophages without the activation of inducible nitric oxide synthase (NOS), an essential enzyme needed for the generation of reactive nitrogen intermediates (RNIs), which regulates survival and multiplication of intracellular bacteria [49,50]. Failure to induce NOS expression in macrophages might be related to the fact that B. pseudomallei possess lipopolysaccharide (LPS) that is different from other Gramnegative bacteria and is a poor activator of macrophage cell responses. In the presence of IFN-g, inducible NOS expression was upregulated resulting in enhanced B. pseudomallei killing.
Infection models One of the difficulties in investigating the pathogenesis of Burkholderia is the lack of suitable animal models of www.sciencedirect.com
Survival and persistence of opportunistic Burkholderia species in host cells Valvano, Keith and Cardona 103
Table 2 Infection models to investigate the pathogenesis of Burkholderia species. Model
Route
Strains
Effects
Reference
BALB/c mice
Intranasal
B. cenocepacia
[58]
C57BL/6 mice
Intranasal Agar bead Agar bead Intranasal Oral Oral
Clearance of pathogen; induction of a strong inflammatory response in the lung; high levels of IL-1b Association with pulmonary macrophages More resistant to infection than BALB/c mice Reduced mortality with an fliCII mutant (major flagellin subunit) Reduced inflammation with a TTSS mutant Chronic severe bronchopneumonia Reduced killing with cep quorum-sensing mutant Intestinal infection; slow and fast killing
Reproduces naturally-acquired glanders Necrosis; lack of root hair and chlorosis (varies according to strain) Survival (varies according to strain) Survival
[51] [55] [28] [54]
Cftr / mice C. elegans
Horse Alfalfa Acanthamoeba
Intratracheally
B. multivorans B. pseudomallei B. cenocepacia B. cenocepacia B. cenocepacia B. cenocepacia B. thailandensis B. pseudomallei B. mallei B. mallei Bcc Bcc B. pseudomallei
infection. Except in the case of B. mallei, where a horse model that reproduces the natural disease has recently been established [51], there is a general lack of infection models that mimic human infection, especially those for chronic infection and long-term bacterial persistence (Table 2). The agar-bead model of lung infection provides the opportunity for a long-term chronic infection with the Bcc (PA Sokol, personal communication) but might not completely mimic CF disease. In contrast, a murine model of chronic pneumonia using Cftr / mice reflects, in part, the situation in human patients and might help to elucidate the mechanisms leading to defective host defense and infection in CF [52]. An interesting way to circumvent some of the problems with animal models is to use ex vivo models involving welldifferentiated cultures established from airway epithelia of CF patients. These models exhibit features resembling the physiopathology of the airways, illustrating differences in the way that epithelia of CF patients and normal subjects handle B. cenocepacia infection. In this model, invading bacteria were free within the cytoplasm and surrounded by intermediate filaments as well as between cells. These results suggest that differences might exist between epithelial and phagocytic cells in the characteristics of the intracellular infection by B. cenocepacia [53]. It is possible that the ability of Burkholderia to survive in host cells reflects the adaptability of these bacteria to many different environments. For instance, the ability of B. pseudomallei and many Bcc isolates to persist within amoeba [29,54] raises the question of the role of protozoa as potential environmental reservoirs for these opportunistic pathogens. Furthermore, nonmammalian infection models using plants [55], amoebae and nematodes such as Caenorhabditis elegans [56,57] might aid to better understand the physiopathology of Burkholderia. www.sciencedirect.com
[58] [59] [60] [7] [52] [56] [57]
Conclusions Burkholderia species are important opportunistic pathogens that have the ability to survive in many different environments, including susceptible human hosts. These bacteria possess large genomes and carry an enormous genetic potential for adaptation to many different environments. The availability of host models for infection, together with the exploitation of genomic sequence data and the development of new genetic tools will afford the possibility to better understand the biology of these opportunistic pathogens.
Acknowledgements We apologise to all researchers whose work has not been cited owing to the lack of space. This work was supported by grants from the Canadian Cystic Fibrosis Foundation and the Canadian Institutes of Health Research to MAV, and STC has been supported by a Fellowship from the Canadian Cystic Fibrosis Foundation. MAV holds a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis.
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