Medical Microbiology and Immunology (2019) 208:25–32 https://doi.org/10.1007/s00430-018-0571-0

REVIEW

Legionella feeleii: pneumonia or Pontiac fever? Bacterial virulence traits and host immune response

Changle Wang1 · Xia Chuai1 · Mei Liang2

Received: 17 July 2018 / Accepted: 27 October 2018 / Published online: 1 November 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Gram-negative bacterium Legionella is able to proliferate intracellularly in mammalian host cells and amoeba, which became known in 1976 since they caused a large outbreak of pneumonia. It had been reported that different strains of Legionella pneumophila, Legionella micdadei, Legionella longbeachae, and Legionella feeleii caused human respiratory diseases, which were known as Pontiac fever or Legionnaires’ disease. However, the differences of the virulence traits among the strains of the single species and the pathogenesis of the two diseases that were due to the bacterial virulence factors had not been well elucidated. L. feeleii is an important pathogenic organism in Legionellae, which attracted attention due to cause an outbreak of Pontiac fever in 1981 in Canada. In published researches, it has been found that L. feeleii serogroup 2 (ATCC 35849, LfLD) possess mono-polar flagellum, and L. feeleii serogroup 1 (ATCC 35072, WRLf) could secrete some exopolysaccharide (EPS) materials to the surrounding. Although the virulence of the L. feeleii strain was evidenced that could be promoted, the EPS might be dispensable for the bacteria that caused Pontiac fever. Based on the current knowledge, we focused on bacterial infection in human and murine host cells, intracellular growth, cytopathogenicity, stimulatory capacity of cytokines secre- tion, and pathogenic effects of the EPS ofL. feeleii in this review.

Keywords Legionella feeleii · Pontiac fever · Legionnaires’ disease · Flagellum · Exopolysaccharide · Bacterial infection

Introduction Legionella, they will cause respiratory diseases by the air- borne agent [4]. Legionellae are gram-negative intracellular pathogenic bac- teria that are capable of living ubiquitously in natural or artificial aquatic environments, such as rivers, lakes, streams, Intracellular growth and Legionella feeleii fountains, air conditioning cooling towers, humidifiers, and whirlpool spas. The organisms live and grow in water Legionella can form a commensal relationship with freshwa- systems at temperatures of 20–50 °C, with the optimum at ter or soil amoebae [5, 6]. The bacteria can be provided with 35 °C [1]. The exception of Legionella living in the natural a niche for multiplication, and the resistance to disadvanta- environment is Legionella longbeachae, which was primar- geous environmental factors can be promoted [7–9]. Differ- ily isolated from the soil [2]. Until now, more than 60 species ent species of Legionella exhibited proliferation capacity in of Legionella have been identified, and at least 20 of them mammalian macrophages and epithelial cells [10–14]. After have been associated with the diseases [3]. Once humans entering the cytoplasm of the eukaryotes, the bacteria estab- inhale aerosolized water droplets that are contaminated by lish Legionella-containing vacuoles (LCVs) to avoid fusion with lysosomes and replicate inside the LCVs. The organ- isms rupture the vacuole membrane and lyse the host cells * Changle Wang [15]. Neighboring cells can be infected with the released [email protected] bacteria for sequential infection [16, 17]. During prolifera- tion, different species of Legionella can even form different 1 Department of Pathogenic Biology, Hebei Medical intracellular colony morphologies in mammalian cells [18]. University, Shijiazhuang 050017, People’s Republic of China Legionella feeleii serogroup 1 (ATCC 35072, hereinaf- 2 School of Basic Medical Science, Hebei Medical University, ter referred to as LfPF) was reported for the first time in Shijiazhuang 050017, People’s Republic of China

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1984, since the bacteria caused an outbreak of Pontiac fever adherence to human epithelial cells [29, 30] and invasion in Canada [19]. The same serogroup bacterium was also of Acanthamoeba atronyxis [31]. A variety of non-flagellar reported that caused pneumonia in two immunosuppressed toxins or effectors and flagellar proteins were demonstrated patients [20]. In 1985 in America, L. feeleii serogroup 2 that could be delivered to the extracellular milieu by the type (ATCC 35849, hereinafter referred to as LfLD) was iso- III-like protein secretion apparatus [32, 33]. lated from a Legionnaires’ disease patient [21]. However, Most of the Legionella species are flagellated pathogenic the pathogenesis of L. feeleii that caused Pontiac fever or organisms [34]. In previous researches, it is identified that Legionnaires’ disease has not been elucidated. L. feeleii flaA was one of the virulence factors that could affect bacte- could proliferate in human macrophages and amoeba at rial virulence in Legionella [35–37]. The flaA mutant strain 37 °C environment [22]. The bacteria can maintain the com- significantly reduced the infectious capacity to the eukaryote position of the cellular fatty acid in a stable status during the cells [38]. LfLD was observed that showed smooth-waved stationary growth phase. Due to the differences of bacterial mono-polar flagellum at 25 and 30 °C; however, LfPF did physiological age, L. feeleii could not grow as rapidly as L. not possess the flagellum [14]. Heuner et al. had reported pneumophila [23]. This organism showed lethal ability to that Western blot analysis for FlaA was positive in L. fee- guinea pigs at the bacterial concentration of ­109/ml. How- leii ATCC 35849 (LfLD), but negative in L. feeleii ATCC ever, there were no death in guinea pigs after the bacteria 35072 (LfPF). They also found that L. feeleii ATCC 35849 were exposed to a series of disadvantageous factors, which (LfLD) was a flagellated bacterium by electron microscopy caused the bacterial toxic activity to be attenuated [24]. [39]. Based on these distinctions, LfLD was found that have enhanced invasion capacity to human epithelial cells and have promoted internalization by human and murine Characteristics and virulence traits macrophages (Fig. 1). While the key role of the flagellum of flagellum in Legionella feeleii for promoting bacterial entry to the host cells has been evi- denced in LfLD, the same flagellar morphology cannot be Flagellum is an appendage of the bacterium whose structure found after the bacteria were cultured between 25 °C and is mainly comprised of three parts, such as a basal body, a 37 °C [14]. The flagellum of L. pneumophila JR32 showed hook structure, and a filament [25, 26]. In the process of the same smooth-waved morphology at 25, 30, and 37 °C assembling the flagellum, a type III-like secretion system (Wang et al., unpublished data). It is hypothesized that there plays a key role in exporting the hook and the filament-form- may be some differences in flagellin arrangement of flagel- ing proteins out of the bacterial cells [27]. In general, the lar production of LfLD at higher temperature. Ott et al. had flagellum can help the bacterium to evade adverse environ- reported that the flagellum of L. pneumophila expressed in a mental conditions, mediate chemotaxis toward favorable and temperature-dependent manner [40]. Actually, the numbers rich nutrimental areas, and promote bacterial virulence [28]. of the flagellated LfLD and L. pneumophila JR32 were the The flagellum was evidenced that it was connected with bac- most at 25 °C of the bacterial culture, moderate at 30 °C, and terial virulence traits, since it could contribute to bacterial less at 37 °C under the electron microscope [14].

Fig. 1 Simple illustration of entry into cytoplasm, intracel- lular proliferation and stimu- lated cytokines secretion of Legionella feeleii LfPF (WRLf anf BTLf) and LFLD. In bacte- rial entry, both flagellum and exopolysaccharide (EPS) play an important role in promoting internalization of bacteria by the host cells. LfLD could induce a great amount of cytokines secretion (direct arrow), while LfPF induced less (indirect arrow). Whether L. feeleii could form Legionella-containing vacuole (LCV, grey color) and Utilize Type 4 Secretion Syatem (T4SS) for proliferation in the cytoplasm, and the pathway of inducing cytokines production should be demonstrated

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The virulence of the flagellum also affected cytopatho- [55]. Proinflammatory cytokines secretion is considered genicity and cytokines secretion, such as the flagellin which as a double-edged sword for the pulmonary cells and tis- were purified from two species of the motile wild type sue. It can contribute to playing a role in host defense when Legionella that triggered host cells death and induced IL-1β produced moderately [56]. Once excess of proinflammatory secretion, while the bacteria failed [41]. In L. pneumoph- cytokines are triggered to secrete, it may play as a patho- ila, the flaA mutant strain expressed deficiency in trigger- genic factor that could exacerbate the damage of the host ing murine bone marrow-derived macrophages death [42]. tissue and develop into pulmonary inflammation. In addi- Similar results were also found in L. feeleii. LfLD exhibited tion, the direct injury of the host pulmonary epithelial tissue stronger cytopathogenicity than LfPF, which killed approxi- that is caused by the intracellular microbes with stronger mately 70% or 93% of human or murine macrophages [14]. pathogenicity was also considered as one of the important The flagellin is the pathogenic protein that is composed pathogeneses of pneumonia. Similar to LfLD, the bacteria of external flagellar filament, which is coded by the gene exhibited stronger infection capacity, triggered more mam- of flaA [38]. It can be monitored by the innate immune sys- malian cells’ death and induced large amounts of cytokines tem of the host cells [43, 44]. L. pneumophila could uti- secretion, which caused more severe disease to humans lize the Type IV Secretion System (T4SS) to translocate (Fig. 2). Hence, according to the knowledge of the current the flagellin into the cytosol of the host cells, which caused researches, human pneumonia may be due to two factors, inflammasome activation 45[ , 46]. Bacterial flagellin can be one is the bacterial virulence and the other is the massive recognized by NLRC4 or NAIP5 receptors, and triggered cytokines secretion. a series of immune cascade reactions, such as IL-1β and IL-18 inflammatory cytokines secretion and pyroptosis of the murine bone marrow-derived macrophages [47–50]. This Exopolysaccharide materials pathogenic protein was also reported that could be ligated to TLR5, which followed by the TLR5-MyD88-NF-κB Gram-negative bacteria are able to secrete exopolysaccha- pathway activation to induce IL-6 and IL-8 production ride (EPS), which had been attributed to promote nutrients in human macrophages or epithelial cells [51–53]. While acquisition, facilitate attachment to surfaces, and provide the human cells that were infected with LfLD induced a protection against detrimental environment [57]. Extracel- great amount of IL-6 and IL-8 cytokines secretion [14], the lular polysaccharide secretion (capsule or slime) is a com- pathway of cytokines production is necessary to elucidate mon phenomenon for many bacteria [58]. The materials (Fig. 1). Based on the immune response of the host cells could not only play a role in altering the transparencies and to the flagellin, it gives a potent support that the flagellum morphologies of the colonies, but also enhance the bacte- played an important role in accelerating the inflammatory rial pathogenicity, thus promoting the resistance to clearance reaction when infected with LfLD. Although the bacterial from mice blood in Vibrio vulnificus [59], and causing high virulence was significantly promoted by the flagellum, both mortality and morbidity in cystic fibrosis in Pseudomonas LfPF and LfLD showed stronger intracellular growth capac- aeruginosa [60]. ity in mammalian cells, which suggested that the flagellum L. feeleii ATCC 35072 (LfPF) was found to secrete some of L. feeleii could not affect the organism proliferation in EPS-like materials, which determined the colony variation the cytosol of the host cells (Fig. 1). Although LfLD and L. [61]. Two types of colonies were isolated and they showed pneumophila JR32 are both flagellated bacteria, LfLD exhib- differences in color, transparency, and morphology. One type ited stronger internalization by the host cells than L. pneu- is white color and rugose morphology, the other is brown mophila JR32 at the initial bacterial infection [14]. There and translucent. The two kinds of colonies were compared might be some other potent virulence factors that facilitate with another L. feeleii strain (ATCC 35849, LfLD), and the entry of LfLD without sharing with L. pneumophila, and found that the white rugose one is the newly formed type. the detailed processes should be elucidated in a further way. Both of the colonies were examined under a transmission electron microscope. Ruthenium red-stained materials were observed around the white rugose L. feeleii (hereinafter Bacterial infection and host defense referred to as WRLf), while they were absent in the brown translucent one (hereinafter referred to as BTLf). WRLf The innate immune system plays a critical role in monitor- and BTLf showed different growth capacities in Buffer ing bacterial entry and infection, and it is also important for Yeast Extract (BYE) broth in vitro, and WRLf was found to acquired immunity [54]. Human pulmonary macrophages transform to BTLf, which showed different percentages of and epithelial cells are considered as the front defenders to transformation in different growth phases [61]. Although the recognize the pathogen, induce the cytokines secretion, and transformation of WRLf to BTLf also could be found during trigger a series of immune response to clear the intruders the intracellular growth [61], the reverse (BTLf transformed

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Fig. 2 Schematic diagram of the virulence traits of Legionella feeleii LfPF and LfLD. Accord- ing to the current knowledge, grey color arrows represent the hypothesis of the pathogenesis of L. feeleii that causes pontiac fever or Legionnaires’ disease. However, the pathogenicity of exopolysaccharide (EPS) is dispensable for contributing Pontiac fever (indirect arrows)

to WRLf) took place unexpectedly, which was found occa- latent period and illness duration, without pneumonia, and sionally after infecting amoebic cells (Wang et al., unpub- the fatality is zero [62]. L. pneumophila [63], Legionella lished data). micdadei [64], Legionella anisa [65], L. feeleii [19], L. A series of assays were performed to investigate the longbeachae [66] and Legionella maceachernii [67] were effects of the EPS on the virulence traits between WRLf reported that caused human Pontiac fever (Table 1). While and BTLf. The material was evidenced that played a role in the process of the illness expresses acutely and has a high resistance to human serum bactericidal action and enhanc- attack rate, until now, there is no clinical report that the ing the internalization by human macrophages and epithe- causative Legionella has been isolated from the patients lial cells (Fig. 1) [61]. The pathogenicity of the EPS was [68]. Pontiac fever may be caused by bacterial toxin or also explored in animal experiments in vivo. ddY mice were robust immune response after infection with L. pneumophila intranasally inoculated with WRLf and BTLf bacterial sus- [69]. In case of L. anisa, it is considered that the bacterial pension, and the animals were sacrificed after 24 and 48 h deficiency of proliferation in human phagocytic cells may post-infection. However, both of the numbers of the colo- be consistent with the developmental process of Pontiac nies that derived from WRLf and BTLf-infected lung tis- fever [65]. However, based on the current knowledge, LfPF sue were reduced (Wang et al., unpublished data). These exhibited a different manner. The virulence of this organism results may indicate that the pathogenicity of the EPS is not was not as strong as L. pneumophila, but it could multiply strong enough to cause the ddY mice disease or death. To in human host cells [14]. Although LfPF showed a novel be an infection model of humans, guinea pigs were infected colony phenotype, both WRLf and BTLf expressed strong with WRLf and BTLf, and the bacteria showed different proliferation capacities in mammalian cells in vitro [61]. pyrogenic effects on the animals [61]. It is the first report Therefore, the pathogenesis of Pontiac fever caused by LfPF that elucidated the EPS material caused the variant colonies was hypothesized that might be attributed to the intracellu- types, and the colonies had different pyrogenic effects to lar growth in human pulmonary macrophages and epithelial guinea pigs in Legionella. cells (Fig. 2). Actually, the EPS was also assumed that may contribute to cause Pontiac fever. However, the guinea pigs infected with WRLf or BTLf were all febrile [61], which Association of legionellosis indicated that the EPS might be dispensable in causing the and the virulence factors of Legionella feeleii disease (Fig. 2). LfPF did not possess flagellum, which may suggest that this organism could not cause severe disease to Legionellosis is caused by the infection of Legionellae, humans as LfLD, since the pathogenicity of LfPF is weak. which majorly contains two different clinical respiratory On the other hand, Legionnaires’ disease is a lethal diseases. One is a mild, self-limiting flu-like disease, termed community-acquired pneumonia (CAP), which caused as Pontiac fever. The other is a severe atypical pneumonia, the first large outbreak among members of the American termed as Legionnaires’ disease. Pontiac fever has short Legion in 1976 [70]. The fatality rate can be increased

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Table 1 Pontiac fever and Legionnaires’ disease

Clinical type Disease Agent [references] Incubation Fatality (%) Attack rate (%)

Non-pneumonia Pontiac fever L. pneumophila [63] 4–60 h (usually 0 − 95 L. micdadei [64] 36–48 h) L. anisa [65] L. feeleii [19] L. longbeachae [66] L. maceachernii [67] Pneumonia Legionnaires’ disease L. pneumophila [72] 2–10 days 10–27 − 30 L. longbeachae [72] L. micdadei [72] L. bozemanii [72] L. feeleii [72] L. anisa [65]

from 10 to 27%, if appropriate therapy is not carried out Conclusions and perspectives in time [71]. The mainly causative agent of Legionnaires’ disease is L. pneumophila, which occupied more than This is the first review that reported the virulence traits of 90% of all reported cases, followed by L. longbeachae, L. feeleii, which was based on the current researches. L. Legionella bozemanii, L. micdadei, Legionella dumoffii, feeleii can be considered as one of the major pathogenic L. feeleii, Legionella wadsworthii and L. anisa [72, 73]. organisms in Legionellae, which was implicated in human The outbreak of Legionnaires’ disease happened in New Pontiac fever and Legionnaires’ disease. However, the York City in 2015, which caused 138 patients to be iden- researches that focused on elucidation and analysis of the tified with the illness and 16 of them died [74]. Legion- pathogenic characteristics of these bacteria are very less. naires’ disease was mentioned that cannot be transmitted In our previous studies, we investigated the virulence traits from person to person [75]. However, in Portugal in 2016, that were due to the absence or presence of the flagellum human-to-human transmission of Legionnaires’ disease in LfPF and LfLD, and the pathogenicity of the EPS in was potent evidenced for the first time [76]. L. feeleii was LfPF. Although L. feeleii strains available were limited, also reported that caused human lethal pneumonia [77]. LfPF and LfLD are the typical strains that investigate the Actually, LfLD was considered that may secret some pathogenicity of non-L. pneumophila, which belong to the proteases, which could break the pulmonary tissue of the same Legionella species that caused different legionello- host and cause pneumonia. Therefore, LfPF, LfLD, and sis. More L. feeleii strains should be collected, and both L. pneumophila JR32 were cultivated on 1% skim milk wild type and mutant strains should be examined in animal agar plates and placed at 37 °C. The contents of the skim experiments to investigate the pathogenicity of the viru- milk agar plates were based on buffered charcoal yeast lence factors in vivo in a further way. To detect whether extract (BCYE) agar, and the charcoal was substituted with L. feeleii could form LCV and utilize T4SS to prolifer- the skim milk. However, except L. pneumophila JR32, ate in the cytoplasm of the host cells are also necessary both LfPF and LfLD could not catalyze the degradation (Fig. 1). Taken together, it should continue to make pro- of the proteins in the milk, which indicated that the bac- gress in analyzing the virulence factors that contribute to teria may have some deficiency in secreting exoprotease cause the diseases in the infection of L. feeleii in future (Wang et al., unpublished data). Some Legionella spe- study, and the pathogenesis of legionellosis still needs to cies have been reported that exhibited the protease and be elucidated. hemolysin activities. A 38 kDa exoprotease was purified from L. pneumophila, which played a role in proteolytic, Acknowledgements We are deeply indebted Dr. Shin-ichi Yoshida, hemolytic and cytotoxic activities, but negative in L. fee- Dr. Mitsumasa Saito, Dr. Kazunobu Amako, Hideko Kameyama for the leii kind guidance and help, scientific discussion and technical assistance strains [78]. Based on the current knowledge, although of previous studies. We also want to thank all the members for the the intracellular proliferation was hypothesized that may kind support when the first author studied at the Department of Bac- contribute to cause disease to humans in L. feeleii, as a teriology, Kyushu University, Japan as a Ph.D. candidate. This work potent important virulence factor, it seems that the flagel- was supported by a Grant from Natural Science Foundation of Hebei Province, China (No. H2018206259). lum may play an important role in making the disease more severe (Fig. 2).

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References 18. Ogawa M, Takade A, Miyamoto H, Taniguchi H, Yoshida S (2001) Morphological variety of intracellular microcolonies of Legionella species in Vero cells. Microbiol Immunol 45(7):557– 1. Newton HJ, Ang DK, van Driel IR, Hartland EL (2010) Molecu- 562. https​://doi.org/10.1111/j.1348-0421.2001.tb026​58.x lar pathogenesis of infections caused by Legionella pneumoph- 19. Herwaldt LA, Gorman GW, McGrath T, Toma S, Brake B, High- ila. Clin Microbiol Rev 23(2):274–298. https://doi.org/10.1128/​ tower AW, Jones J, Reingold AL, Boxer PA, Tang PW et al (1984) CMR.00052​-09 A new Legionella species, Legionella feeleii species nova, causes 2. Steele TW, Moore CV, Sangster N (1990) Distribution of Pontiac fever in an automobile plant. Ann Intern Med 100(3):333– Legionella longbeachae serogroup 1 and other Legionel- 338. https​://doi.org/10.7326/0003-4819-100-3-333 lae in potting soils in Australia. Appl Environ Microbiol 20. Palutke WA, Crane LR, Wentworth BB, Geiger JG, Cardozo L, 56(10):2984–2988 Singhakowinta A, Bartley J, Robinson BE (1986) Legionella 3. Appelt S, Heuner K (2017) The flagellar regulon of feeleii-associated pneumonia in humans. Am J Clin Pathol Legionella—a review. Front Cell Infect Microbiol 7:454. https​ 86(3):348–351 ://doi.org/10.3389/fcimb​.2017.00454​ 21. Thacker WL, Wilkinson HW, Plikaytis BB, Steigerwalt AG, May- 4. Fields BS, Benseton RF, Besser RE (2002) Legionella and berry WR, Moss CW, Brenner DJ (1985) Second serogroup of Legionnaires’ disease: 25 years of investigation. Clin Microbiol Legionella feeleii strains isolated from humans. J Clin Microbiol Rev 15(3):506–526 22(1):1–4 5. Skaliy P, McEachern HV (1979) Survival of the Legionnaires’ 22. Moffat JF, Tompkins LS (1992) A quantitative model of intracel- disease bacterium in water. Ann Intern Med 90(4):662–663 lular growth of Legionella pneumophila in Acanthamoeba castel- 6. Rowbotham TJ (1980) Preliminary report on the pathogenicity lanii. Infect Immun 60(1):296–301 of Legionella pneumophila for freshwater and soil amoebae. J 23. Moss CW, Bibb WF, Karr DE, Guerrant GO, Lambert MA Clin Pathol 33(12):1179–1183 (1983) Cellular fatty acid composition and ubiquinone content of 7. Anand CM, Skinner AR, Malic A, Kurtz JB (1983) Interaction Legionella feeleii sp. nov. J Clin Microbiol 18(4):917–919 of L. pneumophilia and a free living amoeba (Acanthamoeba 24. Fields BS, Barbaree JM, Shotts EB Jr, Feeley JC, Morrill WE, palestinensis). J Hyg (Lond) 91(2):167–178 Sanden GN, Dykstra MJ (1986) Comparison of guinea pig and 8. King CH, Shotts EB Jr, Wooley RE, Porter KG (1988) Survival protozoan models for determining virulence of Legionella species. of coliforms and bacterial pathogens within protozoa during Infect Immun 53(3):553–559 chlorination. Appl Environ Microbiol 54(12):3023–3033 25. Macnab RM (2003) How bacteria assemble flagella. Annu Rev 9. Barker J, Brown MR, Collier PJ, Farrell I, Gilbert P (1992) Rela- Microbiol 57:77–100. https​://doi.org/10.1146/annur​ev.micro​ tionship between Legionella pneumophila and Acanthamoeba .57.03050​2.09083​2 polyphaga: physiological status and susceptibility to chemical 26. Fujii T, Kato T, Hiraoka KD, Miyata T, Minamino T, Chevance inactivation. Appl Environ Microbiol 58(12):2420–2425 FF, Hughes KT, Namba K (2017) Identical folds used for distinct 10. Maruta K, Miyamoto H, Hamada T, Ogawa M, Taniguchi H, mechanical functions of the bacterial flagellar rod and hook. Nat Yoshida S (1988) Entry and intracellular growth of Legionella Commun 8:14276. https​://doi.org/10.1038/ncomm​s1427​6 dumoffii in alveolar epithelial cells. Am J Respir Crit Care 27. Aldridge P, Hughes KT (2002) Regulation of flagellar assembly. Med 157(6 Pt 1):1967–1974. https​://doi.org/10.1164/ajrcc​ Curr Opin Microbiol 5(2):160–165. https://doi.org/10.1016/S1369​ ​ m.157.6.97101​08 -5274(02)00302​-8 11. Alli OA, Zink S, von Lackum NK, Abu-Kwaik Y (2003) 28. Molofsky AB, Shetron-Rama LM, Swanson MS (2005) Compo- Comparative assessment of virulence traits in Legionella spp. nents of the Legionella pneumophila flagellar regulon contribute Microbiology 149(Pt 3):631–641. https​://doi.org/10.1099/ to multiple virulence traits, including lysosome avoidance and mic.0.25980​-0 macrophage death. Infect Immun 73(9):5720–5734. https​://doi. 12. Ohnishi H, Mizunoe Y, Takade A, Tanaka Y, Miyamoto H, org/10.1128/IAI.73.9.5720-5734.2005 Harada M, Yoshida S (2004) Legionella dumoffii DjlA, a mem- 29. Giron JA, Torres AG, Freer E, Kaper JB (2002) The flagella of ber of the DnaJ family, is required for intracellular growth. enteropathogenic Escherichia coli mediate adherence to epithe- Infect Immun 72(6):3592–3603. https​://doi.org/10.1128/ lial cells. Mol Microbiol 44(2):361–379. https​://doi.org/10.104 IAI.72.6.3592-3603.2004 6/j.1365-2958.2002.02899​.x 13. Takekawa Y, Saito M, Wang C, Qin T, Ogawa M, Kanemaru T, 30. Tomich M, Herfst CA, Golden JW, Mohr CD (2002) Role Yoshida S (2012) Characteristic morphology of intracellular of flagella in host cell invasion by Burkholderia cepacia. microcolonies of Legionella oakridgensis OR-10. Can J Micro- Infect Immun 70(4):1799–1806. https​://doi.org/10.1128/ biol 58(2):179–183. https​://doi.org/10.1139/w11-126 IAI.70.4.1799-1806.2002 14. Wang C, Saito M, Tanaka T, Amako K, Yoshida S (2015) Com- 31. Inglis TJ, Robertson T, Woods DE, Dutton N, Chang BJ parative analysis of virulence traits between a Legionella fee- (2003) Flagellum-mediated adhesion by Burkholderia pseu- leii strain implicated in Pontiac Fever and a strain that caused domallei precedes invasion of Acanthamoeba astronyxis. Legionnaires’ disease. Microb Pathog 89:79–86. https​://doi. Infect Immun 71(4):2280–2282. https​://doi.org/10.1128/ org/10.1016/j.micpa​th.2015.09.004 IAI.71.4.2280-2282.2003 15. Isberg RR, O’Connor TJ, Heidtman M (2009) The Legionella 32. Young GM, Schmiel DH, Miller VL (1999) A new pathway pneumophila replication vacuole: making a cosy niche for the secretion of virulence factors by bacteria: the flagellar inside host cells. Nat Rev Microbiol 7(1):13–24. https​://doi. export apparatus functions as a protein-secretion system. Proc org/10.1038/nrmic​ro196​7 Natl Acad Sci USA 96(11):6456–6461. https​://doi.org/10.1073/ 16. Vogel JP, Isberg RR (1999) Cell biology of Legionella pnas.96.11.6456 pneumophila. Curr Opin Microbiol 2(1):30–34. https​://doi. 33. Ghelardi E, Celandroni F, Salvetti S, Beecher DJ, Gominet org/10.1016/S1369​-5274(99)80005​-8 M, Lereclus D, Wong AC, Senesi S (2002) Requirement of 17. Hilbi H, Segal G, Shuman HA (2001) Icm/dot-dependent flhA for swarming differentiation, flagellin export, and secre- upregulation of phagocytosis by Legionella pneumoph- tion of virulence-associated proteins in Bacillus thuringien- ila. Mol Microbiol 42(3):603–617. https​://doi.org/10.104 sis. J Bacteriol 184(23):6424–6433. https​://doi.org/10.1128/ 6/j.1365-2958.2001.02645​.x JB.184.23.6424-6433.2002

1 3 Medical Microbiology and Immunology (2019) 208:25–32 31

34. Bornstein N, Marmet D, Dumaine MH, Surgot M, Fleurette J bacterial ligands in mice. J Exp Med 213(5):647–656. https​:// (1991) Detection of flagella in 278 Legionella strains by latex doi.org/10.1084/jem.20160​006 reagent sensitized with antiflagellum immunoglobulins. J Clin 51. Hawn TR, Verbon A, Lettinga KD, Zhao LP, Li SS, Laws RJ, Mirobiol 29(5):953–956 Skerrett SJ, Beutler B, Schroeder L, Nachman A, Ozinsky A, 35. Dietrich C, Heuner K, Brand BC, Hacker J, Steinert M Smith KD, Aderem A (2003) A common dominant TLR5 stop (2001) Flagellum of Legionella pneumophila positively codon polymorphism abolishes flagellin signaling and is asso- affects the early phase of infection of eukaryotic host cells. ciated with susceptibility to Legionnaires’ disease. J Exp Med Infect Immun 69(4):2116–2122. https​://doi.org/10.1128/ 198(10):1563–1572. https​://doi.org/10.1084/jem.20031​220 IAI.69.4.2116-2122.2001 52. Schmeck B, N’Guessan PD, Ollomang M, Lorenz J, Zahlten J, 36. Silveira TN, Zamboni DS (2010) Pore Formation triggered by Opitz B, Flieger A, Suttorp N, Hippenstiel S (2007) Legionella Legionella spp. is an Nlrc4 inflammasome-dependent host cell pneumophila-induced NF-kappaB- and MAPK-dependent response that precedes pyroptosis. Infect Immun 78(3):1403– cytokine release by lung epithelial cells. Eur Respir J 29(1):25–33. 1413. https​://doi.org/10.1128/IAI.00905​-09 https​://doi.org/10.1183/09031​936.00141​005 37. Pereira MS, Marques GG, Dellama JE, Zamboni DS (2011) The 53. Lorenz J, Zahlten J, Pollok I, Lippmann J, Scharf S, N’Guessan Nlrc4 inflammasome contributes to restriction of pulmonary PD, Opitz B, Flieger A, Suttorp N, Hippenstiel S, Schmeck B infection by flagellated Legionella spp. that trigger pyroptosis. (2011) Legionella pneumophila-induced IκBζ-dependent expres- Front Microbiol 2:33. https​://doi.org/10.3389/fmicb​.2011.00033​ sion of interleukin-6 in lung epithelium. Eur Respir J 37(3):648– 38. Heuner K, Steinert M (2003) The flagellum of Legionella 657. https​://doi.org/10.1183/09031​936.00200​009 pneumophila and its link to the expression of the virulent phe- 54. Takeuchi O, Akira S (2010) Pattern recognition receptors and notype. Int J Med Microbiol 293(2–3):133–143. https​://doi. inflammation. Cell 140(6):805–820. https​://doi.org/10.1016/j. org/10.1078/1438-4221-00259​ cell.2010.01.022 39. Heuner K, Bender-Beck L, Brand BC, Lück PC, Mann KH, Marre 55. Medzhitov R (2007) Recognition of microorganisms and activa- R, Ott M, Hacker J (1995) Cloning and genetic characterization tion of the immune response. Nature 449(7164):819–826. https://​ of the flagellum subunit gene (flaA) of Legionella pneumophila doi.org/10.1038/natur​e0624​6 serogroup 1. Infect Immun 63(7):2499–2507 56. Eisele NA, Anderson DM (2011) Host defense and the airway epi- 40. Ott M, Messner P, Heesemann J, Marre R, Hacker J (1991) Tem- thelium: frontline responses that protect against bacterial invasion perature-dependent expression of flagella in Legionella. J Gen and pneumonia. J Pathog. https​://doi.org/10.4061/2011/24980​2 Microbiol 137(8):1955–1961. https​://doi.org/10.1099/00221​ 57. Wai SN, Mizunoe Y, Takade A, Kawabata SI, Yoshida SI (1998) 287-137-8-1955 Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide 41. Whitfield NN, Byrne BG, Swanson MS (2010) Mouse mac- materials that determine colony morphology, stress resistance, and rophages are permissive to motile Legionella species that fail biofilm formation. Appl Environ Microbiol 64(10):3648–3655 to trigger pyroptosis. Infect Immun 78(1):423–432. https​://doi. 58. Torres-Cabassa A, Gottesman S, Frederick RD, Dolph PJ, Cop- org/10.1128/IAI.00070​-09 lin DL (1987) Control of extracellular polysaccharide synthe- 42. Case CL, Shin S, Roy CR (2009) Asc and Ipaf inflammasomes sis in Erwinia stewartii and Escherichia coli K-12: a common direct distinct pathways for Caspase-1 activation in response to regulatory function. J Bacteriol 169(10):4525–4531. https​://doi. Legionella pneumophila. Infect Immun 77(5):1981–1991. https://​ org/10.1128/jb.169.10.4525-4531.1987 doi.org/10.1128/IAI.01382​-08 59. Yoshida S, Ogawa M, Mizuguchi Y (1985) Relation of capsular 43. Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognition and materials and colony opacity to virulence of Vibrio vulnificus. innate immunity. Cell 124(4):783–801. https://doi.org/10.1016/j.​ Infect Immun 47(2):446–451 cell.2006.02.015 60. Martin DW, Schurr MJ, Mudd MH, Govan JR, Holloway BW, 44. Mariathasan S, Monack DM (2007) Inflammasome adaptors and Deretic V (1993) Mechanism of conversion to mucoidy in Pseu- sensors: intracellular regulators of infection and inflammation. Nat domonas aeruginosa infecting cystic fibrosis patients. Proc Rev Immunol 7(1):31–40. https​://doi.org/10.1038/nri19​97 Natl Acad Sci USA 90(18):8377–8381. https​://doi.org/10.1073/ 45. Molofsky AB, Byrne BG, Whitfield NN, Madigan CA, Fuse ET, pnas.90.18.8377 Tateda K, Swanson MS (2006) Cytosolic recognition of flagellin 61. Wang C, Saito M, Ogawa M, Yoshida S (2016) Colony types and by mouse macrophages restricts Legionella pneumophila infec- virulence traits of Legionella feeleii determined by exopolysac- tion. J Exp Med 203(4):1093–1104. https​://doi.org/10.1084/ chride materials. FEMS Microbiol Lett 363(10):fnw098. https​:// jem.20051​659 doi.org/10.1093/femsl​e/fnw09​8 46. Casson CN, Copenhaver AM, Zwack EE, Nguyen HT, Strowig T, 62. Edelstein PH (2007) Urine antigen tests positive for Pontiac Javdan B, Bradley WP, Fung TC, Flavell RA, Brodsky IE, Shin fever: implications for diagnosis and pathogenesis. Clin Infect S (2013) Caspase-11 activation in response to bacterial secretion Dis 44(2):229–231. https​://doi.org/10.1086/51039​4 systems that access the host cytosol. PLoS Pathog 9(6):e1003400. 63. Mangione EJ, Remis RS, Tait KA, Gee HB, Gorman GW, Went- https​://doi.org/10.1371/journ​al.ppat.10034​00 worth BB, Baron PA, Hightower AW, Barbaree JM, Broome CV 47. Lightfield KL, Persson J, Trinidad NJ, Brubaker SW, Kofoed (1985) An outbreak of Pontiac fever related to whirlpool use, EM, Sauer JD, Dunipace EA, Warren SE, Miao EA, Vance RE Michigan 1982. JAMA 253(4):535–539. https://doi.org/10.1001/​ (2011) Differential requirements for NAIP5 in activation of the jama.1985.03350​28009​1026 NLRC4 inflammasome. Infect Immun 79(4):1606–1614. https​:// 64. Fallon RJ, Rowbotham TJ (1990) Microbiological investigations doi.org/10.1128/IAI.01187​-10 into an outbreak of Pontiac fever due to Legionella micdadei asso- 48. Franchi L, Muñoz-Planillo R, Núñez G (2012) Sensing and ciated with use of a whirlpool. J Clin Pathol 43(6):479–483. https​ reacting to microbes through the inflammasomes. Nat Immunol ://doi.org/10.1136/jcp.43.6.479 13(4):325–332. https​://doi.org/10.1038/ni.2231 65. Fields BS, Barbaree JM, Sanden GN, Morrill WE (1990) Viru- 49. Zhao Y, Shao F (2016) Diverse mechanisms for inflammasome lence of a Legionella anisa strain associated with Pontiac fever: an sensing of cytosolic bacteria and bacterial virulence. Curr Opin evaluation using protozoan, cell culture, and guinea pig models. Microbiol 29:37–42. https​://doi.org/10.1016/j.mib.2015.10.003 Infect Immun 58(9):3139–3142 50. Zhao Y, Shi J, Shi X, Wang Y, Wang F, Shao F (2016) Genetic 66. Cramp GJ, Harte D, Douglas NM, Graham F, Schousboe M, functions of the NAIP family of inflammasome receptors for Sykes K (2010) An outbreak of Pontiac fever due to Legionella

1 3 32 Medical Microbiology and Immunology (2019) 208:25–32

longbeachae serogroup 2 found in potting mix in a horticultural A (2002) Distribution of Legionella species and serogroups iso- nursery in New Zealand. Epidemiol Infect 138(1):15–20. https​:// lated by culture in patients with sporadic community-acquired doi.org/10.1017/S0950​26880​99908​35 legionellosis: an international collaborative survey. J Infect Dis 67. Huhn GD, Adam B, Ruden R, Hilliard L, Kirkpatrick P, Todd 186(1):127–128. https​://doi.org/10.1086/34108​7 J, Crafts W, Passaro D, Dworkin MS (2005) Outbreak of travel- 74. Weiss D, Boyd C, Rakeman JL, Greene SK, Fitzhenry R, McProud related pontiac fever among hotel guests illustrating the need for T, Musser K, Huang L, Kornblum J, Nazarian EJ, Fine AD, better diagnostic tests. J Travel Med 12(4):173–179. https​://doi. Braunstein SL, Kass D, Landman K, Lapierre P, Hughes S, Tran org/10.2310/7060.2005.12401​ A, Taylor J, Baker D, Jones L, Kornstein L, Liu B, Perez R, Luc- 68. Diederen BM (2008) Legionella spp. and Legionnaires’ disease. ero DE, Peterson E, Benowitz I, Lee KF, Ngai S, Stripling M, J Infect 56(1):1–12. https​://doi.org/10.1016/j.jinf.2007.09.010 Varma JK (2017) A large community outbreak of Legionnaires’ 69. Swanson MS, Hammer BK (2000) Legionella pneumophila patho- disease associated with a cooling tower in New York City, 2015. genesis: a fateful journey from amoebae to macrophages. Annu Public Health Rep 132(2):241–250. https://doi.org/10.1177/00333​ ​ Rev Microbiol 54:567–613. https://doi.org/10.1146/annur​ ev.micro​ ​ 54916​68962​0 .54.1.567 75. Personnic N, Striednig B, Hilbi H (2017) Legionella quorum sens- 70. Fraser DW, Tsai TR, Orenstein W, Parkin WE, Beecham HJ, Shar- ing and its role in pathogen–host interactions. Curr Opin Micro- rar RG, Harris J, Mallison GF, Martin SM, McDade JE, Shepard biol 41:29–35. https​://doi.org/10.1016/j.mib.2017.11.010 CC, Brachman PS (1977) Legionnaires’ disease: description of an 76. Correia AM, Ferreira JS, Borges V, Nunes A, Gomes B, Capucho epidemic of pneumonia. N Engl J Med 297(22):1189–1197. https​ R, Gonçalves J, Antunes DM, Almeida S, Mendes A, Guerreiro ://doi.org/10.1056/NEJM1​97712​01297​2201 M, Sampaio DA, Vieira L, Machado J, Simões MJ, Gonçalves 71. Fiumefreddo R, Zaborsky R, Haeuptle J, Christ-Crain M, Tram- P, Gomes JP (2016) Probable person-to-person transmission of puz A, Steffen I, Frei R, Müller B, Schuetz P (2009) Clinical Legionnaires’ disease. N Engl J Med 374(5):497–498. https://doi.​ predictors for Legionella in patients presenting with community- org/10.1056/NEJMc​15053​56 acquired pneumonia to the emergency department. BMC Pulm 77. Lee J, Caplivski D, Wu M, Huprikar S (2009) Pneumonia Med 9:4. https​://doi.org/10.1186/1471-2466-9-4 due to Legionella feeleii: case report and review of the litera- 72. Muder RR, Yu VL (2002) Infection due to Legionella species ture. Transpl Infect Dis 11(4):337–340. https​://doi.org/10.111 other than L. pneumophila. Clin Infect Dis 35(8):990–998. https​ 1/j.1399-3062.2009.00390​.x ://doi.org/10.1086/34288​4 78. Keen MG, Hoffman PS (1989) Characterization of a Legionella 73. Yu VL, Plouffe JF, Pastoris MC, Stout JE, Schousboe M, Widmer pneumophila extracellular protease exhibiting hemolytic and cyto- A, Summersgill J, File T, Heath CM, Paterson DL, Chereshsky toxic activities. Infect Immun 57(3):732–738

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