, Jan. 2005, p. 20–28 Vol. 71, No. 10099-2240/05/$08.00
0 doi:10.1128/AEM.71.1.20–28.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Amoebae as Training Grounds for Intracellular Bacterial Pathogens
and Yousef Abu Kwaik
Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky
; Division of Microbial Ecology, Institute of Ecology and Conservation Biology, University of Vienna, Vienna, Austria
; and Department of Microbiology and Parasitology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
Free-living amoebae are important predators that controlmicrobial communities. They are ubiquitous and have beenisolated from various natural sources such as soil, freshwater,salt water, dust, and air. Although their abundance in soil isonly limited, they have been implicated in the stimulation of phosphorus and nitrogen turnover and thus play an importantrole in soil ecosystems (84). Free-living amoebae are also fre-quently isolated from anthropogenic ecosystems such as tap water, air-conditioning units, and cooling towers, feeding onthe microbial bioﬁlm present in those systems. However, sev-eral bacteria have developed mechanisms to survive phagocy-tosis by free-living amoebae and are able to exploit them ashosts.Transient association with amoebae have been reported fora number of different bacteria including
Legionella pneumo- phila
O157, among others (3, 21, 77, 85). As most of these bacteria are pathogens of humans, amoebae have beensuggested to represent their environmental reservoirs, actingas “Trojan horses” of the microbial world (10). To date, onlythe interaction of
with free-living amoebae hasbeen studied in greater detail.
is an intracel-lular pathogen of humans causing Legionnaires’ disease.
multiplies within its host cell (human macro-phages and amoebae) but can also grow outside its host celland can be cultured by routine methodologies (for reviews, seereferences 19 and 46). Therefore, it is considered as a faculta-tive intracellular pathogen.In addition to those facultative intracellular bacteria, variousobligate endosymbionts have been observed in free-livingamoebae. In this context, it should be noted that the termendosymbiont implies neither a mutualistic nor a parasitic re-lationship between the bacteria and their host cells. Accordingto the broad concept of symbiosis suggested originally by An-ton de Bary (30a) and consistent with the recent awareness of the variability of symbiotic interactions (81), we consider bac-teria as endosymbionts when they manage to establish theirreplicative niche within eukaryotic host cells. Approximately20% of isolates of ubiquitous
spp. recoveredfrom clinical and environmental sources are found to harborsuch bacterial endosymbionts (37). These endosymbionts havebeen shown to maintain a stable interaction with their hosts(37), but in contrast to
, they cannot be culturedoutside their host cells, a primary reason why their identiﬁca-tion and analysis have been hampered.This review examines the impact of the interaction betweenbacteria and amoebae on the evolution of intracellular bacte-rial pathogens of humans (i.e., bacteria capable of causingdisease) by focusing on recent ﬁndings on the stable associa-tion of free-living amoebae with their obligate endosymbiontsand the transient interaction between
andamoebae as a model system.
DIVERSITY OF BACTERIAL SYMBIONTS OF FREE-LIVING AMOEBAE
Bacterial endosymbionts of amoebae were microscopicallyobserved at least three decades ago (83). They have beenshown to maintain a stable interaction with their hosts and areable to outlast encystations of the amoebae (37). However,identiﬁcation and analysis of these endosymbionts have beenseverely hampered by the fact that they cannot be culturedoutside their eukaryotic host cells. Only the advent of molec-ular biological techniques and the establishment of an rRNA-based phylogenetic system for the classiﬁcation of prokaryotesenabled a more detailed characterization of these elusive bac-teria (8). During the past few years, speciﬁc PCR-mediatedampliﬁcation of bacterial rRNA genes, comparative sequenceanalysis, and ﬂuorescence in situ hybridization led to the iden-tiﬁcation of ﬁve novel evolutionary lineages of bacterial endo-symbionts of free-living amoebae (9, 16, 38, 39, 53–55, 57).These lineages have been found to be afﬁliated with (i) the
, (ii) the
, (iii) the
, (iv) the
, and (v) the
(Fig. 1). Interestingly, co-occurrence of phylogenetically differ-ent endosymbionts in a single amoebal isolate has never beenobserved, and signiﬁcant differences regarding the host rangehave been shown for the different endosymbionts (55). Apartfrom one exception (
), spon-taneous loss of endosymbionts from the original amoeba hostor successful eradication of the symbionts from their hosts byantibiotics has never been reported, conﬁrming the stable en-dosymbiosis. Consistent with this ﬁnding, evidence for coevo-lution between endosymbionts and hosts has been reported forone endosymbiont phylogenetic lineage (14). The phylum-level
* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, University of Louisville, Louisville, KY 40202.Phone: (502) 852-4117. Fax: (502) 852-7531. E-mail: firstname.lastname@example.org