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Applied and Environmental Microbiology, July 2003, p . 4268-4271, Vol . 69, No . 7 Complement Resistance Is Essential for Colonization of the Digestive Tract of Hirudo medicinalis by Aeromonas StrainsThomas R . Braschler,1 Susana Merino,2 Juan M . Tomás,2 and Joerg Graf1,3* Institute for Infectious Diseases, University of Berne, CH-3010 Berne, Switzerland,1 Departamento Microbiología, Facultad Biología, Universidad Barcelona, 08071 Barcelona, Spain,2 Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 062693 Received 19 September 2002/ Accepted 14 April 2003
A recent study evaluated the ability of clinical Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus isolates to colonize the medicinal leech and discovered that the strains tested either could not proliferate inside the medicinal leech or were killed (9) . The concentration of the E . coli strain decreased inside the medicinal leech for the first 48 h after feeding, suggesting that the bacteria were killed (9) . Heating the blood or the addition of EDTA or EGTA and Mg2+ to the blood prevented the killing of the E . coli strain in vitro (9) . These treatments indicate that a heat-sensitive and divalent-cation (possibly Ca2+)-requiring property was responsible for the demise of the E . coli strain . This is suggestive of the membrane attack complex of the complement system . Heat treatment of the blood prior to feeding allowed the E . coli strain to proliferate inside the medicinal leech, suggesting that the complement system was also responsible for inhibiting its growth inside the leech . These results indicate that powerful antimicrobial properties of mammals contribute to the unusual specificity of this bacterium-invertebrate symbiosis . If the complement system contributes to the specificity of this symbiotic interaction, then A . veronii bv . sobria mutants that have an increased sensitivity to the complement system would be expected to have a reduced ability to colonize the medicinal leech . Several surface structures are responsible for the resistance of bacteria to complement-mediated killing, including lipopolysaccharide (LPS), outer membrane proteins, polysaccharide capsule, and S layer (15, 22, 24) . A previous study generated serum-sensitive mutants derived from the A . veronii bv . sobria strain AH-1 (also called TF7), which is of the O11 serotype and produces an S layer, and the A . hydrophila strain AH-3, which is of the O34 serotype (15, 17) . The mutants derived from AH-1 have defined defects in the synthesis of the S layer (AH-45) or in both the LPS and S layer (AH-21 and AH-26), and their sensitivities to the membrane attack complex had been previously determined (15, 17) . These phenotypes could be complemented with pLA226 . pLA226 was recovered from a genomic library of strain AH-1 that was constructed in the cosmid pLA2917 (19) . Initial DNA sequencing of cosmid pLA226 revealed open reading frames with high similarity (over 90%) to biosynthetic genes for rhamnose (rmlC, rmlB, and rmlA included in wb clusters for O-antigen biosynthesis [unpublished data]) . The medicinal leeches were obtained from Zentrum Arbeit und Umwelt Giessen, Biebertal, Germany, and maintained at room temperature without feeding (6) . The abilities of the mutants and their parent strains to colonize the medicinal leech were tested as described previously by Indergand and Graf, except that human blood was used instead of sheep blood (9), and the bacteria were grown at 28°C to ensure the expression of the S layer and the O34-antigen LPS (13, 14) . Briefly, the blood meal was inoculated with the test strain (for AH-1 and its derivates the inoculum was 104 CFU/ml and for AH-3 and its derivates the inoculum was 105 CFU/ml) and immediately fed to the animal . A fraction of the inoculated blood was incubated in a microcentrifuge tube at the same temperature as that of the animal (in vitro control) . For heat inactivation, the blood was incubated at 56°C for 30 min (9) . When appropriate, antibiotics were added to the Luria-Bertani (LB) medium (kanamycin, 100 µg/ml; rifampin, 10 µg/ml; and tetracycline, 5 µg/ml) (21) . For the complementation experiment, the strains were grown in LB medium containing the antibiotics to ensure the maintenance of the plasmids, but no antibiotics were added to the blood . The intraluminal fluid recovered from the animal was plated on LB agar plates containing the appropriate antibiotics . The statistical analysis was done using GraphPad Prism3 . The data were log transformed and compared using an unpaired t test with Welch's correction . The analysis of the LPS was done as described previously (15) . Both parent strains, A . veronii bv . sobria strain AH-1 and A . hydrophila strain AH-3, were able to colonize the medicinal leech by 18 h after feeding (1.5 x 108 and 8.0 x 105 CFU/ml, respectively) . These colonization levels are similar to those obtained previously with other Aeromonas strains (6), and in both studies the A . veronii bv . sobria strains reached higher concentrations than did the A . hydrophila strains . Interestingly, the 100-fold difference in the colonization levels between AH-1 and AH-3 disappeared when the blood was heat treated prior to being fed to the animals, and both strains reached a significantly higher concentration (Table 1) . This result suggests that other factors besides complement may affect the colonization of the A . hydrophila strain AH-3, or perhaps there may be a difference in the complement resistance between the two strains .
We wanted to verify the observed colonization defect from one of the mutants with a dramatic colonization defect . The LPS biosynthesis defect of AH-21 could be complemented with plasmid pLA226 (Fig . 1A) (19) . For the complementation experiments, we determined the abilities of AH-1(pLA2917), AH-21(pLA2917), and AH-21(pLA226) to colonize the medicinal leech (Fig . 1B) . AH-21(pLA2917) colonized to a significantly lower level than either the parent strain with the empty control vector, AH-1(pLA2917), or the complemented AH-21(pLA226) strain (Fig . 1B) . These results link the colonization defect to the genes required for the synthesis of the O antigen and the intact LPS . These results further support the hypothesis that the LPS is an essential colonization factor for this symbiosis . The LPS represents the first colonization factor that has been identified for this digestive tract symbiosis .
The dramatically reduced ability of complement-sensitive mutants to colonize the medicinal leech and the restoration of the colonization defect of AH-21 either by heat treating the blood or by complementing the inactivated gene are consistent with our hypothesis that the complement system of the ingested vertebrate blood contributes to the unusual specificity of the digestive tract flora of the medicinal leech . The previous analysis of these mutants linked their genetic defect to an enhanced sensitivity to the complement system (15, 17) . Our results do not exclude the possibility that other heat-sensitive, antimicrobial properties of blood could be active inside the digestive tract of H . medicinalis, and the colonization phenotype of the A . hydrophila strain AH-3 provides support for this hypothesis . From a previous study we know that the microbial symbionts and/or the leech host provides an additional layer(s) that contribute(s) to the specificity (9) . The presence of multiple factors that contribute to the specificity of symbiotic associations has been shown in the well-studied Vibrio fischeri-Euprymna scolopes and Rhizobium-leguminous plant symbioses (8, 23) . These mutants represent, to our knowledge, the first colonization mutants for this symbiosis . In addition, the LPS has been shown to be critical for colonization of germfree chicken gut by A . hydrophila (17) and adherence to Hep2 cells (16) . Both systems were used as models for diarrhea . The addition of purified O antigen and capsular polysaccharides increased the ability of avirulent A . hydrophila strains to survive in tilapia serum (25) . The results from this investigation provide further evidence that some virulence factors are also essential for the benign colonization of host animals (7) and suggest that the study of such alternative animal models can help to discover new aspects of pathogenic digestive tract associations .
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