Complement Resistance Is Essential for Colonization of the Digestive Tract of Hirudo medicinalis by Aeromonas Strains

Abstract

From the crop of the medicinal leech, Hirudo medicinalis, only Aeromonas veronii bv. sobria can be cultured consistently. Serum-sensitive A. veronii mutants were unable to colonize H. medicinalis, indicating the importance of the mammalian complement system for this unusual simplicity. Complementation of one selected mutant restored its ability to colonize. Serum-sensitive mutants are the first mutant class with a colonization defect for this symbiosis.

Aeromonas veronii bv. sobria and Aeromonas hydrophila are pathogens of fish (20, 26) and humans, causing wound infections, septicemia, and probably diarrhea (2-4, 10, 12). In addition to being a pathogen, A. veronii bv. sobria is the digestive tract symbiont of the medicinal leech, Hirudo medicinalis (6). The symbionts are housed in the crop of the digestive tract, apparently to the exclusion of other culturable bacteria (5, 6). The symbionts are thought to aid in the digestion of the ingested blood, provide essential nutrients to the host, or prevent other bacteria from colonizing (5). Interestingly, they can also infect the wounds of patients during medical application if the leeches are attached to tissue with poor blood circulation and the patients do not receive preemptive antibiotics (1, 3, 9, 18).

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 × 10⁸ and 8.0 × 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.

TABLE 1.

Growth of Aeromonas in H. medicinalis and in the in vitro control

Strainb	Phenotypec	Assayd	Pretreatmenta
			None		Heat
			Avge	SD	Avge	SD
AH-1	Wild type	Leech	150,000	±98,000	620,000	±300,000
		In vitro	98,000	±77,000	380,000	±160,000
AH-45	S layer−, serumr	Leech	27,000	±44,000	NDf
		In vitro	48,000	±83,000	280,000	±178,000
AH-26	S layer−, HMW-LPS−, serums	Leech	2.2	±4.3	7,900	±15,000
		In vitro	34	±68	34,000	±20,000
AH-21	S layer−, HMW-LPS−, serums	Leech	0.01	±0.02	150,000	±35,000
		In vitro	<0.01		378,000	±250,000
AH-3	Wild type	Leech	800	±1,300	160,000	±100,000
		In vitro	14	±16	340,000	±160,000
MIT-1	HMW-LPS−, serums	Leech	0.01	±0.01	280,000	±250,000
		In vitro	0.14	±0.2	580,000	±82,000
MIT-6	HMW-LPS−, serums	Leech	<0.01		38,000	±40,000
		In vitro	<0.01		360,000	±36,000

^aPrior to inoculation the blood was either not treated (none) or placed in a water bath at 56°C for 30 min (heat).

^bThe strains used in this study have been described previously (15, 17).

^cAH-45, AH-26, and AH-21 were derived from AH-1 whereas MIT-1 and MIT-6 were derived from AH-3. The phenotypes are as follows: unable to assemble the S layer on the cell surface, S layer⁻; resistant to complement system, serum^r; sensitive to complement system, serum^s; and loss of HMW LPS, HMW-LPS⁻. AH-21 was previously reported as serum^r, but further study showed the strain to be serum^s.

^dA blood sample was inoculated with the test strain and either fed to H. medicinalis (leech) or incubated in a microcentrifuge tube at the same temperature (in vitro).

^eThe mean concentration reached after 18 h is shown (1,000 CFU/ml).

^fND, not done.

Three mutants derived from AH-1 with differing degrees of sensitivity to the complement system were tested for their ability to colonize the medicinal leech. Two complement-sensitive mutants, AH-21 and AH-26 (15), with a defect in the high-molecular-weight (HMW) LPS and without the S layer (13) reached significantly lower concentrations inside the animal and in the in vitro control than did the wild-type strain (Table 1). Strain AH-45 is devoid of the S layer but possesses an intact LPS layer and is complement resistant. AH-45 reached a similar concentration inside the animal and in the in vitro control as did the wild-type strain, suggesting that the S layer is not essential for colonizing the medicinal leech (Table 1). The inactivation of the complement system by heat treatment permitted these mutants to reach similar concentrations inside the animal as those obtained by the wild-type strain (9, 11). The HMW-LPS mutants represent the first isogenic mutants with a colonization defect in this symbiosis and suggest that an intact LPS is the first identified colonization factor.

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.

FIG. 1.

Complementation of AH-21. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the LPSs from different A. veronii bv. sobria strains. Lanes: 1, AH-1; 2, AH-21; 3, AH-21(pLA226). No changes in the LPS on gels were observed when the mutants carried the empty cosmid vector pLA2917. (B) The serum-sensitive mutant AH-21 had a dramatically reduced ability to colonize H. medicinalis, and this defect could be restored by the presence of pLA226 but not the empty control plasmid pLA2917. The concentrations reached 18 h after feeding H. medicinalis are depicted. The P values from the unpaired t test with Welch's correction are shown above a line that indicates the compared data sets.

In addition to the A. veronii strains that we tested, A. hydrophila strain AH-3-derived complement-sensitive mini-Tn5 transposon mutants MIT-1 and MIT-6 (devoid of O34-antigen LPS) were unable to colonize the medical leech. Their colonization level was below the limit of detection (10 CFU/ml, Table 1). The mutants reached similar colonization levels as did the parent strain when the blood was heat treated prior to feeding.

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.