The type II secretion system is essential for erythrocyte lysis and gut colonization by the leech digestive tract symbiont Aeromonas veronii

Abstract

Hemolysin and the type II secretion system (T2SS) have been shown to be important for virulence in many pathogens, but very few studies have shown their importance in beneficial microbes. Here, we investigated the importance of the type II secretion pathway in the beneficial digestive-tract association of Aeromonas veronii and the medicinal leech Hirudo verbana and revealed a critical role for the hemolysis of erythrocytes. A mutant with a miniTn5 insertion in exeM, which is involved in forming the inner membrane platform in the T2SS, was isolated by screening mutants for loss of hemolysis on blood agar plates. A hemolysis assay was used to quantify the mutant's deficiency in lysing sheep erythrocytes and revealed a 99.9% decrease compared to the parent strain. The importance of the T2SS in the colonization of the symbiotic host was assessed. Colonization assays revealed that the T2SS is critical for initial colonization of the leech gut. The defect was tied to the loss of hemolysin production by performing a colonization assay with blood containing lysed erythrocytes. This restored the colonization defect in the mutant. Complementation of the mutant using the promoter region and exeMN revealed that the T2SS is responsible for secreting hemolysin into the extracellular space and that both the T2SS and hemolysin export by the T2SS are critical for initial establishment of A. veronii in the leech gut.

FIG. 1.

Hemolysis activity of A. veronii. The abilities of parent strain Hm21S, the transposon mutant HE-1095, and complemented strain HEC-1334 to lyse erythrocytes were assessed on BA plates. A clear halo around the colony reveals hemolysis of erythrocytes.

FIG. 2.

Measurement of hemolysin activity. The hemolysis activities of parent strain Hm21S, mutant HE-1095, and complemented mutant HEC-1344 were measured and are reported as percentages of complete hemolysis. The percentage of hemolysis was measured by using the following equation: [(A₅₄₀ of samples with hemolysis − A₅₄₀ of negative control)/A₅₄₀ of positive control] × 100 (5, 6).

FIG. 3.

Complete T2SS locus of A. veronii Hm21. The transposon insertion site is indicated by the flag. The solid line indicates 1 kb, and the size of the locus is 11.8 kb.

FIG. 4.

Time courses in vitro and in vivo. (A) Time course of in vitro growth of Hm21S (triangles) and mutant HE-1095 (circles) in blood at 0, 3, 6, 18, 24, and 42 h revealing that the two strains grow to equal levels. Each symbol represents one sample. (B) In vivo assay of the colonization of the medicinal leech gut by Hm21S (triangles) and mutant HE-1095 (circles). Animals were sacrificed at 6, 18, 24, and 42 h after feeding. The data show that the mutant is unable to initially colonize the leech gut. Each symbol represents data from one animal. A statistically significant difference between data sets is indicated by an asterisk (P < 0.05, Mann-Whitney test; n/s, not significantly different). The dotted line represents the limit of detection.

FIG. 5.

Hm21S versus HE-1095 leech competition assay. The ability of the strains to compete in the leech gut was assessed 6 h after feeding by varying the numbers of CFU of Hm21S and HE-1095. A 50:50 (Hm21S:HE-1095) ratio was assessed first, and then 25:75 and finally 15:85, lowering the amount of Hm21S. Each symbol represents one animal. The CI value was calculated for each leech as follows: CI = (mutant_output/competitor_output)/(mutant_input/competitor_input). A CI of 1 indicates that the mutant colonized to the same level as the parent strain, and a CI of <1 indicates that the mutant had a colonization defect. A statistically significant difference between data sets is indicated by an asterisk (P < 0.05 t test; n/s, not significantly different).

FIG. 6.

Complementation of HE-1095 with lysed erythrocytes. Hemolysin was linked to the colonization defect of HE-1095 by feeding leeches different percentages of lysed erythrocytes for a 6-h colonization assay with Hm21S (triangles) or HE-1095 (circle). Each symbol represents one animal. A statistically significant difference between data sets is indicated by an asterisk (P < 0.05, Mann-Whitney test). The dotted line represents the limit of detection.

FIG. 7.

Complementation of HE-1095 with exeMN. The ability of the strains to colonize the leech was assessed 6 h after feeding. Each symbol represents one animal. A colonization assay was performed with Hm21S, the mutant HE-1095, the exeMN-complemented parent strain Hm21C, and the exeMN-complemented mutant HEC-1344. These data show that exeMN reverses the HE-1095 colonization defect in the leech gut. A statistically significant difference between data sets is indicated by an asterisk (P < 0.05, Mann-Whitney test).