Orally administered P22 phage tailspike protein reduces salmonella colonization in chickens: prospects of a novel therapy against bacterial infections

Abstract

One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans.

Figure 1. P22sTsp micro-agglutination assay at 4°C.

P22sTsp agglutinates Salmonella effectively as shown by the diffused cell patterns (wells on the left side of the arrow). No agglutination was observed with Staphylococcus aureus (lower panel) at the highest concentration used (cell sediments appear as round dots). MAC: minimum agglutination concentration.

Figure 2. Protease resistance profile of P22sTsp.

SDS-PAGE analysis of P22sTsp following treatment with A, chicken GI fluid proteases, B, trypsin, C, chymotrypsin and D, pepsin and chymotrypsin in the presence or absence of 10% BSA. A, Lane 1, molecular weight markers; lane 2, untreated P22sTsp; lanes 3–5, P22sTsp incubated with protease solutions at 37°C for 5 min, 20 min and 2 h, respectively. B, C, Lane 1, molecular weight marker; lane 2, untreated P22sTsp; lane 3, trypsin-treated (B) or chymotrypsin-treated (C) P22sTsp (1 h at 37°C). A control protein (single-domain antibody) incubated with the GI fluid (37°C, 2 h), trypsin or chymotrypsin (37°C, 1 h) showed complete digestion (data not shown). D, All reaction samples, including controls (no proteases) were subjected to a purification step with PureProteome™ Nickel Magnetic Beads prior to their analysis by SDS-PAGE (see Materials and Methods). Digestion reactions were carried out for 0 and 60 min. The positions of the molecular weight markers are indicated on the left of the panel D figures.

Figure 3. Schematic overview of the two protocols used for animal studies.

At time point zero, chicks were inoculated with 10⁷ Salmonella. In Protocol 1, chicks were gavaged after inoculation (1 h) with P22sTsp in 10% BSA or with 10% BSA alone. The next two gavages were given at 18 h and 42 h. Chicks were sacrificed at 47 h. In Protocol 2, the first gavage was delayed by 17 h and given at 18 h. The next two gavages were given at 42 h and 66 h. The chicks were subsequently sacrificed at 47 h (Protocol 1) or 71 h (Protocol 2).

Figure 4. Effect of orally administered P22sTsp on Salmonella colonization of chick cecum.

Effect of orally administered P22sTsp on Salmonella colonization of chick cecum (A) or liver and spleen (B). Medians are shown as horizontal bars on graph A. The two protocols were compared and for subsequent studies Protocol 1 was used. Chicks not infected with Salmonella Typhimurium (No S.typh.); chicks infected with S. Typhimurium but did not receive any treatment (S.typh.); infected chicks treated with 10% BSA (S.typh.+BSA); infected chicks treated with P22sTsp in 10% BSA according to Protocol 1 (S.typh.+Tsp/BSA (P1)); infected chicks treated with P22sTsp in 10% BSA according to Protocol 2 (S.typh.+Tsp/BSA (P2)). For cecal results, the overall treatment effect was assessed using the Kruskal-Wallis test with Dunnett's post test to correct for the number of comparisons and was significant (P<0.05). For liver and spleen results, all pairs were compared to each other using the chi square test. Not significant (ns).

Figure 5. Effect of orally administered P22sTsp on Salmonella colonization of chick cecum, liver and spleen at an inoculation level of 10⁴ (Group 1) and 10⁵ (Group 2) bacteria.

Medians are shown as horizontal bars on the graphs. Protocol 1 was followed. The P22sTsp treatment in each group was done in duplicate (cage 1 and cage 2). Non-inoculated chicks remained pathogen free (n = 9) and are not included in the graph. Infected chicks treated with 10% BSA (S.typh.+BSA); infected chicks treated with P22sTsp in 10% BSA (S.typh.+Tsp/BSA). The data from S.typh.+BSA cohorts of group 1 and group 2 were pooled, since there was no significant difference between the two groups (Mann Whitney two-tailed t-test, cecum: P = 0.32; liver: P = 0.23; spleen: P = 0.5). Pooled data for Tsp-treated animals includes 4 cohorts (2 cages, two groups). For comparison within the groups see Table 2.

Figure 6. P22sTsp reduces Salmonella motility.

The spread of Salmonella representative of its motility on soft agar plates was measured at different time points and used to plot a graph of motility diameter versus incubation time.