Evolution of the T4 phage virion is driven by selection pressure from non-bacterial factors

Abstract

Bacteriophages colonize animal and human bodies, propagating on sensitive bacteria that are symbionts, commensals, or pathogens of animals and humans. T4-like phages are dependent on abundant symbionts such as Escherichia coli, commonly present in animal and human gastrointestinal (GI) tracts. Bacteriophage T4 is one of the most complex viruses, and its intricate structure, particularly the capsid head protecting the phage genome, likely contributes substantially to the overall phage fitness in diverse environments. We investigated how individual head proteins-gp24, Hoc, and Soc-affect T4 phage survival under pressure from non-bacterial factors. We constructed a panel of T4 phage variants defective in these structural proteins: T4∆Soc, T4∆24byp24, T4∆Hoc∆Soc, T4∆Hoc∆24byp24, T4∆Soc∆24byp24, and T4∆Hoc∆Soc∆24byp24 (byp = bypass). These variants were investigated for their sensitivity to selected environmental conditions relevant to the microenvironment of the GI tract, including pH, temperature, and digestive enzymes. The simple and "primitive" structure of the phage capsid (∆24byp24) was significantly less stable at low pH and more sensitive to inactivation by digestive enzymes, and the simultaneous lack of gp24 and Soc resulted in a notable decrease in phage activity at 37°C. Gp24 was also found to be highly resistant to thermal and chemical denaturation. Thus, gp24, which was acquired relatively late in evolution, seems to play a key role in T4 withstanding environmental conditions, including those related to the animal/human GI tract, and Soc is a molecular glue that enhances this protective effect. IMPORTANCE Bacteriophages are important components of animal and human microbiota, particularly in the gastrointestinal tract, where they dominate the viral community and contribute to shaping microbial balance. However, interactions with bacterial hosts are not the only element of the equation in phage survival-phages inhabiting the GI tract are constantly exposed to increased temperature, pH fluctuations, or digestive enzymes, which raises the question of whether and how the complex structure of phage capsids contributes to their persistence in the specific microenvironment of human/animal bodies. Here we address this phage-centric perspective, identifying the role of individual head proteins in T4 phage survival in GI tract conditions. The selection pressure driving the evolution of T4-like phages could have come from the external environment that affects phage virions with increased temperature and variable pH; it is possible that in the local microenvironment along the GI tract, the phage benefits from stability-protecting proteins.

Keywords: T4 phage; bacteriophage evolution; gastrointestinal tract; gp24; head proteins; head vertex protein.

Fig 1

Schematic representation of the bacteriophage T4 virion. Four protein components of the head—gp23, gp24, Hoc, and Soc—and their positions within the head structure are indicated.

Fig 2

Stability of T4 phage and its mutants deficient in head proteins at 37°C. Purified phage preparations (N = 6) were diluted in PS to 10⁶ pfu/mL and incubated at 22°C or 37°C for 2 h. Phage titers were determined using spot plating. Log reductions were calculated relative to the control group (22°C). The experiment was repeated twice with concordant results. One representative experiment is presented. *P < 0.0243; ***P < 0.008; ****P < 0.0001 (Welch’s t test).

Fig 3

Thermal denaturation curves and circular dichroism (CD) spectra of recombinant T4 head proteins: major capsid protein gp23 and its two mutants with a single amino acid substitution N381S or N384S, and head vertex protein gp24. Protein solutions in PBS were heated in the range of 25°C–80°C (gp23 and its mutants) or 25°C–95°C (gp24); wavelength: 222 nm. CD spectra were acquired in the range of 200–260 nm at 25°C and 80°C.

Fig 4

Effect of acidic, pH 3.0, and alkaline, pH 10.6, environment on the viability of T4 phage and its mutants defective in head proteins. Purified phage preparations (N = 6) were diluted to 10⁶ pfu/mL in PS, pH 7 and PS, pH 3 or 10.6 and incubated at 37°C for 30 min (pH 3.0) or 1 h (pH 10.6). Phage titers were determined using spot plating. Log reductions were calculated relative to the control group (pH 7). Each experiment was repeated at least twice with concordant results. One representative experiment is presented. *P < 0.05; **P < 0.005 (Mann-Whitney test).

Fig 5

Effects of the proteolytic digestive enzymes pepsin, trypsin, and α-chymotrypsin, and bile extract on the viability of T4 phage and its mutants defective in head proteins. Purified phage preparations (N = 6) (10⁶ pfu/mL) were incubated in PS pH 4 or PS pH 4 with pepsin (1 mg/mL) at 37°C or in PS pH 4 at 22°C for 2 h; in PS + 20 mM CaCl₂ pH 8 or PS + 20 mM CaCl₂ pH 8 with trypsin (2 mg/mL) at 37°C or in PS + 20 mM CaCl₂ pH 8 at 22°C for 2 h; in PS + 20 mM CaCl₂ pH 8 or PS + 20 mM CaCl₂ pH 8 with of α-chymotrypsin (3 mg/mL) at 37°C or in PS + 20 mM CaCl₂ pH 8 at 22°C for 6 h; in PS or 0.7% and 2% porcine bile extract in PS at 37°C or in PS at 22°C for 2 h. Phage titers were determined using spot plating. Log reductions were calculated relative to the relevant control group incubated at 37°C in the absence of the investigated factor (represented with blue bars). Each experiment was repeated at least twice with concordant results. One representative experiment is presented. *P ≤ 0.0332; **P ≤ 0.0021; ***P ≤ 0.0002; ****P < 0.0001 (Brown-Forsythe and Welch ANOVA, Dunnett’s T3 multiple comparisons test).

Fig 6

Schematic representation of the proposed gastrointestinal tract-related factors that may have contributed to the selection pressure on T4 phage.