Prophage Excision in Streptococcus pneumoniae Serotype 19A ST320 Promote Colonization: Insight Into Its Evolution From the Ancestral Clone Taiwan 19F-14 (ST236)

Abstract

Streptococcus pneumoniae 19A ST320, a multidrug-resistant strain with high disease severity that notoriously spread before the use of expanded pneumococcal conjugate vaccines, was derived from a capsular switching event between an international strain Taiwan 19F-14 (ST236) and a serotype 19A strain. However, the molecular mechanisms underlying the adaptive evolution of 19F ST236 to 19A ST320 are unknown. In this study, we compared 19A ST320 to its ancestral clone, 19F ST236, in terms of adherence to respiratory epithelial cells, whole transcriptome, and ability to colonize a young mouse model. Serotype 19A ST320 showed five-fold higher adherence to A549 cells than serotype 19F ST236. High-throughput mRNA sequencing identified a prophage region located between dnaN and ychF in both strains; however, the genes in this region were expressed at significantly higher levels in 19A ST320 than in 19F ST236. Analysis by polymerase chain reaction (PCR) showed that the prophage is able to spontaneously excise from the chromosome and form a circular episome in 19A ST320, but not in 19F ST236. Deletion of the integrase in the prophage of 19A ST320 decreased spontaneous excision and cell adherence, which were restored by complementation. Competition experiments in mice showed that the integrase mutant was six-fold less competitive than the 19A ST320 parent (competitive index [CI]: 0.16; p = 0.02). The 19A ST320 prophage-deleted strain did not change cell adherence capacity, whereas prophage integration strains (integrase mutant and 19F) had decreased expression of the down-stream ychF gene compared to that of 19A ST320. Further deletion of ychF significantly reduced cell adherence. In conclusions, these findings suggest that spontaneous prophage induction confers a competitive advantage to virulent pneumococci.

Keywords: Streptococcus pneumoniae; adherence; integrase; mRNA sequencing; phage.

FIGURE 1

Adherence of serotype 19A ST320 wild type (19A-WT) and serotype 19F ST236 wild type (19F-WT) to A549 cells (p < 0.001, n = 4).

FIGURE 2

Transcriptomic analysis demonstrating spontaneous prophage induction in a serotype 19A ST320 strain. (A) The gray box shows the ratio (19F-WT/19A-WT) of the mRNA levels of genes in the prophage region of the S. pneumoniae serotype 19A ST320 genome. The mRNA expression levels of these genes were lower in 19F ST236 than in 19A ST320. (B) Gene organization of the prophage regions in 19F ST236 (19F-WT) and 19A ST320 (19A-WT) and a model of prophage excision in serotype 19A ST320. There is an additional transposase gene within the prophage region in 19F ST236. The putative integrase is shown as a black arrow. (C) PCR analysis for the presence of prophage excision and circular episomal prophage molecules. The relative positions of the primers on the chromosome are shown. Primer combinations A + B, which are located outside the prophage region, will amplify a product if the prophage is excised; otherwise, no PCR product will be amplified because this region is too long. Primer combination C + D, which are both located inside the prophage, will amplify a product if the prophage is excised and circularized. Primer combinations A + D, which are located inside and outside the prophage region, will amplify a product if the prophage is integrated in the chromosome. In serotype 19A ST320 (19A) strain, all three PCR products (A + B, A + D, and C + D) were detected; therefore, the prophage can excise and reintegrate spontaneously. In serotype 19F ST236 (19F), only A + D product was detected; therefore, the prophage is integrated in the chromosome. The PCR products were separated on 1% agarose gels and stained with ethidium bromide. (D) The percentage of the cell population with excised prophage and in serotype 19A ST320. When bacteria grew into late-log phase population of cells with excised prophage decreased. The average percentage of cells with excised prophage was 17.17 ± 3.19%.

FIGURE 3

Integrase is responsible for prophage excision and higher expression of prophage genes in serotype 19A ST320. (A) PCR analysis for the presence of prophage excision and circular episomal prophage. PCR with primers A and B showed prophage excision in 19A ST320 wild type (19A-WT) and the integrase complementation strain (19A-Δint::int). PCR with primers A and D showed prophage integration in all strains. PCR with primers C and D showed that there was circular prophage in 19A ST320 wild type (19A-WT) and the complementation strain (19A-Δint::int). (B) The mRNA expression levels of HMPREF0837_10263 and HMPREF0837_10266 within the prophage region were significant decreased in the 19A ST320 integrase mutant (19A-Δint) compared to those of 19A ST320 wild type (19A-WT). (p < 0.05, n = 3).

FIGURE 4

Integrase is associated with increased adherence and colonization. (A) Growth curve as measured by both OD₆₀₀ (, left Y-axis) and viable CFUs counts on blood agar (, right Y-axis) of serotype 19A ST320 wild type (19A-WT) and its integrase deletion mutant (19A-Δint). (B) Adherence of serotype 19A ST320 wild type (19A-WT), the integrase mutant (19A-Δint), and the complementation strain (19A-Δint::int) (wild type vs. mutant, p < 0.002, n = 4; wild type vs. complementation, p = 0.8, n = 4). (C) Intranasal challenge of 3-week-old female BALB/c mice with equal inocula of the bacterial strains. Each symbol represents the competitive index (CI) for an individual animal. A competitive index (CI) was calculated based on the ratio of the competing bacterial strains recovered by nasal lavage, normalized to the ratio of the respective bacteria in the inoculum = (output_{test strain}/output_{wild type})/(input_{test strain}/input_{wild type}). The integrase mutant showed a lower CI compared to serotype 19A ST320 (p = 0.02, n = 4).

FIGURE 5

Circularization of prophage DNA affects the mRNA expression of the downstream ychF gene and cellular adhesion ability. (A) Deletion of the prophage region from 19A ST320 (19A-Δphage) did not affect the cell adherence ability, compared to the 19A ST320 wild type (19A-WT) (p = 0.9, n = 4). (B) The mRNA expression level of ychF in the 19A ST320 integrase deletion mutant (19A-Δint) was lower than in the wild-type strain (19A-WT) (p < 0.05, n = 3). The mRNA expression level of dnaN was not different between the wild type and deletion mutant (p = 0.8, n = 3). (C) The cell adhesion ability of prophage downstream gene ychF deletion mutant (19A-ΔychF) was decreased compared to the wild-type strain (p < 0.01, n = 4), but the adhesion ability of the prophage upstream gene dnaN deletion mutant (19A-ΔdnaN) was not (p = 0.6, n = 4). (D) Schematic showing that the promoter region sequence of ychF was rearranged and resulted in a different -35-promoter sequence when the prophage was excised from or integrated into chromosomal DNA of serotype 19A ST320. The -35-promoter sequence and -10-promoter sequence of ychF were predicted using the BPROM program (Softberry Inc., Mount Kisco, NY, United States; www.softberry.com). The 14-bp short sequence of the bacterial DNA attB site (5′-CCC TTT TTG TGT TA-3′) is shown in gray.