Modular prophage interactions driven by capsule serotype select for capsule loss under phage predation

Abstract

Klebsiella species are able to colonize a wide range of environments and include worrisome nosocomial pathogens. Here, we sought to determine the abundance and infectivity of prophages of Klebsiella to understand how the interactions between induced prophages and bacteria affect population dynamics and evolution. We identified many prophages in the species, placing these taxa among the top 5% of the most polylysogenic bacteria. We selected 35 representative strains of the Klebsiella pneumoniae species complex to establish a network of induced phage-bacteria interactions. This revealed that many prophages are able to enter the lytic cycle, and subsequently kill or lysogenize closely related Klebsiella strains. Although 60% of the tested strains could produce phages that infect at least one other strain, the interaction network of all pairwise cross-infections is very sparse and mostly organized in modules corresponding to the strains' capsule serotypes. Accordingly, capsule mutants remain uninfected showing that the capsule is a key factor for successful infections. Surprisingly, experiments in which bacteria are predated by their own prophages result in accelerated loss of the capsule. Our results show that phage infectiousness defines interaction modules between small subsets of phages and bacteria in function of capsule serotype. This limits the role of prophages as competitive weapons because they can infect very few strains of the species complex. This should also restrict phage-driven gene flow across the species. Finally, the accelerated loss of the capsule in bacteria being predated by their own phages, suggests that phages drive serotype switch in nature.

Fig. 1. Prophage distribution in Klebsiella genus.

a Rooted phylogenetic tree of Klebsiella species used in this study based on the core genes. b Average number of prophages per genome. PHASTER prediction for completeness is indicated. Numbers represent the total number of genomes analysed of each species.

Fig. 2. Genomic organization of eleven of the prophages in this study.

Numbers correspond to the host genomes, as displayed in Fig. 3. The number in parenthesis identifies the prophage in the genome. All prophages are classified as “intact” except #63 (1) (“questionable”). Genome boundaries correspond to attL/R sites. Arrows represent predicted ORFs and are oriented according to transcriptional direction. Colors indicate assigned functional categories, tRNAs are represented as red lines and the sequences are oriented based on the putative integrase localization. Local blastn alignments (option dc-megablast) are displayed between pairs of related prophages, colored according to the percentage of identity. The Viral Quotient (VQ) from pVOG is displayed below or on top of each ORF, with gray meaning that there was no match in the pVOG profiles database and thus no associated VQ value. Prophages #62 (2) and #54(1) have inserted the core gene icd, and the boundaries correspond to icd on the right and the most distal att site found, which is likely to be a remnant prophage border. The most proximal att site is also annotated (vertical black line). This figure was generated using the R package GenoPlotR v0.8.9 [103].

Fig. 3. Phylogenetic tree of the 35 Klebsiella strains.

The tree was built using the protein sequences of 3009 families of the core genome of representative strains from the Klebsiella pneumoniae species complex. The first column determines the capsule locus type and the second column provides information about the environment from which it was isolated. The next columns indicate the total, “intact”, “questionable”, and “incomplete” prophages detected in the genomes by PHASTER. The last column shows the final absorbance of a culture after induction by mitomycin C. Background color indicates different Klebsiella spp. The size of the circles along the branches are proportional to bootstrap values ranging between 34 and 100. The gray color indicates other CLTs that are only present once in the dataset. The hash symbol represents the number of the strain in our collection and is used for simplicity throughout the text.

Fig. 4. Some Klebsiella encode viable prophages that can infect and lysogenize other strains.

a Infection matrix indicating the ability of induced and PEG-precipitated supernatants of all strains to form inhibition halos on lawns of all strains. This was repeated three times with three independently produced lysates. The sum of the three experiments is shown. The strains are ordered by phylogeny, and the colors on top of numbers indicate capsule locus types (CLT). Geometric figures along the x-axis indicate the LPS serotype (O-locus). Stars along the y-axis indicate that the genome codes for putative genes involved in colicin production but lacking a lysis gene (white) or has a full colicin operon (yellow). Shades of blue indicate infections between lysates produced by bacteria from the same CLT as the target bacteria, whereas shades of orange indicate cross-CLT infections. b–d Barplots indicate the number of resistant, sensitive, or lysogenized clones from each independent replicate population exposed 24 h to lysates from other strains. Each bar represents the 31 survivor clones from each of three independent experiments. Gray scale indicates whether: (i) the clones are still sensitive to the phage (light gray), (ii) they became resistant by becoming a lysogen (dark gray) as determined by cell death upon addition of MMC and PCR targeting prophage sequences, or (iii) they became resistant by other mechanisms.

Fig. 5. The capsule is required for phage infection.

Lawns of different strains, wild type (Cap+) and capsule null mutant (Cap-) in contact with PEG-precipitated supernatant of other strains. Isogenic capsule mutants of strains #26 and #37 were constructed by an in-frame deletion of wza gene, and a wcaJ deletion for strains #57 and #58 (see Methods).

Fig. 6. Loss of capsule in three Klebsiella strains.

a Ratio of capsulated clones throughout the ten days before daily passages of each culture. Shades of green represent the different environments in which evolution took place. MMC stands for mitomycin C. Full lines represent the average of the independent populations of the same strain and environment (shades of green). Dashed lines represent each of the independent populations. b Area under the curve during the first five days of the experiment.