Phage & Bacteria: A Space for Mutual Co-Existence

by Roberto
 

Phage-bacteria population dynamics can be remarkably complex. This is reflected in the natural history of both; new phage defense systems in bacteria and anti-defense systems in phage seem to be discovered daily. Behind these intricate strategies to help in the reproductive success of each lies the basic problem of any predator-prey relationship. As the predator succeeds, the number of prey can be driven to extinction, leading to the predator's demise. These predator-prey dynamics and possible solutions for survival of both are the subject of myriad models and studies, most of them based on the Lotka-Volterra equations (e.g., the "Kill the Winner" hypothesis).

 

Temperate phages – those able to lysogenize – have solved the problem of extinction because they can preserve their DNA when the host population decreases. But virulent phages – those that rely exclusively on a lytic lifecycle – face a fascinating conundrum. During their proliferation they, by necessity, greatly reduce the numbers of their bacterial hosts. This in turn could lead to their extinction. Most of the models and systems used to understand the survival of virulent phages are based on homogeneously dispersed populations of phage and bacteria. Knowing that many bacteria live as multicellular aggregates (perhaps read that as biofilms...) I was delighted when I read a paper describing a model developed for phage-bacteria interactions based on growing microcolonies of bacteria.

 

Fig. 1. Plaque formed by P1vir. Green spheres are microcolonies of E. coli expressing green fluorescent protein. Note the faint colonies, which are seen across the plaque although less in its central region. Scale bar: 300 µm. Source

I was immediately fascinated by this report because of what served as the authors' inspiration: the careful observation of a plaque formed by a virulent phage (P1_vir) on a lawn of bacteria embedded in soft agar. What could be more routine for a microbiologist than to see such a plaque? We all know virulent phages produce clear plaques and, if we see colonies within the plaque, we immediately pronounce them to be the result of growth of phage-resistant mutants. But the authors of this paper saw the minute details of the microcolonies within the "clear" plaque in an entirely different light (Fig. 1). By close observation, they noticed that the size of the microcolonies increased the further away they were from the plaque center. They recognized that this size gradient of the microcolonies could not be explained solely by the growth of resistant mutants. Neither could it be explained in terms of the cells within these microcolonies having reached stationary phase and thus unable to support phage growth (they showed that cells in such microcolonies are still growing exponentially).

 

To explain the microcolonies the authors hypothesized that "phages may well adsorb in large numbers to susceptible cells on the colony surface and therefore only rarely reach deeper layers of the colony. Thus, the continued growth from the inside of a sufficiently large colony could overwhelm killing on the colony surface." In this way, a microcolony may serve as a spatial refuge of growing phage-sensitive cells at its center while at the same time allowing virulent phage propagation on its surface. The question became: how many cells need to be in a microcolony for this spatial refuge to form?

 

They first addressed the question through mathematical modelling and computer simulations. Their model assumed spherical cells growing exponentially with a doubling time of 20 minutes. Cells could not disperse, which lead to the formation of a spherical microcolony. They then let the surface of the microcolony encounter phages at different times and observed (in their simulations) whether the phages kill all the cells or whether the cells can outgrow the lysis by the phages. The model also includes a latency period between infection and lysis, a burst size (based on values for P1_vir infecting E. coli in liquid culture), and diffusion-limited adsorption of the released phage to the adjacent cells.

 

Fig. 2. Simulations of phage attacks on growing microcolones. Upper row:  A relatively early phage attack (infection at 3.25 h), results in killing of all cells. Lower row: A slightly later attack (infection at 3.75 h), results in continued growth of cells in the center of the microcolony. Blue spheres represent live, uninfected cells. Phage-infected bacteria are yellow to red, the ones closest to lysis are the most red. Source

The results of their simulations proved both striking and, to me at least, surprising (Fig.2.). When the growing microcolony is attacked by phages at early times (up to 3.5 hours), when it has ~220 cells, the phages eventually kill all the cells. But waiting another 30 minutes, when the microcolony has ~500 cells before it is attacked with phages, leads to a completely different result. Phages can only penetrate and kill two layers of cells; thus the center of the microcolony survives while phages persist on the infected surface.

 

The authors then proceeded to put their model to the test by observing the killing effects of P1_vir infecting E. coli embedded in a layer of soft agar. Quite satisfyingly, they observed qualitatively the same results. They allowed microcolonies to grow for various times before spraying them with the virulent phage. When microcolonies pre-grew for up to 5 hours, all cells were killed. After 7 hours of pre-growth, however, they still observed killing at the surface of the microcolony, but cells at the center survived. While some of the surviving cells were indeed phage-resistant mutants, in half of the microcolonies tested they detected no such mutants. Thus, the simple fact of growing cells as an aggregate can result in the co-existence of phage-sensitive cells along with virulent phages. It's nice to see that pursuing a careful observation and then applying principles from "just physics" can lead to such an elegant explanation. I put the phrase "just physics" in quotes because, when you think about it, underlying everything – all the stunning emergent properties of chemistry and biology – there's always physics!

 

 

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