A quorum-sensing-induced bacteriophage defense mechanism

Abstract

One of the key determinants of the size, composition, structure, and development of a microbial community is the predation pressure by bacteriophages. Accordingly, bacteria have evolved a battery of antiphage defense strategies. Since maintaining constantly elevated defenses is costly, we hypothesize that some bacteria have additionally evolved the abilities to estimate the risk of phage infection and to adjust their strategies accordingly. One risk parameter is the density of the bacterial population. Hence, quorum sensing, i.e., the ability to regulate gene expression according to population density, may be an important determinant of phage-host interactions. This hypothesis was investigated in the model system of Escherichia coli and phage λ. We found that, indeed, quorum sensing constitutes a significant, but so far overlooked, determinant of host susceptibility to phage attack. Specifically, E. coli reduces the numbers of λ receptors on the cell surface in response to N-acyl-l-homoserine lactone (AHL) quorum-sensing signals, causing a 2-fold reduction in the phage adsorption rate. The modest reduction in phage adsorption rate leads to a dramatic increase in the frequency of uninfected survivor cells after a potent attack by virulent phages. Notably, this mechanism may apply to a broader range of phages, as AHLs also reduce the risk of χ phage infection through a different receptor. IMPORTANCE To enable the successful manipulation of bacterial populations, a comprehensive understanding of the factors that naturally shape microbial communities is required. One of the key factors in this context is the interactions between bacteria and the most abundant biological entities on Earth, namely, the bacteriophages that prey on bacteria. This proof-of-principle study shows that quorum sensing plays an important role in determining the susceptibility of E. coli to infection by bacteriophages λ and χ. On the basis of our findings in the classical Escherichia coli-λ model system, we suggest that quorum sensing may serve as a general strategy to protect bacteria specifically under conditions of high risk of infection.

FIG 1

AHL quorum-sensing signals reduce λ phage superinfection. The concentrations of free phage from lysogenic cultures with or without AHLs are shown for E. coli BW25113 (wild type [WT]), AHL receptor mutant (sdiA mutant), λ receptor mutant (lamB mutant), and a mutant of a transcriptional regulator for exopolysaccharide (rcsA mutant). The cultures were grown to an OD₆₀₀ of 1.2 in the presence or absence of 5 µM AHLs. Free phage concentration is indicated as PFU per ml of culture. The number of independent cultures tested (n) is indicated. Each error bar indicates 1 standard deviation from the mean. Reported differences were evaluated using a Student’s t test.

FIG 2

Growth in the presence of AHL signals reduces the rate of phage adsorption. ³⁵S-labeled λ phages were added to a shaking culture of wild-type (WT) or AHL receptor mutant (sdiA mutant) cells grown in the presence or absence of 5 µM AHLs. Prior to the addition of phage, chloramphenicol was added to cells to arrest growth at an OD₆₀₀ of 1.0 and prevent phage multiplication. At 2-min intervals, an aliquot of the cell-phage mixture was filtered through a 0.45-µm filter, and the radioactivity of the adsorbed phages retained on the filter was measured. The radioactivity of filters subjected to the identical treatment using lamB mutant cells has been subtracted as background, and each sample has been normalized to the total radioactivity of an unfiltered sample aliquot. The number of independent cultures tested (n) is indicated. Each error bar indicates 1 standard deviation from the mean. Reported differences were evaluated using a two-way analysis of variance (ANOVA). The experiment was repeated on three separate days with similar results. The relative rates of adsorption were calculated as shown in the symbol key to Fig. S1 in the supplemental material and are shown in the figure.

FIG 3

AHL signaling induces downregulation of the λ receptor LamB. (A) Outer membrane protein preparations were separated by SDS-PAGE and stained with Coomassie blue. Outer membrane proteins from wild-type E. coli, AHL receptor mutant (sdiA mutant), and λ receptor mutant (lamB mutant) grown to an OD₆₀₀ of 1.0 with 5 µM AHLs or 0.4% maltose are shown as indicated below the lanes. (B) Quantification of LamB protein. Band intensities of the protein band were quantified using ImageJ software and normalized to the intensity of a LamB band that is not affected by AHLs. To enable pooling of the data from different gels, the intensity of the LamB band in untreated wild-type cells was set at 1 in each gel, and the intensities of the remaining bands relative to that of the untreated wild-type cells are shown. The number of independent outer membrane protein preparations tested (n) is indicated. Each error bar indicates 1 standard deviation from the mean.

FIG 4

AHLs dramatically enhance E. coli’s chances of surviving attack by the lytic phage λ^vir. Wild-type and sdiA mutant E. coli cells were grown to an OD₆₀₀ of 1.0 in the presence or absence of 5 µM AHLs. λ^vir was added at an average phage input of 20 phages per cell. Fifty minutes after the addition of phage, an aliquot of the culture was diluted in M63-salts containing 50 mM sodium citrate to inactivate the free phage. The figure shows the number of colonies formed by cells that survived 50 min in the presence of λ^vir relative to the number of CFU immediately prior to phage addition. The number of independent cultures tested (n) is indicated. Each error bar indicates 1 standard deviation from the mean. Reported differences were evaluated using a Student’s t test.

FIG 5

AHL signaling protects E. coli from χ phage adsorption. Wild-type E. coli was grown to an OD₆₀₀ of 0.75 in the presence or absence of 5 µM AHLs. To arrest growth and prevent phage multiplication, chloramphenicol was added prior to the addition of phage χ. The numbers of free (nonadsorbed) χ phages were measured as PFU on a lawn of sensitive, motile bacteria. The number of independent cultures tested (n) is indicated. Each error bar represents 1 standard deviation from the mean. Reported differences were evaluated using a two-way analysis of variance (ANOVA).