Bacterial Type 6 Secreted Phospholipases Play Family Feud

Abstract

Secreted bacterial phospholipases play many important roles in host-pathogen interactions. In a recent study, Russell et al. (Russell et al., 2013) revealed a new role for highly conserved proteobacterial phospholipases in bacterium-bacterium interactions.

Diverse Gram-negative bacteria have the ability to kill adjacent cells of other species and within species by transferring enzymes across their own cell envelope into intracellular compartments of neighboring bacteria using the type VI (T6) secretion system (Figure 1) (LeRoux et al., 2012). The discovery of these interactions highlight the fact that bacteria are opportunistic; they can switch from cooperative interactions, such as the formation of resistant biofilms or coordinated nutrient access (Elias and Banin, 2012), to deadly antagonism in the blink of an eye (LeRoux et al., 2012). In a recent paper, Russell and colleagues (Russell et al., 2013) present a significant advance in our understanding of the effectors that participate in these complex interactions.

Figure 1. Overview of Type 6 secretion system-mediated transfer of Tle proteins from donor cells to effector cells.

In the donor cell, effectors are neutralized by an immunity protein. It is predicted that Tle proteins act in the periplasm of target cells, and it is not yet known if Tle lipases act on periplasm-exposed faces of the inner membrane, outer membrane, or both membranes. It also remains to be determined whether cell death results from a loss of membrane integrity, or due to perturbation of other pathways triggered by phospholipase activity.

The T6 secretion apparatus includes two proteins with homology to T4 bacteriophage tail spike proteins, including Hcp (haemolysis-co-regulated protein) and VrgG (valine-glycine repeat G), and these proteins are elaborated by the attacking bacterium when in contact with another cell (Silverman et al., 2012). As one would expect for toxins capable of intraspecies killing, the effector molecules have the potential to damage the donor cell, and thus must be co-expressed with neutralizing immunity proteins that repress activity until translocation out of the donor cell (Figure 1). A thorough bioinformatics analysis of the genomes of diverse Proteobacteria revealed an interesting pattern: genes encoding enzymes with predicted esterase or lipase activities were frequently adjacent to both a VrgG homolog and at least one small gene that is predicted to encode a periplasmically-localized immunity protein (Russell et al., 2013). Further analyses revealed that these previously uncharacterized enzymes represent a new superfamily of T6 lipase-like effector (Tle) gene families. The analysis of three different representative Tle family members from Vibrio cholerae, Burkholderia thailandensis, and Pseudomonas aeruginosa indeed found that these lipases are active against bacterial lipids and are lethal to Gram-negative cells (Russell et al., 2013).

Members of the Tle superfamily have different enzymatic activities. For example, a representative Tle1 from B. thailandensis acts as a phospholipase A2 while a Tle2 from V. cholerae has phospholipase A1 activity; phospholipases A1 and A2s release different acyl groups from phosphopholipid substrates (Russell et al., 2013). Mammalian phospholipase A enzymes, such as those in tears and serum (Nevalainen et al., 2008), have been long known for their contribution to bacterial killing, but a role for these enzymes in interactions between bacteria had not previously been described. A Tle5 family member from P. aeruginosa was previously characterized as PldA, a phospholipase D that removes the choline group from phosphatidylcholine (Wilderman et al., 2001). A lipidomics analysis of strains lacking the Tli5 immunity protein that suppresses the activity of PldA/Tle5, in conjunction with biochemical assays, revealed that phosphatidylethanolamine, one of the most abundant bacterial phospholipids, is also a PldA/Tle5 target (Russell et al., 2013).

Tle-containing operons are found across pathogenic, host-associated and free-living bacterial species (Russell et al., 2013), so while T6 secretion systems can play important roles in bacterium-eukaryote interactions (Bernard et al., 2010), these effector proteins likely have roles in microbial populations and communities. One can envision several ways in which T6SS-dependent killing within microbial populations is beneficial within a clonal population. As examples, the lysis of a fraction of cells may cause the release of biofilm-promoting DNA, killing may reduce types of competition that reduce fitness at the population level, or T6 secreted factors may shift the phenotype of a population by eliminating or altering cells with specific behaviors or markers. Alternatively, of course, the attack of clonal cells may be an unintended consequence of a killing mechanism that is most important for the elimination of competing bacterial species.

Insight into the biological roles of T6 secreted effectors can be gained through an understanding of how these pathways are regulated. Numerous stimuli affect the expression of T6 secretion systems and, not surprisingly, regulation across microbial species can differ (Bernard et al., 2010). Matters are further complicated by the fact that bacteria can possess several discrete T6 secretion apparatuses that are encoded at distinct genetic loci, each with a different pattern of induction. For example, in P. aeruginosa, there are three type VI secretion systems (H1-, H2- and H3-T6SS). The H2-T6SS delivers PldA/Tle5, while the H1- and H3-T6SSs transport two peptidoglycan-degrading effector molecules (Tse1 and Tse3) into the periplasm of target Gram-negatives (Russell et al., 2011). All three of these systems are regulated by quorum sensing, but the H1 and H3 systems display different patterns of expression ((Silverman et al., 2012) for review). Expression of the H2-T6SS genes is repressed by iron, likely via the iron-sensing repressor Fur. Regulation by iron availability and quorum sensing may reflect a role in host-pathogen interactions as iron is limiting at most host sites and quorum sensing positively regulates many aspects of virulence. Alternatively, this coordinate regulation could reflect that important nutrients like iron may be scarce in dense microbial communities, and activation of the H2-T6SS in some cells aids access to nutrients via the cannibalism of neighbors. A newly described process termed “T6SS dueling,” whereby P. aeruginosa cells respond to a T6SS-mediated membrane breach with increased expression of their own T6SS machinery to promote T6SS-mediated counterattacks, suggests that it is advantageous to be ever vigilant even within single species populations (Basler et al., 2013). The study of other factors that influence T6SS regulation will likely provide other important insights into the importance of the Tle families and other effectors in different settings.

As the authors point out, the Tle superfamilies from different groups have no detectable intergroup homology beyond some common catalytic motifs, suggesting that phospholipase-degrading enzymes have been incorporated as T6S effectors multiple times. Why could phospholipases be so important in T6S system-mediated interactions? It may be that the membrane is a wonderful target for antibacterial activities. Many antibacterial proteins target the membrane, as its components are conserved across species and thus membrane active factors have broad spectrum activity. Furthermore, the development of resistance to membrane targeting factors is challenging due to complex biosynthetic pathways and the importance of different membrane properties in protection and adaptation to different environments. Some of the newly identified Tle family members may have functions apart from bacterial killing. Phospholipases play very well documented roles in eukaryotic signaling pathways, and their importance in bacterial signaling may be under appreciated. In thinking about the activity of PldA/Tle5 in particular, it is interesting to consider whether the sharing of released products (such as choline, which is useful in osmoprotection, and ethanolamine, a important nutrient source (Garsin, 2010)) may enhance the fitness of a population. We may someday learn that the products of Tle activity in target cells play many different roles, particularly when activities within species and activities between species are considered.

In summary, Russell and colleagues (Russell et al., 2013) have built a compelling case that type VI delivered phospholipases can attack conserved cell membrane components in non-immunized target cells. Whether these molecules are strictly antagonistic, or whether they can serve more subtle roles within a population remains to be seen. The mapping of how multiple inputs converge to control type VI secretion system function in different environmental contexts is a critical next step in achieving a more complete understanding of the role that T6S-phospholipases and other T6S effectors play in both antagonistic and synergistic bacterium-bacterium relationships.