How Symbiotic Bacteria Survive Host Defenses

There’s a surprisingly fine line between bacterial symbiosis and chronic infection. While one is beneficial and the other detrimental, recent findings suggest that they share mechanisms for sidestepping host defenses. Plants in the pea family (legumes) have symbiotic nitrogen-fixing bacteria living in root nodule compartments that also contain antimicrobial compounds. For example, the nitrogenfixer Sinorhizobium meliloti survives antimicrobial compounds called nodule-specific, cysteine-rich (NCR) peptides that are produced by alfalfa and related legumes. Intriguingly, S. meliloti is closely related to Brucella abortus, which causes abortions in cattle and can also cause debilitating chronic infections in people. Moreover, the host legume antimicrobial peptides that S. meliloti evades are similar to defensins, small proteins that help plants and animals kill bacteria. Once inside root nodule compartments, S. meliloti differentiates from the free-living form into elongated bacteroids that fix nitrogen. This differentiation is mediated by antimicrobial NCR peptides on the plant side, but the corresponding factors on the bacterial side are unknown. An S. meliloti cytoplasmic membrane protein called BacA has long been known to be essential for bacteroid development in alfalfa relatives, but its role has remained uncertain. Recently, however, BacA has been linked to NCR peptides: while BacA is required for S. meliloti differentiation in legumes that produce NCR peptides (such as alfalfa and peas), this protein is not required for bacteroid development in legumes that lack these peptides (such as beans and lotus). Now, in this issue of PLoS Biology, Mergaert, Ferguson, and colleagues present compelling evidence that BacA is key to protecting S. meliloti from antimicrobial NCR peptides produced by host legumes. To explore the role of BacA further, the researchers synthesized an antimicrobial peptide (NCR247) of the alfalfa relative Medicago truncatula and compared its effects on cultured S. meliloti with and without BacA. They found that while NCR247 decreases wild-type S. meliloti colony formation, this inhibition is more pronounced in a mutant strain that lacks BacA. In addition, this mutant’s growth was restored by a plasmid carrying the bacA gene. Taken together, these findings suggest that BacA protects cultured S. meliloti against NCR peptides. To test the effects of BacA in living plants, the researchers compared the viability of the wild-type and BacA-deficient S. meliloti strains. Nitrogen-fixing bacteria use infection threads to enter root nodule compartments, which contain NCR peptides that damage the cytoplasmic membrane of S. meliloti. Using a fluorescent dye test that stains dead bacteria red and stains living bacteria green, the researchers showed that both the wild-type and BacA-deficient S. meliloti were alive in the infection threads. In contrast, while the wild-type bacteria were still alive once inside the host root nodule compartments, most of the BacA-deficient mutants were dead. This suggests that without the protection of BacA, S. meliloti is rapidly killed by host antimicrobial NCR peptides in the compartments. To confirm the hypothesis that BacA protects S. meliloti against antimicrobial NCR peptides, the researchers also applied the fluorescent dye test to an M. truncatula mutant that blocks NCR peptide transport into root nodule compartments. As expected, both wild-type and BacAdeficient S. meliloti thrived within this mutant’s host compartments. Extending the similarities between the symbiotic S. meliloti and bacteria that cause chronic infections, BacA-like proteins are also found in B. abortus and Mycobacterium tuberculosis. In addition, the B. abortus BacA is essential for chronic infections in mammals. Previous work had shown that B. abortus BacA protects S. meliloti mutants that lack this protein against antimicrobial peptides in alfalfa relatives, and the researchers likewise found that B. abortus BacA protected these S. meliloti mutants against the synthesized antimicrobial peptide (NCR247) in culture. Altogether, these findings suggest a functional similarity between the S. meliloti and B. abortus BacA proteins, and show that the latter protects against antimicrobial activity like that of the defensins found in its mammalian hosts. This work suggests that by protecting S. meliloti against antimicrobial NCR peptides, BacA is key to establishing the symbiosis between these nitrogen-fixing bacteria and their legume hosts. How does BacA confer protection and limit the damage these compounds cause to the S. meliloti cytoplasmic membrane? Possibilities include a direct role for BacA in NCR transport or an indirect effect of BacA on outer membrane lipids. Because BacA is also key to establishing pathogenic bacteria including B. abortus in animal hosts, the answers could have widespread implications for understanding and ultimately treating such chronic infections in mammals.

There's a surprisingly fine line between bacterial symbiosis and chronic infection. While one is beneficial and the other detrimental, recent findings suggest that they share mechanisms for sidestepping host defenses. Plants in the pea family (legumes) have symbiotic nitrogen-fixing bacteria living in root nodule compartments that also contain antimicrobial compounds. For example, the nitrogenfixer Sinorhizobium meliloti survives antimicrobial compounds called nodule-specific, cysteine-rich (NCR) peptides that are produced by alfalfa and related legumes. Intriguingly, S. meliloti is closely related to Brucella abortus, which causes abortions in cattle and can also cause debilitating chronic infections in people. Moreover, the host legume antimicrobial peptides that S. meliloti evades are similar to defensins, small proteins that help plants and animals kill bacteria.
Once inside root nodule compartments, S. meliloti differentiates from the free-living form into elongated bacteroids that fix nitrogen. This differentiation is mediated by antimicrobial NCR peptides on the plant side, but the corresponding factors on the bacterial side are unknown. An S. meliloti cytoplasmic membrane protein called BacA has long been known to be essential for bacteroid development in alfalfa relatives, but its role has remained uncertain. Recently, however, BacA has been linked to NCR peptides: while BacA is required for S. meliloti differentiation in legumes that produce NCR peptides (such as alfalfa and peas), this protein is not required for bacteroid development in legumes that lack these peptides (such as beans and lotus). Now, in this issue of PLoS Biology, Mergaert, Ferguson, and colleagues present compelling evidence that BacA is key to protecting S. meliloti from antimicrobial NCR peptides produced by host legumes.
To explore the role of BacA further, the researchers synthesized an antimicrobial peptide (NCR247) of the alfalfa relative Medicago truncatula and compared its effects on cultured S. meliloti with and without BacA. They found that while NCR247 decreases wild-type S. meliloti colony formation, this inhibition is more pronounced in a mutant strain that lacks BacA. In addition, this mutant's growth was restored by a plasmid carrying the bacA gene. Taken together, these findings suggest that BacA protects cultured S. meliloti against NCR peptides.
To test the effects of BacA in living plants, the researchers compared the viability of the wild-type and BacA-deficient S. meliloti strains. Nitrogen-fixing bacteria use infection threads to enter root nodule compartments, which contain NCR peptides that damage the cytoplasmic membrane of S. meliloti. Using a fluorescent dye test that stains dead bacteria red and stains living bacteria green, the researchers showed that both the wild-type and BacA-deficient S. meliloti were alive in the infection threads. In contrast, while the wild-type bacteria were still alive once inside the host root nodule compartments, most of the BacA-deficient mutants were dead. This suggests that without the protection of BacA, S. meliloti is rapidly killed by host antimicrobial NCR peptides in the compartments.
To confirm the hypothesis that BacA protects S. meliloti against antimicrobial NCR peptides, the researchers also applied the fluorescent dye test to an M. truncatula mutant that blocks NCR peptide transport into root nodule compartments. As expected, both wild-type and BacAdeficient S. meliloti thrived within this mutant's host compartments.
Extending the similarities between the symbiotic S. meliloti and bacteria that cause chronic infections, BacA-like proteins are also found in B. abortus and Mycobacterium tuberculosis. In addition, the B. abortus BacA is essential for chronic infections in mammals. Previous work had shown that B. abortus BacA protects S. meliloti mutants that lack this protein against antimicrobial peptides in alfalfa relatives, and the researchers likewise found that B. abortus BacA protected these S. meliloti mutants against the synthesized antimicrobial peptide (NCR247) in culture. Altogether, these findings suggest a functional similarity between the S. meliloti and B. abortus BacA proteins, and show that the latter protects against antimicrobial activity like that of the defensins found in its mammalian hosts. This work suggests that by protecting S. meliloti against antimicrobial NCR peptides, BacA is key to establishing the symbiosis between these nitrogen-fixing bacteria and their legume hosts. How does BacA confer protection and limit the damage these compounds cause to the S. meliloti cytoplasmic membrane? Possibilities include a direct role for BacA in NCR transport or an indirect effect of BacA on outer membrane lipids. Because BacA is also key to establishing pathogenic bacteria including B. abortus in animal hosts, the answers could have widespread implications for understanding and ultimately treating such chronic infections in mammals.