Investigating host-induced lipopolysaccharide changes in the legume symbiont Sinorhizobium meliloti

Bacteria can have both beneficial and negative impacts upon life. For example, the interaction between the soil bacterium, Sinorhizobium meliloti, and leguminous plants is beneficial since the bacteria convert nitrogen from the atmosphere into a form which can be utilised by plants. In return, the bacteria obtain nutrients from the plant. The outcome is a symbiosis where the bacteria survive long-term within the plant and promote plant growth. I am interested in understanding the factors, which enable the bacteria to survive long-term within plant cells. This is important since leguminous plants are major food sources and, since the nitrogen source produced by the bacteria is eventually released into the soil, this process alleviates the need to add costly nitrogen fertilisers to the soil. Understanding how processes work, is the first step towards improving processes. Interestingly, the soil bacterium is related to another bacterium, Brucella abortus, which causes abortions in cattle and results in a severe infection in humans known as Brucellosis. The factors important for Brucella to cause disease are poorly understood, but like the soil bacteria within plant cells, Brucella can survive long-term within animal and human cells. Cattle can be vaccinated to prevent Brucella infection, but the vaccine used is largely uncharacterised. Unfortunately, there is no human vaccine against Brucella and this is a problem given that Brucella are potential bioterrorism agents. Thus, understanding more about Brucella infections could help in the development of a human vaccine and improve the exisiting cattle vaccine. Recent evidence suggest that there are commonalities in the strategies used by the soil bacterium and Brucella to survive long-term within plant and animal/human cells, respectively. My research suggests that structures on the outside of the bacterial cell known as lipopolysaccharides (LPS) are important for long-term survival. By using the soil bacterium-plant interaction as a model system, I identified that the bacterial LPS changes during the interaction with the plant. Thus, the overall goal of this proposal is to characterise these plant-induced LPS changes, to determine how the bacterial cell brings about these LPS changes and to determine whether these LPS changes are involved in the long-term surival of the soil bacterium within plant. The outcome of these studies could not only improve our knowledge of the symbiosis but could also provide insights into how bacteria can surive long-term within animal and human cells.

Sinorhizobium meliloti, a beneficial legume symbiont, and Brucella abortus, a phylogenetically related mammalian pathogen, both form chronic intracellular infections in their respective hosts. The BacA protein is essential for host persistence of S. meliloti and B. abortus. I discovered that BacA affects an unusual, very-long-chain fatty acid (VLCFA), modification of the lipid A of both S. meliloti and B. abortus. The lipid A is a component of the lipopolysaccharide (LPS), which forms the outermost leaflet of the outer membrane. In wild-type S. meliloti and B. abortus, every lipid A is modified with a VLCFA. In contrast, aprrox. 50 per cent of the lipid A molecules extracted from S. meliloti and B. abortus bacA null mutants lack a VLCFA. Additionally, analysis of a series of S. meliloti site-directed bacA mutants, which are defective in the legume symbiosis, revealed alterations in their lipid A VLCFA content. Taken together, these data suggest that the VLCFA-modification of the lipid A could be important for host persistence. Based on sequence similarity to a family of eukaryotic peroxisomal membrane proteins, I proposed a model whereby BacA is involved in the transport of activated VLCFAs out of the cytoplasm, where they could then be used to modify the lipid A in the outer membrane. To further investigate the effect of lipid A on host persistence, I constructed and characterised S. meliloti mutants in the acpXL and lpxXL genes, which encode a VLCFA-acyl carrier protein and a VLCFA-acyl transferase protein, respectively. Although the lipid A molecules of acpXL and lpxXL mutants lacked VLCFAs, during growth in complex medium, these mutants could still form a symbiosis, despite being substantially less competitive relative to the wild-type strain. Thus, these data showed that the VLCFA-modified lipid A, observed during growth in complex medium, is important but not essential for the symbiosis. Preliminary studies with Rhizobium leguminosarum, found that LPS hydrophobicity increases during the interaction with the host, due at least in part, to additional VLCFA modifications. Moreover, some of these LPS changes could be reproduced by altering the growth conditions of free-living R. leguminosarum and could also occur in the absence of AcpXL. Since I discovered a similar increase in S. meliloti LPS hydrophobicity during the interaction with alfalfa, these data suggest that S. meliloti lipid A could be further modified with VLCFAs during the symbiosis and that proteins other than AcpXL could be involved. In contrast, BacA may be essential for the host-induced additional lipid A VLCFA modification and, if BacA is directly involved in VLCFA transport, it would provide a means by which the bacterial cell could modify pre-existing lipid A without the need for costly de novo synthesis. Thus, the overall aim of this proposal is to understand the LPS changes that occur in S. meliloti during the legume symbiosis, to identify the proteins involved and then to determine the function of these LPS changes on host persistence. By these combined approaches, I will understand more about the biosynthesis and function of host-induced LPS alterations in S. meliloti. However, these studies could also provide insights into the functions of LPS modifications in other legume symbionts and chronic mammalian pathogens.