Virulence strategies of the respiratory pathogen Legionella pneumophila

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

The bacterium Legionella pneumophila is the causative agent of a potentially life-threatening pneumonia called Legionnaires' disease. Upon inhalation by humans, Legionella enters the lung where it can infect and replicate within alveolar macrophages, specialized immune cells. Instead of being degraded by macrophages, Legionella uses the infected cell for its intracellular replication cycle. If not treated promptly, this respiratory infection ends fatal in up to 30 percent of all cases. The number of Legionnaires' disease cases in the U.S. has increased four-fold over the past 15 years, making Legionella a significant health threat and a considerable economic burden. We are committed to studying how Legionella can bypass our immune system and cause disease so that we can develop better ways to counteract its virulence strategies. Humans are frequently exposed to Legionella since Legionella is ubiquitously found in freshwater habitats such as cooling towers, faucets, shower heads, or water fountains. Major outbreaks of Legionnaires' disease occur when water from contaminated sources is aerosolized and subsequently inhaled by humans. Immune-compromised individuals, infants, or the elderly are at an elevated risk of contracting an infection. Like many other microbial pathogens, Legionella bacteria have developed a variety of strategies to exploit their human host and to cause disease. They use a specialized protein translocation machine called Type IV Secretion System (T4SS) to inject an abundance of proteins, so-called effectors, into the infected host cell. The effectors modulate signaling events within the host to create conditions favorable for Legionella proliferation. Obtaining a detailed understanding of Legionella's effectors and its virulence strategy is essential for the development of novel therapeutics capable of preventing and treating this dangerous pneumonia and will profoundly improve people's lives and wellbeing. Over the past funding period, we have continued to make significant progress in deciphering the virulence strategies of Legionella pneumophila. While studying intracellular growth of Legionella within mammalian cells, we discovered that the host proto-oncoprotein N-Ras accumulated on Legionella-containing vacuoles (LCVs). This was an unusual finding as Ras proteins are best known for their role in cancer rather than microbial infection. NRas transmits extracellular inputs from the plasma membrane along various signaling pathways to promote cell growth, proliferation, and survival. If mutated, Ras GTPases are responsible for 30% of all human cancers, with N-Ras mutations being predominant in 94% of cutaneous melanomas. Human cells encode three highly homologous NRas isoforms, called HRas, KRas4A and KRas4B. To our surprise, we found that neither HRas nor KRas4A or KRas4B colocalized with LCVs, showing that vacuole localization was specific for NRas. Moreover, NRas did not localized to vacuoles containing avirulent bacteria, suggesting that one or more virulence proteins from Legionella were required for NRas recruitment. Using forward genetics, we discovered a single L. pneumophila gene, called denR, that, when mutated, rendered Legionella incapable of hijacking NRas to LCVs. To examine how NRas hijacking by Legionella DenR affected its signaling capability, we compared the global proteome landscape of cells infected with either L. pneumophila wildtype or L. pneumophila ΔdenR (a collaboration with Dr. Aleksandra Nita-Lazar, NIAID). Several pathways known to be associated with Ras downstream signaling were altered by DenR, most notably the well-characterized mitogen-activated protein kinase (MAPK) signaling pathway. In summary, these data provide evidence for a previously unrecognized involvement of the proto-oncoprotein NRas in microbial pathogenesis and show that L. pneumophila encodes the effector DenR to take control of NRas and downregulate MAPK signaling during infection. Lastly, the finding that a virulence protein is capable of downregulating signaling by a proto-oncoprotein will open new avenues for the development of therapeutics aimed at interfering with genetic Ras malignancies. In a follow-up study, we more closely examined how DenR can hijack human NRas during infection. We discovered that DenR is a molecular mimic of human enzymes called palmitoyl transferases (PATS). PATs catalyze the covalent attachment of a fatty acid called palmitic acid to proteins to increase their affinity for membranes. Once NRas has been palmitoylated by DenR, it binds to membranes, including the membrane of the Legionella-containing vacuole, where it can then be utilized by Legionella. The finding that DenR catalyzes a palmitoylation reaction was remarkable since this form of protein lipidation was not known to exist in bacterial, only in eukaryotic proteins. We believe that during evolution DenR was acquired by Legionella from one of its host cells and is now being used by the bacterium against the host. DenR is also special in that it is not an integral membrane proteins like all other known PATs. Instead, DenR is in the watery phase of the cell called the cytosol, and associates with membranes only peripherally to catalyze palmitoylation. In summary, these findings show that bacterial proteins like DenR are capable of catalyzing palmitoylation without being located inside a membrane, that other enzyme like DenR may exist in other bacteria or even human cells that can lipidate proteins, and that protein like NRas that were believed to only play a role in cancer can play a dual role during microbial infection. .