Deciphering microbial virulence mechanisms during Legionella pneumophila infection

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. Previous investigations of Legionella have been confounded by the fact that this bacterium produces nearly 300 effectors, which often have overlapping functions. Functional redundancy among these effectors represents a challenge to investigators to identify the most critical of these effectors the most promising drug targets. We have now developed a novel gene silencing tool in Legionella that harnesses the power of CRISPR-interference (CRISPRi) to suppress not only individual genes but entire groups of bacterial genes. Using this CRISPRi tool, we interrogated more than 200 virulence factors from Legionella pneumophila and are now observing phenotypes in an intracellular pathogen in which few had previously been reported, thus laying the foundation for decrypting the mechanisms of Legionella pneumophila virulence. More recently, we generated an improved CRISPRi tool that allows multiplexed gene silencing to look for genes that, when silenced simultaneously, render Legionella less virulent. In a proof-of-concept study, we used this approach to probe a group of highly conserved transmembrane effectors for their importance during replication of Legionella in human macrophages. Several gene combinations were identified as vital, and those hits have become the focus of our future research with the goal of developing inhibitory compounds. During infection of human immune cells, Legionella resides within a membrane-enclosed compartment, or vacuole, to his from the host cell. Yet, this 'save haven' represents a challenge when the bacteria start to replicate, as the surrounding vacuole has to be expanded as well to give space to the growing number of Legionella progeny. Our studies discovered that Legionella controls vacuole expansion using the virulence factor VpdC. VpdC catalytically modifies the lipid composition of the vacuolar membrane to promote its expansion. Too much or too little VpdC interfered with proper vacuole expansion and rendered Legionella less virulent, suggesting that blocking the coordinated expansion of their vacuole is a novel therapeutic approach to treat infections with Legionella and related pathogens.