Legionella in soil: biotic and abiotic controls of pathogenicity

Soil harbors many pathogens of public health concern, but the ecology of these pathogens in the context of the soil microbiome is far from clear. Amoeba-associated bacteria Legionella spp. are the causal agents of environmentally acquired pneumonia

legionellosis. The exponential increase in legionellosis incidence rates in the past two decades correlated significantly with

temperature and extreme precipitation events in the US, implying augmented risks due to interactions between environmental

Legionella with climate change. However, how climate change affects Legionella’s abundance, distribution, association with amoeba host, and infectivity have not been systematically evaluated.

Our long-term goal is to establish a framework for soil-borne pathogens’ risk assessment based on mechanistic understandings of

microbiome (biotic) and environmental (abiotic) controls on their persistence and proliferation. The specific objectives of this

project are to 1) investigate the distribution of Legionella and host amoeba in terrestrial environments along temperature and

precipitation gradients for risk predictive mapping, and 2) test if these climate factors control Legionella–amoeba association, which

contributes to infectivity. The central hypothesis is that climate/edaphic conditions and interaction with amoeba host co-determine

Legionella’s persistence in terrestrial environments. We will test this hypothesis utilizing the broad range of soil types, ecozones, and environmental gradients on O‘ahu. This project addresses the following specific aims. Specific Aim 1. Map distributions of soil Legionella and host amoeba along environmental gradients.

Approach: Soil samples from a temperature and precipitation gradient on O‘ahu will be collected and analyzed together with

Hawai’i’s microbiome data archive for identification and quantification of Legionella and potential hosts. We will also acquire novel

isolates of environmental Legionella. Legionella distribution and abundance will be linked to climate and edaphic environments. Specific Aim 2. Determine biotic and environmental influence on Legionella’s intracellular lifestyle.

Approach: A size-filtering technique will be developed to concentrate free-living Legionella vs. host amoeba cells for community

characterization. Legionella composition will be compared between the two groups to identify representative sequences and

isolates. Comparative genomics will be performed on representative isolates to find differences in the two groups’ virulence factors.

Representative sequences will be matched to environmental samples to connect Legionella’s lifestyle to environmental conditions. Specific Aim 3. Quantify effects of soil water connectivity on Legionella – amoeba association.

Approach: A transparent micromodel reconstructed based on real soil’s pore structures will be kept at varied water saturation

levels. Legionella and host amoeba cultures will be inoculated at separate locations on the micromodel. Movements of microbial cells and spatially resolved metabolomics will be used to monitor the timing and signaling of interactions.