Legionella pneumophila is a Gram-negative bacterium that can colonize both freshwater protozoa and human alveolar macrophages, the latter infection resulting in Legionnaires’ disease. The intracellular lifecycle of L. pneumophila requires extensive manipulation of its host cell, which is carried out by effector proteins that are translocated into the host cell through the Dot/Icm type IV secretion system. This study focuses on a pair of highly similar type IV substrates called YlfA/LegC7 and YlfB/LegC2 that were initially identified in a screen for proteins that cause growth inhibition in yeast. Analysis of truncation mutants revealed that the hydrophobic residues in the Ylf amino termini were required for localization of each protein to the membranes of host cells. Central and carboxy terminal coiled coil domains were found to mediate binding of YlfA and YlfB to themselves and to each other. In vivo, a ΔylfA ΔylfB double mutant strain of L. pneumophila was shown to be defective in establishing a vacuole that supports bacterial replication. This phenotype was subsequently correlated with a decrease in the association of endoplasmic reticulum (ER)-derived vesicles with vacuoles containing ΔylfA ΔylfB mutant bacteria. These data suggest that the Ylf proteins are membrane-associated effectors that enhance remodeling of the L. pneumophila -containing vacuole by promoting association and possibly fusion of ER-derived membrane vesicles with the bacterial compartment.

Legionella pneumophila is a Gram-negative, facultative intracellular pathogen that causes severe pneumonia known as Legionnaires’ disease. The bacterium causes disease when contaminated water is aerosolized and subsequently inhaled by individuals, which allows the bacteria to gain access to the lungs, where they infect alveolar macrophages. L. pneumophila is ubiquitous in the environment, where it survives by growing in biofilms, intracellularly within protozoa, and planktonically. Biofilms are a major concern for public health because they provide a protective niche that allows for the continuous leaching of bacteria into the water supply. In addition, biofilms enhance the survival of the bacteria by increasing resistance to temperature fluctuations and antimicrobial agents. Currently, there is little known about biofilm formation and regulation by L. pneumophila. Here, we present evidence of a specific gene, bffA, which appears to be involved in the regulation of motility, biofilm formation, cellular replication, and virulence of L. pneumophila. A strain lacking bffA has an enhanced biofilm formation phenotype, forming biofilms that are both faster and thicker than wild type. Additionally, the knockout strain has significantly reduced motility, enhanced uptake into amoebae, and altered growth kinetics on solid media. Our data suggest a potential role for bffA in signaling pathways that govern changes in growth rate and motility in response to environmental conditions.

The OmpA-like protein domain has been associated with peptidoglycan binding proteins, and is often found in virulence factors of bacterial pathogens. The intracellular pathogen Legionella pneumophila encodes for six proteins that contain the OmpA-like domain, among them the highly conserved uncharacterized protein we named CmpA. Here we set out to characterize the CmpA protein and determine its contribution to intracellular survival of L. pneumophila . Secondary structure analysis suggests that CmpA is an inner membrane protein with a peptidoglycan binding domain at the C-teminus. A cmpA mutant was able to replicate normally in broth, but failed to compete with an isogenic wild type strain in an intracellular growth competition assay. The cmpA mutant also displayed significant intracellular growth defects in both the protozoan host Acanthamoeba castellanii , as well as in primary bone marrow derived macrophages, where uptake into the cells was also impaired. The cmpA phenotypes were completely restored upon expression of CmpA in trans . The data presented here establishes CmpA as a novel virulence factor of L. pneumophila that is required for efficient intracellular replication in both mammalian and protozoan hosts.

Background: Legionella are parasites of freshwater protozoa, responsible for Legionellosis. Legionella can be found in a variety of aquatic environments, including rivers, lakes, and springs, as well as in engineered water systems where they can potentially lead to human disease outbreaks. Legionella are considered to be predominantly freshwater organisms with a limited ability to proliferate in saline environments. Exposure of Legionella to high sodium concentrations inhibits growth and virulence of laboratory strains, particularly under elevated temperatures. Nonetheless, Legionella have been identified in some saline environments where they likely interact with various protozoan hosts. In this work, we examine how these selection pressures, sodium and grazing, help shape Legionella ecology within natural environments. Utilizing Legionella-specific primers targeting a variable region of the Legionella 16S rRNA gene, we characterized Legionella abundance, diversity, and community composition in natural spring clusters of varying sodium concentrations, focusing on high sodium concentrations and elevated temperatures. Results: We observed the highest abundance of Legionella in spring clusters of high salinity, particularly in combination with elevated temperatures. Legionella abundance was strongly related to sodium concentrations. The Legionella community structure in saline environments was characterized by relatively low diversity, compared to spring clusters of lower salinity. The community composition in high salinity was characterized by few dominant Legionella genotypes, not related to previously described species. Protozoan microbial community structure and composition patterns resembled those of Legionella, suggesting a common response to similar selection pressures. We examined Legionella co-occurrence with potential protozoan hosts and found associations with Ciliophora and Amoebozoa representatives. Conclusions: Our results indicate that selection forces in saline environments favor a small yet dominant group of Legionella species that are not closely related to known species. These novel environmental genotypes interact with various protozoan hosts, under environmental conditions of high salinity. Our findings suggest that alternative survival mechanisms are utilized by these species, representing mechanisms distinct from those of well-studied laboratory strains. Our study demonstrate how salinity can shape communities of opportunistic pathogens and their hosts, in natural environments, shedding light on evolutionary forces acting within these complex environments.