Mediators of Lipid A Modification, RNA Degradation, and Central Intermediary Metabolism Facilitate the Growth of Legionella pneumophila at Low Temperatures

Abstract

Legionella pneumophila is an aquatic bacterium that is also the agent of Legionnaires' disease pneumonia. Since L. pneumophila is transmitted directly from the environment to the lung, it is important to understand how legionellae survive at low temperatures. To identify genes that are needed for L. pneumophila growth at low temperature, we screened a population of mutagenized legionellae for strains that are specifically impaired for growth at 17°C. From the 7400 mutants tested, eleven displayed defects ranging from ca. 10-fold to a complete inability to grow at the low temperature. PCR and sequence analysis were then utilized to identify the genes whose loss had compromised growth. The proteins thereby implicated in low-temperature growth included components of the type II secretion system (LspE, LspG, LspH), a lipid A biosynthetic enzyme (LpxP), a ribonuclease (RNAse R), an RNA helicase (CsdA/DeaD), TCA cycle enzymes (citrate synthase), enzymes linked to fatty acid (FadB) or amino acid (aspartate aminotransferase) catabolism, and two putative membrane proteins that were, based upon their sequences, unlike previously characterized proteins. Given the magnitude of their mutant's defect, the aspartate aminotransferase, RNA helicase, and one of the putative membrane proteins were the factors most critical for L. pneumophila low-temperature growth. Thus, L. pneumophila not only employs some of the same processes and factors as other bacteria do in order to survive at low temperatures (e.g., LpxP, CsdA) but it also appears to possess novel modes of cold adaptation.Legionella pneumophila is a gram-negative γ-proteobacterium that is ubiquitous in natural and man-made water systems [17,29,45,60]. In its aquatic habitats, it exists planktonically, as an intracellular parasite of protozoa, and as a component of biofilms [35,39,45]. However, the ubiquity of L. pneumophila is also due to its capacity to survive at many temperatures, including those between 63°C and 4°C [17,29,56,60]. Beyond its natural niche, L. pneumophila is also a human pathogen, whereby the inhalation of contaminated water droplets from aerosolgenerating devices results in Legionnaires' disease [15]. Given that L. pneumophila is transmitted to humans directly from water sources, it is important to understand how the legionellae survive in water, including how they adapt to high and low temperature conditions in order to survive in both natural habitats throughout the year, from the warmer to colder months, and man-made systems that provide both warm and cold water. Since L. pneumophila grows best in the laboratory at 32 to 37°C, there are many studies devoted to understanding its physiology at the higher temperatures [56]. In contrast, studies on L. pneumophila at low temperatures are sparse, even though a large number of studies have identified factors that facilitate the low-temperature growth of other bacteria [7,9,43] [12,14,48,54]. Finally, a secreted peptidyl-prolyl isomerase (PpiB), whose secretion is not dependent upon T2S or the known...

Multiple Legionella pneumophila Type II Secretion Substrates, Including a Novel Protein, Contribute to Differential Infection of the Amoebae Acanthamoeba castellanii, Hartmannella vermiformis, and Naegleria lovaniensis

“…The T2S mutants are not impaired for entry into the amoebae, indicating that T2S is promoting bacterial resistance to intracellular killing and/or facilitating replication itself (22). Others of our studies have shown that T2S also promotes the persistence of L. pneumophila in the environment by facilitating extracellular survival in low-temperature water samples (18,(22)(23)(24)(25). Furthermore, L. pneumophila T2S helps to mediate the secretion of a surfactant that confers both sliding motility and antibacterial activity (26,27).…”

Molecular Pathogenesis of Infections Caused byLegionella pneumophila

“…23,2010 LEGIONELLA PATHOGENESIS 283 17°C, and these included mutants with insertions into components of the T2SS. Other major factors included a homologue of the LpxP lipid A acyltransferase; an RNA helicase, CsdA, which was particularly important for bacterial survival; and RNase R, which was implicated previously in replication at low temperatures (70,309). Recently, L. pneumophila was observed to undergo sliding motility on agar plates, and this phenotype was linked to the function of the Lsp secretion system (323).…”

Legionella pneumophila Type II Secretion Dampens the Cytokine Response of Infected Macrophages and Epithelia

“…T2S substrates first translocate across the inner membrane and into the periplasm by the Sec or Tat pathway, and then a pseudopilus acts to push the proteins through a dedicated outer membrane pore (27,58). For environmental L. pneumophila, an intact T2S system (known as Lsp) is required for both intracellular growth in amoebal hosts and extracellular survival in lowtemperature water samples (27,52,61,106). Following their intratracheal inoculation into A/J mice, lsp mutants do not increase in numbers in the lung, indicating that Lsp is also critical for L. pneumophila infection of and pathogenesis in mammalian hosts (27,34,96).…”