RpoS co-operates with other factors to induce Legionella pneumophila virulence in the stationary phase

Legionella pneumophila replicates within amoebae and macrophages and causes the severe pneumonia Legionnaires' disease. When broth cultures enter the post-exponential growth (PE) phase or experience amino acid limitation, L. pneumophila accumulates the stringent response signal (p)ppGpp and expresses traits likely to promote transmission to a new phagocyte. The hypothesis that a stringent response mechanism regulates L. pneumophila virulence was bolstered by our finding that the avirulent mutant Lp120 contains an internal deletion in the gene encoding the stationary phase sigma factor RpoS. To test directly whether RpoS co-ordinates virulence with stationary phase, isogenic wild-type, rpoS-120 and rpoS null mutant strains were constructed and analysed. PE phase L. pneumophila became cytotoxic by an RpoS-independent pathway, but their sodium sensitivity and maximal expression of flagellin required RpoS. Likewise, full induction of sodium sensitivity by experimentally induced (p)ppGpp synthesis required RpoS. To replicate efficiently in macrophages, L. pneumophila used both RpoS-dependent and -independent pathways. Like those containing the dotA type IV secretory apparatus mutant, phagosomes harbouring either rpoS or dotA rpoS mutants rapidly acquired the late endosomal protein LAMP-1, but not the lysosomal marker Texas red-ovalbumin. Together, the data support a model in which RpoS co-operates with other regulators to induce L. pneumophila virulence in the PE phase.     

mRNA translation in yeast during entry into stationary phase

The expression of some Saccharomyces cerevisiae genes is induced as cells enter stationary phase. Their mRNAs are translated during a period in the growth cycle when the translational apparatus is relatively inert, thereby raising the possibility that these mRNAs compete effectively for a limiting pool of translation factors. To test this idea, the translation of mRNAs carrying different 5′-leaders was compared during exponential growth and after entry into stationary phase upon glucose starvation. Closely related sets of lacZ mRNAs, carrying 5′-leaders from the PYK1, PGK1, RpL3, Rp29, HSP12, HSP26 or THI4 mRNAs, were studied. These mRNAs displayed differing translational efficiencies during exponential growth, but their relative translatabilities were not significantly affected by entry into stationary phase, indicating that they compete just as effectively under these conditions. Polysome analysis revealed that the wild-type PYK1, ACT1 and HSP26 mRNAs are all translated efficiently during stationary phase, when the translational apparatus is relatively inert. Also, significant levels of the translation initiation factors eIF-2α, eIF-4E and eIF-4A were maintained during the growth cycle. These data are consistent with the idea that, while translational activity decreases dramatically during entry into stationary phase, yeast cells maintain excess translational capacity under these conditions. Received: 31 March 1998 / Accepted: 4 May 1998     

Legionella pneumophila virulence conserved after multiple single-colony passage on agar

Multiple passage ofLegionella pneumophila on either supplemented Mueller-Hinton or charcoal yeast extract agar by the conventional batch passing technique results in loss of virulence. In this studyL. pneumophila virulence was maintained after multiple passage on buffered charcoal yeast extract (BCYE ) agar by a single-colony transfer technique. Virulence was determined by assessing the growth ofL. pneumophila for thioglycolate-induced susceptible A/J mouse macrophages in vitro and infectivity for susceptible A/J mice in vivo. Passage of the virulentL. pneumophila, 30 times on BCYE agar by the single-colony transfer procedure still resulted in virulence, as compared with the nonpassaged parental bacteria, both in vitro and in vivo. Lethality for susceptible, A/J mice by systemic infection was comparable for the 30th colony-passaged bacteria and the parentalL. pneumophila. These results show that theL. pneumophila phenotype associated with intracellular growth in macrophages or infectivity for susceptible mice is stable following passage by the single-colony transfer procedure on BCYE agar.     

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species.     

Induction of entry into the stationary growth phase in Pseudomonas aeruginosa by N-acylhomoserine lactone

N-acylhomoserine lactone (AHSL, autoinducer) is capable of regulating a set of genes by sensing cell density and developing an intercellular communication in Pseudomonas aeruginosa. Addition of AHSL in the exponential growth phase, regardless of cell density, induces a repression of cell growth of P. aeruginosa, an expression of stationary phase specific factor σ^s in vivo and a morphological change into smaller spherical shape indistinguishable from that in the stationary phase. It is demonstrated that AHSL can trigger an entry of bacteria into stationary phase as a growth controlling signal.     

Iron Acquisition by Legionella pneumophila

For nearly 20 years, it was believed that Legionella pneumophila does not produce siderophores. Yet, we have now determined that L. pneumophila secretes a siderophore (legiobactin) that is detectable by the CAS assay. We have optimized conditions for legiobactin expression, shown its biological activity, and found genes (lbtAB) involved in its production and secretion. LbtA is homologous with siderophore synthetases from E. coli (aerobactin), Sinorhizobium (rhizobactin), and Bordetella (alcaligin), while LbtB is a member of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtAB produce 40–70% less CAS reactivity. The lbtA mutant is also defective for growth in deferrated media containing citrate, indicating that legiobactin is required in conditions of severe iron limitation. lbtAB mutants grow normally in macrophages and amoebae host cells as well as within the lungs of mice. L. pneumophila does express lbtA in macrophages, suggesting that legiobactin has a dispensable role in infection. Legiobactin is iron repressed and does not react in the Csáky and Arnow assays. Anion-exchange HPLC has been used to purify legiobactin, and thus far, structural analysis suggests that the molecule is similar but not identical to rhizobactin, rhizoferrin, and alcaligin. The residual CAS reactivity present in supernatants of the lbtAB mutants suggests that L. pneumophila might produce a second siderophore. Besides siderophores, we have determined that ferrous iron transport, encoded by feoB, is critical for L. pneumophila growth in low-iron conditions, in host cells, and in the mammalian lung. Some of our other studies have discovered a critical, yet undefined, role for the L. pneumophila cytochrome c maturation locus in low-iron growth, intracellular infection, and virulence.     

Legionella pneumophila couples fatty acid flux to microbial differentiation and virulence

During its life cycle, Legionella pneumophila alternates between at least two phenotypes: a resilient, infectious form equipped for transmission and a replicative cell type that grows in amoebae and macrophages. Considering its versatility, we postulated that multiple cues regulate L. pneumophila differentiation. Beginning with a Biolog Phenotype MicroArray screen, we demonstrate that excess short-chain fatty acids (SCFAs) trigger replicative cells to cease growth and activate their panel of transmissive traits. To co-ordinate their response to SCFAs, L. pneumophila utilizes the LetA/LetS two-component system, but not phosphotransacetylase or acetyl kinase, two enzymes that generate high-energy phosphate intermediates. Instead, the stringent response enzyme SpoT appears to monitor fatty acid biosynthesis to govern transmission trait expression, as an altered distribution of acylated acyl carrier proteins correlated with the SpoT-dependent differentiation of cells treated with either excess SCFAs or the fatty acid biosynthesis inhibitors cerulenin and 5-(tetradecyloxy)-2-furoic acid. We postulate that, by exploiting the stringent response pathway to couple cellular differentiation to its metabolic state, L. pneumophila swiftly acclimates to stresses encountered in its host or the environment, thereby enhancing its overall fitness.     

Quorum sensing and starvation: signals for entry into stationary phase

Quorum sensing occurs at high cell density in many microorganisms. It regulates specialized processes such as genetic competence, bioluminescence, virulence, and sporulation. However, recent evidence suggests that quorum-sensing may play a more central role in the physiology of bacteria, where quorum-sensing pathways converge with starvation-sensing pathways to regulate cell entry into stationary phase.     

Phosphatidylcholine synthesis is required for optimal function of Legionella pneumophila virulence determinants

The function of phosphatidylcholine (PC) in the bacterial cell envelope remains cryptic. We show here that productive interaction of the respiratory pathogen Legionella pneumophila with host cells requires bacterial PC. Synthesis of the lipid in L. pneumophila was shown to occur via either phospholipid N -methyltransferase (PmtA) or phosphatidylcholine synthase (PcsA), but the latter pathway was demonstrated to be of predominant importance. Loss of PC from the cell envelope caused lowered yields of L. pneumophila within macrophages as well as loss of high multiplicity cytotoxicity, while mutants defective in PC synthesis could be complemented either by reintroduction of PcsA or by overproduction of PmtA. The lowered yields and reduced cytotoxicity in mutants with defective PC biosynthesis were due to three related defects. First, there was a poorly functioning Dot/Icm apparatus, which delivers substrates required for intracellular growth into the cytosol of infected cells. Second, there was reduced bacterial binding to macrophages, possibly due to loss of PC or a PC derivative on the bacterium that is recognized by the host cell. Finally, strains lacking PC had low steady-state levels of flagellin protein, a deficit that had been previously associated with the phenotypes of lowered cytotoxicity and poor cellular adhesion.     

Inactivation of Legionella pneumophila by monochloramine

Chloramination which is used in South Australia to control the growth of Naegleria fowleri , was investigated to see if it would also control that of Legionella pneumophila . It was found that L. pneumophila was more sensitive than Escherichia coli to monochloramine. At 1.0 mg/l, a 99% kill of L. pneumophila was achieved in 15 min compared with 37 min for a 99% kill of E. coli. Combined with the stability of monochloramine, even at elevated temperatures, the results suggest that this disinfectant would control the growth of L. pneumophila in water distribution systems.     

Inactivation of Legionella pneumophila by monochloramine

Chloramination which is used in South Australia to control the growth of Naegleria fowleri, was investigated to see if it would also control that of Legionella pneumophila. It was found that L. pneumophila was more sensitive than Escherichia coli to monochloramine. At 1.0 mg/l, a 99% kill of L. pneumophila was achieved in 15 min compared with 37 min for a 99% kill of E. coli. Combined with the stability of monochloramine, even at elevated temperatures, the results suggest that this disinfectant would control the growth of L. pneumophila in water distribution systems.     

Implication of proteins containing tetratricopeptide repeats in conditional virulence phenotypes of Legionella pneumophila

Legionella pneumophila, the causative agent of Legionnaires' disease, is a ubiquitous freshwater bacterium whose virulence phenotypes require a type IV secretion system (T4SS). L. pneumophila strain JR32 contains two virulence-associated T4SSs, the Dot/Icm and Lvh T4SSs. Defective entry and phagosome acidification phenotypes of dot/icm mutants are conditional and reversed by incubating broth-grown stationary-phase cultures in water (WS treatment) prior to infection, as a mimic of the aquatic environment of Legionella. Reversal of dot/icm virulence defects requires the Lvh T4SS and is associated with a >10-fold induction of LpnE, a tetratricopeptide repeat (TPR)-containing protein. In the current study, we demonstrated that defective entry and phagosome acidification phenotypes of mutants with changes in LpnE and EnhC, another TPR-containing protein, were similarly reversed by WS treatment. In contrast to dot/icm mutants for which the Lvh T4SS was required, reversal for the ΔlpnE or the ΔenhC mutant required that the other TPR-containing protein be present. The single and double ΔlpnE and ΔenhC mutants showed a hypersensitivity to sodium ion, a phenotype associated with dysfunction of the Dot/Icm T4SS. The ΔlpnE single and the ΔlpnE ΔenhC double mutant showed 3- to 9-fold increases in translocation of Dot/Icm T4SS substrates, LegS2/SplY and LepB. Taken together, these data identify TPR-containing proteins in a second mechanism by which the WS mimic of a Legionella environmental niche can reverse virulence defects of broth-grown cultures and implicate LpnE and EnhC directly or indirectly in translocation of Dot/Icm T4SS protein substrates.     

Cell biology of infection by Legionella pneumophila

Professional phagocytes digest internalized microorganisms by actively delivering them into the phagolysosomal compartment. Intravacuolar bacterial pathogens have evolved a variety of effective strategies to bypass the default pathway of phagosomal maturation to create a niche permissive for their survival and propagation. Here we discuss recent progress in our understanding of the sophisticated mechanisms used by Legionella pneumophila to survive in phagocytes.     

Novel lysophospholipase A secreted by Legionella pneumophila

We show that Legionella pneumophila possesses lysophospholipase A activity, which releases fatty acids from lysophosphatidylcholine. The NH2-terminal sequence of the enzyme contained FGDSLS, corresponding to a catalytic domain in a recently described group of lipolytic enzymes. Culture supernatants of a L. pneumophila pilD mutant lost the ability to cleave lysophosphatidylcholine.