Lpg0393 of Legionella pneumophila Is a Guanine-Nucleotide Exchange Factor for Rab5, Rab21 and Rab22

Legionella pneumophila, a human intracellular pathogen, encodes about 290 effector proteins that are translocated into host cells through a secretion machinery. Some of these proteins have been shown to manipulate or subvert cellular processes during infection, but functional roles of a majority of them remain unknown. Lpg0393 is a newly identified Legionella effector classified as a hypothetical protein. Through X-ray crystallographic analysis, we show that Lpg0393 contains a Vps9-like domain, which is structurally most similar to the catalytic core of human Rabex-5 that activates the endosomal Rab proteins Rab5, Rab21 and Rab22. Consistently, Lpg0393 exhibited a guanine-nucleotide exchange factor activity toward the endosomal Rabs. This work identifies the first example of a bacterial guanine-nucleotide exchange factor that is active towards the Rab5 sub-cluster members, implying that the activation of these Rab proteins might be advantageous for the intracellular survival of Legionella.     

The major outer membrane protein of Legionella pneumophila Lpg1974 shows pore-forming characteristics similar to the human mitochondrial outer membrane pore,hVDAC1

Legionella pneumophila is an aerobic and nonspore-forming pathogenic Gram-negative bacterium of the genus Legionella. It is the causative agent of Legionnaires' disease, also known as Legionellosis. The hosts of this organism are diverse, ranging from simple water borne protozoans such as amoebae to more complex hosts such as macrophages in humans. Genome analyses have shown the presence of genes coding for eukaryotic like proteins in several Legionella species. The presence of these proteins may assist L. pneumophila in its adaptation to the eukaryotic host. We studied the characteristics of a protein (Lpg1974) of L. pneumophila that shows remarkable homologies in length of the primary sequence and for the identity/homology of many amino acids to the voltage dependent anion channel (human VDAC1, Porin 31HL) of human mitochondria. Two different forms of Lpg1974 were overexpressed in Escherichia coli and purified to homogeneity: the one containing a putative N-terminal signal sequence and one without it. Reconstituted protein containing the signal sequence formed ion-permeable pores in lipid bilayer membranes with a conductance of approximately 5.4 nS in 1 M KCl. When the predicted N-terminal signal peptide of Lpg1974 comprising an α-helical structure similar to that at the N-terminus of hVDAC1 was removed, the channels formed in reconstitution experiments had a conductance of 7.6 nS in 1 M KCl. Both Lpg1974 proteins formed pores that were voltage-dependent and anion-selective similar to the pores formed by hVDAC1. These results suggest that Lpg1974 of L. pneumophila is indeed a structural and functional homologue to hVDAC1.     

Osmotic response of individual cells during freezing. II. Membrane permeability analysis

An analytical model is presented to simulate the freezing of individual yeast cells. In addition the model is solved numerically on a digital computer to obtain values for cell volume as a function of temperature, based on the thermal protocol during freezing, and the transport parameters of the cell membrane. The numerical procedure was modified to enable values for the membrane hydraulic permeability reference coefficient, Lpg, and activation energy, ELp, to be deduced by nonlinear analysis of complementary experimental data (10). It was observed that the apparent values of both Lpg and ELp increase with cooling rate, from Lpg = 0.0116 micrometer 3 micrometers-2 atm-1 min-1 and ELp = 19.4 kJ mol-1 for 9 degrees K/min to Lpg = 2.11 micrometers 3 micrometer-2 atm-1 min-1 and ELp = 101 kJ mol-1 for 35 degrees K/min. The deduced permeabilities fall within the range of values determined in a prior study by Levin (6). Analysis with the model also indicates that the turgor pressure exerts a negligible effect on yeast exposed to freezing stress.     

Enzymatic properties of an ecto-nucleoside triphosphate diphosphohydrolase from Legionella pneumophila: substrate specificity and requirement for virulence

Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.     

Expression of Ralstonia solanacearum type III secretion system is dependent on a novel type 4 pili (T4P) assembly protein (TapV) but is T4P independent

Type IV pili (T4P) are virulence factors in various pathogenic bacteria of animals and plants that play important roles in twitching motility, swimming motility, biofilm formation, and adhesion to host cells. Here, we genetically characterized functional roles of a putative T4P assembly protein TapV (Rsc1986 in reference strain GMI1000) and its homologue Rsp0189, which shares 58% amino acid identity with TapV, in Ralstonia solanacearum. Deletion of tapV, but not rsp0189, resulted in significantly impaired twitching motility, swimming motility, and adhesion to tomato roots, which are consistent as phenotypes of the pilA mutant (a known R. solanacearum T4P-deficient mutant). However, unlike the pilA mutant, the tapV mutant produced more biofilm than the wild-type strain. Our gene expression studies revealed that TapV, but not Rsp0189, is important for expression of a type III secretion system (T3SS, a pathogenicity determinant of R. solanacearum) both in vitro and in planta, but it is T4P independent. We further revealed that TapV affected the T3SS expression via the PhcA–TapV–PrhG–HrpB pathway, consistent with previous reports that PhcA positively regulates expression of pilA and prhG. Moreover, deletion of tapV, but not rsp0189, significantly impaired the ability to migrate into and colonize xylem vessels of host plants, but there was no alteration in intercellular proliferation of R. solanacearum in tobacco leaves, which is similar to the pilA mutant. The tapV mutant showed significantly impaired virulence in host plants. This is the first report on the impact of T4P components on the T3SS, providing novel insights into our understanding of various biological functions of T4P and the complex regulatory pathway of T3SS in R. solanacearum.     

Crystal structure of the Legionella pneumophila Lpg2936 in complex with the cofactor S‐adenosyl‐L‐methionine reveals novel insights into the mechanism of RsmE family methyltransferases

The methylation of U1498 located in the 16S ribosomal RNA of Escherichia coli is an important modification affecting ribosomal activity. RsmE methyltransferases methylate specifically this position in a mechanism that requires an S‐adenosyl‐L‐methionine (AdoMet) molecule as cofactor. Here we report the structure of Apo and AdoMet‐bound Lpg2936 from Legionella pneumophila at 1.5 and 2.3 Å, respectively. The protein comprises an N‐terminal PUA domain and a C‐terminal SPOUT domain. The latter is responsible for protein dimerization and cofactor binding. Comparison with similar structures suggests that Lpg2936 is an RsmE‐like enzyme that can target the equivalent of U1498 in the L. pneumophila ribosomal RNA, thereby potentially enhancing ribosomal activity during infection‐mediated effector production. The multiple copies of the enzyme found in both structures reveal a flexible conformation of the bound AdoMet ligand. Isothermal titration calorimetry measurements suggest an asymmetric two site binding mode. Our results therefore also provide unprecedented insights into AdoMet/RsmE interaction, furthering our understanding of the RsmE catalytic mechanism.     

Structure of lpg0406, a carboxymuconolactone decarboxylase family protein possibly involved in antioxidative response from Legionella pneumophila

Lpg0406, a hypothetical protein from Legionella pneumophila, belongs to carboxymuconolactone decarboxylase (CMD) family. We determined the crystal structure of lpg0406 both in its apo and reduced form. The structures reveal that lpg0406 forms a hexamer and have disulfide exchange properties. The protein has an all‐helical fold with a conserved thioredoxin‐like active site CXXC motif and a proton relay system similar to that of alkylhydroperoxidase from Mycobacterium tuberculosis (MtAhpD), suggesting that lpg0406 might function as an enzyme with peroxidase activity and involved in antioxidant defense. A comparison of the size and the surface topology of the putative substrate‐binding region between lpg0406 and MtAhpD indicates that the two enzymes accommodate the different substrate preferences. The structural findings will enhance understanding of the CMD family protein structure and its various functions.     

New Global Insights on the Regulation of the Biphasic Life Cycle and Virulence Via ClpP-Dependent Proteolysis in Legionella pneumophila

Legionella pneumophila, an environmental bacterium that parasitizes protozoa, causes Legionnaires’ disease in humans that is characterized by severe pneumonia. This bacterium adopts a distinct biphasic life cycle consisting of a nonvirulent replicative phase and a virulent transmissive phase in response to different environmental conditions. Hence, the timely and fine-tuned expression of growth and virulence factors in a life cycle–dependent manner is crucial for survival and replication. Here, we report that the completion of the biphasic life cycle and bacterial pathogenesis is greatly dependent on the protein homeostasis regulated by caseinolytic protease P (ClpP)-dependent proteolysis. We characterized the ClpP-dependent dynamic profiles of the regulatory and substrate proteins during the biphasic life cycle of L. pneumophila using proteomic approaches and discovered that ClpP-dependent proteolysis specifically and conditionally degraded the substrate proteins, thereby directly playing a regulatory role or indirectly controlling cellular events via the regulatory proteins. We further observed that ClpP-dependent proteolysis is required to monitor the abundance of fatty acid biosynthesis–related protein Lpg0102/Lpg0361/Lpg0362 and SpoT for the normal regulation of L. pneumophila differentiation. We also found that the control of the biphasic life cycle and bacterial virulence is independent. Furthermore, the ClpP-dependent proteolysis of Dot/Icm (defect in organelle trafficking/intracellular multiplication) type IVB secretion system and effector proteins at a specific phase of the life cycle is essential for bacterial pathogenesis. Therefore, our findings provide novel insights on ClpP-dependent proteolysis, which spans a broad physiological spectrum involving key metabolic pathways that regulate the transition of the biphasic life cycle and bacterial virulence of L. pneumophila, facilitating adaptation to aquatic and intracellular niches.     

A bacterial ecto-triphosphate diphosphohydrolase similar to human CD39 is essential for intracellular multiplication of Legionella pneumophila

As part of its pathogenesis, Legionella pneumophila persists within human alveolar macrophages in non-acidified organelles that do not mature into phagolysosomes. Two L. pneumophila genes, lpg0971 and lpg1905, are predicted to encode ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) that share sequence similarity with human CD39/NTPDase1. The predicted products possess five apyrase conserved domains that are typical of eukaryotic ecto-NTPDases. In this study, we found that an lpg1905 mutant was recovered in lower numbers from macrophages, alveolar epithelial cells and the amoeba, Hartmannella vermiformis compared with wild-type L. pneumophila and an lpg0971 mutant. Similar to human CD39, recombinant purified Lpg1905 exhibited ATPase and ADPase activity and possessed the ability to inhibit platelet aggregation. Mutation of a conserved Glu159 residue that is essential for CD39 activity inhibited ATPase and ADPase activity of Lpg1905. In addition, enzyme activity was inhibited in the presence of the specific ecto-NTPDase inhibitor, ARL67156. The entry and replication defect of the lpg1905 mutant was reversed upon transcomplementation with lpg1905 but not lpg1905E159A encoding an enzymatically inactive form of the protein. Although several protozoan parasites exhibit ecto-NTPDase activity, including Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi, this is the first time a bacterial ecto-NTPDase has been implicated in virulence.     

Transport phenomena during freezing of adipose tissue derived adult stem cells

In the present study a well-established differential scanning calorimeter (DSC) technique is used to measure the water transport phenomena during freezing of stromal vascular fraction (SVF) and adipose tissue derived adult stem (ADAS) cells at different passages (Passages 0 and 2). Volumetric shrinkage during freezing of adipose derived cells was obtained at a cooling rate of 20 degrees C/min in the presence of extracellular ice and two different, commonly used, cryoprotective agents, CPAs (10% DMSO and 10% Glycerol). The adipose derived cells were modeled as spheres of 50 microm diameter with an osmotically inactive volume (Vb) of 0.6Vo, where Vo is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the "best-fit" membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined. The "best-fit" membrane permeability parameters for adipose derived cells in the absence and presence of CPAs ranged from: Lpg=23.1-111.5x10(-15) m3/Ns (0.135-0.652 microm/min-atm) and ELp=43.1-168.8 kJ/mol (9.7-40.4 kcal/mol). Numerical simulations of water transport were then performed under a variety of cooling rates (5-100 degrees C/min) using the experimentally determined membrane permeability parameters. And finally, the simulation results were analyzed to predict the optimal rates of freezing adipose derived cells in the presence and absence of CPAs.     

Variation in the membrane transport properties and predicted optimal rates of freezing for spermatozoa of diploid and tetraploid Pacific oyster, Crassostrea gigas

In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment "ball-on-stick" model with a "ball" 1.66 microm in diameter and a "stick" 41 microm in length and 0.14 microm wide. Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 microm in diameter and a "stick" 53 microm in length and 0.17 microm wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 Vo, where Vo is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: Lpg = 0.30 x 10(-15) m(3)/Ns (0.0017 microm/min-atm) and ELp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.27 x 10(-15) m(3)/Ns (0.0015 microm/min-atm) and ELp[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: Lpg = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp[cpa] = 37.6 kJ/mole (9.0 kcal/mole). The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70 degrees C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C. gigas.     

Freezing response of white bass (Morone chrysops) sperm cells

The water transport response during freezing of sperm cells of Morone chrysops (white bass, WB) was obtained using a shape-independent differential scanning calorimeter (DSC) technique. Sperm cell suspensions were frozen at a cooling rate of 20 degrees C/min in two different media: (1) without cryoprotective agents (CPAs), or (2) with 5% (v/v) dimethyl sulfoxide (Me2SO). For calculations, the sperm cell was modeled as a cylinder of length 24.8 microm and diameter of 0.305 microm, while the osmotically inactive cell volume (Vb) was assumed to be 0.6 Vo, where Vo was the isotonic or the initial cell volume. By fitting a model of water transport to the experimentally determined water transport data, the best fit membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined, and ranged from Lpg = 0.51-1.7 x 10(-15) m3/Ns (0.003-0.01 microm/min-atm), and ELp = 83.6-131.3 kJ/mol (20.0-31.4 kcal/mol). The parameters obtained in this study suggest that the optimal rate of cooling for M. chrysops sperm cells is approximately 22 degrees C/min, a value that compares closely with experimentally determined optimal rates of cooling (approximately 16 degrees C/min).     

Cellular response of adipose derived passage-4 adult stem cells to freezing stress

A differential scanning calorimeter technique was used to generate experimental data for volumetric shrinkage during cooling at 20 degrees C/min in adipose derived adult stem cells (ASCs) in the presence and absence of cryoprotective agents (CPAs). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the membrane permeability parameters of ASCs were obtained. For passage-4 (P4) ASCs, the reference hydraulic conductivity Lpg and the value of the apparent activation energy ELP were determined to be 1.2 X 10(-13) m3/Ns and 177.8 kJ/mole, respectively. We found that the addition of either glycerol or dimethylsulfoxide (DMSO) significantly decreased the value of the reference hydraulic conductivity Lpg(cpa) and the value of the apparent activation energy ELp(cpa) in P4 ASCs. The values of Lpg(cpa) in the presence of glycerol and DMSO were determined as 0.39 x 10(-13) and 0.50 X 109-13) m3/Ns, respectively, while the corresponding values of ELp(cpa) were 51.0 and 61.5 kJ/mole. Numerical simulations of water transport were then performed under a variety of cooling rates (5-100 degreesC/min) using the experimentally determined membrane permeability parameters. And finally, the simulation results were analyzed to predict the optimal rates of freezing P4 adipose derived cells in the presence and absence of CPAs.     

H‐NOX regulation of c‐di‐GMP metabolism and biofilm formation in Legionella pneumophila

Haem Nitric oxide/OXygen (H‐NOX) binding domains are a family of haemoprotein sensors that are widespread in bacterial genomes, but limited information is available on their function. Legionella pneumophila is the only prokaryote found, thus far, to encode two H‐NOX proteins. This paper presents data supporting a role for one of the L. pneumophila H‐NOXs in the regulation of biofilm formation. In summary: (i) unmarked deletions in the hnox1 gene do not affect growth rate in liquid culture or replication in permissive macrophages; (ii) the Δhnox1 strain displays a hyper‐biofilm phenotype; (iii) the gene adjacent to hnox1 is a GGDEF‐EAL protein, lpg1057, and overexpression in L. pneumophila of this protein, or the well‐studied diguanylate cyclase, vca0956, results in a hyper‐biofilm phenotype; (iv) the Lpg1057 protein displays diguanylate cyclase activity in vitro and this activity is inhibited by the Hnox1 protein in the Fe(II)‐NO ligation state, but not the Fe(II) unligated state; and (v) consistent with the Hnox1 regulation of Lpg1057, unmarked deletions of lpg1057 in the Δhnox1 background results in reversion of the hyper‐biofilm phenotype back to wild‐type biofilm levels. Taken together, these results suggest a role for hnox1 in regulating c‐di‐GMP production by lpg1057 and biofilm formation in response to NO.     

Subzero water permeability parameters and optimal freezing rates for sperm cells of the southern platyfish, Xiphophorus maculatus

This study reports the subzero water transport characteristics (and empirically determined optimal rates for freezing) of sperm cells of live-bearing fishes of the genus Xiphophorus, specifically those of the southern platyfish Xiphophorus maculatus. These fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. Water transport during freezing of X. maculatus sperm cell suspensions was obtained using a shape-independent differential scanning calorimeter technique in the presence of extracellular ice at a cooling rate of 20 degrees C/min in three different media: (1) Hanks' balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol, and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder with a length of 52.35 microm and a diameter of 0.66 microm with an osmotically inactive cell volume (Vb) of 0.6 V0, where V0 is the isotonic or initial cell volume. This translates to a surface area, SA to initial water volume, WV ratio of 15.15 microm(-1). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best fit membrane permeability parameters (reference membrane permeability to water at 0 degrees C, Lpg or Lpg [cpa] and the activation energy, E(Lp) or E(Lp) [cpa]) were found to range from: Lpg or Lpg [cpa] = 0.0053-0.0093 microm/minatm; E(Lp) or E(Lp) [cpa] = 9.79-29.00 kcal/mol. By incorporating these membrane permeability parameters in a recently developed generic optimal cooling rate equation (optimal cooling rate, [Formula: see text] where the units of B(opt) are degrees C/min, E(Lp) or E(Lp) [cpa] are kcal/mol, L(pg) or L(pg) [cpa] are microm/minatm and SA/WV are microm(-1)), we determined the optimal rates of freezing X. maculatus sperm cells to be 28 degrees C/min (in HBSS), 47 degrees C/min (in HBSS+14% glycerol) and 36 degrees C/min (in HBSS+10% DMSO). Preliminary empirical experiments suggest that the optimal rate of freezing X. maculatus sperm in the presence of 14% glycerol to be approximately 25 degrees C/min. Possible reasons for the observed discrepancy between the theoretically predicted and experimentally determined optimal rates of freezing X. maculatus sperm cells are discussed.     

The metaeffector MesI regulates the activity of the Legionella effector SidI through direct protein–protein interactions

To create an intracellular niche permissive for its replication, Legionella pneumophila uses hundreds of effectors to target a wide variety of host proteins and manipulate specific host processes such as immune response, and vesicle trafficking. To avoid unwanted disruption of host physiology, this pathogen also imposes precise control of its virulence by the use of effectors called metaeffectors to regulate the activity of other effectors. A number of effector/metaeffector pairs with distinct regulatory mechanisms have been characterized, including abrogation of protein modifications, direct modification of the effector and direct binding to the catalytic pocket of the cognate effector. Recently, MesI (Lpg2505) was found to be a metaeffector of SidI, an effector involved in inhibiting host protein translation. Here we demonstrate that MesI functions by inhibiting the activity of SidI via direct protein–protein interactions. We show that this interaction occurs within L. pneumophila and thus interferes with the translocation of SidI into host cells. We also solved the structure of MesI, which suggests that this protein does not have an active site similar to any known enzymes. Analysis of deletion mutants allowed the identification of regions within SidI and MesI that are important for their interactions.     

The effect of dimethylsulfoxide on the water transport response of rat hepatocytes during freezing.

Successful improvement of cryopreservation protocols for cells in suspension requires knowledge of how such cells respond to the biophysical stresses of freezing (intracellular ice formation, water transport) while in the presence of a cryoprotective agent (CPA). This work investigates the biophysical water transport response in a clinically important cell type--isolated hepatocytes--during freezing in the presence of dimethylsulfoxide (DMSO). Sprague-Dawley rat liver hepatocytes were frozen in Williams E media supplemented with 0, 1, and 2 M DMSO, at rates of 5, 10, and 50 degrees C/min. The water transport was measured by cell volumetric changes as assessed by cryomicroscopy and image analysis. Assuming that water is the only species transported under these conditions, a water transport model of the form dV/dT = f(Lpg([CPA]), ELp([CPA]), T(t)) was curve-fit to the experimental data to obtain the biophysical parameters of water transport--the reference hydraulic permeability (Lpg) and activation energy of water transport (ELp)--for each DMSO concentration. These parameters were estimated two ways: (1) by curve-fitting the model to the average volume of the pooled cell data, and (2) by curve-fitting individual cell volume data and averaging the resulting parameters. The experimental data showed that less dehydration occurs during freezing at a given rate in the presence of DMSO at temperatures between 0 and -10 degrees C. However, dehydration was able to continue at lower temperatures (< -10 degrees C) in the presence of DMSO. The values of Lpg and ELp obtained using the individual cell volume data both decreased from their non-CPA values--4.33 x 10(-13) m3/N-s (2.69 microns/min-atm) and 317 kJ/mol (75.9 kcal/mol), respectively--to 0.873 x 10(-13) m3/N-s (0.542 micron/min-atm) and 137 kJ/mol (32.8 kcal/mol), respectively, in 1 M DMSO and 0.715 x 10(-13) m3/N-s (0.444 micron/min-atm) and 107 kJ/mol (25.7 kcal/mol), respectively, in 2 M DMSO. The trends in the pooled volume values for Lpg and ELp were very similar, but the overall fit was considered worse than for the individual volume parameters. A unique way of presenting the curve-fitting results supports a clear trend of reduction of both biophysical parameters in the presence of DMSO, and no clear trend in cooling rate dependence of the biophysical parameters. In addition, these results suggest that close proximity of the experimental cell volume data to the equilibrium volume curve may significantly reduce the efficiency of the curve-fitting process.     

A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization

Successful infection by the opportunistic pathogen Legionella pneumophila requires the collective activity of hundreds of virulence proteins delivered into the host cell by the Dot/Icm type IV secretion system. These virulence proteins, also called effectors modulate distinct host cellular processes to create a membrane-bound niche called the Legionella containing vacuole (LCV) supportive of bacterial growth. We found that Ceg14 (Lpg0437), a Dot/Icm substrate is toxic to yeast and such toxicity can be alleviated by overexpression of profilin, a protein involved in cytoskeletal structure in eukaryotes. We further showed that mutations in profilin affect actin binding but not other functions such as interactions with poly-l-proline or phosphatidylinositol, abolish its suppressor activity. Consistent with the fact the profilin suppresses its toxicity, expression of Ceg14 but not its non-toxic mutants in yeast affects actin distribution and budding of daughter cells. Although Ceg14 does not detectably interact with profilin, it co-sediments with filamentous actin and inhibits actin polymerization, causing the accumulation of short actin filaments. Together with earlier studies, these results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton.     

Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila

A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter.     

Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy

Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of freezing-induced membrane dehydration was found to be dependent on the ice nucleation temperature, and showed Arrhenius behavior. The presence of glycerol did not prevent the freezing-induced membrane phase transition, but membrane dehydration occurred more gradual and over a wider temperature range. We describe a method to determine membrane hydraulic permeability parameters (E_Lp, Lpg) at subzero temperatures from membrane phase behavior data. In order to do this, it was assumed that the measured freezing-induced shift in wavenumber position of the symmetric CH₂ stretching band arising from the lipid acyl chains is proportional to cellular dehydration. Membrane permeability parameters were also determined by analyzing the H₂O-bending and -libration combination band, which yielded higher values for both E_Lp and Lpg as compared to lipid band analysis. These differences likely reflect differences between transport of free and membrane-bound water. FTIR allows for direct assessment of membrane properties at subzero temperatures in intact cells. The derived biophysical membrane parameters are dependent on intrinsic cell properties as well as freezing extender composition.