Nonredundant Dimethyl Sulfoxide Reductases Influence Salmonella enterica Serotype Typhimurium Anaerobic Growth and Virulence.

Facultative anaerobic enteric pathogens can utilize a diverse array of alternate electron acceptors to support anaerobic metabolism and thrive in the hypoxic conditions within the mammalian gut. Dimethyl sulfoxide (DMSO) is produced by methionine catabolism and can act as an alternate electron acceptor to support anaerobic respiration. The DMSO reductase complex consists of three subunits, DmsA, DmsB, and DmsC, and allows bacteria to grow anaerobically with DMSO as an electron acceptor. The genomes of nontyphoidal Salmonella enterica encode three putative dmsABC operons, but the impact of the apparent genetic redundancy in DMSO reduction on the fitness of nontyphoidal S. enterica during infection remains unknown. We hypothesized that DMSO reduction would be needed for S. enterica serotype Typhimurium to colonize the mammalian gut. We demonstrate that an S. Typhimurium mutant with loss of function in all three putative DMSO reductases (ΔdmsA3) poorly colonizes the mammalian intestine when the microbiota is intact and when inflammation is absent. DMSO reduction enhances anaerobic growth through nonredundant contributions of two of the DMSO reductases. Furthermore, DMSO reduction influences virulence by increasing expression of the type 3 secretion system 2 and reducing expression of the type 3 secretion system 1. Collectively, our data demonstrate that the DMSO reductases of S. Typhimurium are functionally nonredundant and suggest DMSO is a physiologically relevant electron acceptor that supports S. enterica fitness in the gut.

Pathogenic trickery: deception of host cell processes.

Microbial pathogens cause a spectrum of diseases in humans. Although the disease mechanisms vary considerably, most pathogens have developed virulence factors that interact with host molecules, often usurping normal cellular processes, including cytoskeletal dynamics and vesicle targeting. These virulence factors often mimic host molecules, and mediate events as diverse as bacterial invasion, antiphagocytosis, and intracellular parastism.

The evolutionary origins of eukaryotic protein disulfide isomerase domains: new evidence from the Amitochondriate protist Giardia lamblia.

A phylogenetic analysis of protein disulfide isomerase (PDI) domain evolution was performed with the inclusion of recently reported PDIs from the amitochondriate protist Giardia lamblia, yeast PDIs that contain a single thioredoxin-like domain, and PDIs from a diverse selection of protists. We additionally report and include two new giardial PDIs, each with a single thioredoxin-like domain. Inclusion of protist PDIs in our analyses revealed that the evolutionary history of the endoplasmic reticulum may not be simple. Phylogenetic analyses support common ancestry of all eukaryotic PDIs from a thioredoxin ancestor and independent duplications of thioredoxin-like domains within PDIs throughout eukaryote evolution. This was particularly evident for Acanthamoeba PDI, Dictyostelium PDI, and mammalian erp5 domains. In contrast, gene duplication, instead of domain duplication, produces PDI diversity in G. lamblia. Based on our results and the known diversity of PDIs, we present a new hypothesis that the five single-domain PDIs of G. lamblia may reflect an ancestral mechanism of protein folding in the eukaryotic endoplasmic reticulum. The PDI complement of G. lamblia and yeast suggests that a combination of PDIs may be used as a redox chain analogous to that known for bacterial Dsb proteins.

Na(+)-dependent pH regulation by the amitochondriate protozoan parasite Giardia intestinalis.

Giardia intestinalis is a pathogenic fermentative parasite, which inhabits the gastrointestinal tract of animals and humans. G. intestinalis trophozoites are exposed to acidic fluctuations in vivo and must also cope with acidic metabolic endproducts. In this study, a combination of independent techniques ((31)P NMR spectroscopy, distribution of the weak acid pH marker 5,5-dimethyl-2,4-oxazolidinedione (DMO) and the fluorescent pH indicator 2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein (BCECF)) were used to show that G. intestinalis trophozoites exposed to an extracellular pH range of 6.0--7.5 maintain their cytosolic pH (pH(i)) within the range 6.7--7.1. Maintenance of the resting pH(i) was Na(+)-dependent but unaffected by amiloride (or analogs thereof). Recovery of pH(i) from an intracellular acidosis was also Na(+)-dependent, with the rate of recovery varying with the extracellular Na(+) concentration in a saturable manner (K(m) = 18 mm; V(max) = 10 mm H(+) min(-1)). The recovery of pH(i) from an acid load was inhibited by amiloride but unaffected by a number of its analogs. The postulated involvement of one or more Na(+)/H(+) exchanger(s) in the regulation of pH(i) in G. intestinalis is discussed.

Salmonella and apoptosis: to live or let die?

A successful pathogen manipulates its host for its own benefit. One means to establish a successful infection, especially for intracellular pathogens, is to exploit host cell death pathways and alter the viability of host cells. Here we describe the manipulation of apoptosis by Salmonella and discuss the advantages that such actions may confer to the bacteria, and its implications in resistance to disease.

Activation of Akt/protein kinase B in epithelial cells by the Salmonella typhimurium effector sigD.

The serine-threonine kinase Akt is a protooncogene involved in the regulation of cell proliferation and survival. Activation of Akt is initiated by binding to the phospholipid products of phosphoinositide 3-kinase at the inner leaflet of the plasma membranes followed by phosphorylation at Ser(473) and Thr(308). We have found that Akt is activated by Salmonella enterica serovar Typhimurium in epithelial cells. A bacterial effector protein, SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. In HeLa cells, wild type S. typhimurium induced translocation of Akt to membrane ruffles and phosphorylation at residues Thr(308) and Ser(473) and increased kinase activity. In contrast, infection with a SigD deletion mutant did not induce phosphorylation or activity although Akt was translocated to membrane ruffles. Complementation of the SigD deletion strain with a mutant containing a single Cys to Ser mutation (C462S), did not restore the Akt activation phenotype. This residue has previously been shown to be essential for inositol phosphatase activity of the SigD homologue, SopB. Our data indicate a novel mechanism of Akt activation in which the endogenous cellular pathway does not convert membrane-associated Akt into its active form. SigD is also the first bacterial effector to be identified as an activator of Akt.

Poisons, ruffles and rockets: bacterial pathogens and the host cell cytoskeleton.

The cytoskeleton of eukaryotic cells is affected by a number of bacterial and viral pathogens. In this review we consider three recurring themes of cytoskeletal involvement in bacterial pathogenesis: 1) the effect of bacterial toxins on actin-regulating small GTP-binding proteins; 2) the invasion of non-phagocytic cells by the bacterial induction of ruffles at the plasma membrane; 3) the formation of actin tails and pedestals by intracellular and extracellular bacteria, respectively. Considerable progress has been made recently in the characterization of these processes. It is becoming clear that bacterial pathogens have developed a variety of sophisticated mechanisms for utilizing the complex cytoskeletal system of host cells. These bacterially-induced processes are now providing unique insights into the regulation of fundamental eukaryotic mechanisms.

Developmental gene regulation in Giardia lamblia: first evidence for an encystation-specific promoter and differential 5' mRNA processing.

Giardia lamblia must encyst to survive in the environment and subsequently infect new hosts. We investigated the expression of glucosamine-6-phosphate isomerase (Gln6PI), the first enzyme required for biosynthesis of N-acetylgalactosamine, for the major cyst wall polysaccharide. We isolated two Gln6PI genes that encode proteins with large areas of identity, but distinctive central and terminal regions. Both recombinant enzymes have comparable kinetics. Interestingly, these genes have distinct patterns of expression. Gln6PI-A has a conventional, short 5' untranslated region (UTR), and is expressed at a low level during vegetative growth and encystation. The Gln6PI-B gene has two transcripts - one is expressed constitutively and the second species is highly upregulated during encystation. The non-regulated Gln6PI-B transcript has the longest 5'-UTR known for Giardia and is 5' capped or blocked. In contrast, the Gln6PI-B upregulated transcript has a short, non-capped 5'-UTR. A small promoter region (< 56 bp upstream from the start codon) is sufficient for the regulated expression of Gln6PI-B. Gln6PI-B also has an antisense overlapping transcript that is expressed constitutively. A shorter antisense transcript is detected during encystation. This is the first report of a developmentally regulated promoter in Giardia, as well as evidence for a potential role of 5' RNA processing and antisense RNA in differential gene regulation.

Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia.

Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.

Salmonella typhimurium virulence genes are induced upon bacterial invasion into phagocytic and nonphagocytic cells.

Survival and growth of salmonellae within host cells are important aspects of bacterial virulence. We have developed an assay to identify Salmonella typhimurium genes that are induced inside Salmonella-containing vacuoles within macrophage and epithelial cells. A promoterless luciferase gene cassette was inserted randomly into the Salmonella chromosome, and the resulting mutants were screened for genes upregulated in intracellular bacteria compared to extracellular bacteria. We identified four genes in S. typhimurium that were upregulated upon bacterial invasion of both phagocytic and nonphagocytic cells. Expression of these genes was not induced by factors secreted by host cells or media alone. All four genes were induced at early time points (2 to 4 h) postinvasion and continued to be upregulated within host cells at later times (5 to 7 h). One mutant contained an insertion in the ssaR gene, within Salmonella pathogenicity island 2 (SPI-2), which abolished bacterial virulence in a murine typhoid model. Two other mutants contained insertions within SPI-5, one in the sopB/sigD gene and the other in a downstream gene, pipB. The insertions within SPI-5 resulted in the attenuation of S. typhimurium in the mouse model. The fourth mutant contained an insertion within a previously undescribed region of the S. typhimurium chromosome, iicA (induced intracellularly A). We detected no effect on virulence as a result of this insertion. In conclusion, all but one of the genes identified in this study were virulence factors within pathogenicity islands, illustrating the requirement for specific gene expression inside mammalian cells and indicating the key role that virulence factor regulation plays in Salmonella pathogenesis.

Cloning and expression of a prokaryotic enzyme, arginine deiminase, from a primitive eukaryote Giardia intestinalis.

Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolism of arginine to citrulline in the arginine dihydrolase pathway. This pathway has been regarded as restricted to prokaryotic organisms but is an important source of energy to the primitive protozoan Giardia intestinalis. In this paper we report the cloning and expression of the arginine deiminase gene from this parasite. Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene. This was then used as a probe to screen HindIII and PstI "mini" libraries to obtain two overlapping clones that contained the arginine deiminase gene. The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa. Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells. Arginine deiminase was successfully expressed in Escherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme.

L-arginine transport and metabolism in Giardia intestinalis support its position as a transition between the prokaryotic and eukaryotic kingdoms.

Arginine is metabolized by the arginine dihydrolase pathway in Giardia intestinalis trophozoites and is an important metabolic fuel for this parasite. Radiolabelled arginine was used to characterize the transport of arginine into Giardia intestinalis trophozoites. The transporter had a high affinity for arginine (Km 15 microM) and a high activity [Vmax 76 nmol min-1 (mg protein)-1 at 25 degrees C]. Substrate specificity studies indicated an absolute requirement for the alpha-amino and carboxyl groups, but a tolerance for some substitutions in the guanidino group. The use of non-metabolized arginine analogues in combination with HPLC amino acid analysis of intra- and extracellular pools demonstrated that the arginine transporter is an arginine-ornithine antiport. Investigations of the first step of arginine metabolism, involving arginine deiminase, revealed a relatively high affinity for arginine (Km 0.16 mM) and a large maximal velocity [Vmax 550 nmol min-1 (mg protein)-1 at 37 degrees C]. Substrate specificity studies showed that the arginine deiminase had a characteristically different substrate recognition profile to that of the arginine transporter. Overall, the combination of the transporter and the deiminase result in very low intracellular arginine concentrations and their properties are consistent with the rapid transport of arginine for metabolism via the arginine dihydrolase pathway.

The intracellular amino acid pools of Giardia intestinalis, Trichomonas vaginalis, and Crithidia luciliae.

The total intracellular amino acid profiles of Giardia intestinalis trophozoites, Trichomonas vaginalis, and Crithidia luciliae were determined by sensitive amino acid analysis. The three protozoan parasites exhibited distinctively different amino acid profiles, but all three were dominated by high concentrations of intracellular alanine. This common feature suggests that alanine synthesis is a major aspect of intermediary metabolism in these protozoan parasites. There were also distinctively different aspects, particularly those related to arginine metabolism. Ornithine, citrulline, and ammonia were found in G. intestinalis trophozoites, but no intracellular arginine was detected. This pattern is consistent with the high activity of giardial arginine deiminase and the arginine dihydrolase pathway. However, in contrast, both T. vaginalis and C. luciliae contained considerable intracellular pools of arginine. When the G. intestinalis trophozoites were divided into the two populations existing in in vitro culture--attached and nonattached--there were no significant differences between the amino acid profiles of the two populations, with the exception of citrulline, which was found in lower concentrations in the nonattached cells. The T. vaginalis profile was characterised by high concentrations of valine and leucine, whereas the C. luciliae profile was dominated by high levels of glutamate and proline. Overall, the analysis of the total amino acid pool provides a valuable technique to rapidly highlight those amino acids of potential metabolic significance and to provide a rapid technique for defining the nature of amino acid metabolic interactions in situ.

The transport and metabolism of alanine by Giardia intestinalis.

The transport and metabolism of L-alanine by Giardia intestinalis trophozoites was characterised. G. intestinalis formed 14CO2 from L-[1-14C]alanine (1 mM) at a rate of 4.8 nmol min-1 (mg protein)-1 at 30 degrees C. The system was saturable, with an apparent Km of 0.29 mM for alanine, and a maximal rate of 6.1 nmol min-1 (mg protein)-1. L-cycloserine inhibited the metabolism, as did a number of amino acids including glycine, serine and threonine. D-alanine and 2-aminoisobutyrate had no effect. G. intestinalis was shown to have a functional transport system for L-alanine. The transporter was saturable with a Km of 1.5 mM and a maximal velocity of 6.1 nmol min-1 (mg protein)-1 at 23 degrees C. It was temperature dependent, with a Q10 of 2.2 and activation energy of 15.9 kcal mol-1. It was not inhibited by potential inhibitors of energy dependent transport. Glycine, L-serine and L-threonine potently inhibited L-alanine transport, whereas D-alanine, beta-alanine and 2-aminoisobutyrate had no effect. L-serine competitively inhibited L-alanine influx. In trophozoites preloaded with [3H]alanine, rapid exchange occurred with external L-alanine and L-serine, but not with D-alanine confirming that L-alanine and L-serine share a common transport site. These observations indicate that G. intestinalis has a functional alanine transporter, which may be an antiport catalysing the exchange of alanine, serine, glycine and threonine.

Glucose transport in Crithidia luciliae.

The glucose analogue, 2-deoxy-D-glucose, was used to characterise the glucose transport system in Crithidia luciliae choanomastigotes. Uptake was temperature dependent with a Q10 of 2, and saturable with a Km of 0.22 mM and Vmax of 5.5 nmol min-1 (mg protein)-1 at 23 degrees C. Preloaded cells showed rapid exchange of intracellular 2-deoxy-D-glucose when incubated with extracellular D-glucose or 2-deoxy-D-glucose but little exchange with L-glucose. The substrate specificity of the uptake was studied using a number of D-glucose analogues. 6-Deoxy-D-glucose, 3-fluoro-3-deoxy-D-glucose and 4-fluoro-4-deoxy-D-glucose all competed for the transporter and had significant inhibitory effects on 2-deoxy-D-glucose transport. In contrast, 1-thio-beta-D-glucose, trehalose, 3-O-methyl-D-glucose, arginine, thymidine, L-sorbose and L-glucose were not inhibitory. The results imply the existence of a glucose transporter. The transport was blocked by a number of inhibitors and ionophores, including fluoride, azide, cyanide, dinitrophenol, valinomycin and nigericin. Overall, the uptake, exchange and efflux of 2-deoxy-D-glucose is consistent with transport via facilitated diffusion.