Live-cell imaging reveals single-cell and population-level infection strategies of Listeria monocytogenes in macrophages.

Pathogens have developed intricate strategies to overcome the host's innate immune responses. In this paper we use live-cell microscopy with a single bacterium resolution to follow in real time interactions between the food-borne pathogen L. monocytogenes and host macrophages, a key event controlling the infection in vivo. We demonstrate that infection results in heterogeneous outcomes, with only a subset of bacteria able to establish a replicative invasion of macrophages. The fate of individual bacteria in the same host cell was independent from the host cell and non-cooperative, being independent from co-infecting bacteria. A higher multiplicity of infection resulted in a reduced probability of replication of the overall bacterial population. By use of internalisation assays and conditional probabilities to mathematically describe the two-stage invasion process, we demonstrate that the higher MOI compromises the ability of macrophages to phagocytose bacteria. We found that the rate of phagocytosis is mediated via the secreted Listeriolysin toxin (LLO), while the probability of replication of intracellular bacteria remained constant. Using strains expressing fluorescent reporters to follow transcription of either the LLO-encoding hly or actA genes, we show that replicative bacteria exhibited higher PrfA regulon expression in comparison to those bacteria that did not replicate, however elevated PrfA expression per se was not sufficient to increase the probability of replication. Overall, this demonstrates a new role for the population-level, but not single cell, PrfA-mediated activity to regulate outcomes of host pathogen interactions.

Manipulation of host and parasite microbiotas: Survival strategies during chronic nematode infection.

Intestinal dwelling parasites have evolved closely with the complex intestinal microbiota of their host, but the significance of the host microbiota for metazoan pathogens and the role of their own intestinal microbiota are still not fully known. We have found that the parasitic nematode Trichuris muris acquired a distinct intestinal microbiota from its host, which was required for nematode fitness. Infection of germ-free mice and mice monocolonized with Bacteroides thetaiotaomicron demonstrated that successful T. muris infections require a host microbiota. Following infection, T. muris-induced alterations in the host intestinal microbiota inhibited subsequent rounds of infection, controlling parasite numbers within the host intestine. This dual strategy could promote the long-term survival of the parasite within the intestinal niche necessary for successful chronic nematode infection.

Three tandem promoters, together with IHF, regulate growth phase dependent expression of the Escherichia coli kps capsule gene cluster.

In this study we characterise three tandem promoters (PR1-1, PR1-2 and PR1-3) within the PR1 regulatory region of the Escherichia coli kps capsule gene cluster. Transcription from promoter PR1-2 was dependent on the activity of the upstream promoter PR1-1, which activated PR1-2 via transcription coupled DNA supercoiling. During growth at 37 °C a temporal pattern of transcription from all three promoters was observed with maximum transcriptional activity evident during mid-exponential phase followed by a sharp decrease in activity as the cells enter stationary phase. The growth phase dependent transcription was regulated by Integration Host Factor (IHF), which bound within the PR1 region to repress transcription from PR1-2 and PR1-3. This pattern of transcription was mirrored by growth phase dependent expression of the K1 capsule. Overall these data reveal a complex pattern of transcriptional regulation for an important virulence factor with IHF playing a role in regulating growth phase expression.

Listeria monocytogenes Has Both Cytochrome bd-Type and Cytochrome aa 3-Type Terminal Oxidases, Which Allow Growth at Different Oxygen Levels, and Both Are Important in Infection.

Listeria monocytogenes is a foodborne pathogen responsible for a number of life-threatening infections of humans. During an infection, it invades epithelial cells before spreading from the intestine to the cells of the liver and spleen. This requires an ability to adapt to varying oxygen levels. Here, we demonstrate that L. monocytogenes has two terminal oxidases, a cytochrome bd-type (CydAB) and a cytochrome aa 3-type menaquinol (QoxAB) oxidase, and that both are used for respiration under different oxygen tensions. Furthermore, we show that possession of both terminal oxidases is important in infection. In air, the CydAB bd-type oxidase is essential for aerobic respiration and intracellular replication, and cydAB mutants are highly attenuated in mice. In contrast, the QoxAB aa 3-type oxidase is required neither for aerobic respiration in air nor for intracellular growth. However, the qoxAB mutants are attenuated in mice, with a delay in the onset of disease signs and with increased survival time, indicating a role for the QoxAB aa 3-type oxidase in the initial stages of infection. Growth of bacteria under defined oxygen conditions revealed that at 1% (vol/vol), both oxidases are functional, and the presence of either is sufficient for aerobic respiration and intracellular replication. However, at 0.2% (vol/vol), both oxidases are necessary for maximum growth. These findings are consistent with the ability of L. monocytogenes to switch between terminal oxidases under different oxygen conditions, providing exquisite adaptation to different conditions encountered within the infected host.

Lamellipodin Is Important for Cell-to-Cell Spread and Actin-Based Motility in Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen capable of invading a broad range of cell types and replicating within the host cell cytoplasm. This paper describes the colocalization of host cell lamellipodin (Lpd) with intracellular L. monocytogenes detectable 6 h postinfection of epithelial cells. The association was mediated via interactions between both the peckstrin homology (PH) domain in Lpd and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] on the bacterial surface and by interactions between the C-terminal EVH1 (Ena/VASP [vasodilator-stimulated phosphoprotein] homology domain 1) binding domains of Lpd and the host VASP (vasodilator-stimulated phosphoprotein) recruited to the bacterial cell surface by the listerial ActA protein. Depletion of Lpd by short interfering RNA (siRNA) resulted in reduced plaque size and number, indicating a role for Lpd in cell-to-cell spread. In contrast, overexpression of Lpd resulted in an increase in the number of L. monocytogenes-containing protrusions (listeriopods). Manipulation of the levels of Lpd within the cell also affected the intracellular velocity of L. monocytogenes, with a reduction in Lpd corresponding to an increase in intracellular velocity. These data, together with the observation that Lpd accumulated at the interface between the bacteria and the developing actin tail at the initiation of actin-based movement, indicate a possible role for Lpd in the actin-based movement and the cell-to-cell spread of L. monocytogenes.

Recombinant plants provide a new approach to the production of bacterial polysaccharide for vaccines.

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections.

Inhibition of calpain blocks the phagosomal escape of Listeria monocytogenes.

Listeria monocytogenes is a gram-positive facultative intracellular bacterium responsible for the food borne infection listeriosis, affecting principally the immunocompromised, the old, neonates and pregnant women. Following invasion L. monocytogenes escapes the phagosome and replicates in the cytoplasm. Phagosome escape is central to L. monocytogenes virulence and is required for initiating innate host-defence responses such as the secretion of the cytokine interleukin-1. Phagosome escape of L. monocytogenes is reported to depend upon host proteins such as γ-interferon-inducible lysosomal thiol reductase and the cystic fibrosis transmembrane conductance regulator. The host cytosolic cysteine protease calpain is required in the life cycle of numerous pathogens, and previous research reports an activation of calpain by L. monocytogenes infection. Thus we sought to determine whether host calpain was required for the virulence of L. monocytogenes. Treatment of macrophages with calpain inhibitors blocked escape of L. monocytogenes from the phagosome and consequently its proliferation within the cytosol. This was independent of any direct effect on the production of bacterial virulence factors or of a bactericidal effect. Furthermore, the secretion of interleukin-1ß, a host cytokine whose secretion induced by L. monocytogenes depends upon phagosome escape, was also blocked by calpain inhibition. These data indicate that L. monocytogenes co-opts host calpain to facilitate its escape from the phagosome, and more generally, that calpain may represent a cellular Achilles heel exploited by pathogens.

The K5 lyase KflA combines a viral tail spike structure with a bacterial polysaccharide lyase mechanism.

K5 lyase A (KflA) is a tail spike protein (TSP) encoded by a K5A coliphage, which cleaves K5 capsular polysaccharide, a glycosaminoglycan with the repeat unit [-4)-betaGlcA-(1,4)- alphaGlcNAc(1-], displayed on the surface of Escherichia coli K5 strains. The crystal structure of KflA reveals a trimeric arrangement, with each monomer containing a right-handed, single-stranded parallel beta-helix domain. Stable trimer formation by the intertwining of strands in the C-terminal domain, followed by proteolytic maturation, is likely to be catalyzed by an autochaperone as described for K1F endosialidase. The structure of KflA represents the first bacteriophage tail spike protein combining polysaccharide lyase activity with a single-stranded parallel beta-helix fold. We propose a catalytic site and mechanism representing convergence with the syn-beta-elimination site of heparinase II from Pedobacter heparinus.

The K5 capsule of Escherichia coli strain Nissle 1917 is important in stimulating expression of Toll-like receptor 5, CD14, MyD88, and TRIF together with the induction of interleukin-8 expression via the mitogen-activated protein kinase pathway in epithelial cells.

Escherichia coli strain Nissle 1917, which has been widely used as a probiotic for the treatment of inflammatory bowel disorders, expresses a K5 capsule, the expression of which is often associated with extraintestinal and urinary tract isolates of E. coli. Previously, it had been shown that the expression of a K5 capsule by Nissle 1917 was important in mediating interactions with epithelial cells and the extent of chemokine expression. In this paper, we show that infection with Nissle 1917 induces expression of Toll-like receptor 4 (TLR4) and TLR5 in Caco-2 cells and that maximal induction of TLR5 required the K5 capsule. In addition, purified K5 polysaccharide was capable of inducing expression of TLR5 and mCD14 and potentiated the activity of both TLR4 and TLR5 agonists to increase the proinflammatory response. Infection with Nissle 1917 also increased the expression of the adaptor molecules MyD88 and TRIF, which was K5 capsule dependent. By Western blot analysis, it was possible to show that induction of interleukin-8 by Nissle 1917 was predominantly through the mitogen-activated protein (MAP) kinase pathway and that expression of the K5 capsule was important for activation of the MAP kinase pathway. This paper provides new information on the function of the K5 capsule in mediating interactions between Nissle 1917 and epithelial cells and the mechanisms that underlie the probiotic properties of Nissle 1917.

Structure-based mechanism of CMP-2-keto-3-deoxymanno-octulonic acid synthetase: convergent evolution of a sugar-activating enzyme with DNA/RNA polymerases.

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2beta-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2beta-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg(2+) ion (Mg-B). Both ligands occupy conformations compatible with an S(n)2-type attack on the alpha-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg(2+) ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp(100) and Asp(235) in addition to the CTP alpha-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn(2+)-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg(2+) to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the alpha-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group.

The K5 capsule of Escherichia coli strain Nissle 1917 is important in mediating interactions with intestinal epithelial cells and chemokine induction.

Escherichia coli strain Nissle 1917 has been widely used as a probiotic for the treatment of inflammatory bowel disorders and shown to have immunomodulatory effects. Nissle 1917 expresses a K5 capsule, the expression of which often is associated with extraintestinal and urinary tract isolates of E. coli. In this paper, we investigate the role of the K5 capsule in mediating interactions between Nissle 1917 and intestinal epithelial cells. We show that the loss of capsule significantly reduced the level of monocyte chemoattractant protein 1 (MCP-1), RANTES, macrophage inflammatory protein 2alpha (MIP-2alpha), MIP-2beta, interleukin-8, and gamma interferon-inducible protein 10 induction by Nissle 1917 in both Caco-2 cells and MCP-1 induction in ex vivo mouse small intestine. The complementation of the capsule-minus mutation confirmed that the effects on chemokine induction were capsule specific. The addition of purified K5, but not K1, capsular polysaccharide to the capsule-minus Nissle 1917 at least in part restored chemokine induction to wild-type levels. The purified K5 capsular polysaccharide alone was unable to stimulate chemokine production, indicating that the K5 polysaccharide was acting to mediate interactions between Nissle 1917 and intestinal epithelial cells. The induction of chemokine by Nissle 1917 was generated predominantly by interaction with the basolateral surface of Caco-2 cells, suggesting that Nissle 1917 will be most effective in inducing chemokine expression where the epithelial barrier is disrupted.

The Escherichia coli K5 capsule is not synthesized in a protected compartment within the cytoplasm.

The intracellular expression of the K5 lyase enzyme, which degrades the K5 polysaccharide, decreased cell surface expression of the Escherichia coli K5 capsule. This indicates that biosynthesis of K5 polysaccharide in the cytoplasm is accessible to the action of K5 lyase and is not synthesized within a protected cytoplasmic compartment.

Regulation of expression of the region 3 promoter of the Escherichia coli K5 capsule gene cluster involves H-NS, SlyA, and a large 5' untranslated region.

Escherichia coli group 2 capsule gene clusters are temperature regulated, being expressed at 37 degrees C but not at 20 degrees C. Expression is regulated at the level of transcription by two convergent promoters, PR1 and PR3. In this paper, we show that regulation of transcription from PR3 involves a number of novel features including H-NS, SlyA, and a large 741-bp 5' untranslated region (UTR). H-NS represses transcription from PR3 at 20 degrees C and binds both 5' and 3' of the transcription start site. The 3' downstream regulatory element (DRE) was essential for temperature-dependent H-NS repression. At 37 degrees C, SlyA activates transcription independent of H-NS but maximal transcription requires H-NS. The UTR is present between the transcription start site and the first gene in the operon, kpsM. We demonstrate that the UTR, as well as containing the H-NS DRE, functions to moderate the extent of transcription that reaches kpsM and allows the binding of antitermination factor RfaH.

Mutations in the waaR gene of Escherichia coli which disrupt lipopolysaccharide outer core biosynthesis affect cell surface retention of group 2 capsular polysaccharides.

On the basis of increased resistance to K5 capsule-specific bacteriophage, a waaR transposon mutant defective in the biosynthesis of lipopolysaccharide outer core was isolated. In a K1-expressing strain the mutation equally affected sensitivity to K1 capsule-specific bacteriophage, indicating a general effect on group 2 capsules. The waaR mutation affected retention on the cell surface of the K5 polysaccharide, with increased polysaccharide accumulating in the culture supernatant. This indicates that interactions between the outer core of lipopolysaccharide and group 2 capsular polysaccharides are important for the stabilization of group 2 capsular polysaccharides on the cell surface.

Analysis of the role of HP0208, a phase-variable open reading frame, and its homologues HP1416 and HP0159 in the biosynthesis of Helicobacter pylori lipopolysaccharide.

The roles of the three ORFs HP0208, HP0159 and HP1416 in the biosynthesis of Helicobacter pylori 26695 LPS were investigated in this study. These ORFs represent a paralogous family of genes with homology to the Salmonella enterica serovar Typhimurium (hereafter referred to as S. typhimurium) waaJ gene, which encodes an alpha-1,2-glycosyltransferase required for core LPS biosynthesis. HP0208 contains multiple tandem repeats of the dimer 5'GA at its 5' end and its expression is predicted to be subject to phase variation. The number of 5'GA repeats present in this ORF was found to be non-permissive for the expression of HP0208 in the majority of H. pylori strains examined. To determine a role for this ORF in LPS biosynthesis a non-phase-variable, constitutively expressed variant of HP0208 was constructed and introduced into the genome of H. pylori 26695. Analysis of the LPS profile of this strain by Tricine-SDS-PAGE and immunoblotting with anti-Lewis Y antigen (Le(y)) mAbs confirmed a role for HP0208 in the biosynthesis of core LPS. A role for HP0159 and HP1416 in the biosynthesis of core LPS was also established. Although homologous to waaJ, H. pylori HP0208, HP0159 and HP1416 failed to complement an S. typhimurium waaJ mutant, suggesting that these ORFs encode functionally different enzymes.