Background: Infections with Salmonella cause significant morbidity and mortality worldwide. Replication of Salmonella typhimurium inside its host cell is a model system for studying the pathogenesis of intracellular bacterial infections. Genome-scale modeling of bacterial metabolic networks provides a powerful tool to identify and analyze pathways required for successful intracellular replication during host-pathogen interaction.

SummaryThe secretion of pathogenicity factors by Salmonella typhimurium is mediated by a type III secretion system that includes an outer membrane protein of the secretin family. Related secretins are also required for f1 phage assembly and type II secretion. When the Cterminal 43 amino acids of the S. typhimurium secretin InvG are added to f1 pIV, the chimeric f1 pIV-ЈInvG 43 protein becomes dependent on the co-expression of another gene, invH, for function in phage assembly.[ 3 H]-palmitic acid labelling, globomycin sensitivity and density gradient flotation were used to demonstrate that InvH is an outer membrane lipoprotein that is processed by signal peptidase II. A complex between chimeric f1 pIV-ЈInvG 43 and InvH was demonstrated in vivo. InvH was shown to be required for the proper localization of InvG in the outer membrane and for the secretion of the virulence factor SipC. These results suggest that InvH and InvG are part of the functional outer membrane translocation complex in type III secretion systems.

SummaryRelated outer membrane proteins, termed secretins, participate in the secretion of macromolecules across the outer membrane of many Gram-negative bacteria. In the pullulanase-secretion system, PulS, an outer membrane-associated lipoprotein, is required both for the integrity and the proper outer membrane localization of the PulD secretin. Here we show that the PulS-binding site is located within the C-terminal 65 residues of PulD. Addition of this domain to the filamentous phage secretin, pIV, or to the unrelated maltose-binding protein rendered both proteins dependent on PulS for stability. A chimeric protein composed of bacteriophage f1 pIV and the C-terminal domain of PulD required properly localized PulS to support phage assembly. An in vivo complex formed between the pIV-PulD 65 chimera and PulS was detected by co-immunoprecipitation and by affinity chromatography.

BackgroundIntracellular bacterial pathogens depend on acquisition of iron for their success as pathogens. The host cell requires iron as an essential component for cellular functions that include innate immune defense mechanisms. The transferrin receptor TfR1 plays an important part for delivering iron to the host cell during infection. Its expression can be modulated by infection, but its essentiality for bacterial intracellular survival has not been directly investigated.ResultsWe identified two distinct iron-handling scenarios for two different bacterial pathogens. Francisella tularensis drives an active iron acquisition program via the TfR1 pathway program with induction of ferrireductase (Steap3), iron membrane transporter Dmt1, and iron regulatory proteins IRP1 and IRP2, which is associated with a sustained increase of the labile iron pool inside the macrophage. Expression of TfR1 is critical for Francisella's intracellular proliferation. This contrasts with infection of macrophages by wild-type Salmonella typhimurium, which does not require expression of TfR1 for successful intracellular survival. Macrophages infected with Salmonella lack significant induction of Dmt1, Steap3, and IRP1, and maintain their labile iron pool at normal levels.ConclusionThe distinction between two different phenotypes of iron utilization by intracellular pathogens will allow further characterization and understanding of host-cell iron metabolism and its modulation by intracellular bacteria.

SummarySalmonella enterica uses a type III secretion system encoded by SPI-2 to target specific virulence factors into the host cytosol of macrophages to inhibit the phagosomal-lysosomal maturation pathway. This ensures survival of Salmonella inside its intracellular niche, the Salmonella -containing vacuole (SCV). One such virulence factor is SpiC, which was previously shown to interfere with intracellular vesicular trafficking. In this study we have used a yeast two-hybrid assay to identify a NIPSNAP homologue as host cell target for SpiC that we have termed TassC. In vitro and in vivo co-purification of SpiC and TassC confirm the specificity of this interaction. Suppression of TassC production compensates a SpiC production deficit and allows spiC -Salmonella to survive within macrophages at levels comparable to wild-type Salmonella . We hypothesize that TassC is a host cell factor that determines vesicular trafficking in macrophages and is inactivated by Salmonella SpiC.

Francisella tularensis is a pathogen optimally adapted to efficiently invade its respective host cell and to proliferate intracellularly. We investigated the role of host cell membrane microdomains in the entry of F. tularensis subspecies holarctica vaccine strain (F. tularensis live vaccine strain) into murine macrophages. F. tularensis live vaccine strain recruits cholesterol-rich lipid domains (“lipid rafts”) with caveolin-1 for successful entry into macrophages. Interference with lipid rafts through the depletion of plasma membrane cholesterol, through induction of raft internalization with choleratoxin, or through removal of raft-associated GPI-anchored proteins by treatment with phosphatidylinositol phospholipase C significantly inhibited entry of Francisella and its intracellular proliferation. Lipid raft-associated components such as cholesterol and caveolin-1 were incorporated into Francisella-containing vesicles during entry and the initial phase of intracellular trafficking inside the host cell. These findings demonstrate that Francisella requires cholesterol-rich membrane domains for entry into and proliferation inside macrophages.

Many surface proteins are thought to be anchored to the cell wall of gram-positive organisms via their C termini, while the N-terminal domains of these molecules are displayed on the bacterial surface. Cell wall anchoring of surface proteins in Staphylococcus aureus requires both an N-terminal leader peptide and a C-terminal cell wall sorting signal. By fusing the cell wall sorting signal of protein A to the C terminus of staphylococcal ␤-lactamase, we demonstrate here that lipoproteins can also be anchored to the cell wall of S. aureus. The topology of cell wall-anchored ␤-lactamase is reminiscent of that described for Braun's murein lipoprotein in that the N terminus of the polypeptide chain is membrane anchored whereas the C-terminal end is tethered to the bacterial cell wall.Anchoring of surface proteins to the cell wall of Staphylococcus aureus occurs by a mechanism that requires an N-terminal leader peptide and a C-terminal cell wall sorting signal (30,31). The sorting signal consists of an LPXTG sequence motif followed by a hydrophobic domain and a positively charged domain (30). Surface proteins of S. aureus are first directed into the secretory pathway by an N-terminal leader peptide that is cleaved during the translocation of the polypeptide chain across the cytoplasmic membrane (31). The hydrophobic and the charged domains of the sorting signal function to retain surface proteins within the secretory pathway, which allows proteolytic cleavage between the threonine (T) and the glycine (G) of the LPXTG motif (18). The carboxyl group of threonine is subsequently amide linked to the free amino group of the pentaglycine peptidoglycan cross bridge, thereby linking the C-terminal end of surface proteins to the staphylococcal cell wall (29).More than 100 surface proteins of many different grampositive bacteria harbor an N-terminal leader peptide and a C-terminal cell wall sorting signal (7,31). After N-terminal leader peptide cleavage and C-terminal cell wall anchoring, the N-terminal domains of all these molecules are thought to be displayed freely on the bacterial surface whereas the C-terminal anchor structures are buried in the thick peptidoglycan layer (32, 33). The cellular location of lipoproteins in grampositive organisms is reciprocal to that of surface proteins: the N termini of lipoproteins are tethered to the cytoplasmic membrane by means of a lipid modification, while the C-terminal domains are thought to fulfill a variety of functions similar to those of periplasmic proteins in gram-negative bacteria (9). We wondered whether lipid anchoring and cell wall linkage of polypeptide chains in S. aureus were mutually exclusive events or whether they could occur in the same molecule. To test this possibility, we fused the cell wall sorting signal of protein A to the C terminus of staphylococcal ␤-lactamase, a well-characterized lipoprotein (1,20).The N-terminal lipid modification of staphylococcal ␤-lactamase (BlaZ) is specified by a type II leader peptide (6, 39) and consists of a glyceride thioether l...

Donor-derived bacterial infection is a recognized complication of solid organ transplantation (SOT). The present report describes the clinical details and successful outcome in a liver transplant recipient despite transmission of methicillin-resistant Staphylococcus aureus (MRSA) from a deceased donor with MRSA endocarditis and bacteremia. We further describe whole genome sequencing (WGS) and complete de novo assembly of the donor and recipient MRSA isolate genomes, which confirms that both isolates are genetically 100% identical. We propose that similar application of WGS techniques to future investigations of donor bacterial transmission would strengthen the definition of proven bacterial transmission in SOT, particularly in the presence of highly clonal bacteria such as MRSA. WGS will further improve our understanding of the epidemiology of bacterial transmission in SOT and the risk of adverse patient outcomes when it occurs.