Endocytic recycling and vesicular transport systems mediate transcytosis of Leptospira interrogans across cell monolayer.

Many bacterial pathogens can cause septicemia and spread from the bloodstream into internal organs. During leptospirosis, individuals are infected by contact with Leptospira-containing animal urine-contaminated water. The spirochetes invade internal organs after septicemia to cause disease aggravation, but the mechanism of leptospiral excretion and spreading remains unknown. Here, we demonstrated that Leptospira interrogans entered human/mouse endothelial and epithelial cells and fibroblasts by caveolae/integrin-ß1-PI3K/FAK-mediated microfilament-dependent endocytosis to form Leptospira (Lep)-vesicles that did not fuse with lysosomes. Lep-vesicles recruited Rab5/Rab11 and Sec/Exo-SNARE proteins in endocytic recycling and vesicular transport systems for intracellular transport and release by SNARE-complex/FAK-mediated microfilament/microtubule-dependent exocytosis. Both intracellular leptospires and infected cells maintained their viability. Leptospiral propagation was only observed in mouse fibroblasts. Our study revealed that L. interrogans utilizes endocytic recycling and vesicular transport systems for transcytosis across endothelial or epithelial barrier in blood vessels or renal tubules, which contributes to spreading in vivo and transmission of leptospirosis.

A SUMOylation-dependent switch of RAB7 governs intracellular life and pathogenesis of Salmonella Typhimurium.

Salmonella Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Aided by a battery of effector proteins, S. Typhimurium resides intracellularly in a specialized vesicle, called the Salmonella-containing vacuole (SCV) that utilizes the host endocytic vesicular transport pathway (VTP). Here, we probed the possible role of SUMOylation, a post-translation modification pathway, in SCV biology. Proteome analysis by complex mass-spectrometry (MS/MS) revealed a dramatically altered SUMO-proteome (SUMOylome) in S. Typhimurium-infected cells. RAB7, a component of VTP, was key among several crucial proteins identified in our study. Detailed MS/MS assays, in vitro SUMOylation assays and structural docking analysis revealed SUMOylation of RAB7 (RAB7A) specifically at lysine 175. A SUMOylation-deficient RAB7 mutant (RAB7K175R) displayed longer half-life, was beneficial to SCV dynamics and functionally deficient. Collectively, the data revealed that RAB7 SUMOylation blockade by S. Typhimurium ensures availability of long-lived but functionally compromised RAB7, which was beneficial to the pathogen. Overall, this SUMOylation-dependent switch of RAB7 controlled by S. Typhimurium is an unexpected mode of VTP pathway regulation, and unveils a mechanism of broad interest well beyond Salmonella-host crosstalk. This article has an associated First Person interview with the first author of the paper.

Interaction of microbial pathogens with host exocytic pathways.

Many microbial pathogens co-opt or perturb host membrane trafficking pathways. This review covers recent examples in which microbes interact with host exocytosis, the fusion of intracellular vesicles with the plasma membrane. The bacterial pathogens Listeria monocytogenes and Staphylococcus aureus subvert recycling endosomal pathways of exocytosis in order to induce their entry into human cells. By contrast, entry of the protozoan pathogen Trypanosoma cruzi or the virus adenovirus into host cells involves exploitation of lysosomal exocytosis. Toxins produced by Bacillus anthracis or Vibrio cholerae interfere with exocytosis pathways mediated by the GTPase Rab11 and the exocyst complex. By doing so, anthrax or cholera toxins impair recycling of cadherins to cell-cell junctions and disrupt the barrier properties of endothelial cells or intestinal epithelial cells, respectively. Uropathogenic Escherichia coli (UPEC) is expelled from bladder epithelial cells through two different exocytic routes that involve sensing of bacteria in vacuoles by host Toll-like receptor 4 (TLR4) or monitoring of the pH of lysosomes harbouring UPEC. The TLR4 pathway is mediated by multiple Rab GTPases and the exocyst, whereas the other pathway involves exocytosis of lysosomes. Expulsion of UPEC through these pathways is thought to benefit the host.

The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier.

The Gram-positive bacterium Listeria monocytogenes can enter the human central nervous system and cause life-threatening meningitis. During this process the pathogen has to invade and cross diverse cellular barriers involving the functions of the surface proteins Internalin (InlA) and InlB. Whereas the internalin-dependent crossing of the intestinal epithelium and the fetoplacental barrier have been subject to intensive investigation, limited research elucidating the crossing of the human blood-cerebrospinal fluid barrier (BCSFB) has been reported. We have recently established a functional in vitro model of the BCSFB based on human choroid plexus papilloma (HIBCPP) cells. We show polarized expression of receptors involved in listerial invasion (i.e. E-Cadherin, Met) in HIBCPP cells. Infecting HIBCPP cells with the L. monocytogenes strain EGD, we demonstrate polar invasion exclusively from the in vivo relevant basolateral cell side. Intracellular listeria were found in vacuoles and the cytoplasm, where they were often associated with "actin tail"-like structures. Furthermore, the L. monocytogenes wild type strain shows significantly higher internalization rates than isogenic mutants lacking either InlA, InlB or both surface proteins. Deletion of either one or both proteins leads to a similarly decreased invasion, suggesting an interdependent function of InlA and InlB during invasion of choroid plexus epithelial cells.

Enterocyte microvillus-derived vesicles detoxify bacterial products and regulate epithelial-microbial interactions.

The continuous monolayer of intestinal epithelial cells (IECs) lining the gut lumen functions as the site of nutrient absorption and as a physical barrier to prevent the translocation of microbes and associated toxic compounds into the peripheral vasculature. IECs also express host defense proteins such as intestinal alkaline phosphatase (IAP), which detoxify bacterial products and prevent intestinal inflammation. Our laboratory recently showed that IAP is enriched on vesicles that are released from the tips of IEC microvilli and accumulate in the intestinal lumen. Here, we show that these native "lumenal vesicles" (LVs) (1) contain catalytically active IAP that can dephosphorylate lipopolysaccharide (LPS), (2) cluster on the surface of native lumenal bacteria, (3) prevent the adherence of enteropathogenic E. coli (EPEC) to epithelial monolayers, and (4) limit bacterial population growth. We also find that IECs upregulate LV production in response to EPEC and other Gram-negative pathogens. Together, these results suggest that microvillar vesicle shedding represents a novel mechanism for distributing host defense machinery into the intestinal lumen and that microvillus-derived LVs modulate epithelial-microbial interactions.

Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking.

Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes.

Salmonella enterica replication in hemophagocytic macrophages requires two type three secretion systems.

Salmonella enterica serotype Typhimurium is a natural pathogen of mice, which acquire the bacteria orally and develop systemic acute infections that can become subacute to chronic infections. S. Typhimurium can reside within hemophagocytic macrophages (HMs) in SV129S6 mice, an Slc11a1/Nramp1(+/+) inbred strain. HMs are activated macrophages which have ingested viable hematopoietic cells and are a key characteristic of infectious and inflammatory diseases. Here we show that modest S. Typhimurium replication in HMs begins at 18 h postinfection, while activated macrophages kill the bacteria. For bacterial replication to occur, the phagocytosed viable cells must be grown to a low cell density and the multiplicity of infection must be low. HMs are able to kill phagocytosed Escherichia coli, produce reactive nitrogen species, and retain S. Typhimurium within membrane-bound vesicles. S. Typhimurium does not rescue E. coli upon coinfection of HMs. This indicates that S. Typhimurium does not cause HMs to become permissive for other microbes; rather, S. Typhimurium is especially equipped to survive within HMs. Two type three secretion systems (T3SS) encoded by S. Typhimurium are required for replication within HMs. While the T3SS within Salmonella pathogenicity island 2 (SPI-2) has been previously shown to be important for bacterial survival in cells, a role for SPI-1 in replication in macrophages has not been reported. The requirement for SPI-1 in HMs may help explain the role of SPI-1 during long-term colonization of mice.

Bacterial particle endocytosis by epithelial cells is selective and enhanced by tumor necrosis factor receptor ligands.

Bacterial pathogens use virulence strategies to invade epithelial barriers, but active processes of epithelial cells may also contribute to the endocytosis of microbial particles. To focus on the latter, we studied the uptake of fixed and fluorescently labeled bacterial particles in intestinal and bronchoepithelial cell cultures and found it to be enhanced in Caco-2BBe and NCI-H292 cells after treatment with tumor necrosis factor alpha and an agonist antibody against the lymphotoxin beta receptor. Confocal fluorescence microscopy, flow cytometry, and transmission electron microscopy revealed that Staphylococcus aureus and Yersinia enterocolitica were readily endocytosed, although there was scant uptake of Shigella sonnei, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae particles. Endocytosed Staphylococcus was often associated with cytoplasmic claudin-4 vesicles; this was not found for Yersinia, suggesting that cytokine treatment upregulated two distinct endocytosis pathways. Interestingly, when Staphylococcus and Yersinia were coincubated with epithelial monolayers, the cells were unlikely to take up Yersinia unless they had also endocytosed large numbers of Staphylococcus particles, although the two bacteria were apparently processed in distinct compartments. Cytokine treatment induced an upregulation and redistribution of beta1 integrin to the apical surface of NCI-H292 cells; consistent with this effect, treatment with anti-beta1 integrin antibody blocked uptake of both Yersinia and Staphylococcus in NCI-H292 and Caco-2BBe cells. Our results suggest that capture of bacterial particles by mucosal epithelial cells is selective and that different endocytic mechanisms are enhanced by proinflammatory cytokines.

Francisella targets cholesterol-rich host cell membrane domains for entry into macrophages.

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.

Late endocytic multivesicular bodies intersect the chlamydial inclusion in the absence of CD63.

Chlamydiae are obligate intracellular bacterial pathogens that replicate solely within a membrane-bound vacuole termed an inclusion. Within the confines of the inclusion, the replicating bacteria acquire amino acids, nucleotides, and other precursors from the host cell. Trafficking from CD63-positive multivesicular bodies to the inclusion was previously identified as a novel interaction that provided essential precursors for the maintenance of a productive intracellular infection. The present study analyzes the direct delivery of resident protein and lipid constituents of multivesicular bodies to the intracellular chlamydiae. The manipulation of this trafficking pathway with an inhibitor of multivesicular body transport and the delivery of exogenous antibodies altered protein and cholesterol acquisition and delayed the maturation of the chlamydial inclusion. Although inhibitor studies and ultrastructural analyses confirmed a novel interaction between CD63-positive multivesicular bodies and the intracellular chlamydiae, neutralization with small interfering RNAs and anti-CD63 Fab fragments revealed that CD63 itself was not required for this association. These studies confirm CD63 as a constituent in multivesicular body-to-inclusion transport; however, other requisite components of these host cell compartments must control the delivery of key nutrients that are essential to intracellular bacterial development.

Invasion and biofilm formation of Burkholderia dolosa is comparable with Burkholderia cenocepacia and Burkholderia multivorans.

BACKGROUND: Colonisation with Burkholderia cepacia complex pathogens has been associated with accelerated decline in cystic fibrosis (CF) patients. The two most common species among the CF community are Burkholderia cenocepacia and Burkholderia multivorans. However, Burkholderia dolosa has recently been causing concern due to its transmissibility and virulence in CF patients. METHODS: We have compared the ability of five B. dolosa strains to invade lung epithelial cells in vitro with other members of the Bcc. The bacterial epithelial cell interaction was visualised by transmission electron microscopy. We have also examined the ability of these strains to form biofilms in vitro. RESULTS: We have found that members of this species can invade pulmonary epithelial cells in vitro as readily as those from B. cenocepacia and B. multivorans. Confirmation of intracellular invasion was obtained by transmission electron microscopy. B. dolosa strains were readily observed in membrane bound vesicles inside the lung epithelial cells. In addition, strains from this species were capable of forming strong biofilms at a level comparable to the more clinically relevant species. CONCLUSIONS: B. dolosa shows comparable virulence characteristics in vitro to the two most clinically relevant species indicating precautions should be taken when this species is identified in the CF population.

Degradation of Chlamydia pneumoniae by peripheral blood monocytic cells.

Chlamydia pneumoniae is a common human respiratory pathogen that has been associated with a variety of chronic diseases, including atherosclerosis. The role of this organism in the pathogenesis of atherosclerosis remains unknown. A key question is how C. pneumoniae is transferred from the site of primary infection to a developing atherosclerotic plaque. It has been suggested that circulating monocytes could be vehicles for dissemination of C. pneumoniae since the organism has been detected in peripheral blood monocytic cells (PBMCs). In this study we focused on survival of C. pneumoniae within PBMCs isolated from the blood of healthy human donors. We found that C. pneumoniae does not grow and multiply in cultured primary monocytes. In C. pneumoniae-infected monocyte-derived macrophages, growth of the organism was very limited, and the majority of the bacteria were eradicated. We also found that the destruction of C. pneumoniae within infected macrophages resulted in a gradual diminution of chlamydial antigens, although some of these antigens could be detected for days after the initial infection. The detected antigens present in infected monocytes and monocyte-derived macrophages represented neither chlamydial inclusions nor intact organisms. The use of {N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]}-6-aminocaproyl-d-erythro-sphingosine as a vital stain for chlamydiae proved to be a sensitive method for identifying rare C. pneumoniae inclusions and was useful in the detection of even aberrant developmental forms.

Salmonella modulates vesicular traffic by altering phosphoinositide metabolism.

Salmonella enterica, the cause of food poisoning and typhoid fever, induces actin cytoskeleton rearrangements and membrane ruffling to gain access into nonphagocytic cells, where it can replicate and avoid innate immune defenses. Here, we found that SopB, a phosphoinositide phosphatase that is delivered into host cells by a type III secretion system, was essential for the establishment of Salmonella's intracellular replicative niche. SopB mediated the formation of spacious phagosomes following bacterial entry and was responsible for maintaining high levels of phosphatidylinositol-three-phosphate [PtdIns(3)P] in the membrane of the bacteria-containing vacuoles. Absence of SopB caused a significant defect in the maturation of the Salmonella-containing vacuole and impaired bacterial intracellular growth.

Dynein-mediated vesicle transport controls intracellular Salmonella replication.

Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.