Newly Isolated Animal Pathogen Corynebacterium silvaticum Is Cytotoxic to Human Epithelial Cells.

Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.

Acinetobacter baumannii outer membrane protein A induces HeLa cell autophagy via MAPK/JNK signaling pathway.

Autophagy is an evolutionary conserved self-balancing process that plays an important role in maintaining cellular homeostasis via the clearance of damaged organelles and misfolded proteins. Infection-triggered autophagy specifically inhibits the invasion of intracellular bacterial replication and hence protects the cells from microbial infections. It has been reported that Acinetobacter baumannii trigger cell autophagy. However, the role of its virulence protein OmpA remains unclear. Therefore, this study aimed to explore the effects of Acinetobacter baumannii OmpA on cell autophagy and its underlying molecular mechanisms. The results showed that OmpA induced autophagy in HeLa and RAW264.7 cells, increased LC3BII expression, and hindered p62 degradation. Moreover, OmpA triggered incomplete autophagy by interfering the fusion of autophagosomes with lysosomes. Besides, OmpA activated MAPK/JNK signaling pathway and enhanced the phosphorylation levels of JNK, p38, and ERK, c-Jun. Inhibition of JNK signaling pathway suppressed OmpA-induced autophagy in HeLa cells. Ab wild-type strains carrying OmpA triggered incomplete autophagy and resulted in a large number of IL-1ß production. Ab-△OmpA strain (OmpA gene mutation) restored autophagic flux and reduced the accumulation of p62 and the release of IL-1ß in HeLa cells. Rapamycin activated autophagy to inhibit OmpA-induced IL-1ß secretion and protect HeLa cells from inflammatory damage. Collectively, these results suggest that OmpA can induce autophagy in HeLa cells through MAPK/JNK signaling pathway. Pre-treatment with Rapamycin activates autophagy and protects against cell death.

Chlamydia trachomatis utilizes the mammalian CLA1 lipid transporter to acquire host phosphatidylcholine essential for growth.

Phosphatidylcholine is a constituent of Chlamydia trachomatis membranes that must be acquired from its mammalian host to support bacterial proliferation. The CLA1 (SR-B1) receptor is a bi-directional phosphatidylcholine/cholesterol transporter that is recruited to the inclusion of Chlamydia-infected cells along with ABCA1. C. trachomatis growth was inhibited in a dose-dependent manner by BLT-1, a selective inhibitor of CLA1 function. Expression of a BLT-1-insensitive CLA1(C384S) mutant ameliorated the effect of the drug on chlamydial growth. CLA1 knockdown using shRNAs corroborated an important role for CLA1 in the growth of C. trachomatis. Trafficking of a fluorescent phosphatidylcholine analogue to Chlamydia was blocked by the inhibition of CLA1 or ABCA1 function, indicating a critical role for these transporters in phosphatidylcholine acquisition by this organism. Our analyses using a dual-labelled fluorescent phosphatidylcholine analogue and mass spectrometry showed that the phosphatidylcholine associated with isolated Chlamydia was unmodified host phosphatidylcholine. These results indicate that C. trachomatis co-opts host phospholipid transporters normally used to assemble lipoproteins to acquire host phosphatidylcholine essential for growth.

The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells.

Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.

Shigella flexneri modulates stress granule composition and inhibits stress granule aggregation.

Invasion and multiplication of the facultative, cytosolic, enteropathogen Shigella flexneri within the colonic epithelial lining leads to an acute inflammatory response, fever and diarrhea. During the inflammatory process, infected cells are subjected to numerous stresses including heat, oxidative stress and genotoxic stress. The evolutionarily conserved pathway of cellular stress management is the formation of stress granules that store translationally inactive cellular mRNAs and interfere with cellular signalling pathways by sequestering signalling components. In this study, we investigated the ability of S. flexneri-infected cells to form stress granules in response to exogenous stresses. We found that S. flexneri infection inhibits movement of the stress granule markers eIF3 and eIF4B into stress granules and prevents the aggregation of G3BP1 and eIF4G-containing stress granules. This inhibition occurred only with invasive, but not with non-invasive bacteria and occurred in response to stresses that induce translational arrest through the phosphorylation of eIF2α and by treating cells with pateamine A, a drug that induces stress granules by inhibiting the eIF4A helicase. The S. flexneri-mediated stress granule inhibition could be largely phenocopied by the microtubule-destabilizing drug nocodazole and while S. flexneri infection did not lead to microtubule depolymerization, infection greatly enhanced acetylation of alpha-tubulin. Our data suggest that qualitative differences in the microtubule network or subversion of the microtubule-transport machinery by S. flexneri may be involved in preventing the full execution of this cellular stress response.

Lipid exchange between Borrelia burgdorferi and host cells.

Borrelia burgdorferi, the agent of Lyme disease, has cholesterol and cholesterol-glycolipids that are essential for bacterial fitness, are antigenic, and could be important in mediating interactions with cells of the eukaryotic host. We show that the spirochetes can acquire cholesterol from plasma membranes of epithelial cells. In addition, through fluorescent and confocal microscopy combined with biochemical approaches, we demonstrated that B. burgdorferi labeled with the fluorescent cholesterol analog BODIPY-cholesterol or (3)H-labeled cholesterol transfer both cholesterol and cholesterol-glycolipids to HeLa cells. The transfer occurs through two different mechanisms, by direct contact between the bacteria and eukaryotic cell and/or through release of outer membrane vesicles. Thus, two-way lipid exchange between spirochetes and host cells can occur. This lipid exchange could be an important process that contributes to the pathogenesis of Lyme disease.

Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

Influence of oligomerization state on the structural properties of invasion plasmid antigen B from Shigella flexneri in the presence and absence of phospholipid membranes.

Shigella flexneri causes bacillary dysentery, an important cause of mortality among children in the developing world. Shigella secretes effector proteins via its type III secretion system (T3SS) to promote bacterial uptake into human colonic epithelial cells. The T3SS basal body spans the bacterial cell envelope anchoring a surface-exposed needle. A pentamer of invasion plasmid antigen D lies at the nascent needle tip and invasion plasmid antigen B (IpaB) is recruited into the needle tip complex on exposure to bile salts. From here, IpaB forms a translocon pore in the host cell membrane. Although the mechanism by which IpaB inserts into the membrane is unknown, it was recently shown that recombinant IpaB can exist as either a monomer or tetramer. Both of these forms of IpaB associate with membranes, however, only the tetramer forms pores in liposomes. To reveal differences between these membrane-binding events, Cys mutations were introduced throughout IpaB, allowing site-specific fluorescence labeling. Fluorescence quenching was used to determine the influence of oligomerization and/or membrane association on the accessibility of different IpaB regions to small solutes. The data show that the hydrophobic region of tetrameric IpaB is more accessible to solvent relative to the monomer. The hydrophobic region appears to promote membrane interaction for both forms of IpaB, however, more of the hydrophobic region is protected from solvent for the tetramer after membrane association. Limited proteolysis demonstrated that changes in IpaB's oligomeric state may determine the manner by which it associates with phospholipid membranes and the subsequent outcome of this association.

Fusaric acid modulates Type Three Secretion System of Salmonella enterica serovar Typhimurium.

Natural small-molecule products are promising lead compounds for developing a generation of novel antimicrobials agents to meet the challenge of antibiotic-resistant pathogens. To facilitate the search for novel anti-virulence agents, we chose a virulence factor of Type Three Secretion System (T3SS) as a drug target to screen candidates from a small-molecule library in our laboratory. This study demonstrated fusaric acid had dramatically inhibitory effects on secretion of Salmonella island 1 (SPI-1) effector proteins and invasion of Salmonella into HeLa cells. Moreover, fusaric acid had no inhibitory effects on bacterial growth and viability of host cells. Protein HilA is a key regulator of SPI-1 in Salmonella, which affects transcription of SPI-1 effectors and SPI-1 apparatus genes. In this study, fusaric acid (FA) did not affect secretion of SPI-1 effectors in HilA over-expressed strain, suggesting it did not affect the transcription of SPI-1. In addition, fusaric acid did not affect the protein level of apparatus protein PrgH in SPI-1 needle complex. As a result, we proposed fusaric acid had an inhibitory effect on SPI-1 probably depending on its influence on SicA/InvF. In summary, fusaric acid is a novel inhibitor of T3SS with potential for further developing novel anti-virulence agents.

Iron-regulated surface determinant B (IsdB) promotes Staphylococcus aureus adherence to and internalization by non-phagocytic human cells.

Staphylococcus aureus is a human pathogen that causes invasive and recurring infections. The ability to internalize into and persist within host cells is thought to contribute to infection. Here we report a novel role for the well-characterized iron-regulated surface determinant B (IsdB) protein which we have shown can promote adhesion of 293T, HeLa cells and platelets to immobilized bacteria independently of its ability to bind haemoglobin. IsdB bound to the active form of the platelet integrin αIIb ß3 , both on platelets and when the integrin was expressed ectopically in CHO cells. IsdB also promoted bacterial invasion into human cells. This was clearly demonstrated with bacteria lacking fibronectin-binding proteins (FnBPs), which are known to promote invasion in the presence of fibronectin. However, IsdB also contributed significantly to invasion by cells expressing FnBPs in the presence of serum. Thus IsdB appears to be able to interact with the broader family of integrins that bind ligands with the RGD motif and to act as a back up mechanism to promote interactions with mammalian cells.

Apoptosis in HEp-2 cells infected with Ureaplasma diversum.

BACKGROUND: Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. RESULTS: The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. CONCLUSIONS: The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.

The actin-polymerizing activity of SipA is not essential for Salmonella enterica serovar Typhimurium-induced mucosal inflammation.

Salmonella enterica serovar Typhimurium depends on type III secretion systems to inject effector proteins into host cells to promote bacterial invasion and to induce intestinal inflammation. SipA, a type III effector, is known to play important roles in both the invasion and the elicitation of intestinal inflammation. The actin-modulating activity of SipA has been shown to promote Salmonella entry into epithelial cells. To investigate whether the actin-modulating activity of SipA is required for its ability to induce an inflammatory response in vivo, we generated the SipA(K635A E637W) mutant, which is deficient in actin-modulating activity. Salmonella strains expressing the chromosomal SipA(K635A E637W) point mutation had reduced invasion abilities but still caused colitis similar to that caused by the wild-type strain in a mouse model of infection. Our data indicate that the SipA actin-polymerizing activity is not essential for the SipA-induced inflammatory response in the mouse model of infection.

Activities of first-choice antimicrobials against gamma interferon-treated Chlamydia trachomatis differ in hypoxia.

Gamma interferon (IFN-γ)-mediated host responses play a central role in resolving genital Chlamydia trachomatis infections but may also result in persistence of the pathogen, which shows reduced susceptibility to antimicrobials. The antichlamydial function of IFN-γ is oxygen dependent, and the efficacy of antimicrobials against C. trachomatis is reduced in a low-oxygen environment. In this study, we show that the antichlamydial efficacies of azithromycin and doxycycline differ in IFN-γ-treated cells under hypoxia.

Salmonella Enteritidis RfbD enhances bacterial colonization and virulence through inhibiting autophagy.

Autophagy is a crucial innate immune response that clears pathogens intracellularly. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. Here, we reported that dTDP-4-dehydro-ß-ւ-rhamnose reductase (RfbD) was able to enhance bacterial colonization in vivo and in vitro by regulating autophagy. We screened the transposon mutant library of Salmonella Enteritidis strain Z11 by High-Content Analysis System, found that rfbD gene has an effect on autophagy. The Z11ΔrfbD-infected group showed greater expression of LC3-II than the Z11-infected group in HeLa, RAW264.7, and J774A.1 cells. Overall, the survival of Z11ΔrfbD in RAW264.7 cells was reduced after 8 h of infection compared to that of the Z11 wild-type strain. In addition, we observed that inhibition of autophagic flux significantly increased the survival of Z11ΔrfbD in RAW264.7 cells. Mice infection experiments revealed that Z11ΔrfbD virulence was significantly reduced, and bacterial load was reduced in the liver and cecum in mice model, and LC3-II expression was significantly increased. These findings indicate an important role of Salmonella Enteritidis protein as a strategy to suppress autophagy and provides new ideas for manipulating autophagy as a novel strategy to treat infectious diseases.

The conserved Tarp actin binding domain is important for chlamydial invasion.

The translocated actin recruiting phosphoprotein (Tarp) is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.

[Two multidrug-resistant Salmonella infantis isolates behave like hypo-invasive strains but have high intracellular proliferation]. / Dos aislamientos de Salmonella infantis multirresistentes se comportan como hipoinvasivos pero con elevada proliferación intracelular.

In this work, plasmid-encoded virulence factors in two Salmonella Infantis isolates carrying multiresistance plasmids were investigated. In addition, their invasion and proliferative ability in non-phagocytic cells was studied. None of them showed the typical determinants of virulence plasmids (spy operon). The invasion assays of S. Infantis isolates on eukaryotic cells showed a decreased ability to invade but they remained and proliferated in the cytoplasm regardless of having used a permissive (HeLa) or non-permissive (NRK) cell line. Finally, there was no microscopic evidence suggesting a bactericidal effect of these eukaryotic cell lines on the isolates tested.

Eps15 and Epsin1 are crucial for enteropathogenic Escherichia coli pedestal formation despite the absence of adaptor protein 2.

Enteropathogenic Escherichia coli (EPEC) are primarily extracellular pathogens that generate actin-rich structures known as pedestals during their pathogenesis. Surprising evidence has demonstrated that despite maintaining an extracellular location, EPEC require the endocytic protein, clathrin, for pedestal formation. To evaluate the strategies EPEC use to usurp endocytic machinery, we investigated the roles of a number of clathrin-coated pits components, adaptor protein 2 (AP-2), Eps15 and epsin1, during EPEC infections. We demonstrated that in conjunction with clathrin, pedestal formation also required the recruitment of Eps15 and epsin1 but not AP-2. Because AP-2 orchestrates the recruitment of clathrin, Eps15, and epsin1, as well as other adaptors, during assembly of clathrin-coated pits at the plasma membrane, our findings reveal a novel internalization subversion strategy employed by EPEC. These results further emphasize the recent paradigm that endocytic proteins are important for EPEC-mediated disease.

BslA, the S-layer adhesin of B. anthracis, is a virulence factor for anthrax pathogenesis.

Microbial pathogens use adhesive surface proteins to bind to and interact with host tissues, events that are universal for the pathogenesis of infectious diseases. A surface adhesin of Bacillus anthracis, the causative agent of anthrax, required to mediate these steps has not been discovered. Previous work identified BslA, an S-layer protein, to be necessary and sufficient for adhesion of the anthrax vaccine strain, Bacillus anthracis Sterne, to host cells. Here we asked whether encapsulated bacilli require BslA for anthrax pathogenesis in guinea pigs. Compared with the highly virulent parent strain B. anthracis Ames, bslA mutants displayed a dramatic increase in the lethal dose and in mean time-to-death. Whereas all tissues of animals infected with B. anthracis Ames contained high numbers of bacilli, only few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant. Surface display of BslA occurred at the poles of encapsulated bacilli and enabled the binding of vegetative forms to host cells. Together these results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames.

The type VI secretion system plays a role in type 1 fimbria expression and pathogenesis of an avian pathogenic Escherichia coli strain.

Avian pathogenic Escherichia coli (APEC) strains frequently cause extraintestinal infections and are responsible for significant economic losses in the poultry industry worldwide. APEC isolates are closely related to human extraintestinal pathogenic E. coli (ExPEC) strains and may also act as pathogens for humans. Known APEC virulence factors include adhesins such as type 1 fimbriae and curli, iron acquisition systems, and cytotoxins. Here we show that APEC strain SEPT362, isolated from a septicemic hen, expresses a type VI secretion system (T6SS); causes cytoskeleton rearrangements; and invades epithelial cells, replicates within macrophages, and causes lethal disease in chicks. To assess the contribution of the T6SS to SEPT362 pathogenesis, we generated two mutants, hcp (which encodes a protein suggested to be both secreted and a structural component of the T6SS) and clpV (encoding the T6SS ATPase). Both mutants showed decreased adherence and actin rearrangement on epithelial cells. However, only the hcp mutant presented a mild decrease in its ability to invade epithelial cells, and none of these mutants were defective for intramacrophage replication. Transcriptome studies showed that the level of expression of type 1 fimbriae was decreased in these mutants, which may account for the diminished adhesion and invasion of epithelial cells. The T6SS seems to be important for the disease process, given that both mutants were attenuated for infection in chicks. These results suggest that the T6SS influences the expression of type 1 fimbriae and contributes to APEC pathogenesis.

Higher expression of ferritin protects Chlamydia trachomatis infected HeLa 229 cells from reactive oxygen species mediated cell death.

Apoptosis plays an important role in modulating the pathogenesis of a variety of infectious diseases. Chlamydial infection protects cells against different forms of apoptosis: extrinsic, intrinsic, and granzyme B mediated. Redox reactions are central to the life and death decision of cells and pathogens and an intimate relationship exists between oxidative stress and iron metabolism. The link between redox status and ferritin was largely unexplored in chlamydia-infected cells. In the present study, we showed that Chlamydia trachomatis (CT) infection induced FHC protein in HeLa cells. FHC induction by CT-infected cells stably expressing FHC blunted ROS production compared with mock infected cells, and the infected cells were relatively resistant to apoptosis induced by H2O2. We also demonstrated that endogenous FHC overexpression correlates well with the stabilization of the mitochondrial membrane potential in CT-infected cells. Increased expression of FHC is independent of iron supplementation (FAC) and depletion (DFO) in CT-infected cells. These data suggest that FHC up-regulation is an acute response of HeLa cells against CT infection and that FHC exerts anti-apoptotic activity against oxidative stress.