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Intracellular Pathogens (intracellular + pathogen)
Kinds of Intracellular Pathogens Selected AbstractsMolecular motors hijacking by intracellular pathogensCELLULAR MICROBIOLOGY, Issue 1 2006Thomas Henry Summary Cargoes are transported intracellularly along cytoskeletal tracks composed of actin or tubulin. Their movement involves the action of molecular motor proteins that generate directed movement along microtubules or actin filaments. The three classes of molecular motors , kinesins, dyneins and myosins , are involved in a multiplicity of biological movements such as mitosis, positioning of organelles, intracellular transports and also vesicular sorting through membrane tubulation and fission and delivery to their target compartment. Intracellular pathogens use this molecular machinery to reach their site of replication, to leave their host or to control the dynamics of membrane exchanges with their replication compartment. [source] Immunity to vacuolar pathogens: What can we learn from Legionella?CELLULAR MICROBIOLOGY, Issue 11 2004Annie L. Neild Summary Intracellular pathogens can manipulate host cellular pathways to create specialized organelles. These pathogen-modified vacuoles permit the survival and replication of bacterial and protozoan microorganisms inside of the host cell. By establishing an atypical organelle, intracellular pathogens present unique challenges to the host immune system. To understand pathogenesis, it is important to not only investigate how these organisms create unique subcellular compartments, but to also determine how mammalian immune systems have evolved to detect and respond to pathogens sequestered in specialized vacuoles. Recent studies have identified genes in the respiratory pathogen Legionella pneumophila that are essential for establishing a unique endoplasmic reticulum-derived organelle inside of mammalian macrophages, making this pathogen an attractive model system for investigations on host immune responses that are specific for bacteria that establish vacuoles disconnected from the endocytic pathway. This review will focus on the host immune response to Legionella and highlight areas of Legionella research that should help elucidate host strategies to combat infections by intracellular pathogens. [source] Viability of Listeria monocytogenes in co-culture with Acanthamoeba spp.FEMS MICROBIOLOGY ECOLOGY, Issue 1 2009Alisha Akya Abstract Listeria monocytogenes is a human pathogen, ubiquitous in the environment, and can grow and survive under a wide range of environmental conditions. It contaminates foods via raw materials or food-processing environments. However, the current knowledge of its ecology and, in particular, the mode of environmental survival and transmission of this intracellular pathogen remains limited. Research has shown that several intracellular pathogens are able to survive or replicate within free-living amoebae. To examine the viability of L. monocytogenes in interaction with Acanthamoeba spp., bacteria were co-cultured with three freshly isolated amoebae, namely Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba lenticulata. The survival of bacteria and amoebae was determined using culture techniques and microscopy. Under the experimental conditions used, all amoebae were able to eliminate bacteria irrespective of the hly gene. Bacteria did not survive or replicate within amoeba cells. However, extra-amoebic bacteria grew saprophytically on materials released from amoebae, which may play an important role in the survival of bacteria under extreme environmental conditions. [source] The contribution of both oxygen and nitrogen intermediates to the intracellular killing mechanisms of C1q-opsonized Listeria monocytogenes by the macrophage-like IC-21 cell lineIMMUNOLOGY, Issue 1 2000C. Álvarez-Domínguez Summary Listeria monocytogenes is a facultative intracellular pathogen which is internalized by host mammalian cells upon binding to their surface. Further listerial growth occurs in the cytosol after escape from the phagosomal,endosomal compartment. We have previously reported that C1q is able to potentiate L. monocytogenes phagocytosis upon bacterial opsonization by ingestion through C1q-binding structures. In this report, we analysed the post-phagocytic events upon internalization of C1q-opsonized L. monocytogenes and found an induction of macrophage (M,)-like IC-21 cell bactericidal mechanisms displayed by the production of oxygen and nitrogen metabolites. Both types of molecules are effective in L. monocytogenes killing. Further analysis of the cellular responses promoted by interaction of C1q with its surface binding structures, leads us to consider C1q as a collaborative molecule involved in M, activation. Upon interaction with surface binding structures, C1q was able to trigger and/or amplify the production of reactive oxygen and nitrogen intermediates induced by stimuli such as interferon-, and L. monocytogenes phagocytosis. [source] General and specific host responses to bacterial infection in Peyer's patches: a role for stromelysin-1 (matrix metalloproteinase-3) during Salmonella enterica infectionMOLECULAR MICROBIOLOGY, Issue 1 2007Scott A. Handley Summary Salmonella enterica serovar Typhimurium (S. typhimurium) and Yersinia enterocolitica are enteric pathogens capable of colonizing and inducing inflammatory responses in Peyer's patches (PPs) and mesenteric lymph nodes (MLNs). Although the tissue colonization pattern is similar between these two pathogens, their pathogenic lifestyles are quite different. For example, while S. typhimurium is primarily an intracellular pathogen, Y. enterocolitica survives primarily extracellularly. We determined and compared the transcriptional changes occurring in response to S. typhimurium and Y. enterocolitica colonization of PP using Affymetrix GeneChip technology. Both pathogens elicited a general inflammatory response indicated by the upregulation of cytokines and chemokines. However, specific differences were also observed, most notably in the transcriptional regulation of gamma interferon (IFN-,) and IFN-,-regulated genes in response to S. typhimurium but not Y. enterocolitica. Of particular note, a group of genes encoding matrix metalloproteinases (MMPs) had increased transcript numbers in the PPs following infection with both pathogens. The experiments described here compare oral S. typhimurium or Y. enterocolitica infection in stromelysin-1 (MMP-3)-deficient mice (mmp-3,/,) with mice possessing functional MMP-3 (mmp-3+/+). There was little difference in the survival of MMP-3-deficient mice infected with Y. enterocolitica when compared with littermate controls. Surprisingly though, mmp-3,/, mice were markedly more resistant to S. typhimurium infection than the control mice. S. typhimurium was able to colonize mmp-3,/, mice, albeit in a delayed fashion, to equivalent levels as mmp-3+/+ mice. Nevertheless, significantly lower levels of inflammatory cytokines were detected in tissues and serum in the mmp-3,/, mice in comparison with mmp-3+/+ mice. We hypothesize that MMP-3 is involved in initiating an early and lethal cytokine response to S. typhimurium colonization. [source] The RNA chaperone Hfq is essential for the virulence of Salmonella typhimuriumMOLECULAR MICROBIOLOGY, Issue 1 2007Alexandra Sittka Summary The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Q, RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, Salmonella typhimurium. A Salmonella hfq deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages in vitro. Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non-coding RNAs, placing Hfq at the centre of post-transcriptional regulation of virulence gene expression in Salmonella. In addition, the hfq mutation appears to cause a chronic activation of the RpoE-mediated envelope stress response which is likely due to a misregulation of membrane protein expression. [source] Plasticity of a transcriptional regulation network among alpha-proteobacteria is supported by the identification of CtrA targets in Brucella abortusMOLECULAR MICROBIOLOGY, Issue 4 2002Anne-Flore Bellefontaine Summary CtrA is a master response regulator found in many alpha-proteobacteria. In Caulobacter crescentus and Sinorhizobium meliloti, this regulator is essential for viability and is transcriptionally autoregulated. In C. crescentus, it is required for the regulation of multiple cell cycle events, such as DNA methylation, DNA replication, flagella and pili biogenesis and septation. Here, we report the characterization of the ctrA gene homologue in the ,2 -proteobacteria Brucella abortus, a facultative intracellular pathogen responsible for brucellosis. We detected CtrA expression in the main Brucella species, and its overproduction led to a phenotype typical of cell division defect, consistent with its expected role. A purified B. abortus CtrA recombinant protein (His6,CtrA) was shown to protect the B. abortus ctrA promoter from DNase I digestion, suggesting transcriptional autoregulation, and this protection was enhanced under CtrA phosphorylation on a conserved Asp residue. Despite the similarities shared by B. abortus and C. crescentus ctrA, the pathway downstream from CtrA may be distinct, at least partially, in both bacteria. Indeed, beside ctrA itself, only one (the ccrM gene) out of four B. abortus homologues of known C. crescentus CtrA targets is bound in vitro by phosphorylated B. abortus CtrA. Moreover, further footprinting experiments support the hypothesis that, in B. abortus, CtrA might directly regulate the expression of the rpoD, pleC, minC and ftsE homologues. Taken together, these results suggest that, in B. abortus and C. crescentus, similar cellular processes are regulated by CtrA through the control of distinct target genes. The plasticity of the regulation network involving CtrA in these two bacteria may be related to their distinct lifestyles. [source] DNA and protein transfer from bacteria to eukaryotes, the agrobacterium storyMOLECULAR PLANT PATHOLOGY, Issue 1 2000The 18th Bateson Memorial Lecture Agrobacterium is a well-studied plant pathogen, which has the unique ability to transfer DNA and protein into a number of eukaryotes. The DNA is integrated randomly into the plant genome where it is expressed, thereby leading to the disease crown gall. This system is a paradigm for the interaction of a number of plant and animal pathogens which transfer proteins into their host cells. In Agrobacterium, the tumour inducing (Ti) plasmid codes for the functions specifically required for the transfer process. These genes, termed virulence or vir genes, are activated by plant signal molecules acting through a two component regulatory system. A key structure coded by 11 genes of the vir B operon is a pilus, synthesized at 20 °C, but poorly at 25 °C. How this pilus functions in DNA and protein transfer is unclear, but homologous genes are found in many animal pathogens. In addition to Ti plasmid-encoded vir genes, chromosomal virulence genes have also been identified. However, these mutations are often pleiotropic because they involve both the normal physiology of Agrobacterium as well as the metabolism of Agrobacterium when it is associated with plant cells. Based on 16S ribosomal RNA sequencing, Agrobacterium is closely related to the intracellular pathogen of animals, Brucella. Several chromosomal mutations of Agrobacterium required for virulence in plants are also required for invasion of animal host cells by Brucella. [source] Quantitative proteomics of intracellular Porphyromonas gingivalisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2007Qiangwei Xia Abstract Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18,h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were overexpressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be underexpressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction. [source] Do mitochondrial DNA haplogroups play a role in susceptibility to tuberculosis?RESPIROLOGY, Issue 6 2007Massoud HOUSHMAND Background and objectives: Mitochondrial DNA has a unique role in ATP production and subsequent mitochondrial reactive oxygen species (ROS) production in eukaryotic cells and there is a potential role for ROS and oxygen burst against Mycobacterium tuberculosis, an intracellular pathogen. This study aimed to determine whether the frequency of different mitochondrial haplogroups was significantly different in patients with tuberculosis (TB) compared with a normal population. Methods: Mitochondrial DNA haplogroups M, N, J and K were studied by PCR-restriction fragment length polymorphism and sequencing. Cases were 54 patients with confirmed smear positive pulmonary TB. Controls were 256 healthy persons. Results: There were no statistically significant differences between those with TB and the control group. Conclusions: There was no statistically significant association between mtDNA haplogroups and the presence of TB infection. [source] Dietary fish oil impairs induction of ,-interferon and delayed-type hypersensitivity during a systemic Salmonella enteritidis infection in ratsAPMIS, Issue 8 2010Johannes Snel Snel J, Born L, van der Meer R. Dietary fish oil impairs induction of ,-interferon and delayed-type hypersensitivity during a systemic Salmonella enteritidis infection in rats. APMIS 2010; 118: 578,84. Fish oil that is rich in n-3 polyunsaturated fatty acids markedly modulates immunological responses. Literature data indicate that the fish oil reduces cellular immunity and therefore impairs resistance to infections. We have investigated how dietary fish oil affects the immune response against a facultative intracellular pathogen, Salmonella enteritidis. Wistar rats were fed a diet containing 16% (w/w) of either fish oil or corn oil. After a 4-week adaptation period, rats were intraperitoneally challenged with 4 × 105 cfu of S. enteritidis. During the 14-day infection period, urine was collected on a daily basis. At days 2 and 14, eight rats per group were sacrificed. Urinary nitrate, used as a marker for NO production, was lower on a fish oil diet during days 3,8. At day 2, serum ,-interferon was 48 ± 7 pg/mL in the fish oil-fed rats compared with 162 ± 52 pg/mL in the corn oil-fed rats. No effects were found on living salmonella in liver and spleen. At day 14, as markers of an impaired T-helper 1 (Th-1) response, a 38% lower delayed-type hypersensitivity responses and a lower salmonella-specific IgG2b were observed in the fish oil-fed rats. Although here dietary fish oil has affected only immune parameters, this impairment of the innate and Th-1-mediated immune response may have implications for the host resistance against other intracellular pathogens. [source] Fibronectin-binding proteins secreted by Mycobacterium aviumAPMIS, Issue 9 2000HIDEKI Kitaura Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Fibronectin is an extracellular matrix protein and is a virulence factor for several extracellular pathogenic bacteria binding to mucosal surfaces. We investigated the fibronectin (FN)-binding proteins in the culture filtrate of M. avium by two-dimensional electrophoresis (2DE). Proteins in Sauton medium of M. avium after 3 weeks were separated by 2DE. The proteins were blotted onto polyvinylidene difluoride membrane and incubated with FN. FN-binding proteins were detected by Western blotting using anti-FN antibody. FN bound to five spots (33 kDa, 32 kDa, 31 kDa, 30 kDa and 25 kDa). N-terminal amino acids of these were determined. The 33 kDa spot corresponded to antigen 85 (Ag 85) C. The 32 and 31 kDa spots were either Ag 85 A or Ag 85 B. The 30 kDa spot corresponded to Ag 85 B of M. avium. The 25 kDa spot corresponded to MPA51 (M. avium MPB51). Thus, FN bound exclusively to the Ag 85 complex and MPA51. [source] Microreview: Type IV secretion in the obligatory intracellular bacterium Anaplasma phagocytophilumCELLULAR MICROBIOLOGY, Issue 9 2010Yasuko Rikihisa Summary Anaplasma phagocytophilum is an obligatory intracellular bacterium that infects neutrophils, the primary host defence cells. Consequent effects of infection on host cells result in a potentially fatal systemic disease called human granulocytic anaplasmosis. Despite ongoing reductive genome evolution and deletion of most genes for intermediary metabolism and amino acid biosynthesis, Anaplasma has also experienced expansion of genes encoding several components of the type IV secretion (T4S) apparatus. Two A. phagocytophilum T4S effector molecules are currently known; Anaplasma translocated substrate 1 (Ats-1) and ankyrin repeat domain-containing protein A (AnkA) have C-terminal positively charged amino acid residues that are recognized by the T4S coupling protein, VirD4. AnkA and Ats-1 contain eukaryotic protein motifs and are uniquely evolved in the family Anaplasmataceae; Ats-1 contains a mitochondria-targeting signal. They are abundantly produced and secreted into the host cytoplasm, are not toxic to host cells, and manipulate host cell processes to aid in the infection process. At the cellular level, the two effectors have distinct subcellular localization and signalling in host cells. Thus in this obligatory intracellular pathogen, the T4S system has evolved as a host-subversive survival factor. [source] Sorting nexin 3 (SNX3) is a component of a tubular endosomal network induced by Salmonella and involved in maturation of the Salmonella -containing vacuoleCELLULAR MICROBIOLOGY, Issue 9 2010Virginie Braun Summary Salmonella enterica serovar Typhimurium is an intracellular pathogen that grows within a modified endomembrane compartment, the Salmonella -containing vacuole (SCV). Maturation of nascent SCVs involves the recruitment of early endosome markers and the remodelling of phosphoinositides at the membrane of the vacuole, in particular the production of phosphatidylinositol 3-phosphate [PI(3)P]. Sorting nexins (SNXs) are a family of proteins characterized by the presence of a phox homology (PX) domain that binds to phosphoinositides and are involved in intracellular trafficking in eukaryotic cells. We therefore studied whether sorting nexins, particularly sorting nexin 3 (SNX3), play a role in Salmonella infection. We found that SNX3 transiently localized to SCVs at early times post invasion (10 min) and presented a striking tubulation phenotype in the vicinity of SCVs at later times (30,60 min). The bacterial effector SopB, which is known to promote PI(3)P production on SCVs, was required for the formation of SNX3 tubules. In addition, RAB5 was also required for the formation of SNX3 tubules. Depletion of SNX3 by siRNA impaired RAB7 and LAMP1 recruitment to the SCV. Moreover, the formation of Salmonella -induced filaments (Sifs) was altered by SNX3 knock-down. Therefore, SNX3 plays a significant role in regulating the maturation of SCVs. [source] Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophagesCELLULAR MICROBIOLOGY, Issue 7 2009Tara D. Wehrly Summary The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis ssp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression profiles. Phagocytosed bacteria rapidly responded to their intracellular environment and subsequently altered their transcriptional profile. Differential gene expression profiles were revealed that correlated with specific intracellular locale of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of transport and metabolic genes characterized the cytosolic replication stage. Expression of the Francisella Pathogenicity Island (FPI) genes, which are required for intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci encoding putative hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated. Among these, deletion of FTT0383, FTT0369c or FTT1676 abolished the ability of Schu S4 to survive or proliferate intracellularly and cause lethality in mice, therefore identifying novel determinants of Francisella virulence from their intracellular expression profile. [source] Multiple MyD88-dependent responses contribute to pulmonary clearance of Legionella pneumophilaCELLULAR MICROBIOLOGY, Issue 1 2009Kristina A. Archer Summary MyD88-dependent signalling is important for secretion of early inflammatory cytokines and host protection in response to Legionella pneumophila infection. Although toll-like receptor (TLR)2 contributes to MyD88-dependent clearance of L. pneumophila, TLR-independent functions of MyD88 could also be important. To determine why MyD88 is critical for host protection to L. pneumophila, the contribution of multiple TLRs and IL-18 receptor (IL-18R)-dependent interferon-gamma (IFN-,) production in a mouse was examined. Mice deficient for TLR5 or TLR9, or deficient for TLR2 along with either TLR5 or TLR9, were competent for controlling bacterial replication and had no apparent defects in cytokine production compared with control mice. MyD88-dependent production of IFN-, in the lung was mediated primarily by natural killer cells and required IL-18R signalling. Reducing IFN-, levels did not greatly affect the kinetics of L. pneumophila replication or clearance in infected mice. Additionally, IFN-,-deficient mice did not have a susceptibility phenotype as severe as the MyD88-deficient mice and were able to control a pulmonary infection by L. pneumophila. Thus, MyD88-dependent innate immune responses induced by L. pneumophila involve both TLR-dependent responses and IL-18R-dependent production of IFN-, by natural killer cells, and these MyD88-dependent pathways can function independently to provide host protection against an intracellular pathogen. [source] Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survivalCELLULAR MICROBIOLOGY, Issue 10 2008Oscar Zaragoza Summary Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H2O2 that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H2O2. Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to ,-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells. [source] Protein kinase E of Mycobacterium tuberculosis has a role in the nitric oxide stress response and apoptosis in a human macrophage model of infectionCELLULAR MICROBIOLOGY, Issue 2 2008Deepak Jayakumar Summary Mycobacterium tuberculosis, an intracellular pathogen, inhibits macrophage apoptosis to support survival and replication inside the host cell. We provide evidence that the functional serine/threonine kinase, PknE, is important for survival of M. tuberculosis that enhances macrophage viability by inhibiting apoptosis. A promoter of PknE identified in this study was shown to respond to nitric oxide stress. Deletion of pknE in virulent M. tuberculosis, H37Rv, resulted in a strain that has increased resistance to nitric oxide donors and increased sensitivity to reducing agents. The deletion mutant created by specialized transduction induced enhanced apoptosis while inhibiting necrosis. The pknE mutant also modifies the innate immune response as shown by the marked decline in the pro-inflammatory cytokines in a macrophage model of infection. These findings suggest a novel mechanism, by which PknE senses nitric oxide stress and prevents apoptosis by interfering with host signalling pathways. [source] iNOS activity is critical for the clearance of Burkholderia mallei from infected RAW 264.7 murine macrophagesCELLULAR MICROBIOLOGY, Issue 2 2008Paul J. Brett Summary Burkholderia mallei is a facultative intracellular pathogen that can cause fatal disease in animals and humans. To better understand the role of phagocytic cells in the control of infections caused by this organism, studies were initiated to examine the interactions of B. mallei with RAW 264.7 murine macrophages. Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of , 1. In contrast, the organism was efficiently cleared by the macrophages when infected at an moi of 10. Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-,, IL-6, IL-10, GM-CSF, RANTES and IFN-, when infected at an moi of 10. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that moi significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells. [source] Anaplasma phagocytophilum specifically induces tyrosine phosphorylation of ROCK1 during infectionCELLULAR MICROBIOLOGY, Issue 7 2007Venetta Thomas Summary Anaplasma phagocytophilum, an obligate intracellular pathogen that persists within polymorphonuclear leucocytes, is the second most common tick-borne agent in North America. We now show that infection of a promyelocytic cell line and neutrophils with A. phagocytophilum results in pathogen-specific tyrosine phosphorylation of ROCK1. Phosphorylation is associated with PSGL-1 and Syk, because PSGL-1 blocking antibodies and siRNA targeting Syk interfere with ROCK1 phosphorylation in A. phagocytophilum -infected cells. Knockdown of either Syk or ROCK1 also markedly impaired A. phagocytophilum infection. These data demonstrate a role for A. phagocytophilum -mediated ROCK1 phosphorylation in infection, and suggests that inhibiting this pathway may lead to new, non-antibiotic strategies to treat human granulocytic anaplasmosis. [source] Anaplasma phagocytophilum AnkA is tyrosine-phosphorylated at EPIYA motifs and recruits SHP-1 during early infectionCELLULAR MICROBIOLOGY, Issue 5 2007Jacob W. IJdo Summary Anaplasma phagocytophilum is an intracellular pathogen that infects and survives in neutrophilic granulocytes. The A. phagocytophilum genome encodes a type four secretion system (T4SS) that may facilitate intracellular survival by translocation of virulence factors, but to date, no such factors have been identified. Because T4SS-translocated proteins of several intracellular organisms undergo tyrosine phosphorylation by host cell kinases, we investigated tyrosine phosphorylation of A. phagocytophilum proteins during infection. Within minutes after incubation of A. phagocytophilum with HL-60 cells or PMN, a 190 kDa bacterial protein, AnkA, was increasingly tyrosine-phosphorylated. A. phagocytophilum binding to host cells without entry was sufficient for AnkA tyrosine phosphorylation. An in vitro Src kinase assay demonstrated that purified AnkA expressed in Escherichia coli was phosphorylated at tyrosines located at the C-terminal portion of AnkA. Similarly, AnkA expressed in COS-7 cells underwent tyrosine phosphorylation by Src at the C-terminus. The phosphorylated tyrosines were located in EPIYA motifs that display the consensus sequence for binding to SH2 domains. Immunoprecipitation studies demonstrated AnkA binding to the host cell phosphatase SHP-1 during early infection. Phosphorylation of the EPIYA motifs and the presence of the SH2 domains were necessary for AnkA,SHP-1 interaction. We conclude that AnkA is a translocated virulence factor that is tyrosine-phosphorylated by host cell kinases upon translocation into the host cell early during infection. A. phagocytophilum may manipulate the host cell through SHP-1 recruitment. [source] Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetiiCELLULAR MICROBIOLOGY, Issue 4 2007Daniel E. Voth Summary Most intracellular parasites employ sophisticated mechanisms to direct biogenesis of a vacuolar replicative niche that circumvents default maturation through the endolysosomal cascade. However, this is not the case of the Q fever bacterium, Coxiella burnetii. This hardy, obligate intracellular pathogen has evolved to not only survive, but to thrive, in the harshest of intracellular compartments: the phagolysosome. Following internalization, the nascent Coxiella phagosome ultimately develops into a large and spacious parasitophorous vacuole (PV) that acquires lysosomal characteristics such as acidic pH, acid hydrolases and cationic peptides, defences designed to rid the host of intruders. However, transit of Coxiella to this environment is initially stalled, a process that is apparently modulated by interactions with the autophagic pathway. Coxiella actively participates in biogenesis of its PV by synthesizing proteins that mediate phagosome stalling, autophagic interactions, and development and maintenance of the mature vacuole. Among the potential mechanisms mediating these processes is deployment of a type IV secretion system to deliver effector proteins to the host cytosol. Here we summarize our current understanding of the cellular events that occur during parasitism of host cells by Coxiella. [source] New concepts in Salmonella virulence: the importance of reducing the intracellular growth rate in the hostCELLULAR MICROBIOLOGY, Issue 7 2005Alberto Tierrez Summary The literature refers to Salmonella enterica as an intracellular bacterial pathogen that proliferates within vacuoles of mammalian cells. However, recent in vivo studies have revealed that the vast majority of infected cells contain very few intracellular bacteria (three to four organisms). Salmonella intracellular growth is also limited in cultured dendritic cells and fibroblasts, two cell types abundant in tissues located underneath the intestinal epithelium. Recently, a Salmonella factor previously known for its role as a negative regulator of intracellular growth has been shown to tightly repress certain pathogen functions upon host colonization and to be critical for virulence. The connection between virulence and the negative control of intracellular growth is further sustained by the fact that some attenuated mutants overgrow in non-phagocytic cells located in the intestinal lamina propria. These findings are changing our classical view of Salmonella as a fast growing intracellular pathogen and suggest that this pathogen may trigger responses directed to reduce the growth rate within the infected cell. These responses could play a critical role in modulating the delicate balance between disease and persistence. [source] Listeria monocytogenes -infected bone marrow myeloid cells promote bacterial invasion of the central nervous systemCELLULAR MICROBIOLOGY, Issue 2 2005Olivier F. Join-Lambert Summary Listeria monocytogenes is a facultative intracellular pathogen that is able to invade the central nervous system causing meningoencephalitis and brain abscesses. The mechanisms allowing bacteria to cross the blood,brain barrier are poorly understood. In this work, we used an experimental model of acute listeriosis in the mouse inducing a reproducible invasion of the central nervous system. At the early phase of infection, we find that bacteria invade and rapidly grow in bone marrow cells identified as bone marrow myelomonocytic cells expressing the phenotype CD31pos:Ly-6Cpos:CD11bpos:LY-6Glow. We demonstrate that central nervous system invasion is facilitated by injecting L. monocytogenes- infected bone marrow cells in comparison with free bacteria or infected spleen cells. In mice transplanted with bone marrow cells from transgenic donor mice expressing the green fluorescent protein (GFP), we show that infected myeloid GFP+ cells adhere to activated brain endothelial cells, accumulate in brain vessels and participate to the pathogenesis of meningoencephalitis and brain abscesses. Our results demonstrate that bone marrow, the main haematopoietic tissue, is a previously unrecognized reservoir of L. monocytogenes -infected myeloid cells, which can play a crucial role in the pathophysiology of meningoencephalitis by releasing infected cells into the circulation that ultimately invade the central nervous system. [source] Leishmania donovani infection down-regulates TLR2-stimulated IL-12p40 and activates IL-10 in cells of macrophage/monocytic lineage by modulating MAPK pathways through a contact-dependent mechanismCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2008Dinesh Chandra Summary The failure of Leishmania, an intracellular pathogen, to stimulate a pro-inflammatory response following entry into macrophages has been well reported. This occurs in spite of the fact that ligands for the toll-like receptors (TLR) have been recently shown on the parasite surface and their role in disease protection well documented. The outcome of infection in leishmaniasis is determined by the Th1 versus Th2 nature of the effector response and the generation of IL-12 and IL-10 by the infected macrophages is important for this decision. We evaluated the effect of L. donovani infection of monocytes (cell line THP-1, and monocytes derived from human peripheral blood) on Pam3cys (TLR2 ligand) and lipopolysaccharide (TLR4 ligand) stimulated production of IL-12p40 and IL-10. L. donovani infection caused suppression of TLR2 and TLR4-stimulated IL-12p40, with an increase in IL-10 production. Parasites also modulated the TLR2-stimulated mitogen-activated protein kinase (MAPK) pathway by suppressing MAPK P38 phosphorylation and activating extracellular regulated kinase (ERK)1/2 phosphorylation. These effects could be reversed either by using a MAPK P38 activator, anisomycin, or ERK1/2 inhibitor, U0126. L. donovani caused modulation of TLR2-stimulated MAPK pathways in a contact-dependent mechanism. In addition parasite structural integrity but not viability was required for suppression of TLR2-stimulated IL-12p40 and activation of IL-10. These observations suggest that L. donovani has evolved survival strategies that subvert the pro-inflammatory response generated through TLRs. [source] From protozoa to mammalian cells: a new paradigm in the life cycle of intracellular bacterial pathogensENVIRONMENTAL MICROBIOLOGY, Issue 3 2000Minireview It is becoming apparent that several intracellular bacterial pathogens of humans can also survive within protozoa. This interaction with protozoa may protect these pathogens from harsh conditions in the extracellular environment and enhance their infectivity in mammals. This relationship has been clearly established in the case of the interaction between Legionella pneumophila and its protozoan hosts. In addition, the adaptation of bacterial pathogens to the intracellular life within the primitive eukaryotic protozoa may have provided them with the means to infect the more evolved mammalian cells. This is evident from the existence of several similarities, at both the phenotypic and the molecular levels, between the infection of mammalian and protozoan cells by L. pneumophila. Thus, protozoa appear to play a central role in the transition of bacteria from the environment to mammals. In essence, protozoa may be viewed as a ,biological gym', within which intracellular bacterial pathogens train for their encounters with the more evolved mammalian cells. Thus, intracellular bacterial pathogens have benefited from the structural and biochemical conservation of cellular processes in eukaryotes. The interaction of intracellular bacterial pathogens and protozoa highlights this conservation and may constitute a simplified model for the study of these pathogens and the evolution of cellular processes in eukaryotes. Furthermore, in addition to being environmental reservoirs for known intracellular pathogens of humans and animals, protozoa may be sources of emerging pathogenic bacteria. It is thus critical to re-examine the relationship between bacteria and protozoa to further our understanding of current human bacterial pathogenesis and, possibly, to predict the appearance of emerging pathogens. [source] How B cells shape the immune response against Mycobacterium tuberculosisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2009Paul J. Maglione Abstract Extensive work illustrating the importance of cellular immune mechanisms for protection against Mycobacterium tuberculosis has largely relegated B-cell biology to an afterthought within the tuberculosis (TB) field. However, recent studies have illustrated that B lymphocytes, through a variety of interactions with the cellular immune response, play previously underappreciated roles in shaping host defense against non-viral intracellular pathogens, including M. tuberculosis. Work in our laboratory has recently shown that, by considering these lymphocytes more broadly within their variety of interactions with cellular immunity, B cells have a significant impact on the outcome of airborne challenge with M. tuberculosis as well as the resultant inflammatory response. In this review, we advocate for a revised view of TB immunology in which roles of cellular and humoral immunity are not mutually exclusive. In the context of our current understanding of host defense against non-viral intracellular infections, we review recent data supporting a more significant role of B cells during M. tuberculosis infection than previously thought. [source] Immunisation with BCG and recombinant MVA85A induces long-lasting, polyfunctional Mycobacterium tuberculosis -specific CD4+ memory T lymphocyte populationsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2007Natalie Abstract In the search for effective vaccines against intracellular pathogens such as HIV, tuberculosis and malaria, recombinant viral vectors are increasingly being used to boost previously primed T cell responses. Published data have shown prime-boost vaccination with BCG-MVA85A (modified vaccinia virus Ankara expressing antigen 85A) to be highly immunogenic in humans as measured by ex vivo IFN-, ELISPOT. Here, we used polychromatic flow cytometry to investigate the phenotypic and functional profile of these vaccine-induced Mycobacterium tuberculosis (M.tb) antigen 85A-specific responses in greater detail. Promisingly, antigen 85A-specific CD4+ T cells were found to be highly polyfunctional, producing IFN-,, TNF-,, IL-2 and MIP-1,. Surface staining showed the responding CD4+ T cells to be relatively immature (CD45RO+ CD27intCD57,); this observation was supported by the robust proliferative responses observed following antigenic stimulation. Furthermore, these phenotypic and functional properties were independent of clonotypic composition and epitope specificity, which was maintained through the different phases of the vaccine-induced immune response. Overall, these data strongly support the use of MVA85A in humans as a boosting agent to expand polyfunctional M.tb -specific CD4+ T cells capable of significant secondary responses. [source] Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class,I invariant regionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2007Linda Wooldridge Abstract CD8+ cytotoxic T,lymphocytes (CTL) are key determinants of immunity to intracellular pathogens and neoplastic cells. Recognition of specific antigens in the form of peptide-MHC class,I complexes (pMHCI) presented on the target cell surface is mediated by T cell receptor (TCR) engagement. The CD8 coreceptor binds to invariant domains of pMHCI and facilitates antigen recognition. Here, we investigate the biological effects of a Q115E substitution in the ,2,domain of human leukocyte antigen (HLA)-A*0201 that enhances CD8 binding by,,50% without altering TCR/pMHCI interactions. Soluble and cell surface-expressed forms of Q115E HLA-A*0201 exhibit enhanced recognition by CTL without loss of specificity. These CD8-enhanced antigens induce greater CD3 ,,chain phosphorylation in cognate CTL leading to substantial increases in cytokine production, proliferation and priming of naive T cells. This effect provides a fundamental new mechanism with which to enhance cellular immunity to specific T cell antigens. [source] Treatment of neonatal mice with Flt3 ligand leads to changes in dendritic cell subpopulations associated with enhanced IL-12 and IFN-, productionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2004Sabine Vollstedt Abstract Treatment with the hematopoietic growth factor Flt3 ligand (FL) increases DC numbers in neonatal mice and enhances their resistance against intracellular pathogens. Flow cytometric analysis showed the presence of conventional DC (cDC) and plasmacytoid pre-DC (pDC) in neonatal spleens from untreated and FL-treated mice. CD8, and MHC class,II expression on cDC and pDC was higher on DC from FL-treated mice than on DC from control littermates. After FL treatment, two additional subpopulations of DC-lineage cells were found that were able to produce IL-12 and IFN-,. The IL-12 production of cDC from FL-treated animals was more than 50-fold increased and their ability to stimulate T,cell proliferation was also increased. We conclude that the enhanced resistance against intracellular pathogens was due to increased numbers of DC-lineage cells and their increased ability to produce the essential cytokines. [source] | |||||||||||||||||||||||||||||||