Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages.
ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) causes invasive, drug-resistant skin and soft tissue infections. Reports that S. aureus bacteria survive inside macrophages suggest that the intramacrophage environment may be a niche for persistent infection; however, mechanisms by which the bacteria might evade macrophage phagosomal defenses are unclear. We examined the fate of the S. aureus-containing phagosome in THP-1 macrophages by evaluating bacterial intracellular survival and phagosomal acidification and maturation and by testing the impact of phagosomal conditions on bacterial viability. Multiple strains of S. aureus survived inside macrophages, and in studies using the MRSA USA300 clone, the USA300-containing phagosome acidified rapidly and acquired the late endosome and lysosome protein LAMP1. However, fewer phagosomes containing live USA300 bacteria than those containing dead bacteria associated with the lysosomal hydrolases cathepsin D and β-glucuronidase. Inhibiting lysosomal hydrolase activity had no impact on intracellular survival of USA300 or other S. aureus strains, suggesting that S. aureus perturbs acquisition of lysosomal enzymes. We examined the impact of acidification on S. aureus intramacrophage viability and found that inhibitors of phagosomal acidification significantly impaired USA300 intracellular survival. Inhibition of macrophage phagosomal acidification resulted in a 30-fold reduction in USA300 expression of the staphylococcal virulence regulator agr but had little effect on expression of sarA, saeR, or sigB. Bacterial exposure to acidic pH in vitro increased agr expression. Together, these results suggest that S. aureus survives inside macrophages by perturbing normal phagolysosome formation and that USA300 may sense phagosomal conditions and upregulate expression of a key virulence regulator that enables its intracellular survival.
Project description:Invading pathogens provoke the autophagic machinery and, in a process termed xenophagy, the host cell survives because autophagy is employed as a safeguard for pathogens that escaped phagosomes. However, some pathogens can manipulate the autophagic pathway and replicate within the niche of generated autophagosome-like vesicles. By automated fluorescence-based high content analyses, we demonstrate that Staphylococcus aureus strains (USA300, HG001, SA113) stimulate autophagy and become entrapped in intracellular PtdIns(3)P-enriched vesicles that are decorated with human WIPI-1, an essential PtdIns(3)P effector of canonical autophagy and membrane protein of both phagophores and autophagosomes. Further, agr-positive S. aureus (USA300, HG001) strains were more efficiently entrapped in WIPI-1 positive autophagosome-like vesicles when compared to agr-negative cells (SA113). By confocal and electron microscopy we provide evidence that single- and multiple-Staphylococci entrapped undergo cell division. Moreover, the number of WIPI-1 positive autophagosome-like vesicles entrapping Staphylococci significantly increased upon (i) lysosomal inhibition by bafilomycin A(1) and (ii) blocking PIKfyve-mediated PtdIns(3,5)P(2) generation by YM201636. In summary, our results provide evidence that the PtdIns(3)P effector function of WIPI-1 is utilized during xenophagy of Staphylococcus aureus. We suggest that invading S. aureus cells become entrapped in autophagosome-like WIPI-1 positive vesicles targeted for lysosomal degradation in nonprofessional host cells.
Project description:While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (?pscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages.
Project description:Intracellular chloride channel protein 1 (CLIC1) is a 241 amino acid protein of the glutathione S transferase fold family with redox- and pH-dependent membrane association and chloride ion channel activity. Whilst CLIC proteins are evolutionarily conserved in Metazoa, indicating an important role, little is known about their biology. CLIC1 was first cloned on the basis of increased expression in activated macrophages. We therefore examined its subcellular localisation in murine peritoneal macrophages by immunofluorescence confocal microscopy. In resting cells, CLIC1 is observed in punctate cytoplasmic structures that do not colocalise with markers for endosomes or secretory vesicles. However, when these macrophages phagocytose serum-opsonised zymosan, CLIC1 translocates onto the phagosomal membrane. Macrophages from CLIC1(-/-) mice display a defect in phagosome acidification as determined by imaging live cells phagocytosing zymosan tagged with the pH-sensitive fluorophore Oregon Green. This altered phagosomal acidification was not accompanied by a detectable impairment in phagosomal-lysosomal fusion. However, consistent with a defect in acidification, CLIC1(-/-) macrophages also displayed impaired phagosomal proteolytic capacity and reduced reactive oxygen species production. Further, CLIC1(-/-) mice were protected from development of serum transfer induced K/BxN arthritis. These data all point to an important role for CLIC1 in regulating macrophage function through its ion channel activity and suggest it is a suitable target for the development of anti-inflammatory drugs.
Project description:Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain pre-exposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in future.
Project description:The emergence of serious infections due to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has fueled interest in the contributions of specific staphylococcal virulence factors to clinical disease. To assess the contributions of agr-dependent factors to the fate of organisms in polymorphonuclear neutrophils (PMN), we examined the consequences for organism and host cells of feeding PMN with wild-type CA-MRSA (LAC) or CA-MRSA (LAC agr KO) at different multiplicities of infection (MOIs). Phagocytosed organisms rapidly increased the transcription of RNAIII in a time- and MOI-dependent fashion; extracellular USA300 (LAC) did not increase RNAIII expression despite having the capacity to respond to autoinducing peptide-enriched culture medium. HOCl-mediated damage and intracellular survival were the same in the wild-type and USA300 (LAC agr KO). PMN lysis by ingested USA300 (LAC) was time- and MOI-dependent and, at MOIs >1, required ?-hemolysin (hla) as USA300 (LAC agr KO) and USA300 (LAC hla KO) promoted PMN lysis only at high MOIs. Taken together, these data demonstrate activation of the agr operon in human PMN with the subsequent production of ?-hemolysin and PMN lysis. The extent to which these events in the phagosomes of human PMN contribute to the increased morbidity and mortality of infections with USA300 (LAC) merits further study.
Project description:Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus subsp. massiliense CRM0019 was obtained during an epidemic of postsurgical infections and was characterized by increased persistence in vivo. To better understand the successful survival strategies of this microorganism, we evaluated its infectivity and proliferation in macrophages (RAW and BMDM) and alveolar epithelial cells (A549). For that, we assessed the following parameters, for both M. abscessus CRM0019 as well as the reference strain M. abscessus ATCC 19977: internalization, intracellular survival for up 3 days, competence to subvert lysosome fusion and the intracellular survival after cell reinfection.CRM0019 and ATCC 19977 strains showed the same internalization rate (approximately 30% after 6 h infection), in both A549 and RAW cells. However, colony forming units data showed that CRM0019 survived better in A549 cells than the ATCC 19977 strain. Phagosomal characteristics of CRM0019 showed the bacteria inside tight phagosomes in A549 cells, contrasting to the loosely phagosomal membrane in macrophages. This observation holds for the ATCC 19977 strain in both cell types. The competence to subvert lysosome fusion was assessed by acidification and acquisition of lysosomal protein. For M. abscessus strains the phagosomes were acidified in all cell lines; nevertheless, the acquisition of lysosomal protein was reduced by CRM0019 compared to the ATCC 19977 strain, in A549 cells. Conversely, in macrophages, both M. abscessus strains were located in mature phagosomes, however without bacterial death. Once recovered from macrophages M. abscessus could establish a new intracellular infection. Nevertheless, only CRM0019 showed a higher growth rate in A549, increasing nearly 10-fold after 48 and 72 h.M. abscessus CRM0019 creates a protective and replicative niche in alveolar epithelial cells mainly by avoiding phagosome maturation. Once recovered from infected macrophages, CRM0019 remains infective and displays greater intracellular growth in A549 cells compared to the ATCC 19977 strain. This evasion strategy in alveolar epithelial cells may contribute to the long survival of the CRM0019 strain in the host and thus to the inefficacy of in vivo treatment.
Project description:Staphylococcus aureus is a Gram-positive human pathogen that is readily internalized by professional phagocytes such as macrophages and neutrophils but also by non-professional phagocytes such as epithelial or endothelial cells. Intracellular bacteria have been proposed to play a role in evasion of the innate immune system and may also lead to dissemination within migrating phagocytes. Further, S.?aureus efficiently lyses host cells with a battery of cytolytic toxins. Recently, phenol-soluble modulins (PSM) have been identified to comprise a genus-specific family of cytolytic peptides. Of these the PSM? peptides have been implicated in killing polymorphonuclear leucocytes after phagocytosis. We questioned if the peptides were active in destroying endosomal membranes to avoid lysosomal killing of the pathogen and monitored integrity of infected host cell endosomes by measuring the acidity of the intracellular bacterial microenvironment via flow cytometry and by a reporter recruitment technique. Isogenic mutants of the methicillin-resistant S.?aureus (MRSA) strains USA300 LAC, USA400 MW2 as well as the strongly cytolytic methicillin-sensitive strain 6850 were compared with their respective wild type strains. In all three genetic backgrounds, PSM? mutants were unable to escape from phagosomes in non-professional (293, HeLa, EAhy.926) and professional phagocytes (THP-1), whereas mutants in PSM? and ?-toxin as well as ?-toxin, phosphatidyl inositol-dependent phospholipase C and Panton Valentine leucotoxin escaped with efficiencies of the parental strains. S.?aureus replicated intracellularly only in presence of a functional PSM? operon thereby illustrating that bacteria grow in the host cell cytoplasm upon phagosomal escape.
Project description:Macrophages are critical effectors of the early innate response to bacteria in tissues. Phagocytosis and killing of bacteria are interrelated functions essential for bacterial clearance but the rate-limiting step when macrophages are challenged with large numbers of the major medical pathogen Staphylococcus aureus is unknown. We show that macrophages have a finite capacity for intracellular killing and fail to match sustained phagocytosis with sustained microbial killing when exposed to large inocula of S. aureus (Newman, SH1000 and USA300 strains). S. aureus ingestion by macrophages is associated with a rapid decline in bacterial viability immediately after phagocytosis. However, not all bacteria are killed in the phagolysosome, and we demonstrate reduced acidification of the phagolysosome, associated with failure of phagolysosomal maturation and reduced activation of cathepsin D. This results in accumulation of viable intracellular bacteria in macrophages. We show macrophages fail to engage apoptosis-associated bacterial killing. Ultittop mately macrophages with viable bacteria undergo cell lysis, and viable bacteria are released and can be internalized by other macrophages. We show that cycles of lysis and reuptake maintain a pool of viable intracellular bacteria over time when killing is overwhelmed and demonstrate intracellular persistence in alveolar macrophages in the lungs in a murine model.
Project description:Autophagy is an important innate immune defense mechanism that controls Mycobacterium tuberculosis (Mtb) growth inside macrophages. Autophagy machinery targets Mtb-containing phagosomes via xenophagy after damage to the phagosomal membrane due to the Type VII secretion system Esx-1 or via LC3-associated phagocytosis without phagosomal damage. Conversely, Mtb restricts autophagy-related pathways via the production of various bacterial protein factors. Although bacterial lipids are known to play strategic functions in Mtb pathogenesis, their role in autophagy manipulation remains largely unexplored. Here, we report that the lipid virulence factors sulfoglycolipids (SLs) and phthiocerol dimycocerosates (DIMs) control autophagy-related pathways through distinct mechanisms in human macrophages. Using knock-out and knock-in mutants of Mtb and Mycobacterium bovis BCG (Bacille Calmette Guerin) and purified lipids, we found that (i) Mtb mutants with DIM and SL deficiencies promoted functional autophagy via an MyD88-dependent and phagosomal damage-independent pathway in human macrophages; (ii) SLs limited this pathway by acting as TLR2 antagonists; (iii) DIMs prevented phagosomal damage-independent autophagy while promoting Esx-1-dependent xenophagy; (iv) and DIMs, but not SLs, limited the acidification of LC3-positive Mtb compartments. In total, our study reveals an unexpected and intricate role for Mtb lipid virulence factors in controlling autophagy-related pathways in human macrophages, thus providing further insight into the autophagy manipulation tactics deployed by intracellular bacterial pathogens.
Project description:BACKGROUND: Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification. CONCLUSIONS/SIGNIFICANCE: Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23-32 min, 3-4 min and 74-120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply require fluorophore conjugation to a particle of interest, such as a pathogen or mimetic, in combination with common cell labeling dyes. As such, these methods hold promise for future measurements of receptor-mediated internalization in other cell systems, e.g. pathogen-host systems.