Early Acidification of Phagosomes ContainingBrucella suis Is Essential for Intracellular Survival in Murine Macrophages

ABSTRACT Brucella suis is a facultative intracellular pathogen of mammals, residing in macrophage vacuoles. In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication ofB. suis. Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence intensity of opsonized, carboxyfluorescein-rhodamine- and Oregon Green 488-rhodamine-labeled bacteria. Compartments containing live B. suis acidified to a pH of about 4.0 to 4.5 within 60 min. Acidification of B. suis-containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A1, a specific inhibitor of vacuolar proton-ATPases. This neutralization at 1 h postinfection resulted in a 2- to 34-fold reduction of opsonized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfection, respectively. Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability. Addition of ammonium chloride at 7 h after the beginning of infection, however, did not affect intracellular multiplication of B. suis, in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. Thus, we conclude that phagosomes with B. suis acidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage.

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Targeting Salmonella Typhimurium Invasion and Intracellular Survival Using Pyrogallol

Frontiers in Microbiology ◽

10.3389/fmicb.2021.631426 ◽

2021 ◽

Vol 12 ◽

Author(s):

Biruk Tesfaye Birhanu ◽

Eon-Bee Lee ◽

Seung-Jin Lee ◽

Seung-Chun Park

Keyword(s):

Inhibitory Concentration ◽

Minimum Bactericidal Concentration ◽

Efflux Pump ◽

Intracellular Survival ◽

Host Cells ◽

Intracellular Bacteria ◽

Intracellular Pathogen ◽

Intracellular Accumulation ◽

Protection Assay ◽

Serovar Typhimurium

Salmonella enterica serovar Typhimurium, an intracellular pathogen, evades the host immune response mechanisms to cause gastroenteritis in animals and humans. After invading the host cells, the bacteria proliferate in Salmonella-containing vacuole (SCV) and escapes from antimicrobial therapy. Moreover, Salmonella Typhimurium develops resistance to various antimicrobials including, fluoroquinolones. Treating intracellular bacteria and combating drug resistance is essential to limit the infection rate. One way of overcoming these challenges is through combination therapy. In this study, Pyrogallol (PG), a polyphenol, is combined with marbofloxacin (MAR) to investigate its effect on Salmonella Typhimurium invasion and intracellular survival inhibition. The Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PG against Salmonella Typhimurium were 128 and 256 μg/mL, respectively. The lowest fractional inhibitory concentration (FIC) index for a combination of PG and MAR was 0.5. The gentamycin protection assay revealed that PG (30 μg/mL) alone and in combination with sub-MIC of MAR inhibited 72.75 and 76.18% of the invading bacteria in Caco-2 cells, respectively. Besides, the intracellular survival of Salmonella Typhimurium was reduced by 7.69 and 74.36% in treatment with PG alone and combined with sub-MIC of MAR, respectively, which was visualized by the confocal microscopy. PG has also shown to increase the intracellular accumulation of fluoroquinolone by 15.2 and 34.9% at 30 and 100 μg/mL concentration, respectively. Quantitative real-time PCR demonstrated PG suppressed the genetic expression of hilA, invF, sipB, and acrA by 14.6, 15.4, 13.6, and 36%, respectively. However, the downregulation of hilA, invF, sipB, and acrA increased to 80, 74.6, 78, and 70.1%, in combination with sub-MIC of MAR, respectively. Similarly, PG combined with MAR inhibited the expression of sdiA, srgE, and rck genes by 78.6, 62.8, and 61.8%, respectively. In conclusion, PG has shown antimicrobial activity against Salmonella Typhimurium alone and in combination with MAR. It also inhibited invasion and intracellular survival of the bacteria through downregulation of quorum sensing, invading virulence, and efflux pump genes. Hence, PG could be a potential antimicrobial candidate which could limit the intracellular survival and replication of Salmonella Typhimurium.

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Opsonin-independent adherence and intracellular development of Legionella pneumophila within U-937 cells

Canadian Journal of Microbiology ◽

10.1139/m93-103 ◽

1993 ◽

Vol 39 (7) ◽

pp. 718-722 ◽

Cited By ~ 28

Author(s):

Frank G. Rodgers ◽

Frank C. Gibson III

Keyword(s):

Host Cell ◽

Legionella Pneumophila ◽

Surface Interactions ◽

Host Cells ◽

Intracellular Bacteria ◽

Suitable Model ◽

Intracellular Replication ◽

Cell Monolayers ◽

Histiocytic Lymphoma ◽

Intracellular Multiplication

Legionella pneumophila adhered to and multiplied intracellularly in the human histiocytic lymphoma U-937 cell line. The infectious process was evaluated by viable bacterial cell colony counts and documented by transmission and scanning electron microscopy. In the absence of opsonins, wash-resistant bacterial adherence to host cells occurred within 1 h and attachment of 1 or 2 organisms per U-937 host cell involved close surface interactions at the prokaryotic and eukaryotic membranes. Intracellular multiplication of bacteria was maximal by 24 h after inoculation of cell monolayers. Release of L. pneumophila from these cells appeared as a lytic process that resulted in an increase in the numbers of microorganisms in the extracellular fluids and a concomitant decline in the number of intracellular bacteria. The course of cellular infection was completed by 72 h. The cellular and ultrastructural events of L. pneumophila adherence and uptake by U-937 cells in the absence of antibody or complement have been defined. In addition, this work further establishes the U-937 cell as a suitable model for investigating Legionella – host cell interactions.Key words: Legionella, U-937 cells, intracellular replication, opsonin-independent uptake.

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Analysis of the Behavior of eryC Mutants of Brucella suis Attenuated in Macrophages

Infection and Immunity ◽

10.1128/iai.73.10.6782-6790.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6782-6790 ◽

Cited By ~ 20

Author(s):

Sonja Burkhardt ◽

Maria P. Jiménez de Bagüés ◽

Jean-Pierre Liautard ◽

Stephan Köhler

Keyword(s):

Carbon Source ◽

Sole Carbon Source ◽

Intracellular Pathogen ◽

Wild Type ◽

Allelic Exchange ◽

Brucella Suis ◽

Lower Extent ◽

Intracellular Multiplication

ABSTRACT The facultatively intracellular pathogen Brucella, characterized by its capacity to replicate in professional and non professional phagocytes, also causes abortion in ruminants. This property has been linked to the presence of erythritol in the placenta, as brucellae preferentially utilize erythritol. The ery operon encodes enzymes involved in erythritol metabolism, and a link with virulence has since been discussed. Allelic exchange mutants in eryC of Brucella suis were erythritol sensitive in vitro with a MIC of 1 to 5 mM of erythritol. Their multiplication in macrophage-like cells was 50- to 90-fold reduced, but complementation of the mutant restored wild-type levels of intracellular multiplication and the capacity to use erythritol as a sole carbon source. In vivo, the eryC mutant colonized the spleens of infected BALB/c mice to a significantly lower extent than the wild type and the complemented strain. Interestingly, eryC mutants that were in addition spontaneously erythritol tolerant nevertheless exhibited wild-type-like intramacrophagic and intramurine replication. We concluded from our results that erythritol was not an essential carbon source for the pathogen in the macrophage host cell but that the inactivation of the eryC gene significantly reduced the intramacrophagic and intramurine fitness of B. suis.

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Effect of nitric oxide on the growth ofChlamydophila pneumoniae

Canadian Journal of Microbiology ◽

10.1139/w05-080 ◽

2005 ◽

Vol 51 (11) ◽

pp. 941-947 ◽

Cited By ~ 13

Author(s):

Caterina Romano Carratelli ◽

Antonietta Rizzo ◽

Rossella Paolillo ◽

Maria Rosaria Catania ◽

Piergiorgio Catalanotti ◽

...

Keyword(s):

Nitric Oxide ◽

Control Mechanism ◽

Chlamydophila Pneumoniae ◽

Host Cells ◽

Intracellular Pathogen ◽

Immune Control ◽

Systemic Spread ◽

J774 Cells ◽

Dose Dependent

Chlamydophila pneumoniae is an important human intracellular pathogen; however, the pathogenesis of C. pneumoniae infection is poorly understood and the immune control mechanism versus host cells is not completely known. The role of the nitric oxide (NO) synthase pathway in inhibiting the ability of C. pneumoniae to infect macrophage J774 cells and the ability of NO to damage isolated C. pneumoniae were investigated. Exposure of infected cultures to recombinant murine gamma interferon (MurIFN-γ) resulted in increased production of NO and reduced viability. Addition of 2-(N,N-diethylamino)-diazenolase-2-oxide before infection of J774 cells or during chlamydial cultivation released NO, both resulting in a reduction in the viability of C. pneumoniae in a dose-dependent way. These results indicate that immune control of chlamydial growth in murine macrophage cells may trigger a mechanism that includes NO release with effects on the multiplication of the microorganism, thus suggesting that NO may play a role in preventing the systemic spread of Chlamydia.Key words: Chlamydophila pneumoniae, J774 cells, NO.

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An infection-induced RhoB-Beclin 1-Hsp90 complex enhances clearance of uropathogenic Escherichia coli

Nature Communications ◽

10.1038/s41467-021-22726-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chunhui Miao ◽

Mingyu Yu ◽

Geng Pei ◽

Zhenyi Ma ◽

Lisong Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Treatment Strategies ◽

Uropathogenic Escherichia Coli ◽

Beclin 1 ◽

Host Cells ◽

Infection Model ◽

Intracellular Bacteria ◽

Bladder Epithelium ◽

Binding Residue

AbstractHost cells use several anti-bacterial pathways to defend against pathogens. Here, using a uropathogenic Escherichia coli (UPEC) infection model, we demonstrate that bacterial infection upregulates RhoB, which subsequently promotes intracellular bacteria clearance by inducing LC3 lipidation and autophagosome formation. RhoB binds with Beclin 1 through its residues at 118 to 140 and the Beclin 1 CCD domain, with RhoB Arg133 being the key binding residue. Binding of RhoB to Beclin 1 enhances the Hsp90-Beclin 1 interaction, preventing Beclin 1 degradation. RhoB also directly interacts with Hsp90, maintaining RhoB levels. UPEC infections increase RhoB, Beclin 1 and LC3 levels in bladder epithelium in vivo, whereas Beclin 1 and LC3 levels as well as UPEC clearance are substantially reduced in RhoB+/− and RhoB−/− mice upon infection. We conclude that when stimulated by UPEC infections, host cells promote UPEC clearance through the RhoB-Beclin 1-HSP90 complex, indicating RhoB may be a useful target when developing UPEC treatment strategies.

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Saccharomyces boulardii Preserves the Barrier Function and Modulates the Signal Transduction Pathway Induced in Enteropathogenic Escherichia coli-Infected T84 Cells

Infection and Immunity ◽

10.1128/iai.68.10.5998-6004.2000 ◽

2000 ◽

Vol 68 (10) ◽

pp. 5998-6004 ◽

Cited By ~ 115

Author(s):

Dorota Czerucka ◽

Stephanie Dahan ◽

Baharia Mograbi ◽

Bernard Rossi ◽

Patrick Rampal

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Signal Transduction Pathway ◽

Specific Inhibitor ◽

Saccharomyces Boulardii ◽

Intracellular Bacteria ◽

T84 Cells ◽

Protein Map ◽

Mitogen Activated Protein ◽

Map Kinase Pathway

ABSTRACT Use of the nonpathogenic yeast Saccharomyces boulardiiin the treatment of infectious diarrhea has attracted growing interest. The present study designed to investigate the effect of this yeast on enteropathogenic Escherichia coli (EPEC)-associated disease demonstrates that S. boulardii abrogated the alterations induced by an EPEC strain on transepithelial resistance, [3H]inulin flux, and ZO-1 distribution in T84 cells. Moreover, EPEC-mediated apoptosis of epithelial cells was delayed in the presence of S. boulardii. The yeast did not modify the number of adherent bacteria but lowered by 50% the number of intracellular bacteria. Infection by EPEC induced tyrosine phosphorylation of several proteins in T84 cells, including p46 and p52 SHC isoforms, that was attenuated in the presence of S. boulardii. Similarly, EPEC-induced activation of the ERK1/2 mitogen-activated protein (MAP) kinase pathway was diminished in the presence of the yeast. Interestingly, inhibition of the ERK1/2 pathway with the specific inhibitor PD 98059 decreased EPEC internalization, suggesting that modulation of the ERK1/2 MAP pathway might account for the lowering of the number of intracellular bacteria observed in the presence of S. boulardii. Altogether, this study demonstrated that S. boulardii exerts a protective effect on epithelial cells after EPEC adhesion by modulating the signaling pathway induced by bacterial infection.

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Initial Characterization of the Two ClpP Paralogs ofChlamydia trachomatisSuggests Unique Functionality for Each

Journal of Bacteriology ◽

10.1128/jb.00635-18 ◽

2018 ◽

Vol 201 (2) ◽

Cited By ~ 5

Author(s):

Nicholas A. Wood ◽

Krystal Y. Chung ◽

Amanda M. Blocker ◽

Nathalia Rodrigues de Almeida ◽

Martin Conda-Sheridan ◽

...

Keyword(s):

Host Cells ◽

Developmental Cycle ◽

Intracellular Bacteria ◽

Clp Protease ◽

Content Type ◽

Sexually Transmitted ◽

Obligate Intracellular ◽

Developmental Form ◽

Obligate Intracellular Bacteria

ABSTRACTMembers ofChlamydiaare obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other.Chlamydiaspp. have five uncharacterizedclpgenes,clpX,clpC, twoclpPparalogs, andclpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactiveclpPmutants inChlamydiaspp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detectedin vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.IMPORTANCEChlamydia trachomatisis the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression inChlamydiaspp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

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Methodology for Recognition of Invasive Potential of Escherichia coli

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/60.3.546 ◽

1977 ◽

Vol 60 (3) ◽

pp. 546-562 ◽

Cited By ~ 3

Author(s):

Ira J Mehlman ◽

Elmer L Eide ◽

Arvey C Sanders ◽

Morris Fishbein ◽

Calvin C G Aulisio

Keyword(s):

Mammalian Cell ◽

Mammalian Cell Culture ◽

Brain Heart Infusion ◽

Host Cells ◽

Minimal Essential Medium ◽

Invasive Potential ◽

Host Culture ◽

Fetal Bovine ◽

Intracellular Multiplication ◽

Positive Results

Abstract Surveillance for dysentery-related invasive potential in bacteria using the Sereny keratoconjunctivitis test is restricted by expense, time factor, and necessity for confirmation. Primary screening of isolates in a standardized mammalian cell culture system is recommended. Bacteria are grown 20 hr in veal infusion, washed, and resuspended in 20% heat-inactivated fetal bovine serum (FBS) supplemented with 0.12% brain heart infusion and 0.1% bile salts. The HeLa culture is grown 20 hr as a monolayer in chamber slides with 90% minimal essential medium (MEM)-10% FBS. The host culture is infected at a ratio of 10 bacteria/ mammalian cell for 3 hr at 35°C. The infection medium is replaced with MEM-FBS supplemented with 300 μg lysozyme and 5 μg gentamycin/ml. The infected monolayer is incubated 5 hr at 35°C to permit intracellular multiplication. Specimens arc washed, fixed with methanol, and stained successively with May-Grunwald and Giemsa dyes. Bacteria occur within the cytoplasm if invasion has occurred. The criterion for a positive test is that 1% of the host cells possesses at least 5 bacteria in 2 of 3 trials. Invasiveness is correlated with and possibly pre-conditioned by cytotoxic principle(s). Infectivity rates vary from 0 to 30%. The cytopathic effect is noted in 5–50% of HeLa cells. Positive results must be confirmed by the Sereny test.

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Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of Yersinia pseudotuberculosis by Macrophages

Infection and Immunity ◽

10.1128/iai.00183-08 ◽

2008 ◽

Vol 76 (9) ◽

pp. 4299-4310 ◽

Cited By ~ 24

Author(s):

Yue Zhang ◽

James Murtha ◽

Margaret A. Roberts ◽

Richard M. Siegel ◽

James B. Bliska

Keyword(s):

Cell Death ◽

Type Iii Secretion ◽

Yersinia Pseudotuberculosis ◽

De Novo ◽

Host Cells ◽

Host Responses ◽

Intracellular Bacteria ◽

Wild Type ◽

Macrophage Response ◽

Type Iii

ABSTRACT Yersinia pseudotuberculosis uses a plasmid (pYV)-encoded type III secretion system (T3SS) to translocate a set of effectors called Yops into infected host cells. YopJ functions to induce apoptosis, and YopT, YopE, and YopH act to antagonize phagocytosis in macrophages. Because Yops do not completely block phagocytosis and Y. pseudotuberculosis can replicate in macrophages, it is important to determine if the T3SS modulates host responses to intracellular bacteria. Isogenic pYV-cured, pYV+ wild-type, and yop mutant Y. pseudotuberculosis strains were allowed to infect bone marrow-derived murine macrophages at a low multiplicity of infection under conditions in which the survival of extracellular bacteria was prevented. Phagocytosis, the intracellular survival of the bacteria, and the apoptosis of the infected macrophages were analyzed. Forty percent of cell-associated wild-type bacteria were intracellular after a 20-min infection, allowing the study of the macrophage response to internalized pYV+ Y. pseudotuberculosis. Interestingly, macrophages restricted survival of pYV+ but not pYV-cured or ΔyopB Y. pseudotuberculosis within phagosomes: only a small fraction of the pYV+ bacteria internalized replicated by 24 h. In addition, ∼20% of macrophages infected with wild-type pYV+ Y. pseudotuberculosis died of apoptosis after 20 h. Analysis of yop mutants expressing catalytically inactive effectors revealed that YopJ was important for apoptosis, while a role for YopE, YopH, and YopT in modulating macrophage responses to intracellular bacteria could not be identified. Apoptosis was reduced in Toll-like receptor 4-deficient macrophages, indicating that cell death required signaling through this receptor. Treatment of macrophages harboring intracellular pYV+ Y. pseudotuberculosis with chloramphenicol reduced apoptosis, indicating that the de novo bacterial protein synthesis was necessary for cell death. Our finding that the presence of a functional T3SS impacts the survival of both bacterium and host following phagocytosis of Y. pseudotuberculosis suggests new roles for the T3SS in Yersinia pathogenesis.

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Efficient Killing of Multidrug-Resistant Intracellular Bacteria by AIEgens in Vivo

10.26434/chemrxiv.12579611 ◽

2020 ◽

Author(s):

Ying Li ◽

Fei Liu ◽

Jiangjiang Zhang ◽

Xiaoye Liu ◽

Peihong Xiao ◽

...

Keyword(s):

Membrane Damage ◽

Multidrug Resistant ◽

Host Cells ◽

Comparable Efficacy ◽

Intracellular Bacteria ◽

New Agents ◽

Immune Clearance ◽

Infected Cells ◽

Dose Levels

<a>Bacteria infected cells acting as “Trojan horses” not only protect bacteria from antibiotic therapies and immune clearance, but also increase the dissemination of pathogens from the initial sites of infection. Antibiotics are hard and insufficient to treat such hidden intracellular bacteria, especially the multidrug</a>-resistant (MDR) bacteria. Herein, aggregation-induced emission luminogens (AIEgens) such as TBPs showed potent broad-spectrum bactericidal activity against both <a></a><a>extracellular and intracellular</a> Gram-positive pathogens at low-dose levels. TBPs triggered reactive oxygen species (ROS)-mediated membrane damage to kill bacteria, regardless of light irradiation. Additionally, such AIEgens activated mitochondria dependent autophagy to eliminate intracellular bacteria in host cells. Compared to the routinely used vancomycin in clinics, TBPs showed comparable efficacy against methicillin-resistant Staphylococcus aureus (MRSA) in vivo. Our studies demonstrate that AIEgens are promising new agents for the treatment of MDR bacteria associated infections.

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