scholarly journals Targeting Salmonella Typhimurium Invasion and Intracellular Survival Using Pyrogallol

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.

scholarly journals Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Jin-Hahn Kim ◽  
Akhilesh Kumar Chaurasia ◽  
Nayab Batool ◽  
Kwan Soo Ko ◽  
Kyeong Kyu Kim
Keyword(s):  
Host Cells ◽  
Intracellular Bacteria ◽  
Protection Assay ◽  
Host Cell Membrane ◽  
Bacterial Killing ◽  
Content Type ◽  
Gentamicin Protection Assay ◽  
Enzyme Protection ◽  
The Difference ◽  
Kinetics Of

ABSTRACTPrecise enumeration of living intracellular bacteria is the key step to estimate the invasion potential of pathogens and host immune responses to understand the mechanism and kinetics of bacterial pathogenesis. Therefore, quantitative assessment of host-pathogen interactions is essential for development of novel antibacterial therapeutics for infectious disease. The gentamicin protection assay (GPA) is the most widely used method for these estimations by counting the CFU of intracellular living pathogens. Here, we assess the longstanding drawbacks of the GPA by employing an antistaphylococcal endopeptidase as a bactericidal agent to kill extracellularStaphylococcus aureus. We found that the difference between the two methods for the recovery of intracellular CFU ofS. aureuswas about 5 times. We prove that the accurate number of intracellular CFU could not be precisely determined by the GPA due to the internalization of gentamicin into host cells during extracellular bacterial killing. We further demonstrate that lysostaphin-mediated extracellular bacterial clearance has advantages for measuring the kinetics of bacterial internalization on a minute time scale due to the fast and tunable activity and the inability of protein to permeate the host cell membrane. From these results, we propose that accurate quantification of intracellular bacteria and measurement of internalization kinetics can be achieved by employing enzyme-mediated killing of extracellular bacteria (enzyme protection assay [EPA]) rather than the host-permeative drug gentamicin, which is known to alter host physiology.

scholarly journals MdsABC-Mediated Pathway for Pathogenicity in Salmonella enterica Serovar Typhimurium

2015 ◽  
Vol 83 (11) ◽  
pp. 4266-4276 ◽  
Author(s):  
Saemee Song ◽  
Boeun Lee ◽  
Ji-Hyun Yeom ◽  
Soonhye Hwang ◽  
Ilnam Kang ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Disulfide Bonds ◽  
Efflux Pump ◽  
Host Cells ◽  
Deletion Strain ◽  
Bacterial Number ◽  
Phagocytic Cells ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Key Factor

ABSTRACTMdsABC is aSalmonella-specific tripartite efflux pump that has been implicated in the virulence ofSalmonella entericaserovar Typhimurium; however, little is known about the virulence factors associated with this pump. We observed MdsABC expression-dependent alterations in the degree of resistance to extracellular oxidative stress and macrophage-mediated killing. Thin-layer chromatography and tandem mass spectrometry analyses revealed that overexpression of MdsABC led to increased secretion of 1-palmitoyl-2-stearoyl-phosphatidylserine (PSPS), affecting the ability of the bacteria to invade and survive in host cells. Overexpression of MdsABC and external addition of PSPS similarly rendered themdsABCdeletion strain resistant to diamide. Diagonal gel analysis showed that PSPS treatment reduced the diamide-mediated formation of disulfide bonds, particularly in the membrane fraction of the bacteria.Salmonellainfection of macrophages induced the upregulation of MdsABC expression and led to an increase of intracellular bacterial number and host cell death, similar to the effects of MdsABC overexpression and PSPS pretreatment on themdsABCdeletion strain. Our study shows that MdsABC mediates a previously uncharacterized pathway that involves PSPS as a key factor for the survival and virulence ofS. Typhimurium in phagocytic cells.

scholarly journals Construction of Aminoglycoside-Sensitive Burkholderia cenocepacia Strains for Use in Studies of Intracellular Bacteria with the Gentamicin Protection Assay

2010 ◽  
Vol 76 (10) ◽  
pp. 3170-3176 ◽  
Author(s):  
Mohamad A. Hamad ◽  
Alexander M. Skeldon ◽  
Miguel A. Valvano
Keyword(s):  
Efflux Pump ◽  
Opportunistic Pathogen ◽  
Multidrug Resistant ◽  
Eukaryotic Cell ◽  
Burkholderia Cenocepacia ◽  
Intracellular Bacteria ◽  
Raw 264.7 ◽  
Protection Assay ◽  
Intracellular Infection ◽  
Gentamicin Protection Assay

ABSTRACT Burkholderia cenocepacia is a multidrug-resistant opportunistic pathogen that infects the airways of patients with cystic fibrosis (CF) and can survive intracellularly in macrophages and epithelial cells. The gentamicin protection assay, which relies on the poor ability of gentamicin or other aminoglycosides to permeate eukaryotic cell membranes, is traditionally employed to quantify intracellular bacteria. However, the high resistance of these bacteria to aminoglycosides hampers the use of the gentamicin protection assay to investigate intracellular infection by B. cenocepacia. Here, we report the construction of gentamicin-sensitive strains of B. cenocepacia carrying a deletion of the BCAL1674, BCAL1675, and BCAL1676 genes that form an operon encoding an AmrAB-OprA-like efflux pump. We show that bacteria carrying this deletion are hypersensitive to gentamicin and also delay phagolysosomal fusion upon infection of RAW 264.7 murine macrophages, as previously demonstrated for the parental strain. We also demonstrate for the first time that low concentrations of gentamicin can be used to effectively kill extracellular bacteria and reliably quantify the intracellular infection by B. cenocepacia, which can replicate in RAW 264.7 macrophages.

scholarly journals Eradication of Intracellular Salmonellaenterica Serovar Typhimurium with a Small-Molecule, Host Cell-Directed Agent

2009 ◽  
Vol 53 (12) ◽  
pp. 5236-5244 ◽  
Author(s):  
Hao-Chieh Chiu ◽  
Samuel K. Kulp ◽  
Shilpa Soni ◽  
Dasheng Wang ◽  
John S. Gunn ◽  
...  
Keyword(s):  
Host Cell ◽  
Small Molecule ◽  
Pathogenic Bacteria ◽  
Intracellular Survival ◽  
Resistant Bacteria ◽  
Intracellular Bacteria ◽  
Akt Kinase ◽  
Antibiotic Resistant ◽  
Serovar Typhimurium ◽  
Host Cell Factors

ABSTRACTEradication of intracellular pathogenic bacteria with host-directed chemical agents has been an anticipated innovation in the treatment of antibiotic-resistant bacteria. We previously synthesized and characterized a novel small-molecule agent, AR-12, that induces autophagy and inhibits the Akt kinase in cancer cells. As both autophagy and the Akt kinase have been shown recently to play roles in the intracellular survival of several intracellular bacteria, includingSalmonellaentericaserovar Typhimurium, we investigated the effect of AR-12 on the intracellular survival ofSalmonellaserovar Typhimurium in macrophages. Our results show that AR-12 induces autophagy in macrophages, as indicated by increased autophagosome formation, and potently inhibits the survival of serovar Typhimurium in macrophages in association with increased colocalization of intracellular bacteria with autophagosomes. Intracellular bacterial growth was partially rescued in the presence of AR-12 by the short hairpin RNA-mediated knockdown of Beclin-1 or Atg7 in macrophages. Moreover, AR-12 inhibits Akt kinase activity in infected macrophages, which we show to be important for its antibacterial effect as the enforced expression of constitutively activated Akt1 in these cells reverses the AR-12-induced inhibition of intracellular serovar Typhimurium survival. Finally, oral administration of AR-12 at 2.5 mg/kg/day to serovar Typhimurium-infected mice reduced hepatic and splenic bacterial burdens and significantly prolonged survival. These findings show that AR-12 represents a proof of principle that the survival of intracellular bacteria can be suppressed by small-molecule agents that target both innate immunity and host cell factors modulated by bacteria.

scholarly journals Disruption of the Salmonella-Containing Vacuole Leads to Increased Replication of Salmonella enterica Serovar Typhimurium in the Cytosol of Epithelial Cells

2002 ◽  
Vol 70 (6) ◽  
pp. 3264-3270 ◽  
Author(s):  
John H. Brumell ◽  
Patrick Tang ◽  
Michelle L. Zaharik ◽  
B. Brett Finlay
Keyword(s):  
Epithelial Cells ◽  
Host Cell ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Vacuolar Compartment ◽  
Serovar Typhimurium ◽  
Endosomal System

ABSTRACT Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that inhabits a vacuolar compartment, called the Salmonella-containing vacuole (SCV), in infected host cells. Maintenance of the SCV is accomplished by SifA, and mutants of this Salmonella pathogenicity island 2 type III effector replicate more efficiently in epithelial cells. Here we demonstrate that enhanced replication of sifA mutants occurs in the cytosol of these cells. Increased replication of wild-type bacteria was also observed in cells treated with wortmannin or expressing Rab5 Q79L or Rab7 N125I, all of which caused a loss of SCV integrity. Our findings demonstrate the requirement of the host cell endosomal system for maintenance of the SCV and that loss of this compartment allows increased replication of serovar Typhimurium in the cytosol of epithelial cells.

scholarly journals Flagella Facilitate Escape of Salmonella from Oncotic Macrophages

2007 ◽  
Vol 189 (22) ◽  
pp. 8224-8232 ◽  
Author(s):  
Gen-ichiro Sano ◽  
Yasunari Takada ◽  
Shinichi Goto ◽  
Kenta Maruyama ◽  
Yutaka Shindo ◽  
...  
Keyword(s):  
Cell Death ◽  
Actin Filaments ◽  
Systemic Disease ◽  
Eukaryotic Cell ◽  
Host Cells ◽  
Dependent Manner ◽  
Intracellular Parasite ◽  
Intracellular Accumulation ◽  
Serovar Typhimurium ◽  
Entire Cell

ABSTRACT The intracellular parasite Salmonella enterica serovar Typhimurium causes a typhoid-like systemic disease in mice. Whereas the survival of Salmonella in phagocytes is well understood, little has been documented about the exit of intracellular Salmonella from host cells. Here we report that in a population of infected macrophages Salmonella induces “oncosis,” an irreversible progression to eukaryotic cell death characterized by swelling of the entire cell body. Oncotic macrophages (OnMφs) are terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling negative and lack actin filaments (F-actin). The plasma membrane of OnMφs filled with bacilli remains impermeable, and intracellular Salmonella bacilli move vigorously using flagella. Eventually, intracellular Salmonella bacilli intermittently exit host cells in a flagellum-dependent manner. These results suggest that induction of macrophage oncosis and intracellular accumulation of flagellated bacilli constitute a strategy whereby Salmonella escapes from host macrophages.

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

1999 ◽  
Vol 67 (8) ◽  
pp. 4041-4047 ◽  
Author(s):  
Françoise Porte ◽  
Jean-Pierre Liautard ◽  
Stephan Köhler
Keyword(s):  
Ammonium Chloride ◽  
Fluorescence Intensity ◽  
Specific Inhibitor ◽  
Host Cells ◽  
Intracellular Bacteria ◽  
Intracellular Pathogen ◽  
Fold Reduction ◽  
Brucella Suis ◽  
J774 Cells ◽  
Intracellular Multiplication

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.

scholarly journals Intracellular survival of Salmonella enterica serovar Typhi in human macrophages is independent of Salmonella pathogenicity island (SPI)-2

Microbiology ◽  
2010 ◽  
Vol 156 (12) ◽  
pp. 3689-3698 ◽  
Author(s):  
Chantal G. Forest ◽  
Elyse Ferraro ◽  
Sébastien C. Sabbagh ◽  
France Daigle
Keyword(s):  
Salmonella Enterica ◽  
Systemic Disease ◽  
Distinct Type ◽  
Intracellular Survival ◽  
Effector Proteins ◽  
Host Cells ◽  
Secretion Systems ◽  
Human Macrophages ◽  
Successful Infection ◽  
Serovar Typhimurium

For successful infection, Salmonella enterica secretes and injects effector proteins into host cells by two distinct type three secretion systems (T3SSs) located on Salmonella pathogenicity islands (SPIs)-1 and -2. The SPI-2 T3SS is involved in intracellular survival of S. enterica serovar Typhimurium and systemic disease. As little is known regarding the function of the SPI-2 T3SS from S. enterica serovar Typhi, the aetiological agent of typhoid fever, we investigated its role for survival in human macrophages. Mutations in the translocon (sseB), basal secretion apparatus (ssaR) and regulator (ssrB) did not result in any reduction in survival under many of the conditions tested. Similar results were obtained with another S. Typhi strain or by using human primary cells. Results were corroborated based on complete deletion of the SPI-2 T3SS. Surprisingly, the data suggest that the SPI-2 T3SS of S. Typhi is not required for survival in human macrophages.

scholarly journals Salmonella Suppresses the TRIF-Dependent Type I Interferon Response in Macrophages

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Katherine A. Owen ◽  
C. J. Anderson ◽  
James E. Casanova
Keyword(s):  
Immune Responses ◽  
Salmonella Enterica ◽  
Intracellular Survival ◽  
Innate Immune ◽  
Host Cells ◽  
Type I ◽  
Innate Immune Responses ◽  
Beta Interferon ◽  
Serovar Typhimurium ◽  
Ifn Γ

ABSTRACT Salmonella enterica is an intracellular pathogen that causes diseases ranging from gastroenteritis to typhoid fever. Salmonella bacteria trigger an autophagic response in host cells upon infection but have evolved mechanisms for suppressing this response, thereby enhancing intracellular survival. We recently reported that S. enterica serovar Typhimurium actively recruits the host tyrosine kinase focal adhesion kinase (FAK) to the surface of the Salmonella -containing vacuole (SCV) (K. A. Owen et al., PLoS Pathog 10:e1004159, 2014). FAK then suppresses autophagy through activation of the Akt/mTORC1 signaling pathway. In FAK −/− macrophages, bacteria are captured in autophagosomes and intracellular survival is attenuated. Here we show that the cell-autonomous bacterial suppression of autophagy also suppresses the broader innate immune response by inhibiting production of beta interferon (IFN-β). Induction of bacterial autophagy (xenophagy), but not autophagy alone, triggers IFN-β production through a pathway involving the adapter TRIF and endosomal Toll-like receptor 3 (TLR3) and TLR4. Selective FAK knockout in macrophages resulted in rapid bacterial clearance from mucosal tissues after oral infection. Clearance correlated with increased IFN-β production by intestinal macrophages and with IFN-β-dependent induction of IFN-γ by intestinal NK cells. Blockade of either IFN-β or IFN-γ increased host susceptibility to infection, whereas experimental induction of IFN-β was protective. Thus, bacterial suppression of autophagy not only enhances cell-autonomous survival but also suppresses more-systemic innate immune responses by limiting type I and type II interferons. IMPORTANCE Salmonella enterica serovar Typhimurium represents one of the most commonly identified bacterial causes of foodborne illness worldwide. S . Typhimurium has developed numerous strategies to evade detection by the host immune system. Autophagy is a cellular process that involves the recognition and degradation of defective proteins and organelles. More recently, autophagy has been described as an important means by which host cells recognize and eliminate invading intracellular pathogens and plays a key role in the production of cytokines. Previously, we determined that Salmonella bacteria are able to suppress their own autophagic capture and elimination by macrophages. Building on that study, we show here that the inhibition of autophagy by Salmonella also prevents the induction of a protective cytokine response mediated by beta interferon (IFN-β) and IFN-γ. Together, these findings identify a novel virulence strategy whereby Salmonella bacteria prevent cell autonomous elimination via autophagy and suppress the activation of innate immune responses.

scholarly journals Genome Expression Analysis of Nonproliferating Intracellular Salmonella enterica Serovar Typhimurium Unravels an Acid pH-Dependent PhoP-PhoQ Response Essential for Dormancy

2012 ◽  
Vol 81 (1) ◽  
pp. 154-165 ◽  
Author(s):  
Cristina Núñez-Hernández ◽  
Alberto Tierrez ◽  
Álvaro D. Ortega ◽  
M. Graciela Pucciarelli ◽  
Marta Godoy ◽  
...  
Keyword(s):  
Metabolic Reprogramming ◽  
Host Cells ◽  
Infection Model ◽  
Intracellular Bacteria ◽  
Virulence Plasmid ◽  
Intracellular Growth ◽  
Content Type ◽  
Serovar Typhimurium

Genome-wide expression analyses have provided clues on howSalmonellaproliferates inside cultured macrophages and epithelial cells. However,in vivostudies show thatSalmonelladoes not replicate massively within host cells, leaving the underlying mechanisms of such growth control largely undefined.In vitroinfection models based on fibroblasts or dendritic cells reveal limited proliferation of the pathogen, but it is presently unknown whether these phenomena reflect events occurringin vivo. Fibroblasts are distinctive, since they represent a nonphagocytic cell type in whichS. entericaserovar Typhimurium actively attenuates intracellular growth. Here, we show in the mouse model thatS. Typhimurium restrains intracellular growth within nonphagocytic cells positioned in the intestinal lamina propria. This response requires a functional PhoP-PhoQ system and is reproduced in primary fibroblasts isolated from the mouse intestine. The fibroblast infection model was exploited to generate transcriptome data, which revealed that ∼2% (98 genes) of theS. Typhimurium genome is differentially expressed in nongrowing intracellular bacteria. Changes include metabolic reprogramming to microaerophilic conditions, induction of virulence plasmid genes, upregulation of the pathogenicity islands SPI-1 and SPI-2, and shutdown of flagella production and chemotaxis. Comparison of relative protein levels of several PhoP-PhoQ-regulated functions (PagN, PagP, and VirK) in nongrowing intracellular bacteria and extracellular bacteria exposed to diverse PhoP-PhoQ-inducing signals denoted a regulation responding to acidic pH. These data demonstrate thatS. Typhimurium restrains intracellular growthin vivoand support a model in which dormant intracellular bacteria could sense vacuolar acidification to stimulate the PhoP-PhoQ system for preventing intracellular overgrowth.

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