scholarly journals Autophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate-specific phospholipase A1 activity

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Shivani Agarwal ◽  
Hyunjin Kim ◽  
Robin B. Chan ◽  
Shivangi Agarwal ◽  
Rebecca Williamson ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Host Immune System ◽  
Endosomal Trafficking ◽  
Phospholipase A1 ◽  
Effector Domains ◽  
Pla1 Activity ◽  
Phosphatidylinositol 3

Abstract Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small intestine. Unlike other effector domains of the multifunctional toxin that target cytoskeleton, the function of alpha-beta hydrolase (ABH) remained elusive. This study demonstrates that ABH is an esterase/lipase with catalytic Ser–His–Asp triad. ABH binds with high affinity to phosphatidylinositol-3-phosphate (PtdIns3P) and cleaves the fatty acid in PtdIns3P at the sn1 position in vitro making it the first PtdIns3P-specific phospholipase A1 (PLA1). Expression of ABH in vivo reduces intracellular PtdIns3P levels and its PtdIns3P-specific PLA1 activity blocks endosomal and autophagic pathways. In accordance with recent studies acknowledging the potential of extracellular pathogens to evade or exploit autophagy to prevent their clearance and facilitate survival, this is the first report highlighting the role of ABH in inhibiting autophagy and endosomal trafficking induced by extracellular V. cholerae.

scholarly journals Role of DnaK in In Vitro and In Vivo Expression of Virulence Factors of Vibrio cholerae

1999 ◽  
Vol 67 (3) ◽  
pp. 1025-1033 ◽  
Author(s):  
Sabyasachi Chakrabarti ◽  
Nilanjan Sengupta ◽  
Rukhsana Chowdhury
Keyword(s):  
Heat Shock ◽  
Vibrio Cholerae ◽  
Virulence Factors ◽  
Heat Shock Gene ◽  
Insertion Mutant ◽  
In Vivo Expression ◽  
Shock Gene

ABSTRACT The dnaK gene of Vibrio cholerae was cloned, sequenced, and used to construct a dnaK insertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathogen. The central regulator of several virulence genes of V. choleraeis ToxR, a transmembrane DNA binding protein. The V. cholerae dnaK mutant grown in standard laboratory medium exhibited phenotypes characteristic of cells deficient in ToxR activity. Using Northern blot analysis and toxR transcriptional fusions, we demonstrated a reduction in expression of the toxR gene in the dnaK mutant strain together with a concomitant increase in expression of a htpG-like heat shock gene that is located immediately upstream and is divergently transcribed fromtoxR. This may be due to increased heat shock induction in the dnaK mutant. In vivo, however, although expression from heat shock promoters in the dnaK mutant was similar to that observed in vitro, expression of both toxR andhtpG was comparable to that by the parental strain. In both strains, in vivo expression of toxR was significantly higher than that observed in vitro, but no reciprocal decrease inhtpG expression was observed. These results suggest that the modulation of toxR expression in vivo may be different from that observed in vitro.

scholarly journals Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 922
Author(s):  
Gaurav Sinsinbar ◽  
Sushanth Gudlur ◽  
Kevin J Metcalf ◽  
Milan Mrksich ◽  
Madhavan Nallani ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Protective Role ◽  
Binding Motif ◽  
Host Immune System ◽  
E Coli ◽  
Sds Page ◽  
Lps Binding

Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT’s activity than previously proposed.

Distinct courses of infection withLeishmania(L.)amazonensisare observed in BALB/c, BALB/c nude and C57BL/6 mice

Parasitology ◽  
2016 ◽  
Vol 143 (6) ◽  
pp. 692-703 ◽  
Author(s):  
LEONARDO G. VELASQUEZ ◽  
MARIANA K. GALUPPO ◽  
ELOIZA DE REZENDE ◽  
WESLEY N. BRANDÃO ◽  
JEAN PIERRE PERON ◽  
...  
Keyword(s):  
Parasite Burden ◽  
Inflammatory Cells ◽  
The Body ◽  
Mouse Strains ◽  
Host Immune System ◽  
Cutaneous Form ◽  
Inflammatory Infiltrates

SUMMARYLeishmania(L.)amazonensis[L.(L.)amazonensis] is widely distributed in Brazil and its symptomatic infections usually lead to few localized lesions and sometimes to diffuse cutaneous form, with nodules throughout the body, anergy to parasite antigens and poor therapeutic response. The variability of these manifestations draws attention to the need for studies on the pathophysiology of infection by this species. In this study, we analysed the course and immunological aspects ofL.(L.)amazonensisinfection in BALB/c and C57BL/6 strains, both susceptible, but displaying different clinical courses, and athymic BALB/c nude, to illustrate the role of T cell dependent responses. We analysed footpad thickness and parasite burden byin vivoimaging. Furthermore, we evaluated the cellular profile and cytokine production in lymph nodes and the inflammatory infiltrates of lesions. Nude mice showed delayed lesion development and less inflammatory cells in lesions, but higher parasite burden than BALB/c and C57BL/6. BALB/c and C57BL/6 mice had similar parasite burdens, lesion sizes and infiltrates until 6 weeks after infection, and after that C57BL/6 mice controlled the infection. Small differences in parasite numbers were observed in C57BL/6 macrophagesin vitro, indicating thatin vivomilieu accounts for most differences in infection. We believe our results shed light on the role of host immune system in the course ofL.(L.)amazonensisinfection by comparing three mouse strains that differ in parasitaemia and inflammatory cells.

scholarly journals Erratum: Autophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate-specific phospholipase A1 activity

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Shivani Agarwal ◽  
Hyunjin Kim ◽  
Robin B. Chan ◽  
Shivangi Agarwal ◽  
Rebecca Williamson ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Endosomal Trafficking ◽  
Phospholipase A1 ◽  
Phosphatidylinositol 3

scholarly journals Role of Cyclic Di-GMP during El Tor Biotype Vibrio cholerae Infection: Characterization of the In Vivo-Induced Cyclic Di-GMP Phosphodiesterase CdpA

2008 ◽  
Vol 76 (4) ◽  
pp. 1617-1627 ◽  
Author(s):  
Rita Tamayo ◽  
Stefan Schild ◽  
Jason T. Pratt ◽  
Andrew Camilli
Keyword(s):  
Small Intestine ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Virulence Gene ◽  
Virulence Gene Expression ◽  
Infant Mouse

ABSTRACT In Vibrio cholerae, the second messenger cyclic di-GMP (c-di-GMP) positively regulates biofilm formation and negatively regulates virulence and is proposed to play an important role in the transition from persistence in the environment to survival in the host. Herein we describe a characterization of the infection-induced gene cdpA, which encodes both GGDEF and EAL domains, which are known to mediate diguanylate cyclase and c-di-GMP phosphodiesterase (PDE) activities, respectively. CdpA is shown to possess PDE activity, and this activity is regulated by its inactive degenerate GGDEF domain. CdpA inhibits biofilm formation but has no effect on colonization of the infant mouse small intestine. Consistent with these observations, cdpA is expressed during in vitro growth in a biofilm but is not expressed in vivo until the late stage of infection, after colonization has occurred. To test for a role of c-di-GMP in the early stages of infection, we artificially increased c-di-GMP and observed reduced colonization. This was attributed to a significant reduction in toxT transcription during infection. Cumulatively, these results support a model of the V. cholerae life cycle in which c-di-GMP must be down-regulated early after entering the small intestine and maintained at a low level to allow virulence gene expression, colonization, and motility at appropriate stages of infection.

scholarly journals A Recombinant Live Attenuated Strain of Vibrio cholerae Induces Immunity against Tetanus Toxin andBordetella pertussis Tracheal Colonization Factor

1998 ◽  
Vol 66 (4) ◽  
pp. 1648-1653 ◽  
Author(s):  
Inês Chen ◽  
Theresa M. Finn ◽  
Liu Yanqing ◽  
Qi Guoming ◽  
Rino Rappuoli ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Tetanus Toxin ◽  
Bacterial Colonization ◽  
Lethal Dose ◽  
Lethal Challenge ◽  
Colonization Factor ◽  
Heterologous Antigens

ABSTRACT An attenuated strain of Vibrio cholerae was used as a carrier for the expression of heterologous antigens such as fragment C from tetanus toxin (TetC) and tracheal colonization factor fromBordetella pertussis (Tcf). In vitro, high levels of protein were obtained when the Escherichia coli nirBpromoter was used and the bacteria were grown with low aeration. Intranasal immunization of mice with IEM101 expressing TetC elicited serum vibriocidal activity and induced antibodies against tetanus toxin which were protective against lethal challenge with 10 times the 50% lethal dose of tetanus toxin. Bacterial viability was essential for the induction of anti-TetC antibodies. Intranasal administration of IEM101 expressing Tcf induced a significant reduction in bacterial colonization of the tracheas of mice challenged with wild-type B. pertussis. These data are in agreement with the putative role of Tcf in Bordetellatracheal colonization. In conclusion, we have demonstrated thatV. cholerae may be used as a live vector to deliver heterologous antigens in vivo and that protection to both systemic and local challenge may be achieved.

scholarly journals Multi-Functional MPT Protein as a Therapeutic Agent against Mycobacterium tuberculosis

Biomedicines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 545
Author(s):  
Jae-Sung Kim ◽  
Euni Cho ◽  
Seok-Jun Mun ◽  
Sojin Kim ◽  
Sun-Young Kim ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Virulence Factors ◽  
Therapeutic Agent ◽  
Effective Vaccine ◽  
Host Immune System ◽  
Host Proteins ◽  
Signaling Systems

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), avoids the host immune system through its virulence factors. MPT63 and MPT64 are the virulence factors secreted by MTB which regulate host proteins for the survival and proliferation of MTB in the host. Here, we found that MPT63 bound directly with TBK1 and p47phox, whereas MPT64 interacted with TBK1 and HK2. We constructed a MPT63/64-derived multifunctional recombinant protein (rMPT) that was able to interact with TBK1, p47phox, or HK2. rMPT was shown to regulate IFN-β levels and increase inflammation and concentration of reactive oxygen species (ROS), while targeting macrophages and killing MTB, both in vitro and in vivo. Furthermore, the identification of the role of rMPT against MTB was achieved via vaccination in a mouse model. Taken together, we here present rMPT, which, by regulating important immune signaling systems, can be considered an effective vaccine or therapeutic agent against MTB.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Anti-obesity role of Aster glehni extract: in vivo and in vitro effects

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
HM Lee ◽  
TG Ahn ◽  
CW Kim ◽  
HJ An
Keyword(s):  
In Vitro Effects

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

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