scholarly journals Small Molecule Inhibitors of LcrF, a Yersinia pseudotuberculosis Transcription Factor, Attenuate Virulence and Limit Infection in a Murine Pneumonia Model

2010 ◽  
Vol 78 (11) ◽  
pp. 4683-4690 ◽  
Author(s):  
Lynne K. Garrity-Ryan ◽  
Oak K. Kim ◽  
Joan-Miquel Balada-Llasat ◽  
Victoria J. Bartlett ◽  
Atul K. Verma ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Secretion System ◽  
Bacterial Burden ◽  
Small Molecule Library ◽  
Null Mutants

ABSTRACT LcrF (VirF), a transcription factor in the multiple adaptational response (MAR) family, regulates expression of the Yersinia type III secretion system (T3SS). Yersinia pseudotuberculosis lcrF-null mutants showed attenuated virulence in tissue culture and animal models of infection. Targeting of LcrF offers a novel, antivirulence strategy for preventing Yersinia infection. A small molecule library was screened for inhibition of LcrF-DNA binding in an in vitro assay. All of the compounds lacked intrinsic antibacterial activity and did not demonstrate toxicity against mammalian cells. A subset of these compounds inhibited T3SS-dependent cytotoxicity of Y. pseudotuberculosis toward macrophages in vitro. In a murine model of Y. pseudotuberculosis pneumonia, two compounds significantly reduced the bacterial burden in the lungs and afforded a dramatic survival advantage. The MAR family of transcription factors is well conserved, with members playing central roles in pathogenesis across bacterial genera; thus, the inhibitors could have broad applicability.

scholarly journals The transcription factor IscR promotes Yersinia type III secretion system activity by antagonizing the repressive H-NS-YmoA histone-like protein complex

2021 ◽  
Author(s):  
David Balderas ◽  
Pablo Alvarez ◽  
Mané Ohanyan ◽  
Erin Mettert ◽  
Natasha Tanner ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Complex ◽  
Type Iii Secretion ◽  
Defense Mechanisms ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
Transcription In Vitro

The type III secretion system (T3SS) is a appendage used by many bacterial pathogens, such as pathogenic Yersinia, to subvert host defenses. However, because the T3SS is energetically costly and immunogenic, it must be tightly regulated in response to environmental cues to enable survival in the host. Here we show that expression of the Yersinia Ysc T3SS master regulator, LcrF, is orchestrated by the opposing activities of the repressive YmoA/H-NS histone-like protein complex and induction by the iron and oxygen-regulated IscR transcription factor. Although IscR has been shown to bind the lcrF promoter and is required for in vivo expression of lcrF, in this study we show IscR alone fails to enhance lcrF transcription in vitro. Rather, we find that in a ymoA mutant, IscR is no longer required for LcrF expression or T3SS activity. Additionally, a mutation in YmoA that prevents H-NS binding (ymoAD43N) rescues the T3SS defect of a ∆iscR mutant, suggesting that a YmoA/H-NS complex is needed for this repressive activity. Furthermore, chromatin immunoprecipitation analysis revealed that H-NS is enriched at the lcrF promoter at environmental temperatures, while IscR is enriched at this promoter at mammalian body temperature under aerobic conditions. Importantly, CRISPRi knockdown of H-NS leads to increased lcrF transcription. Collectively, our data suggest that as IscR levels rise with iron limitation and oxidative stress, conditions Yersinia experiences during extraintestinal infection, IscR antagonizes YmoA/H-NS-mediated repression of lcrF transcription to drive T3SS activity and manipulate host defense mechanisms.

scholarly journals An NF-κB-Based High-Throughput Screen Identifies Piericidins as Inhibitors of the Yersinia pseudotuberculosis Type III Secretion System

2013 ◽  
Vol 58 (2) ◽  
pp. 1118-1126 ◽  
Author(s):  
Miles C. Duncan ◽  
Weng Ruh Wong ◽  
Allison J. Dupzyk ◽  
Walter M. Bray ◽  
Roger G. Linington ◽  
...  
Keyword(s):  
Natural Products ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Block Type ◽  
Content Type ◽  
Type Iii

ABSTRACTThe type III secretion system (T3SS) is a bacterial appendage used by dozens of Gram-negative pathogens to subvert host defenses and cause disease, making it an ideal target for pathogen-specific antimicrobials. Here, we report the discovery and initial characterization of two related natural products with T3SS-inhibitory activity that were derived from a marine actinobacterium. Bacterial extracts containing piericidin A1 and the piericidin derivative Mer-A 2026B inhibitedYersinia pseudotuberculosisfrom triggering T3SS-dependent activation of the host transcription factor NF-κB in HEK293T cells but were not toxic to mammalian cells. As theYersiniaT3SS must be functional in order to trigger NF-κB activation, these data indicate that piericidin A1 and Mer-A 2026B block T3SS function. Consistent with this, purified piericidin A1 and Mer-A 2026B dose-dependently inhibited translocation of theY. pseudotuberculosisT3SS effector protein YopM inside CHO cells. In contrast, neither compound perturbed bacterial growthin vitro, indicating that piericidin A1 and Mer-A 2026B do not function as general antibiotics inYersinia. In addition, whenYersiniawas incubated under T3SS-inducing culture conditions in the absence of host cells, Mer-A 2026B and piericidin A1 inhibited secretion of T3SS cargo as effectively as or better than several previously described T3SS inhibitors, such as MBX-1641 and aurodox. This suggests that Mer-A 2026B and piericidin A1 do not block type III secretion by blocking the bacterium-host cell interaction, but rather inhibit an earlier stage, such as T3SS needle assembly. In summary, the marine-derived natural products Mer-A 2026B and piericidin A1 possess previously uncharacterized activity against the bacterial T3SS.

scholarly journals Design of small molecule inhibitors of type III secretion system ATPase EscN from enteropathogenic Escherichia coli.

1970 ◽  
Vol 64 (1) ◽  
Author(s):  
Lukasz Bzdzion ◽  
Hanna Krezel ◽  
Karol Wrzeszcz ◽  
Irmina Grzegorek ◽  
Katarzyna Nowinska ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Small Molecule ◽  
Active Site ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Small Molecule Inhibitors ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Type Iii

Enteropathogenic E. coli (EPEC) is a human pathogen using type III secretion system for delivery of proteins directly into the human host. The system contains a single ATPase, EscN, which is essential for uncoupling of proteins from their complexes with chaperones before the delivery. The structure of EscN ATPase (PDB code: 2obm) was used to screen computationally for small molecule inhibitors blocking its active site. Two lead candidates were examined but only one, Compound 54, was selected for further optimization. After extended QSAR optimization, two derivatives were found to be competitive inhibitors of EscN capable of blocking ATPase activity with a Ki below 50 µM. One candidate, WEN05-03, with a Ki=16±2 µM, was also minimally toxic to mammalian cells as determined by other assays. In the cell infection model of HeLa cells with EPEC, Compound WEN05-03 completely blocked actin cluster formation at 100 µM concentration, when analyzed by confocal microscopy. The second best inhibitor of EscN ATPase activity was WEN04-34 with a Ki=46±2 µM. However, the compound was highly toxic to the BALB/3T3 cell line. In summary, the work identifies a compound blocking bacterial ATPase in its active site without causing cellular toxicity to the host cells. It is the first report showing feasibility of using bacterial virulence system ATPase as a target for safe, non-toxic compounds and offering a proof-of-concept for non-antibiotic alternatives.

Inhibition of NGLY1 inactivates the transcription factor Nrf1 and potentiates proteasome inhibitor cytotoxicity

2017 ◽  
Author(s):  
Carolyn Bertozzi ◽  
Fred Tomlin ◽  
Ulla Gerling-Driessen ◽  
Yi-Chang Liu ◽  
Ryan Flynn ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
Proteasome Inhibitor ◽  
Activation Mechanism ◽  
Cancer Drugs ◽  
Small Molecule Library ◽  
Library Screen ◽  
In Cells

We discovered that the proteostasis modulating transcription factor Nrf1 requires cytosolic de-N-glycosylation by the N-glycanase NGly1 as part of its activation mechanism. Through a covalent small molecule library screen, we discovered an inhibitor of NGly1 that blocks Nrf1 activation in cells and potentiates the activity of proteasome inhibitor cancer drugs. The requirement of NGly1 for Nrf1 activity likely underlies several pathologies associated with a rare hereditary deficiency in NGly1.

scholarly journals Diminished LcrV Secretion Attenuates Yersinia pseudotuberculosis Virulence

2007 ◽  
Vol 189 (23) ◽  
pp. 8417-8429 ◽  
Author(s):  
Jeanette E. Bröms ◽  
Matthew S. Francis ◽  
Åke Forsberg
Keyword(s):  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Signal Sequence ◽  
Infection Model ◽  
Target Cells ◽  
Cell Infection ◽  
Host Cell Membrane ◽  
Type Iii ◽  
A Cell

ABSTRACT Many gram-negative bacterial pathogenicity factors that function beyond the outer membrane are secreted via a contact-dependent type III secretion system. Two types of substrates are predestined for this mode of secretion, namely, antihost effectors that are translocated directly into target cells and the translocators required for targeting of the effectors across the host cell membrane. N-terminal secretion signals are important for recognition of the protein cargo by the type III secretion machinery. Even though such signals are known for several effectors, a consensus signal sequence is not obvious. One of the translocators, LcrV, has been attributed other functions in addition to its role in translocation. These functions include regulation, presumably via interaction with LcrG inside bacteria, and immunomodulation via interaction with Toll-like receptor 2. Here we wanted to address the significance of the specific targeting of LcrV to the exterior for its function in regulation, effector targeting, and virulence. The results, highlighting key N-terminal amino acids important for LcrV secretion, allowed us to dissect the role of LcrV in regulation from that in effector targeting/virulence. While only low levels of exported LcrV were required for in vitro effector translocation, as deduced by a cell infection assay, fully functional export of LcrV was found to be a prerequisite for its role in virulence in the systemic murine infection model.

scholarly journals A Salmonella Virulence Factor Activates the NOD1/NOD2 Signaling Pathway

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
A. Marijke Keestra ◽  
Maria G. Winter ◽  
Daisy Klein-Douwel ◽  
Mariana N. Xavier ◽  
Sebastian E. Winter ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Intestinal Inflammation ◽  
Inflammatory Responses ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Content Type ◽  
Type Iii

ABSTRACTThe invasion-associated type III secretion system (T3SS-1) ofSalmonella entericaserotype Typhimurium (S. Typhimurium) activates the transcription factor NF-κB in tissue culture cells and induces inflammatory responses in animal models through unknown mechanisms. Here we show that bacterial delivery or ectopic expression of SipA, a T3SS-1-translocated protein, led to the activation of the NOD1/NOD2 signaling pathway and consequent RIP2-mediated induction of NF-κB-dependent inflammatory responses. SipA-mediated activation of NOD1/NOD2 signaling was independent of bacterial invasionin vitrobut required an intact T3SS-1. In the mouse colitis model, SipA triggered mucosal inflammation in wild-type mice but not in NOD1/NOD2-deficient mice. These findings implicate SipA-driven activation of the NOD1/NOD2 signaling pathway as a mechanism by which the T3SS-1 induces inflammatory responsesin vitroandin vivo.IMPORTANCESalmonella entericaserotype Typhimurium (S. Typhimurium) deploys a type III secretion system (T3SS-1) to induce intestinal inflammation and benefits from the ensuing host response, which enhances growth of the pathogen in the intestinal lumen. However, the mechanisms by which the T3SS-1 triggers inflammatory responses have not been resolved. Here we show that the T3SS-1 effector protein SipA induces NF-κB activation and intestinal inflammation by activating the NOD1/NOD2 signaling pathway. These data suggest that the T3SS-1 escalates innate responses through a SipA-mediated activation of pattern recognition receptors in the host cell cytosol.

scholarly journals Autoproteolysis of YscU of Yersinia pseudotuberculosis Is Important for Regulation of Expression and Secretion of Yop Proteins

2009 ◽  
Vol 191 (13) ◽  
pp. 4259-4267 ◽  
Author(s):  
Ann-Catrin Björnfot ◽  
Moa Lavander ◽  
Åke Forsberg ◽  
Hans Wolf-Watz
Keyword(s):  
Type Iii Secretion ◽  
Calcium Concentration ◽  
Wild Type Strain ◽  
Yersinia Pseudotuberculosis ◽  
Secretion System ◽  
Wild Type ◽  
Regulation Of Expression ◽  
Significant Difference ◽  
In The Wild ◽  
Needle Protein

ABSTRACT YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscUCC. Autoproteolysis occurs at the conserved N↓PTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca2+ conditions) and calcium-depleted medium (−Ca2+ conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca2+-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca2+ conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under −Ca2+ conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.

scholarly journals Salmonella-Induced Caspase-2 Activation in Macrophages

2000 ◽  
Vol 192 (7) ◽  
pp. 1035-1046 ◽  
Author(s):  
Veronika Jesenberger ◽  
Katarzyna J. Procyk ◽  
Junying Yuan ◽  
Siegfried Reipert ◽  
Manuela Baccarini
Keyword(s):  
Type Iii Secretion ◽  
Caspase 3 ◽  
Secretion System ◽  
Caspase 1 ◽  
Chemical Inhibition ◽  
Induction Of Apoptosis ◽  
Caspase 2 ◽  
Induced Apoptosis

The enterobacterial pathogen Salmonella induces phagocyte apoptosis in vitro and in vivo. These bacteria use a specialized type III secretion system to export a virulence factor, SipB, which directly activates the host's apoptotic machinery by targeting caspase-1. Caspase-1 is not involved in most apoptotic processes but plays a major role in cytokine maturation. We show that caspase-1–deficient macrophages undergo apoptosis within 4–6 h of infection with invasive bacteria. This process requires SipB, implying that this protein can initiate the apoptotic machinery by regulating components distinct from caspase-1. Invasive Salmonella typhimurium targets caspase-2 simultaneously with, but independently of, caspase-1. Besides caspase-2, the caspase-1–independent pathway involves the activation of caspase-3, -6, and -8 and the release of cytochrome c from mitochondria, none of which occurs during caspase-1–dependent apoptosis. By using caspase-2 knockout macrophages and chemical inhibition, we establish a role for caspase-2 in both caspase-1–dependent and –independent apoptosis. Particularly, activation of caspase-1 during fast Salmonella-induced apoptosis partially relies on caspase-2. The ability of Salmonella to induce caspase-1–independent macrophage apoptosis may play a role in situations in which activation of this protease is either prevented or uncoupled from the induction of apoptosis.

scholarly journals Expression of ExsA in trans Confers Type III Secretion System-Dependent Cytotoxicity on Noncytotoxic Pseudomonas aeruginosa Cystic Fibrosis Isolates

2001 ◽  
Vol 69 (1) ◽  
pp. 538-542 ◽  
Author(s):  
Denis Dacheux ◽  
Ina Attree ◽  
Bertrand Toussaint
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Transcriptional Activation ◽  
Type Iii Secretion ◽  
Ex Vivo ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
In Trans

ABSTRACT Twelve Pseudomonas aeruginosa cystic fibrosis isolates that are not able to exert a type III secretion system (TTSS)-dependent cytotoxicity towards phagocytes have been further studied. The strains, although possessing TTSS genes and exsA, which encodes a positive regulator of the TTSS regulon, showed no transcriptional activation of the exsCBA regulatory operon. The expression of exsA in trans restored the in vitro secretion of TTSS proteins and ex vivo cytotoxicity.

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