scholarly journals Murine AML12 hepatocytes allow Salmonella Typhimurium T3SS1-independent invasion and intracellular fate

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. Holbert ◽  
E. Barilleau ◽  
S. M. Roche ◽  
J. Trotereau ◽  
S. Georgeault ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Rho Gtpases ◽  
Wild Type Strain ◽  
Secretion System ◽  
Wild Type ◽  
Common Pathway ◽  
Hepatocyte Cell Line ◽  
Murine Hepatocyte ◽  
Intracellular Fate

AbstractNumerous studies have demonstrated the key role of the Salmonella Pathogenicity Island 1-encoded type III secretion system (T3SS1) apparatus as well as its associated effectors in the invasion and intracellular fate of Salmonella in the host cell. Several T3SS1 effectors work together to control cytoskeleton networks and induce massive membrane ruffles, allowing pathogen internalization. Salmonella resides in a vacuole whose maturation requires that the activity of T3SS1 subverts early stages of cell signaling. Recently, we identified five cell lines in which Salmonella Typhimurium enters without using its three known invasion factors: T3SS1, Rck and PagN. The present study investigated the intracellular fate of Salmonella Typhimurium in one of these models, the murine hepatocyte cell line AML12. We demonstrated that both wild-type Salmonella and T3SS1-invalidated Salmonella followed a common pathway leading to the formation of a Salmonella containing vacuole (SCV) without classical recruitment of Rho-GTPases. Maturation of the SCV continued through an acidified phase that led to Salmonella multiplication as well as the formation of a tubular network resembling Salmonella induced filaments (SIF). The fact that in the murine AML12 hepatocyte, the T3SS1 mutant induced an intracellular fate resembling to the wild-type strain highlights the fact that Salmonella Typhimurium invasion and intracellular survival can be completely independent of T3SS1.

scholarly journals Evidence for a Type III Secretion System in Aeromonas salmonicida subsp. salmonicida

2002 ◽  
Vol 184 (21) ◽  
pp. 5966-5970 ◽  
Author(s):  
Sarah E. Burr ◽  
Katja Stuber ◽  
Thomas Wahli ◽  
Joachim Frey
Keyword(s):  
Type Iii Secretion ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Aeromonas Salmonicida ◽  
Broad Host Range ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iii

ABSTRACT Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis, is an important fish pathogen. We have screened this bacterium with a broad-host-range probe directed against yscV, the gene that encodes the archetype of a highly conserved family of inner membrane proteins found in every known type III secretion system. This has led to the identification of seven open reading frames that encode homologues to proteins functioning within the type III secretion systems of Yersinia species. Six of these proteins are encoded by genes comprising a virA operon. The A. salmonicida subsp. salmonicida yscV homologue, ascV, was inactivated by marker replacement mutagenesis and used to generate an isogenic ascV mutant. Comparison of the extracellular protein profiles from the ascV mutant and the wild-type strain indicates that A. salmonicida subsp. salmonicida secretes proteins via a type III secretion system. The recently identified ADP-ribosylating toxin AexT was identified as one such protein. Finally, we have compared the toxicities of the wild-type A. salmonicida subsp. salmonicida strain and the ascV mutant against RTG-2 rainbow trout gonad cells. While infection with the wild-type strain results in significant morphological changes, including cell rounding, infection with the ascV mutant has no toxic effect, indicating that the type III secretion system we have identified plays an important role in the virulence of this pathogen.

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 enterica subspecies enterica serovar Enteritidis Salmonella pathogenicity island 2 type III secretion system: role in intestinal colonization of chickens and systemic spread

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2770-2781 ◽  
Author(s):  
Amanda L. S. Wisner ◽  
Taseen S. Desin ◽  
Birgit Koch ◽  
Po-King S. Lam ◽  
Emil M. Berberov ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Wild Type ◽  
Type Iii ◽  
Systemic Spread

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. For the purposes of our studies we used a chicken isolate of S. Enteritidis (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen-free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type strain Sal18: Sal18 attTn7 : : tet and Sal18 attTn7 : : cat, while the other two groups received the wild-type strain (Sal18 attTn7 : : tet) and one of two mutant strains. From this study, we concluded that S. Enteritidis strains deficient in the SPI-1 and SPI-2 systems were outcompeted by the wild-type strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains: the wild-type strain, and three other strains lacking either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge, we observed a reduced systemic spread of the SPI-2 mutants, but by day 3, the systemic distribution levels of the mutants matched that of the wild-type strain. Based on these two studies, we conclude that the S. Enteritidis SPI-2 T3SS facilitates invasion and systemic spread in chickens, although alternative mechanisms for these processes appear to exist.

scholarly journals The Edwardsiella piscicida Type III Translocon Protein EseC Inhibits Biofilm Formation by Sequestering EseE

2019 ◽  
Vol 85 (8) ◽  
Author(s):  
Ying Li Liu ◽  
Tian Tian He ◽  
Lu Yi Liu ◽  
Jia Yi ◽  
Pin Nie ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Wild Type ◽  
Content Type ◽  
Type Iii ◽  
Edwardsiella Piscicida

ABSTRACT The type III secretion system (T3SS) is one of the most important virulence factors of the fish pathogen Edwardsiella piscicida. It contains three translocon proteins, EseB, EseC, and EseD, required for translocation of effector proteins into host cells. We have previously shown that EseB forms filamentous appendages on the surface of E. piscicida, and these filamentous structures mediate bacterial cell-cell interactions promoting autoaggregation and biofilm formation. In the present study, we show that EseC, but not EseD, inhibits the autoaggregation and biofilm formation of E. piscicida. At 18 h postsubculture, a ΔeseC strain developed strong autoaggregation and mature biofilm formation, accompanied by enhanced formation of EseB filamentous appendages. This is in contrast to the weak autoaggregation and immature biofilm formation seen in the E. piscicida wild-type strain. EseE, a protein that directly binds to EseC and also positively regulates the transcription of the escC-eseE operon, was liberated and showed increased levels in the absence of EseC. This led to augmented transcription of the escC-eseE operon, thereby increasing the steady-state protein levels of intracellular EseB, EseD, and EseE, as well as biofilm formation. Notably, the levels of intracellular EseB and EseD produced by the ΔeseE and ΔeseC ΔeseE strains were similar but remarkably lower than those produced by the wild-type strain at 18 h postsubculture. Taken together, we have shown that the translocon protein EseC inhibits biofilm formation through sequestering EseE, a positive regulator of the escC-eseE operon. IMPORTANCE Edwardsiella piscicida, previously known as Edwardsiella tarda, is a Gram-negative intracellular pathogen that mainly infects fish. The type III secretion system (T3SS) plays a pivotal role in its pathogenesis. The T3SS translocon protein EseB is required for the assembly of filamentous appendages on the surface of E. piscicida. The interactions between the appendages facilitate autoaggregation and biofilm formation. In this study, we explored the role of the other two translocon proteins, EseC and EseD, in biofilm formation. We have demonstrated that EseC, but not EseD, inhibits the autoaggregation and biofilm formation of E. piscicida, providing new insights into the regulatory mechanism involved in E. piscicida biofilm formation.

scholarly journals Two-Component Sensor RhpS Promotes Induction of Pseudomonas syringae Type III Secretion System by Repressing Negative Regulator RhpR

2007 ◽  
Vol 20 (3) ◽  
pp. 223-234 ◽  
Author(s):  
Yanmei Xiao ◽  
Lefu Lan ◽  
Chuntao Yin ◽  
Xin Deng ◽  
Douglas Baker ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Deletion Strain ◽  
Wild Type ◽  
Two Component ◽  
In The Wild

The Pseudomonas syringae type III secretion system (T3SS) is induced during interaction with the plant or culture in minimal medium (MM). How the bacterium senses these environments to activate the T3SS is poorly understood. Here, we report the identification of a novel two-component system (TCS), RhpRS, that regulates the induction of P. syringae T3SS genes. The rhpR and rhpS genes are organized in an operon with rhpR encoding a putative TCS response regulator and rhpS encoding a putative biphasic sensor kinase. Transposon insertion in rhpS severely reduced the induction of P. syringae T3SS genes in the plant as well as in MM and significantly compromised the pathogenicity on host plants and hypersensitive response-inducing activity on nonhost plants. However, deletion of the rhpRS locus allowed the induction of T3SS genes to the same level as in the wild-type strain and the recovery of pathogenicity upon infiltration into plants. Overexpression of RhpR in the ΔrhpRS deletion strain abolished the induction of T3SS genes. However, overexpression of RhpR in the wild-type strain or overexpression of RhpR(D70A), a mutant of the predicted phosphorylation site of RhpR, in the ΔrhpRS deletion strain only slightly reduced the induction of T3SS genes. Based on these results, we propose that the phosphorylated RhpR represses the induction of T3SS genes and that RhpS reverses phosphorylation of RhpR under the T3SS-inducing conditions. Epistasis analysis indicated that rhpS and rhpR act upstream of hrpR to regulate T3SS genes.

scholarly journals Secretion and Function of Salmonella SPI-2 Effector SseF Require Its Chaperone, SscB

2004 ◽  
Vol 186 (15) ◽  
pp. 5078-5086 ◽  
Author(s):  
Shipan Dai ◽  
Daoguo Zhou
Keyword(s):  
Type Iii Secretion ◽  
Type Strain ◽  
Wild Type Strain ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Transcriptional Level ◽  
Wild Type ◽  
And Function

ABSTRACT Salmonella strains utilize a type III secretion system for their successful survival and replications inside host cells. SseF is one of the several effector proteins that are required for conferring this survival ability by altering the trafficking of the Salmonella-containing vacuoles. These effector proteins often require appropriate chaperones to maintain their stabilities inside the bacteria. These chaperones are also known to assist the subsequent secretion and translocation of their substrates. We report here that SscB acts as the chaperone for SseF, an effector for the Salmonella pathogenicity island 2 (SPI-2). We found that the sscB gene is required for the formation of Salmonella sp.-induced continuous filaments in epithelial cells. Efficient Salmonella replication in macrophages requires SscB function. Intracellular and secretion levels of SseF are greatly reduced in an sscB mutant strain compared to the wild-type strain. A protein stability assay demonstrated that the half-life of SseF is significantly shortened in the absence of SscB. Transcriptional analysis of the sseF gene showed that the effect of SscB on the SseF level is not at the transcriptional level. A coprecipitation experiment indicated that SscB interacts with SseF. In summary, our results indicate that SscB is a chaperone for SPI-2 effector SseF to facilitate its secretion and function inside the host cells.

scholarly journals Formate Acts as a Diffusible Signal To Induce Salmonella Invasion

2008 ◽  
Vol 190 (12) ◽  
pp. 4233-4241 ◽  
Author(s):  
Yanyan Huang ◽  
Mitsu Suyemoto ◽  
Cherilyn D. Garner ◽  
Kellie M. Cicconi ◽  
Craig Altier
Keyword(s):  
Type Iii Secretion ◽  
Culture Medium ◽  
Wild Type Strain ◽  
Sodium Formate ◽  
Secretion System ◽  
Intestinal Epithelial ◽  
Wild Type ◽  
Intestinal Epithelial Barrier ◽  
Cultured Epithelial Cells ◽  
Serovar Typhimurium

ABSTRACT To infect an animal host, Salmonella enterica serovar Typhimurium must penetrate the intestinal epithelial barrier. This process of invasion requires a type III secretion system encoded within Salmonella pathogenicity island I (SPI1). We found that a mutant with deletions of the acetate kinase and phosphotransacetylase genes (ackA-pta) was deficient in invasion and SPI1 expression but that invasion gene expression was completely restored by supplying medium conditioned by growth of the wild-type strain, suggesting that a signal produced by the wild type, but not by the ackA-pta mutant, was required for invasion. This mutant also excreted 68-fold-less formate into the culture medium, and the addition of sodium formate to cultures restored both the expression of SPI1 and the invasion of cultured epithelial cells by the mutant. The effect of formate was pH dependent, requiring a pH below neutrality, and studies in mice showed that the distal ileum, the preferred site of Salmonella invasion in this species, had the appropriate formate concentration and pH to elicit invasion, while the cecum contained no detectable formate. Furthermore, we found that formate affected the major regulators of SPI1, hilA and hilD, but that the primary routes of formate metabolism played no role in its activity as a signal.

scholarly journals Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

2021 ◽  
Author(s):  
Chang-Hun Shin ◽  
Hang Soo Cho ◽  
Hyung-Jin Won ◽  
Ho Jeong Kwon ◽  
Chan-Wha Kim ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Mutant Selection ◽  
Enhanced Production ◽  
Industrial Strains ◽  
Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

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