Biology subjects

Kuwae, A.

Publications and source records attributed to Kuwae, A..

4 recordsLinked to original sources

bioRxiv2025

Bordetella BcrH1 and BcrH2 are specific chaperones for the pore-forming complex.

Bordetella has a type III secretion system that secretes virulence proteins crucial to the establishment of infection. The genes encoding components of the Bordetella type III secretion system are located in the bsc region on the chromosome. This region includes the bcrH1 and bcrH2 genes, which respectively encode the proteins BcrH1 and BcrH2. In this study, we analyzed the roles of BcrH1 and BcrH2 in Bordetella infection. First, we created a BcrH1/BcrH2-double deficient strains, and analyzed the amounts of the type III secreted proteins BopB and BopD, which make a complex that forms pores in the host membrane, in bacterial cells of each protein-deficient strain. The results showed that the BopB and BopD signals were weakened in the whole cell fraction of the BcrH1/BcrH2-double deficient strains, respectively. The hemolytic activity and cell toxicity of each BcrH protein-deficient strain were significantly lower than those of the wild-type strain. When anti-BcrH1 and anti-BcrH2 antibodies were used for the immunoprecipitation assay, the BopB and BopD signals were detected in the precipitated fractions, respectively. These results strongly suggest that BcrH1 and BcrH2 are specific chaperones for maintaining the stability of BopB and BopD, respectively.

microbiology↗

bioRxiv2021

Bcr4 is a Chaperone for the Inner Rod Protein in the Bordetella Type III Secretion System

Bordetella bronchiseptica injects virulence proteins called effectors into host cells via a type III secretion system (T3SS) conserved among many Gram-negative bacteria. Small proteins called chaperones are required for stabilizing some T3SS components or localizing them to the T3SS machinery. In a previous study, we identified a chaperone-like protein named Bcr4 that regulates T3SS activity in B. bronchiseptica. Bcr4 does not show strong sequence similarity to well-studied T3SS proteins of other bacteria, and its function remains to be elucidated. Here, we investigated the mechanism by which Bcr4 controls T3SS activity. A pull-down assay revealed that Bcr4 interacts with BscI, based on its homology to other bacterial proteins, to be an inner rod protein of the T3SS machinery. An additional pull-down assay using truncated Bcr4 derivatives and secretion profiles of B. bronchiseptica producing truncated Bcr4 derivatives showed that the Bcr4 C-terminal region is necessary for the interaction with BscI and activation of the T3SS. Moreover, the deletion of BscI abolished the secretion of type III secreted proteins from B. bronchiseptica and the translocation of a cytotoxic effector into cultured mammalian cells. Finally, we showed that BscI is unstable in the absence of Bcr4. These results suggest that Bcr4 supports the construction of the T3SS machinery by stabilizing BscI. This is the first demonstration of a chaperone for the T3SS inner rod protein among the virulence bacteria possessing the T3SS. ImportanceThe type III secretion system (T3SS) is a needle-like complex that projects outward from bacterial cells. Bordetella bronchiseptica uses the T3SS to inject virulence proteins into host cells. Our previous study reported that a protein named Bcr4 is essential for the secretion of virulence proteins from B. bronchiseptica bacterial cells and delivery through the T3SS. Because other bacteria lack a Bcr4 homologue, the function of Bcr4 has not been elucidated. In this study, we discovered that Bcr4 interacts with BscI, a component of the T3SS machinery. We showed that a B. bronchiseptica BscI-deficient strain was unable to secrete type III secreted proteins. Furthermore, in a B. bronchiseptica strain that overproduces T3SS component proteins, Bcr4 is required to maintain BscI in bacterial cells. These results suggest that Bcr4 stabilizes BscI to allow construction of the T3SS in B. bronchiseptica.

microbiology↗

bioRxiv2020

Downregulation of a Type III Secretion System under Reducing Conditions in Bordetella pertussis

Bordetella pertussis uses a type III secretion system (T3SS) to inject virulence proteins into host cells. Although the B. pertussis T3SS was presumed to be involved in host colonization, the efficient secretion of type III secreted proteins from B. pertussis has not been observed. To investigate the roles of type III secreted proteins during infection, we attempted to optimize culture conditions for the production and secretion of a type III secreted protein, BteA, in B. pertussis. We observed that B. pertussis efficiently secretes BteA in ascorbic acid-depleted (AsA-) medium. When L2 cells, a rat lung epithelial cell line, were infected with B. pertussis cultured in the AsA- medium, BteA-dependent cytotoxicity was observed. We also performed an immunofluorescence assay of L2 cells infected with B. pertussis. The clear fluorescence signals of Bsp22, a needle structure of T3SS, were detected on the bacterial surface of B. pertussis cultured in the AsA- medium. Since ascorbic acid is known as a reducing agent, we cultured B. pertussis in liquid medium containing other reducing agents such as 2-mercaptoethanol and dithioerythritol. Under these reducing conditions, the production of type III secreted proteins was repressed. These results suggest that in B. pertussis, the production and secretion of type III secreted proteins are downregulated under reducing conditions. IMPORTANCEThe type III secretion system (T3SS) of Bordetella pertussis forms a needle-like structure that protrudes from the bacterial cell surface. B. pertussis uses T3SS to translocate virulence proteins called effectors into host cells. The culture conditions for effector production in B. pertussis have not been investigated. We attempted to optimize culture medium compositions for producing and secreting type III secreted proteins. We found that B. pertussis secretes type III secreted proteins in reducing agent-deprived liquid medium, and that BteA-secreting B. pertussis provokes cytotoxicity against cultured mammalian cells. These results suggest that redox signaling is involved in the regulation of B. pertussis T3SS.

microbiology↗

bioRxiv2018

Tandem tyrosine residues in the EPEC multicargo chaperone CesT support differential type III effector translocation and early host colonization

Enteropathogenic Escherichia coli (EPEC) are worldwide human enteric pathogens inflicting significant morbidity and causing large economic losses. A type 3 secretion system (T3SS) is critical for EPEC intestinal colonization, and injection of effectors into host cells contributes to cellular subversion and innate immune evasion. Here, we demonstrate that two strictly conserved C-terminal tyrosine residues, Y152 and Y153, within the multicargo T3SS chaperone CesT serve differential roles in regulating effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine attenuated EPEC type 3 effector injection into host cells, and significantly limited Tir effector mediated intimate adherence, a key feature of attaching and effacing pathogenesis. Whereas CesT Y153 supported normal levels of Tir translocation, CesT Y152 was strictly required for the effector NleA to be expressed and subsequently translocated into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y152, Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating Y152 in CesT functionality. A mouse infection model of EPEC intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for strictly conserved tandem tyrosine residues within CesT. Tyrosine 152 of CesT is implicated in NleA expression, providing functional relevance for localized amino acid conservation. Therefore, CesT via its novel C-terminal domain, has relevant roles beyond typical T3SC that interact and stabilize effector proteins.

microbiology↗