Single, double and triple mutants of Salmonella enterica serovar Typhimurium degP (htrA), degQ (hhoA) and degS (hhoB) have diverse phenotypes on exposure to elevated temperature and their growth in vivo is attenuated to different extents

Mo, Elaine; Peters, Sarah; Willers, Chrissie; Maskell, Duncan J.; Charles, Ian G.

doi:10.1016/j.micpath.2006.07.004

Cited by 48 publications

(42 citation statements)

References 57 publications

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“…HtrA is the primary periplasmic protease that has previously been implicated in Salmonella pathogenesis (29). We tested possible SodCII degradation by HtrA during macrophage infection by repeating the above experiment in an htrA null background.…”

Section: Resultsmentioning

confidence: 99%

Protecting against Antimicrobial Effectors in the Phagosome Allows SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium

Kim¹,

Richards

Slauch

2010

J Bacteriol

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Salmonella enterica serovar Typhimurium replicates in macrophages, where it is subjected to antimicrobial substances, including superoxide, antimicrobial peptides, and proteases. The bacterium produces two periplasmic superoxide dismutases, SodCI and SodCII. Although both are expressed during infection, only SodCI contributes to virulence in the mouse by combating phagocytic superoxide. The differential contribution to virulence is at least partially due to inherent differences in the SodCI and SodCII proteins that are independent of enzymatic activity. SodCII is protease sensitive, and like other periplasmic proteins, it is released by osmotic shock. In contrast, SodCI is protease resistant and is retained within the periplasm after osmotic shock, a phenomenon that we term "tethering." We hypothesize that in the macrophage, antimicrobial peptides transiently disrupt the outer membrane. SodCII is released and/or phagocytic proteases gain access to the periplasm, and SodCII is degraded. SodCI is tethered within the periplasm and is protease resistant, thereby remaining to combat superoxide. Here we test aspects of this model. SodCII was released by the antimicrobial peptide polymyxin B or a mouse macrophage antimicrobial peptide (CRAMP), while SodCI remained tethered within the periplasm. A Salmonella pmrA constitutive mutant no longer released SodCII in vitro. Moreover, in the constitutive pmrA background, SodCII could contribute to survival of Salmonella during infection. SodCII also provided a virulence benefit in mice genetically defective in production of CRAMP. Thus, consistent with our model, protecting the outer membrane against antimicrobial peptides allows SodCII to contribute to virulence in vivo. These data also suggest direct in vivo cooperative interactions between macrophage antimicrobial effectors.

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Section: Resultsmentioning

confidence: 99%

Protecting against Antimicrobial Effectors in the Phagosome Allows SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium

Kim¹,

Richards

Slauch

2010

J Bacteriol

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“…In prokaryotes, Deg proteases are usually located in the periplasm and are implicated in the response to a variety of stresses, such as heat (3,30,33), oxidative stress (3,47), and high-light stress (3,39). Deg proteases are also essential for virulence in several pathogenic bacteria (33,47).…”

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confidence: 99%

“…Deg proteases are also essential for virulence in several pathogenic bacteria (33,47). An intriguing feature of this protease family is its functional diversity; the enzymes range from general proteases to highly specific regulatory enzymes.…”

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confidence: 99%

The Serine Protease HhoA from Synechocystis sp. Strain PCC 6803: Substrate Specificity and Formation of a Hexameric Complex Are Regulated by the PDZ Domain

2007

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Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg 2؉ or Ca 2؉ . We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled ␤-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a ⌬hhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.Proteolysis is an essential process in every living cell and is involved in protein quality control (45), as well as in regulation of diverse cellular events (11). In the cyanobacterium Synechocystis sp. strain PCC 6803, which is a widely used model system for studying photosynthesis and acclimation to abiotic stresses, 77 proteases are encoded in the genome (http://merops.sanger .ac.uk/). Three genes encode ATP-independent serine endopeptidases belonging to the Deg (HtrA) family (14,19,21,40). The protease domain of these enzymes harbors the hallmark catalytic triad consisting of His, Asp, and Ser responsible for the proteolytic activity and may additionally confer a chaperone function (16,42). Deg proteases usually contain one or two protein-protein interaction domains of the PDZ type C terminal of the protease domain (7). Studies of Escherichia coli DegP (also designated HtrA), E. coli DegS (also designated HhoB), and human HtrA2 showed that PDZ domains regulate the proteolytic activity (16,18,27,38,42,46), are involved in the formation of homooligomeric complexes (16,18,38), and play a role in substrate recognition (7,16,23,35).In prokaryotes, Deg proteases are usually located in the periplasm and are implicated in the response to a variety of stresses, such as heat (3, 30, 33), oxidative stress (3, 47), and high-light stress (3, 39). Deg proteases are also essential for virulence in several pathogenic bacteria (33,47). An intriguing fea...

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“…This could explain why the mucD mutant is not affected by temperature stress in X. campestris pv. campestris, whereas in most bacterial species degP mutants show a thermosensitive phenotype in vitro (47,74).…”

Section: Discussionmentioning

confidence: 99%

Insights into the Extracytoplasmic Stress Response of Xanthomonas campestris pv. campestris : Role and Regulation of σ ^E -Dependent Activity

et al. 2011

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Xanthomonas campestris pv. campestris is an epiphytic bacterium that can become a vascular pathogen responsible for black rot disease of crucifers. To adapt gene expression in response to ever-changing habitats, phytopathogenic bacteria have evolved signal transduction regulatory pathways, such as extracytoplasmic function (ECF) factors. The alternative sigma factor E , encoded by rpoE, is crucial for envelope stress response and plays a role in the pathogenicity of many bacterial species. Here, we combine different approaches to investigate the role and mechanism of E -dependent activation in X. campestris pv. campestris. We show that the rpoE gene is organized as a single transcription unit with the anti-gene rseA and the protease gene mucD and that rpoE transcription is autoregulated. rseA and mucD transcription is also controlled by a highly conserved E -dependent promoter within the E gene sequence. The E -mediated stress response is required for stationary-phase survival, resistance to cadmium, and adaptation to membrane-perturbing stresses (elevated temperature and ethanol). Using microarray technology, we started to define the E regulon of X. campestris pv. campestris. These genes encode proteins belonging to different classes, including periplasmic or membrane proteins, biosynthetic enzymes, classical heat shock proteins, and the heat stress factor H . The consensus sequence for the predicted E -regulated promoter elements is GGAACTN 15-17 GTCNNA. Determination of the rpoH transcription start site revealed that rpoH was directly regulated by E under both normal and heat stress conditions. Finally, E activity is regulated by the putative regulated intramembrane proteolysis (RIP) proteases RseP and DegS, as previously described in many other bacteria. However, our data suggest that RseP and DegS are not only dedicated to RseA cleavage and that the proteolytic cascade of RseA could involve other proteases.Bacteria often encounter diverse and rapidly changing environments. To overcome harmful situations, they must be capable of sensing external changes and transmitting this information across biological membranes into the cell, which results in the appropriate redirection of gene expression to prevent or repair cellular damages caused by stress. Extracytoplasmic function (ECF) factors provide one common means of bacterial signal transduction to regulate gene expression in response to various extracellular changes (65). ECF factors represent the largest and most diverse subfamily of 70 proteins. They generally recognize a Ϫ35 box with a clear bias toward a GAAC in their target promoters, while the Ϫ10 region tends to be highly variable between ECF subfamily members (65). One of the best-studied ECF factors is the key regulator of the extracytoplasmic stress response factor E from Escherichia coli, encoded by the rpoE gene (56). ECF proteins were recently divided into 43 major phylogenetically distinct groups named ECF01 to ECF43 (65). RpoE-like ECF factors are part of one predominant subgroup found in most b...

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Single, double and triple mutants of Salmonella enterica serovar Typhimurium degP (htrA), degQ (hhoA) and degS (hhoB) have diverse phenotypes on exposure to elevated temperature and their growth in vivo is attenuated to different extents

Cited by 48 publications

References 57 publications

Protecting against Antimicrobial Effectors in the Phagosome Allows SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium

Protecting against Antimicrobial Effectors in the Phagosome Allows SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium

The Serine Protease HhoA from Synechocystis sp. Strain PCC 6803: Substrate Specificity and Formation of a Hexameric Complex Are Regulated by the PDZ Domain

Insights into the Extracytoplasmic Stress Response of Xanthomonas campestris pv. campestris : Role and Regulation of σ ^E -Dependent Activity

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Single, double and triple mutants of Salmonella enterica serovar Typhimurium degP (htrA), degQ (hhoA) and degS (hhoB) have diverse phenotypes on exposure to elevated temperature and their growth in vivo is attenuated to different extents

Cited by 48 publications

References 57 publications

Protecting against Antimicrobial Effectors in the Phagosome Allows SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium

Protecting against Antimicrobial Effectors in the Phagosome Allows SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium

The Serine Protease HhoA from Synechocystis sp. Strain PCC 6803: Substrate Specificity and Formation of a Hexameric Complex Are Regulated by the PDZ Domain

Insights into the Extracytoplasmic Stress Response of Xanthomonas campestris pv. campestris : Role and Regulation of σ E -Dependent Activity

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Insights into the Extracytoplasmic Stress Response of Xanthomonas campestris pv. campestris : Role and Regulation of σ ^E -Dependent Activity