Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia

Khodai-Kalaki, Maryam; Aubert, Daniel; Valvano, Miguel A.

doi:10.1074/jbc.m113.489914

Cited by 14 publications

(15 citation statements)

References 45 publications

(62 reference statements)

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“…Since cAMP levels are reduced under high-nutrient conditions, it is expected that these EepR levels would increase, leading to greater production of these nutrient acquisition factors. CRP is also a major virulence factor in a number of organisms, and the cAMP-EepR pathway may be of importance in the virulence of other organisms with EepR-like proteins such as AtsT from Burkholderia species (59). Furthermore, our observations suggest that CRP and EepR have opposing roles in regulating desiccation stress survival through a mechanism that is unknown at this time.…”

Section: Discussionmentioning

confidence: 64%

EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model

et al. 2015

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Serratia marcescens is a soil-and water-derived bacterium that secretes several host-directed factors and causes hospital infections and community-acquired ocular infections. The putative two-component regulatory system composed of EepR and EepS regulates hemolysis and swarming motility through transcriptional control of the swrW gene and pigment production through control of the pigA-pigN operon. Here, we identify and characterize a role for EepR in regulation of exoenzyme production, stress survival, cytotoxicity to human epithelial cells, and virulence. Genetic analysis supports the model that EepR is in a common pathway with the widely conserved cyclic-AMP receptor protein that regulates protease production. Together, these data introduce a novel regulator of host-pathogen interactions and secreted-protein production. Secreted enzymes such as proteases can act as immune system and physiology modulators and virulence factors during infection by a wide variety of pathogens (1-5). Serratia marcescens is a Gram-negative coccobacillus bacterium isolated from a variety of environmental niches and the human gut. It is an important opportunistic pathogen in hospitals (6-9), and isolates collected in intensive care units became more resistant to antibiotics between 2004 and 2009 (10). S. marcescens is also a leading contaminant of contact lens cases and contact lenses and can cause vision-threatening microbial keratitis and other ocular infections (11-13). S. marcescens synthesizes several secreted enzymes, including proteases, a potent nuclease, chitinases, and other enzymes that have been used for biotechnological applications or have been implicated in cytotoxicity to mammalian cells and pathogenesis (14-16). However, the regulation of these enzymes is poorly understood.Known regulators of S. marcescens proteolysis include the crp gene that encodes the cyclic-AMP (cAMP) receptor protein CRP. Mutation of crp yields bacteria with elevated production of the protease serralysin (PrtS; also known as PrtA) and a loss of secreted-chitinase and -lipase activities (17). Matsuyama and colleagues observed reduced expression of extracellular protease activity when the hexS gene, an lhrA homolog, was expressed from a multicopy plasmid, suggesting a negative regulatory role for this protein (63).Extracellular proteases, such as the S. marcescens PrtS metalloprotease, are considered important virulence factors. In vitro studies indicate that serralysin is cytotoxic to a variety of cell types (16,21,22). PrtS contributed to tissue damage in corneal infections using a rabbit model (23), altered immune cell interactions using a silkworm model (20,24), and enhanced secondary infections by a respiratory virus in mice (25). Those studies provided the impetus to determine regulators of S. marcescens protease production.A putative regulatory system that includes the response regulator-like protein EepR and a putative hybrid-histidine kinase, EepS, was recently identified for its role in controlling expression of swarming motility, pigme...

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Section: Discussionmentioning

confidence: 64%

EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model

et al. 2015

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“…This phenotype reflects a collapse of the actin filaments in the lamellipodia and defective retraction during migration, and is T6SS dependent (Aubert et al, 2008;Flannagan et al, 2012;Rosales-Reyes et al, 2012). The T6SS activity can be stimulated by deleting AtsR, a hybrid sensor kinase of a two-component system and a negative regulator of T6SS gene expression in B. cenocepacia (Aubert et al, 2008(Aubert et al, , 2010(Aubert et al, , 2013Khodai-Kalaki et al, 2013). Deletion of atsR provides higher experimental reproducibility, especially in strain K56-2 that has a low macrophage infection index.…”

Section: Resultsmentioning

confidence: 99%

A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome and Trigger Inflammation

Aubert

Yang

et al. 2016

Cell Host & Microbe

151

143

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Burkholderia cenocepacia is an opportunistic pathogen of the cystic fibrosis lung that elicits a strong inflammatory response. B. cenocepacia employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho-type GTPases, causing actin cytoskeletal defects. Here, we identified TecA, a non-VgrG T6SS effector responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces caspase-1 inflammasome activation, which is mediated by the familial Mediterranean fever disease protein Pyrin. In mouse infection, the deamidase activity of TecA is necessary and sufficient for B. cenocepacia-triggered lung inflammation and also protects mice from lethal B. cenocepacia infection. Therefore, Burkholderia TecA is a T6SS effector that modifies a eukaryotic target through an asparagine deamidase activity, which in turn elicits host cell death and inflammation through activation of the Pyrin inflammasome.

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“…Study of B. cenocepacia T6SS in vitro was rendered possible by the discovery of AtsR (adhesion and type six secretion system regulator), a hybrid sensor kinase that negatively regulates the expression of B. cenocepacia virulence factors, including the T6SS (Aubert et al, 2008(Aubert et al, , 2010(Aubert et al, , 2013Khodai-Kalaki et al, 2013). Deletion of atsR causes a significant increase in T6SS activity, as denoted by increased amounts of Hcp released into bacterial culture supernatant (Aubert et al, 2008), induction of actin cytoskeletal rearrangements in infected macrophages (Aubert et al, 2008;Flannagan et al, 2012;Rosales-Reyes et al, 2012), and delayed assembly of the NADPH oxidase complex at the membrane of the B. cenocepacia-containing vacuole (Keith et al, 2009;Rosales-Reyes et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

Aubert

Valvano

2015

Microbiology

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The Gram-negative bacterial type VI secretion system (T6SS) delivers toxins to kill or inhibit the growth of susceptible bacteria, while other secretion systems target eukaryotic cells. Deletion of atsR, a negative regulator of virulence factors in B. cenocepacia K56-2, increases T6SS activity. Macrophages infected with a K56-2 DatsR mutant display dramatic alterations in their actin cytoskeleton architecture that rely on the T6SS, which is responsible for the inactivation of multiple Rho-family GTPases by an unknown mechanism. We employed a strategy to standardize the bacterial infection of macrophages and densitometrically quantify the T6SS-associated cellular phenotype, which allowed us to characterize the phenotype of systematic deletions of each gene within the T6SS cluster and ten vgrG genes in K56-2 DatsR. None of the genes from the T6SS core cluster nor the individual vgrG genes were directly responsible for the cytoskeletal changes in infected cells. However, a mutant strain with all vgrG genes deleted was unable to cause macrophage alterations. Despite not being able to identify a specific effector protein responsible for the cytoskeletal defects in macrophages, our strategy resulted in the identification of the critical core components and accessory proteins of the T6SS assembly machinery and provides a screening method to detect T6SS effectors targeting the actin cytoskeleton in macrophages by random mutagenesis.

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Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia

Cited by 14 publications

References 45 publications

EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model

EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model

A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome and Trigger Inflammation

Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

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