Heran Darwin, Ph.D.

Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli
Cerda-Maira, Francisca A; McAllister, Fiona; Bode, Nadine J; Burns, Kristin E; Gygi, Steven P; Darwin, K Heran
2011 ;12(8):863-870, EMBO reports
Prokaryotic ubiquitin-like protein (Pup) is a post-translational modifier that attaches to more than 50 proteins in Mycobacteria. Proteasome accessory factor A (PafA) is responsible for Pup conjugation to substrates, but the manner in which proteins are selected for pupylation is unknown. To address this issue, we reconstituted the pupylation of model Mycobacterium proteasome substrates in Escherichia coli, which does not encode Pup or PafA. Surprisingly, Pup and PafA were sufficient to pupylate at least 51 E. coli proteins in addition to the mycobacterial proteins. These data suggest that pupylation signals are intrinsic to targeted proteins and might not require Mycobacterium-specific cofactors for substrate recognition by PafA in vivo
— id: 135563, year: 2011, vol: 12, page: 863, stat: Journal Article,

A novel copper-responsive regulon in Mycobacterium tuberculosis
Festa, Richard A; Jones, Marcus B; Butler-Wu, Susan; Sinsimer, Daniel; Gerads, Russell; Bishai, William R; Peterson, Scott N; Darwin, K Heran
2011 Jan;79(1):133-148, Molecular microbiology
In this work we describe the identification of a copper-inducible regulon in Mycobacterium tuberculosis (Mtb). Among the regulated genes was Rv0190/MT0200, a paralogue of the copper metalloregulatory repressor CsoR. The five-locus regulon, which includes a gene that encodes the copper-protective metallothionein MymT, was highly induced in wild-type Mtb treated with copper, and highly expressed in an Rv0190/MT0200 mutant. Importantly, the Rv0190/MT0200 mutant was hyper-resistant to copper. The promoters of all five loci share a palindromic motif that was recognized by the gene product of Rv0190/MT0200. For this reason we named Rv0190/MT0200 RicR for regulated in copper repressor. Intriguingly, several of the RicR-regulated genes, including MymT, are unique to pathogenic Mycobacteria. The identification of a copper-responsive regulon specific to virulent mycobacterial species suggests copper homeostasis must be maintained during an infection. Alternatively, copper may provide a cue for the expression of genes unrelated to metal homeostasis, but nonetheless necessary for survival in a host
— id: 116213, year: 2011, vol: 79, page: 133, stat: Journal Article,

"Depupylation" of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates
Burns, Kristin E; Cerda-Maira, Francisca A; Wang, Tao; Li, Huilin; Bishai, William R; Darwin, K Heran
2010 Sep 10;39(5):821-827, Molecular cell
Ubiquitin (Ub) provides the recognition and specificity required to deliver proteins to the eukaryotic proteasome for destruction. Prokaryotic ubiquitin-like protein (Pup) is functionally analogous to Ub in Mycobacterium tuberculosis (Mtb), as it dooms proteins to the Mtb proteasome. Studies suggest that Pup and Ub do not share similar mechanisms of activation and conjugation to target proteins. Dop (deamidase of Pup; Mtb Rv2112c/MT2172) deamidates the C-terminal glutamine of Pup to glutamate, preparing it for ligation to target proteins by proteasome accessory factor A (PafA). While studies have shed light on the conjugation of Pup to proteins, it was not known if Pup could be removed from substrates in a manner analogous to the deconjugation of Ub from eukaryotic proteins. Here, we show that Mycobacteria have a 'depupylase' activity provided by Dop. The discovery of a depupylase strengthens the parallels between the Pup- and Ub-tagging systems of prokaryotes and eukaryotes, respectively
— id: 138125, year: 2010, vol: 39, page: 821, stat: Journal Article,

Pupylation : A Signal for Proteasomal Degradation in Mycobacterium tuberculosis
Burns, Kristin E; Darwin, K Heran
2010 ;54:149-157, Sub-cellular biochemistry
This chapter describes the identification of the first prokaryotic ubiquitin-like protein modifier, Pup, which covalently attaches to proteins to target them for destruction by a bacterial proteasome in a manner akin to ubiquitin in eukaryotes. Despite using a proteasome as the end point for proteolysis, Pup and ubiquitin differ in sequence, structure and method of activation and conjugation to protein substrates. Pup is so far the only known posttranslational protein modifier in prokaryotes and its discovery opens the door to the possibility that others are present not only for proteolysis, but also to regulate protein function or localization. Here, we discuss the putative mechanism of activation and conjugation of Pup (termed 'pupylation') to target proteins. In addition, because it is unclear whether or not Pup, like ubiquitin, is recycled or degraded during substrate targeting to the proteasome, we propose methods that may identify Pup deconjugation enzymes ('depupylases'). Finally, we outline future directions for Pup research and anti-tuberculosis drug discovery
— id: 119237, year: 2010, vol: 54, page: 149, stat: Journal Article,

Pupylation versus ubiquitylation: tagging for proteasome-dependent degradation
Burns, Kristin E; Darwin, K Heran
2010 Apr 1;12(4):424-431, Cellular microbiology
Prokaryotic ubiquitin-like protein (Pup) is the first identified prokaryotic protein that is functionally analogous to ubiquitin. Despite using the proteasome as the end-point for proteolysis, Pup differs from ubiquitin both biochemically and structurally. We will discuss these differences that have been highlighted by several recent studies. Finally, we will speculate on the possible interactions between the two analogous pathways in pathogen and host
— id: 109512, year: 2010, vol: 12, page: 424, stat: Journal Article,

Prokaryotic ubiquitin-like protein provides a two-part degron to Mycobacterium proteasome substrates
Burns, Kristin E; Pearce, Michael J; Darwin, K Heran
2010 Jun;192(11):2933-2935, Journal of bacteriology
Prokaryotic ubiquitin-like protein (Pup) is a posttranslational modifier that targets proteins for degradation by the mycobacterial proteasome. We show that the disordered amino terminus of Pup is required for degradation, while the helical carboxyl terminus mediates its attachment to proteins. Thus, Pup has distinct regions that either interact with pupylation enzymes or initiate proteasomal degradation
— id: 109670, year: 2010, vol: 192, page: 2933, stat: Journal Article,

Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis
Cerda-Maira, Francisca A; Pearce, Michael J; Fuortes, Michele; Bishai, William R; Hubbard, Stevan R; Darwin, K Heran
2010 Sep;77(5):1123-1135, Molecular microbiology
Proteins targeted for degradation by the Mycobacterium proteasome are post-translationally tagged with prokaryotic ubiquitin-like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed 'pupylation', Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild-type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site-directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins
— id: 114585, year: 2010, vol: 77, page: 1123, stat: Journal Article,

SAMPyling proteins in archaea
Darwin, K Heran; Hofmann, Kay
2010 Jun;35(6):348-351, Trends in biochemical sciences
For some time, post-translational small protein modifications were found only in eukaryotes; much later, such modifications were identified in some species of bacteria. The recent discovery of ubiquitin-like proteins that form polymeric chains and covalently modify proteins in archaea finally closes the evolutionary gap among the domains of life
— id: 110081, year: 2010, vol: 35, page: 348, stat: Journal Article,

Prokayrotic ubiquitin-like protein (Pup) proteome of Mycobacterium tuberculosis
Festa, Richard A; McAllister, Fiona; Pearce, Michael J; Mintseris, Julian; Burns, Kristin E; Gygi, Steven P; Darwin, K Heran
2010 ;5(1):e8589-e8589, PLoS ONE
Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its 'pupylome'. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtbresponse regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation
— id: 106208, year: 2010, vol: 5, page: e8589, stat: Journal Article,

Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation
Wang, Tao; Darwin, K Heran; Li, Huilin
2010 Nov;17(11):1352-1357, Nature structural & molecular biology
Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an alpha-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis
— id: 133840, year: 2010, vol: 17, page: 1352, stat: Journal Article,

The Mycobacterium tuberculosis Proteasome: More Than Just a Barrel-shaped Protease
Cerda-Maira, Francisca; Darwin, K Heran
2009 Dec;11(14-15):1150-1155, Microbes & infection
In eukaryotes the proteasome is responsible for the degradation of many proteins that are targeted for turnover by post-translational modification with ubiquitin. A similar system was identified in Mycobacterium tuberculosis and has shown to be essential for the pathogenesis of this bacterium. Here, we overview the current information of the Mtb proteasome and discuss the role of this protease in pathogenesis
— id: 101566, year: 2009, vol: 11, page: 1150, stat: Journal Article,

Prokaryotic Ubiquitin-Like Protein Pup Is Intrinsically Disordered
Chen, Xiang; Solomon, William C; Kang, Yang; Cerda-Maira, Francisca; Darwin, K Heran; Walters, Kylie J
2009 Sep 11;392(1):208-217, Journal of molecular biology
The prokaryotic ubiquitin-like protein Pup targets substrates for degradation by the Mycobacterium tuberculosis proteasome through its interaction with Mpa, an ATPase that is thought to abut the 20S catalytic subunit. Ubiquitin, which is assembled into a polymer to similarly signal for proteasomal degradation in eukaryotes, adopts a stable and compact structural fold that is adapted into other proteins for diverse biological functions. We used NMR spectroscopy to demonstrate that, unlike ubiquitin, the 64-amino-acid protein Pup is intrinsically disordered with small helical propensity in the C-terminal region. We found that the Pup:Mpa interaction involves an extensive contact surface that spans S21-K61 and that the binding is in the 'slow exchange' regime on the NMR time scale, thus demonstrating higher affinity than most ubiquitin:ubiquitin receptor pairs. Interestingly, during the titration experiment, intermediate Pup species were observable, suggesting the formation of one or more transient state(s) upon binding. Moreover, Mpa selected one configuration for a region undergoing chemical exchange in the free protein. These findings provide mechanistic insights into Pup's functional role as a degradation signal
— id: 101567, year: 2009, vol: 392, page: 208, stat: Journal Article,

Prokaryotic ubiquitin-like protein (Pup), proteasomes and pathogenesis
Darwin, K Heran
2009 Jul;7(7):485-491, Nature reviews. Microbiology
Proteasomes are ATP-dependent, multisubunit proteases that are found in all eukaryotes and archaea and some bacteria. In eukaryotes, the small protein ubiquitin is covalently attached in a post-translational manner to proteins that are targeted for proteasomal degradation. Despite the presence of proteasomes in many prokaryotes, ubiquitin or other post-translational protein modifiers were presumed to be absent from these organisms. Recently a prokaryotic ubiquitin-like protein, Pup, was found to target proteins for proteolysis by the Mycobacterium tuberculosis proteasome. The discovery of this ubiquitin-like modifier opens up the possibility that other bacteria may also have small post-translational protein tagging systems, with the ability to affect cellular processes
— id: 100188, year: 2009, vol: 7, page: 485, stat: Journal Article,

Structural insights on the Mycobacterium tuberculosis proteasomal ATPase Mpa
Wang, Tao; Li, Hua; Lin, Gang; Tang, Chunyan; Li, Dongyang; Nathan, Carl; Darwin, K Heran; Li, Huilin
2009 Oct 14;17(10):1377-1385, Structure
Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPgammaS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double beta barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome
— id: 112096, year: 2009, vol: 17, page: 1377, stat: Journal Article,

Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis
Pearce, Michael J; Mintseris, Julian; Ferreyra, Jessica; Gygi, Steven P; Darwin, K Heran
2008 Nov 14;322(5904):1104-1107, Science
The protein modifier ubiquitin is a signal for proteasome-mediated degradation in eukaryotes. Proteasome-bearing prokaryotes have been thought to degrade proteins via a ubiquitin-independent pathway. We have identified a prokaryotic ubiquitin-like protein, Pup (Rv2111c), which was specifically conjugated to proteasome substrates in the pathogen Mycobacterium tuberculosis. Pupylation occurred on lysines and required proteasome accessory factor A (PafA). In a pafA mutant, pupylated proteins were absent and substrates accumulated, thereby connecting pupylation with degradation. Although analogous to ubiquitylation, pupylation appears to proceed by a different chemistry. Thus, like eukaryotes, bacteria may use a small-protein modifier to control protein stability
— id: 93375, year: 2008, vol: 322, page: 1104, stat: Journal Article,

Characterization of the proteasome accessory factor (paf) operon in Mycobacterium tuberculosis
Festa, Richard A; Pearce, Michael J; Darwin, K Heran
2007 Apr;189(8):3044-3050, Journal of bacteriology
In a previous screen for Mycobacterium tuberculosis mutants that are hypersusceptible to reactive nitrogen intermediates (RNI), two genes associated with the M. tuberculosis proteasome were identified. One of these genes, pafA (proteasome accessory factor A), encodes a protein of unknown function. In this work, we determined that pafA is in an operon with two additional genes, pafB and pafC. In order to assess the contribution of these genes to RNI resistance, we isolated mutants with transposon insertions in pafB and pafC. In contrast to the pafA mutant, the pafB and pafC mutants were not severely sensitized to RNI, but pafB and pafC were nonetheless required for full RNI resistance. We also found that PafB and PafC interact with each other and that each is likely required for the stability of the other protein in M. tuberculosis. Finally, we show that the presence of PafA, but not PafB or PafC, regulates the steady-state levels of three proteasome substrates. Taken together, these data demonstrate that PafA, but not PafB or PafC, is critical for maintaining the steady-state levels of known proteasome substrates, whereas all three proteins appear to play a role in RNI resistance.
— id: 72728, year: 2007, vol: 189, page: 3044, stat: Journal Article,

Self-compartmentalized bacterial proteases and pathogenesis
Butler, Susan M; Festa, Richard A; Pearce, Michael J; Darwin, K Heran
2006 May;60(3):553-562, Molecular microbiology
Protein degradation is required for homeostasis of all living organisms. Self-compartmentalized ATP-dependent proteases are required for virulence of several pathogenic bacteria. Among the proteases implicated are ClpP and Lon, as well as the more recently identified bacterial proteasome. It is generally assumed that when a pathogen invades a host, microbial proteins become irreversibly damaged and need to be degraded. However, recent data suggest that proteolysis is also essential for virulence gene regulation. In this review, we will discuss what is known about the relationship between ATP-dependent proteolysis and pathogenesis. In addition, we will propose other potential roles these chambered proteases may have in bacterial virulence. Importantly, these proteases show promise as targets for antimicrobial therapy
— id: 64667, year: 2006, vol: 60, page: 553, stat: Journal Article,

Identification of substrates of the Mycobacterium tuberculosis proteasome
Pearce, Michael J; Arora, Pooja; Festa, Richard A; Butler-Wu, Susan M; Gokhale, Rajesh S; Darwin, K Heran
2006 Nov 15;25(22):5423-5432, EMBO journal
The putative proteasome-associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co-factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome-dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice
— id: 69696, year: 2006, vol: 25, page: 5423, stat: Journal Article,

Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue
Darwin, K Heran; Lin, Gang; Chen, Zhiqiang; Li, Huilin; Nathan, Carl F
2005 Jan;55(2):561-571, Molecular microbiology
A screen for Mycobacterium tuberculosis (Mtb) mutants sensitive to reactive nitrogen intermediates identified transposon insertions in the presumptive proteasomal ATPase gene mpa (mycobacterium proteasome ATPase; Rv2115c). mpa mutants are attenuated in both wild type and nitric oxide synthase 2 deficient mice. In this work, we show that attenuation of mpa mutants is severe, and that Mpa is an ATPase associated with various cellular activities (AAA) ATPase that forms hexameric rings resembling the eukaryotic complex p97/valosin-containing protein (VCP). Point mutations in the conserved Walker box ATPase motifs of Mpa greatly reduced or abolished ATPase activity in vitro and abrogated protection of Mtb against acidified nitrite. A mutant Mpa protein missing only its last two amino acids retained ATPase activity, yet failed to protect Mtb against nitrite. The corresponding strain was attenuated in mice. Thus, Mpa is an ATPase whose enzymatic activity is necessary but not sufficient to protect against reactive nitrogen intermediates
— id: 56015, year: 2005, vol: 55, page: 561, stat: Journal Article,

Role for nucleotide excision repair in virulence of Mycobacterium tuberculosis
Darwin, K Heran; Nathan, Carl F
2005 Aug;73(8):4581-4587, Infection & immunity
Mutations in Mycobacterium tuberculosis uvrB result in severe sensitivity to acidified nitrite, a source of nitric oxide (6). In this study, we show that a uvrB mutant is exquisitely sensitive to UV light but not to several sources of reactive oxygen species in vitro. Furthermore, a uvrB mutant was attenuated in mice as judged by an extension of life span. Attenuation in mice was partially reversed by genetic inactivation of nitric oxide synthase 2 (iNOS) and almost completely reversed in mice lacking both iNOS and phagocyte oxidase. Thus, a gene predicted to encode a key element of DNA repair is required for resistance of M. tuberculosis to both reactive nitrogen and reactive oxygen species in mice
— id: 96290, year: 2005, vol: 73, page: 4581, stat: Journal Article,

A glutamate-alanine-leucine (EAL) domain protein of Salmonella controls bacterial survival in mice, antioxidant defence and killing of macrophages: role of cyclic diGMP
Hisert, Katherine B; MacCoss, Michael; Shiloh, Michael U; Darwin, K Heran; Singh, Shaneen; Jones, Roger A; Ehrt, Sabine; Zhang, Zhaoying; Gaffney, Barbara L; Gandotra, Sheetal; Holden, David W; Murray, Diana; Nathan, Carl
2005 Jun;56(5):1234-1245, Molecular microbiology
Signature-tagged transposon mutagenesis of Salmonella with differential recovery from wild-type and immunodeficient mice revealed that the gene here named cdgR[for c-diguanylate (c-diGMP) regulator] is required for the bacterium to resist host phagocyte oxidase in vivo. CdgR consists solely of a glutamate-alanine-leucine (EAL) domain, a predicted cyclic diGMP (c-diGMP) phosphodiesterase. Disruption of cdgR decreased bacterial resistance to hydrogen peroxide and accelerated bacterial killing of macrophages. An ultrasensitive assay revealed c-diGMP in wild-type Salmonella with increased levels in the CdgR-deficient mutant. Thus, besides its known role in regulating cellulose synthesis and biofilm formation, bacterial c-diGMP also regulates host-pathogen interactions involving antioxidant defence and cytotoxicity
— id: 96291, year: 2005, vol: 56, page: 1234, stat: Journal Article,

The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide
Darwin, K Heran; Ehrt, Sabine; Gutierrez-Ramos, Jose-Carlos; Weich, Nadine; Nathan, Carl F
2003 Dec 12;302(5652):1963-1966, Science
The production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages helps to control infection by Mycobacterium tuberculosis (Mtb). However, the protection is imperfect and infection persists. To identify genes that Mtb requires to resist RNI, we screened 10,100 Mtb transposon mutants for hypersusceptibility to acidified nitrite. We found 12 mutants with insertions in seven genes representing six pathways, including the repair of DNA (uvrB) and the synthesis of a flavin cofactor (fbiC). Five mutants had insertions in proteasome-associated genes. An Mtb mutant deficient in a presumptive proteasomal adenosine triphosphatase was attenuated in mice, and exposure to proteasomal protease inhibitors markedly sensitized wild-type Mtb to RNI. Thus, the mycobacterial proteasome serves as a defense against oxidative or nitrosative stress
— id: 45183, year: 2003, vol: 302, page: 1963, stat: Journal Article,

Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium
Darwin KH; Miller VL
2001 Apr 17;20(8):1850-1862, EMBO journal
Invasion of the intestinal epithelium by Salmonella sp. requires a type III secretion system (TTSS) common in many bacterial pathogens. TTSS translocate effector proteins from bacteria into eukaryotic cells. These effectors manipulate cellular functions in order to benefit the pathogen. In the human and animal pathogen Salmonella typhimurium, the expression of genes encoding the secreted effector molecules Sip/Ssp ABCD, SigD, SptP and SopE requires both the AraC/XylS-like regulator InvF and the secretion chaperone SICA: In this work, an InvF binding site was identified in the promoter regions of three operons. SicA does not appear to affect InvF stability nor to bind DNA directly. However, SicA could be co-purified with InvF, suggesting that InvF and SicA interact with each other to activate transcription from the effector gene promoters. This is the first demonstration of a contact between a protein cofactor and an AraC/XylS family transcriptional regulator and, moreover, is the first direct evidence of an interaction between a transcriptional regulator and a TTSS chaperone. The regulation of effector genes described here for InvF and SicA may represent a new paradigm for regulation of virulence in a wide variety of pathogens
— id: 45184, year: 2001, vol: 20, page: 1850, stat: Journal Article,

SigE is a chaperone for the Salmonella enterica serovar Typhimurium invasion protein SigD
Darwin KH; Robinson LS; Miller VL
2001 Feb;183(4):1452-1454, Journal of bacteriology
SigD is translocated into eucaryotic cells by a type III secretion system. In this work, evidence that the putative chaperone SigE directly interacts with SigD is presented. A bacterial two-hybrid system demonstrated that SigE can interact with itself and SigD. In addition, SigD was specifically copurified with SigE-His(6) on a nickel column
— id: 45185, year: 2001, vol: 183, page: 1452, stat: Journal Article,

The putative invasion protein chaperone SicA acts together with InvF to activate the expression of Salmonella typhimurium virulence genes
Darwin KH; Miller VL
2000 Feb;35(4):949-960, Molecular microbiology
SigD and SigE (Salmonella invasion gene) are proteins needed for optimal invasion of Salmonella typhimurium into eukaryotic cells in vitro. SigD is a secreted protein and SigE is a putative chaperone required for SigD stability and/or secretion. SigD is secreted by a type III secretion apparatus encoded within a pathogenicity island on the Salmonella chromosome known as Salmonella pathogenicity island 1 (SPI1). The expression of sigDE, which is not linked to SPI1, is co-ordinately regulated with the SPI1 genes and is dependent on the transcriptional regulators SirA, HilA and InvF. These three proteins alone are unable to activate transcription from the sigD promoter in Escherichia coli, therefore it is likely that other factors are needed for expression. A screen for genes required for the expression of a sigD-lacZYA reporter fusion found a mutant with a transposon insertion in spaS, an SPI1 gene which encodes a putative inner-membrane component of the type III secretion system. The expression of a SPI1 operon encoding a putative chaperone (SicA) and several secreted proteins (Sips B, C, D and A) was also reduced in this mutant. The regulation defect of the spaS mutant was complemented by sicA and not by spaS. Because sicA is encoded immediately downstream of spaS, the mutation in spaS was likely to be polar on the expression of sicA. In addition, a sicA disruption mutant was as defective as an invF deletion mutant for the expression of sigD, sicA and sipC reporter fusions. The introduction of plasmids encoding invF and sicA into a non-pathogenic E. coli K-12 strain stimulated the transcription of both a sicA- and a sigD-lacZYA promoter fusion. This result suggests that InvF and SicA are sufficient for the expression of these genes. This is the first demonstration of a positive regulatory role for a putative type III secretion system chaperone in the expression of virulence genes
— id: 45186, year: 2000, vol: 35, page: 949, stat: Journal Article,

InvF is required for expression of genes encoding proteins secreted by the SPI1 type III secretion apparatus in Salmonella typhimurium
Darwin KH; Miller VL
1999 Aug;181(16):4949-4954, Journal of bacteriology
The expression of genes encoding proteins secreted by the SPI1 (Salmonella pathogenicity island) type III secretion apparatus is known to require the transcriptional activators SirA and HilA. However, neither SirA nor HilA is believed to directly activate the promoters of these genes. invF, the first gene of the inv-spa gene cluster, is predicted to encode an AraC-type transcriptional activator and is required for invasion into cultured epithelial cells. However, the genes which are regulated by InvF have not been identified. In this work, an in-frame deletion in invF was constructed and tested for the expression of Phi(sigD-lacZYA), sipC::Tn5lacZY, and a plasmid-encoded Phi(sicA-lacZYA). SigD (Salmonella invasion gene) is a secreted protein required for the efficient invasion of Salmonella typhimurium into cultured eucaryotic cells. sicA (Salmonella invasion chaperone) is the first gene of a putative operon encoding the Sip/Ssp (Salmonella invasion/Salmonella secreted proteins) invasion proteins secreted by the SPI1 type III export apparatus. invF was required for the expression of the sigD, sicA, and sipC fusions. This is the first demonstration that there is a functional promoter in the intergenic sequence between spaS and sicA. In addition, several proteins were either absent from or found in reduced amounts in the culture supernatants of the invF mutant. Therefore, invF is required for the optimal expression of several genes encoding SPI1-secreted proteins. Genetic evidence is also presented suggesting there is HilA-dependent readthrough transcription from the invF promoter at least through sipC
— id: 45187, year: 1999, vol: 181, page: 4949, stat: Journal Article,

Molecular basis of the interaction of Salmonella with the intestinal mucosa
Darwin KH; Miller VL
1999 Jul;12(3):405-428, Clinical microbiology reviews
Salmonella is one of the most extensively characterized bacterial pathogens and is a leading cause of bacterial gastroenteritis. Despite this, we are only just beginning to understand at a molecular level how Salmonella interacts with its mammalian hosts to cause disease. Studies during the past decade on the genetic basis of virulence of Salmonella have significantly advanced our understanding of the molecular basis of the host-pathogen interaction, yet many questions remain. In this review, we focus on the interaction of enterocolitis-causing salmonellae with the intestinal mucosa, since this is the initiating step for most infections caused by Salmonella. Animal and in vitro cell culture models for the interaction of these bacteria with the intestinal epithelium are reviewed, along with the bacterial genes that are thought to affect this interaction. Lastly, recent studies on the response of epithelial cells to Salmonella infection and how this might promote diarrhea are discussed
— id: 45188, year: 1999, vol: 12, page: 405, stat: Journal Article,