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1.  From screen to target: insights and approaches for the development of anti-virulence compounds 
A detailed understanding of host-pathogen interactions provides exciting opportunities to interfere with the infection process. Anti-virulence compounds aim to modulate or pacify pathogenesis by reducing expression of critical virulence determinants. In particular, prevention of attachment by inhibiting adhesion mechanisms has been the subject of intense research. Whilst it has proven relatively straightforward to develop robust screens for potential anti-virulence compounds, understanding their precise mode of action has proven much more challenging. In this review we illustrate this challenge from our own experiences working with the salicylidene acylhydrazide group of compounds. We aim to provide a useful perspective to guide researchers interested in this field and to avoid some of the obvious pitfalls.
PMCID: PMC4179734  PMID: 25325019
infection; anti-virulence; secretion; inhibitor; Escherichia coli
2.  The structure of an orthorhombic crystal form of a ‘forced reduced’ thiol peroxidase reveals lattice formation aided by the presence of the affinity tag 
The crystal structure of the C61S mutant of Tpx from E. coli is reported at 1.97 Å resolution. A brief structural comparison with homologues is presented.
Thiol peroxidase (Tpx) is an atypical 2-Cys peroxiredoxin, which has been suggested to be important for cell survival and virulence in Gram-negative pathogens. The structure of a catalytically inactive version of this protein in an orthorhombic crystal form has been determined by molecular replacement. Structural alignments revealed that Tpx is conserved. Analysis of the crystal packing shows that the linker region of the affinity tag is important for formation of the crystal lattice.
PMCID: PMC3374505  PMID: 22691780
thiol peroxidases; peroxiredoxins; C61S mutant; reduced
3.  Express Your LOV: An Engineered Flavoprotein as a Reporter for Protein Expression and Purification 
PLoS ONE  2012;7(12):e52962.
In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an “effector” protein normally produced by enterohemorrhagic E. coli strains and “injected” into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.
PMCID: PMC3531456  PMID: 23300834
4.  Use of a Dual Reporter Plasmid to Demonstrate Bactofection with an Attenuated AroA- Derivative of Pasteurella multocida B:2 
PLoS ONE  2013;8(8):e71524.
A reporter plasmid pSRG has been developed which expresses red fluorescent protein (RFP) from a constitutive prokaryotic promoter within Pasteurella multocida B:2 and green fluorescent protein (GFP) from a constitutive eukaryotic promoter within mammalian cells. This construct has been used to determine the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of mammalian cells. Invasion assays with embryonic bovine lung (EBL) cells and an attenuated AroA- derivative of Pasteurella multocida B:2 (strain JRMT12), harbouring the plasmid pSRG, showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously, indicating release of the plasmid into the intracellular environment. At 5 h post-invasion, more EBL cells were expressing GFP, while still harbouring RFP-expressing bacteria. Concurrently, some EBL cells were shown to express only GFP, indicating loss of viable bacteria within these cells. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine.
PMCID: PMC3741130  PMID: 23951183
5.  Increased S-Nitrosylation and Proteasomal Degradation of Caspase-3 during Infection Contribute to the Persistence of Adherent Invasive Escherichia coli (AIEC) in Immune Cells 
PLoS ONE  2013;8(7):e68386.
Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn’s disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients.
PMCID: PMC3701656  PMID: 23861899
6.  Expression, purification, crystallization and initial X-ray diffraction analysis of thiol peroxidase from Yersinia pseudotuberculosis  
Recombinant thiol peroxidase from Y. pseudotuberculosis has been purified and crystallized in three crystal forms.
Thiol peroxidase is an atypical 2-Cys peroxiredoxin that reduces alkyl hydroperoxides. Wild-type and C61S mutant protein have been recombinantly expressed in Escherichia coli and purified using nickel-affinity chromatography. Initial crystallization trials yielded three crystal forms in three different space groups (P21, P64 and P212121) both in the presence and the absence of DTT.
PMCID: PMC2998365  PMID: 21139206
Yersinia pseudotuberculosis; thiol peroxidases; Tpx; peroxiredoxins
7.  Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system 
In this work we describe protocols for the generation of gene deletions and gene replacements using a temperature sensitive plasmid in Escherichia coli O157:H7. This technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. A number of examples are used to illustrate the flexibility of the system which has been used to create novel gene replacements including fusions for protein localisation work and reporters for transcriptional analyses. In this paper we describe protocols which can be used with a high degree of success when applied to E. coli O157. The deletion and replacement of the LEE4 operon of E. coli O157 is detailed to show the advantages and limitations of the technology.
PMCID: PMC1592459  PMID: 17033696
Gene Deletion; Escherichia coli O157:H7
8.  Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline 
As-p18, an unusual fatty-acid-binding protein from a parasitic nematode, was expressed in bacteria, purified and crystallized. The use of a microfocus beamline was essential for data collection.
As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum. Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystals were obtained, but initial diffraction data analysis revealed that they were composed of a number of ‘microdomains’. Interpretable data could only be collected using a microfocus beamline with a beam size of 12 × 8 µm.
PMCID: PMC3412778  PMID: 22869127
fatty-acid-binding proteins; parasitic nematodes; Ascaris suum; microfocus beamlines
9.  Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus  
Na-FAR-1, a fatty acid- and retinol-binding protein, was expressed in bacteria, purified and crystallized. Crystals grew in two different morphologies under the same conditions.
Na-FAR-1 is an unusual α-helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = β = γ = 90°) and diffracted to 2.5 Å resolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = β = γ = 90°) and diffracted to 3.2 Å resolution. Crystal form 2 showed signs of significant twinning.
PMCID: PMC3388935  PMID: 22750878
fatty acid- and retinol-binding proteins; parasitic nematodes; Necator americanus; Na-FAR-1
10.  Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli 
PLoS Pathogens  2012;8(5):e1002672.
Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins.
Author Summary
Many significant infectious diseases that impact human health evolve in animal hosts. Our work focuses on infections caused by strains of enterohemorrhagic Escherichia coli (EHEC) that cause bloody diarrhoea and life threatening kidney and brain damage in humans as an incidental host, while ruminants are a reservoir host. EHEC strains are infected with bacteriophages that can integrate their genetic material into the bacterial chromosome. This includes genes for the production of Shiga toxins (Stx) that are responsible for the severe pathology in humans. It has been demonstrated that certain EHEC strains are more likely to be associated with human disease and ‘supershedding’ animals. The current study has shown that these EHEC strains are more likely to contain two related Stx bacteriophages, rather than one, and that the intercalating bacteriophages take control of the bacterial type III secretion system that is essential for ruminant colonization. We propose that this regulation favours co-acquisition of other genetic regions that encode type III-secreted proteins and regulators that can overcome this control. This finding helps our understanding of EHEC strain evolution and indicates that selection of more toxic strains may be occurring in the ruminant host with important implications for human health.
PMCID: PMC3355084  PMID: 22615557
11.  FolX from Pseudomonas aeruginosa is octameric in both crystal and solution 
Febs Letters  2012;586(8):1160-1165.
► Structural investigation of FolX from Pseudomonas aeruginosa. ► A combination of AUC and SAXS allows the X-ray structure to be ‘improved’. ► FolX is shown an octamer in both crystal and in solution. ► A network of 16 hydrogen bonds and 8 salt bridges provide octamer stability.
FolX encodes an epimerase that forms one step of the tetrahydrofolate biosynthetic pathway, which is of interest as it is an established target for important drugs. Here we report the crystal structure of FolX from the bacterial opportunistic pathogen Pseudomonas aeruginosa, as well as a detailed analysis of the protein in solution, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). In combination, these techniques confirm that the protein is an octamer both in the crystal structure, and in solution.
Structured summary of protein interactions
FolX and FolXbind by x-ray crystallography (View interaction)
FolX and FolXbind by cosedimentation in solution (View interaction)
FolX and FolXbind by x ray scattering (View interaction)
PMCID: PMC3405516  PMID: 22575651
FolX, 7,8-dihydroneopterin-triphosphate-epimerase; AUC, analytical ultracentrifugation; SAXS, small-angle X-ray scattering; SV, sedimentation velocity; SE, sedimentation equilibrium; Pteridine biosynthesis; X-ray structure; Solution structure
12.  Expression and Regulation of the Escherichia coli O157:H7 Effector Proteins NleH1 and NleH2 
PLoS ONE  2012;7(3):e33408.
E. coli O157 carries two genes encoding the effector proteins NleH1 and NleH2 which are 87% identical. Despite the similarity between the proteins, the promoter regions upstream of the genes encoding the effectors are more divergent suggesting that the actual expression of the genes may be differentially regulated. This was tested by creating reporter fusions and examining their expression in different genetic backgrounds, media and on contact with host cells. The function of the proteins was also tested following transfection into host cells.
Principal Findings
Expression of both NleH1 and NleH2 was enhanced when cultured under conditions that stimulated expression of the Type Three Secretion System (T3SS) and was influenced by the regulators Ler and GrlA. Maximal expression of NleH1 required 531 bp of the upstream untranslated region but NleH2 required only 113 bp. Interestingly, contact with host cells strongly repressed expression of both NleH1 and NleH2. Following transfection, both proteins produced only minor effects on NF-κB activation when assessed using a NF-κB luciferase reporter assay, a result that is consistent with the recent report demonstrating the dependence on RPS3 for NleH1 modulation of NF-κB.
This study demonstrates the importance of considering gene regulation when studying bacterial effector proteins. Despite their sequence similarity, NleH1 and NleH2 are expressed differentially and may, therefore, be translocated at distinct times during an infection.
PMCID: PMC3299786  PMID: 22428045
13.  Structural Characterisation of Tpx from Yersinia pseudotuberculosis Reveals Insights into the Binding of Salicylidene Acylhydrazide Compounds 
PLoS ONE  2012;7(2):e32217.
Thiol peroxidase, Tpx, has been shown to be a target protein of the salicylidene acylhydrazide class of antivirulence compounds. In this study we present the crystal structures of Tpx from Y. pseudotuberculosis (ypTpx) in the oxidised and reduced states, together with the structure of the C61S mutant. The structures solved are consistent with previously solved atypical 2-Cys thiol peroxidases, including that for “forced” reduced states using the C61S mutant. In addition, by investigating the solution structure of ypTpx using small angle X-ray scattering (SAXS), we have confirmed that reduced state ypTpx in solution is a homodimer. The solution structure also reveals flexibility around the dimer interface. Notably, the conformational changes observed between the redox states at the catalytic triad and at the dimer interface have implications for substrate and inhibitor binding. The structural data were used to model the binding of two salicylidene acylhydrazide compounds to the oxidised structure of ypTpx. Overall, the study provides insights into the binding of the salicylidene acylhydrazides to ypTpx, aiding our long-term strategy to understand the mode of action of this class of compounds.
PMCID: PMC3288085  PMID: 22384182
14.  Identification of Bacterial Target Proteins for the Salicylidene Acylhydrazide Class of Virulence-blocking Compounds* 
The Journal of Biological Chemistry  2011;286(34):29922-29931.
A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.
PMCID: PMC3191033  PMID: 21724850
Bacteria; Bacterial Genetics; Gene Regulation; Protein Drug Interactions; Protein Secretion; Protein Targeting; Transcription
15.  The EspF Effector, a Bacterial Pathogen's Swiss Army Knife▿  
Infection and Immunity  2010;78(11):4445-4453.
Central to the pathogenesis of many bacterial pathogens is the ability to deliver effector proteins directly into the cells of their eukaryotic host. EspF is one of many effector proteins exclusive to the attaching and effacing pathogen family that includes enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Work in recent years has revealed EspF to be one of the most multifunctional effector proteins known, with defined roles in several host cellular processes, including disruption of the epithelial barrier, antiphagocytosis, microvillus effacement, host membrane remodelling, modulation of the cytoskeleton, targeting and disruption of the nucleolus, intermediate filament disruption, cell invasion, mitochondrial dysfunction, apoptosis, and inhibition of several important epithelial transporters. Surprisingly, despite this high number of functions, EspF is a relatively small effector protein, and recent work has begun to decipher the molecular events that underlie its multifunctionality. This review focuses on the activities of EspF within the host cell and discusses recent findings and molecular insights relating to the virulence functions of this fascinating bacterial effector.
PMCID: PMC2976335  PMID: 20679436
16.  Characterization of the Effects of Salicylidene Acylhydrazide Compounds on Type III Secretion in Escherichia coli O157:H7 ▿ †  
Infection and Immunity  2009;77(10):4209-4220.
Recent work has highlighted a number of compounds that target bacterial virulence by affecting gene regulation. In this work, we show that small-molecule inhibitors affect the expression of the type III secretion system (T3SS) of Escherichia coli O157:H7 in liquid culture and when this bacterium is attached to bovine epithelial cells. Inhibition of T3SS expression resulted in a reduction in the capacity of the bacteria to form attaching and effacing lesions. Our results show that there is marked variation in the abilities of four structurally related compounds to inhibit the T3SS of a panel of isolates. Using transcriptomics, we performed a comprehensive analysis of the conserved and inhibitor-specific transcriptional responses to these four compounds. These analyses of gene expression show that numerous virulence genes, located on horizontally acquired DNA elements, are affected by the compounds, but the number of genes significantly affected varied markedly for the different compounds. Overall, we highlight the importance of assessing the effect of such “antivirulence” agents on a range of isolates and discuss the possible mechanisms which may lead to the coordinate downregulation of horizontally acquired virulence genes.
PMCID: PMC2747932  PMID: 19635828
18.  Regulators Encoded in the Escherichia coli Type III Secretion System 2 Gene Cluster Influence Expression of Genes within the Locus for Enterocyte Effacement in Enterohemorrhagic E. coli O157:H7  
Infection and Immunity  2004;72(12):7282-7293.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 subverts host cells through a type III secretion system encoded by the locus for enterocyte effacement (LEE). Genome sequencing of this pathotype revealed the existence of a gene cluster encoding components of a second cryptic type III secretion system, E. coli type III secretion system 2 (ETT2). Recently, we showed that the ETT2 gene cluster is present in whole or in part in the majority of E. coli strains but is unable to encode a functional secretion system in most strains, including EHEC O157:H7. However, here we show that mutational inhibition of two regulatory genes (ECs3720 or etrA and ECs3734 or eivF) from the ETT2 cluster in EHEC O157:H7 leads to greatly increased secretion of proteins encoded by the LEE and to increased adhesion to human intestinal cells. Studies in which transcriptional fusions and microarrays were used indicated that EtrA and EivF exert profound negative effects on gene transcription within the LEE. Consistent with these observations, expression of these regulators in an EHEC O26:H- strain led to suppression of protein secretion under LEE-inducing conditions. These findings provide fresh examples of the influence of mobile genetic elements on regulation of the LEE and of cross talk between type III secretion system gene clusters. In addition, they provide a cautionary tale because they show that the effects of regulatory genes can outlive widespread decay of other genes in a functionally coherent gene cluster, a phenomenon that we have named the “Cheshire cat effect.” It also seems likely that variations in the ETT2 regulator repertoire might account for strain-to-strain variation in secretion of LEE-encoded proteins.
PMCID: PMC529121  PMID: 15557654
19.  Mutation of toxB and a Truncated Version of the efa-1 Gene in Escherichia coli O157:H7 Influences the Expression and Secretion of Locus of Enterocyte Effacement-Encoded Proteins but not Intestinal Colonization in Calves or Sheep  
Infection and Immunity  2004;72(9):5402-5411.
Enterohemorrhagic Escherichia coli (EHEC) strains comprise a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in humans and animals. Non-O157:H7 EHEC strains contain the gene efa-1 (referred to in previous publications as efa1), which influences adherence to cultured epithelial cells. An almost identical gene in enteropathogenic E. coli (lifA) mediates the inhibition of lymphocyte proliferation and proinflammatory cytokine synthesis. We have shown previously that significantly lower numbers of EHEC O5 and O111 efa-1 mutants are shed in feces following experimental infection in calves and that these mutants exhibit reduced adherence to intestinal epithelia compared with isogenic wild-type strains. E. coli O157:H7 strains lack efa-1 but encode a homolog on the pO157 plasmid (toxB/l7095) and contain a truncated version of the efa-1 gene (efa-1′/z4332 in O island 122 of the EDL933 chromosome). Here we report that E. coli O157:H7 toxB and efa-1′ single and double mutants exhibit reduced adherence to cultured epithelial cells and show reduced expression and secretion of proteins encoded by the locus of enterocyte effacement (LEE), which plays a key role in the host-cell interactions of EHEC. The activity of LEE1, LEE4, and LEE5 promoters was not significantly altered in E. coli O157:H7 strains harboring toxB or efa-1′ mutations, indicating that the effect on the expression of LEE-encoded secreted proteins occurs at a posttranscriptional level. Despite affecting type III secretion, mutation of toxB and efa-1′ did not significantly affect the course of fecal shedding of E. coli O157:H7 following experimental inoculation of 10- to 14-day-old calves or 6-week-old sheep. Mutation of tir caused a significant reduction in fecal shedding of E. coli O157:H7 in calves, indicating that the formation of AE lesions is important for colonization of the bovine intestine.
PMCID: PMC517426  PMID: 15322038
20.  Heterogeneous Surface Expression of EspA Translocon Filaments by Escherichia coli O157:H7 Is Controlled at the Posttranscriptional Level  
Infection and Immunity  2003;71(10):5900-5909.
Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as “effectors” such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both β-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEE1-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEE1-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.
PMCID: PMC201059  PMID: 14500511
21.  Differences in Levels of Secreted Locus of Enterocyte Effacement Proteins between Human Disease-Associated and Bovine Escherichia coli O157 
Infection and Immunity  2001;69(8):5107-5114.
Ongoing extensive epidemiological studies of verotoxin-carrying Escherichia coli O157 (stx+ eae+) have shown this bacterial pathogen to be common in cattle herds in the United States and the United Kingdom. However, the incidence of disease in humans due to this pathogen is still very low. This study set out to investigate if there is a difference between strains isolated from human disease cases and those isolated from asymptomatic cattle which would account for the low disease incidence of such a ubiquitous organism. The work presented here has compared human disease strains from both sporadic and outbreak cases with a cross-section, as defined by pulsed-field gel electrophoresis, of E. coli O157 strains from cattle. Human (n = 22) and bovine (n = 31) strains were genotyped for carriage of the genes for Shiga-like toxin types 1, 2, and 2c; E. coli secreted protein genes espA, espB, and espP; the enterohemolysin gene; eae (intimin); ast (enteroaggregative E. coli stable toxin [EAST]); and genes for common E. coli adhesins. Strains were also phenotyped for hemolysin, EspP, Tir, and EspD expression as well as production of actin and cytoskeletal rearrangement associated with attaching and effacing (A/E) lesions on HeLa cells. The genotyping confirmed that there was little difference between the two groups, including carriage of stx2 and stx2c, which was similar in both sets. ast alleles were confirmed to all contain mutations that would prevent EAST expression. espP mutations were found only in cattle strains (5 of 30). Clear differences were observed in the expression of locus of enterocyte effacement (LEE)-encoded factors between strains and in different media. EspD, as an indicator of LEE4 (espA, -B, and -D) expression, and Tir levels in supernatants were measured. Virtually all strains from both sources could produce EspD in Luria-Bertani broth, although at very different levels. Standard trichloroacetic acid precipitation of secreted proteins from tissue culture medium produced detectable levels of EspD from the majority of strains of human origin (15 of 20) compared with only a few (4 of 20) bovine strains (P < 0.001), which is indicative of much higher levels of protein secretion from the human strains. Addition of bovine serum albumin carrier protein before precipitation and enhanced detection techniques confirmed that EspD could be detected after growth in tissue culture medium for all strains, but levels from strains of human origin were on average 90-fold higher than those from strains of bovine origin. In general, levels of secretion also correlated with ability to form A/E lesions on HeLa cells, with only the high-level protein secretors in tissue culture medium exhibiting a localized adherence phenotype. This research shows significant differences between human- and bovine-derived E. coli O157 (stx+ eae+) strains and their production of certain LEE-encoded virulence factors. These data support the recent finding of Kim et al. (J. Kim, J. Nietfeldt, and A. K. Benson, Proc. Natl. Acad. Sci. USA 96:13288–13293, 1999) proposing different E. coli O157 lineages in cattle and humans and extend the differential to the regulation of virulence factors. Potentially only a subset of E. coli O157 isolates (stx+ eae+) in cattle may be capable of causing severe disease in humans.
PMCID: PMC98606  PMID: 11447192
22.  Glutathione Is Involved in Environmental Stress Responses in Rhizobium tropici, Including Acid Tolerance 
Journal of Bacteriology  2000;182(6):1748-1753.
The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.
PMCID: PMC94474  PMID: 10692382
23.  Perturbation of Anion Balance during Inhibition of Growth of Escherichia coli by Weak Acids 
Journal of Bacteriology  1998;180(4):767-772.
During inhibition of cell growth by weak acids, there is substantial accumulation of the weak acid anions in the cytoplasm. This study was undertaken to determine the impact of anion accumulation on cellular pools. At pH 6, growth in the presence of 8 mM acetate led to an internal pool of greater than 240 mM acetate anion and resulted in reduced levels of glutamate in the cell, but there were no significant changes in K+ and Na+ levels. At low osmolarity, the change in the glutamate pool compensated for only a small fraction of the accumulated acetate anion. However, at high osmolarity, glutamate compensated for over half of the accumulated acetate. Recovery of the normal cytoplasmic pH after the removal of acetate was dependent on the synthesis of glutamate.
PMCID: PMC106953  PMID: 9473028
24.  The metabolic enzyme AdhE controls the virulence of Escherichia coli O157:H7 
Molecular Microbiology  2014;93(1):199-211.
Classical studies have focused on the role that individual regulators play in controlling virulence gene expression. An emerging theme, however, is that bacterial metabolism also plays a key role in this process. Our previous work identified a series of proteins that were implicated in the regulation of virulence. One of these proteins was AdhE, a bi-functional acetaldehyde-CoA dehydrogenase and alcohol dehydrogenase. Deletion of its gene (adhE) resulted in elevated levels of extracellular acetate and a stark pleiotropic phenotype: strong suppression of the Type Three Secretion System (T3SS) and overexpression of non-functional flagella. Correspondingly, the adhE mutant bound poorly to host cells and was unable to swim. Furthermore, the mutant was significantly less virulent than its parent when tested in vivo, which supports the hypothesis that attachment and motility are central to the colonization process. The molecular basis by which AdhE affects virulence gene regulation was found to be multifactorial, involving acetate-stimulated transcription of flagella expression and post-transcriptional regulation of the T3SS through Hfq. Our study reveals fascinating insights into the links between bacterial physiology, the expression of virulence genes, and the underlying molecular mechanism mechanisms by which these processes are regulated.
PMCID: PMC4249723  PMID: 24846743

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