Andrew J. Roe

Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity

The mechanism by which methionine relieves the growth inhibition of Escherichia coli K-12 that is caused by organic weak acid food preservatives was investigated. In the presence of 8 mM acetate the specific growth rate of E. coli Frag1 (in MacIlvaines minimal medium pH 6.0) is reduced by 50%. Addition of methionine restores growth to 80% of that observed in untreated controls. Similar relief was seen with cultures treated with either benzoate or propionate. Mutants with an elevated intracellular methionine pool were almost completely resistant to the inhibitory effects of acetate, suggesting that the methionine pool becomes limiting for growth in acetate-treated cells. Measurement of the intracellular concentrations of pathway intermediates revealed that the homocysteine pool is increased dramatically in acetate-treated cells, suggesting that acetate inhibits a biosynthetic step downstream from this intermediate. Supplementation of the medium with homocysteine inhibits the growth of E. coli cells. Acetate inhibition of growth arises from the depletion of the intracellular methionine pool with the concomitant accumulation of the toxic intermediate homocysteine and this augments the effect of lowering cytoplasmic pH.

Glutathione Is Involved in Environmental Stress Responses in Rhizobium tropici, Including Acid Tolerance

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.

An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157: H7 with bovine intestinal epithelium

Enterohaemorrhagic Escherichia coli O157 : H7 is a bacterial pathogen that can cause haemorrhagic colitis and haemolytic uremic syndrome. In the primary reservoir host, cattle, the terminal rectum is the principal site of E. coli O157 colonization. In this study, bovine terminal rectal primary epithelial cells were used to examine the role of H7 flagella in epithelial adherence. Binding of a fliCH7 mutant O157 strain to rectal epithelium was significantly reduced as was binding of the flagellated wild‐type strain following incubation with H7‐specific antibodies. Complementation of fliCH7 mutant O157 strain with fliCH7 restored the adherence to wild‐type levels; however, complementation with fliCH6 did not restore it. High‐resolution ultrastructural and imunofluorescence studies demonstrated the presence of abundant flagella forming physical contact points with the rectal epithelium. Binding to terminal rectal epithelium was specific to H7 by comparison with other flagellin types tested. In‐cell Western assays confirmed temporal expression of flagella during O157 interaction with epithelium, early expression was suppressed during the later stages of microcolony and attaching and effacing lesion formation. H7 flagella are expressed in vivo by individual bacteria in contact with rectal mucosa. Our data demonstrate that the H7 flagellum acts as an adhesin to bovine intestinal epithelium and its involvement in this crucial initiating step for colonization indicates that H7 flagella could be an important target in intervention strategies.

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

ABSTRACT 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.

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

ABSTRACT 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.

Differences in Levels of Secreted Locus of Enterocyte Effacement Proteins between Human Disease-Associated and Bovine Escherichia coli O157

ABSTRACT Ongoing extensive epidemiological studies of verotoxin-carryingEscherichia 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 genesespA, 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 andstx2c, 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. coliO157 (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.

Salmonella Transforms Follicle-Associated Epithelial Cells into M Cells to Promote Intestinal Invasion

Salmonella Typhimurium specifically targets antigen-sampling microfold (M) cells to translocate across the gut epithelium. Although M cells represent a small proportion of the specialized follicular-associated epithelium (FAE) overlying mucosa-associated lymphoid tissues, their density increases during Salmonella infection, but the underlying molecular mechanism remains unclear. Using in vitro and in vivo infection models, we demonstrate that the S. Typhimurium type III effector protein SopB induces an epithelial-mesenchymal transition (EMT) of FAE enterocytes into M cells. This cellular transdifferentiation is a result of SopB-dependent activation of Wnt/β-catenin signaling leading to induction of both receptor activator of NF-κB ligand (RANKL) and its receptor RANK. The autocrine activation of RelB-expressing FAE enterocytes by RANKL/RANK induces the EMT-regulating transcription factor Slug that marks epithelial transdifferentiation into M cells. Thus, via the activity of a single secreted effector, S. Typhimurium transforms primed epithelial cells into M cells to promote host colonization and invasion.

Heterogeneous surface expression of EspA translocon filaments by Escherichia coli O157:H7 is controlled at the posttranscriptional level

ABSTRACT 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.

LOV to BLUF Flavoprotein Contributions to the Optogenetic Toolkit

Optogenetics is an emerging field that combines optical and genetic approaches to non-invasively interfere with cellular events with exquisite spatiotemporal control. Although it arose originally from neuroscience, optogenetics is widely applicable to the study of many different biological systems and the range of applications arising from this technology continues to increase. Moreover, the repertoire of light-sensitive proteins used for devising new optogenetic tools is rapidly expanding. Light, Oxygen, or Voltage sensing (LOV) and Blue-Light-Utilizing flavin adenine dinucleotide (FAD) (BLUF) domains represent new contributors to the optogenetic toolkit. These small (100-140-amino acids) flavoprotein modules are derived from plant and bacterial photoreceptors that respond to UV-A/blue light. In recent years, considerable progress has been made in uncovering the photoactivation mechanisms of both LOV and BLUF domains. This knowledge has been applied in the design of synthetic photoswitches and fluorescent reporters with applications in cell biology and biotechnology. In this review, we summarize the photochemical properties of LOV and BLUF photosensors and highlight some of the recent advances in how these flavoproteins are being employed to artificially regulate and image a variety of biological processes.

Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence blocking compounds

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.