Richard D. Haigh

Virulence of Enteropathogenic Escherichia coli, a Global Pathogen

SUMMARY Enteropathogenic Escherichia coli (EPEC) remains an important cause of diarrheal disease worldwide. Research into EPEC is intense and provides a good virulence model of other E. coli infections as well as other pathogenic bacteria. Although the virulence mechanisms are now better understood, they are extremely complex and much remains to be learnt. The pathogenesis of EPEC depends on the formation of an ultrastructural lesion in which the bacteria make intimate contact with the host apical enterocyte membrane. The formation of this lesion is a consequence of the ability of EPEC to adhere in a localized manner to the host cell, aided by bundle-forming pili. Tyrosine phosphorylation and signal transduction events occur within the host cell at the lesion site, leading to a disruption of the host cell mechanisms and, consequently, to diarrhea. These result from the action of highly regulated EPEC secreted proteins which are released via a type III secretion system, many genes of which are located within a pathogenicity island known as the locus of enterocyte effacement. Over the last few years, dramatic increases in our knowledge of EPEC virulence have taken place. This review therefore aims to provide a broad overview of and update to the virulence aspects of EPEC.

Microbial endocrinology: how stress influences susceptibility to infection

A holistic approach to understanding the mechanisms by which stress influences the pathogenesis of infectious disease has resulted in the development of the field of microbial endocrinology. This transdisciplinary field represents the intersection of microbiology with mammalian endocrinology and neurophysiology, and is based on the tenet that microorganisms have evolved systems for using neurohormones, which are widely distributed throughout nature, as environmental cues to initiate growth and pathogenic processes. This review reveals that responsiveness to human stress hormones is widespread in the microbial world and documents recent advances in microbial endocrinology.

The Mammalian Neuroendocrine Hormone Norepinephrine Supplies Iron for Bacterial Growth in the Presence of Transferrin or Lactoferrin

Norepinephrine stimulates the growth of a range of bacterial species in nutritionally poor SAPI minimal salts medium containing 30% serum. Addition of size-fractionated serum components to SAPI medium indicated that transferrin was required for norepinephrine stimulation of growth of Escherichia coli. Since bacteriostasis by serum is primarily due to the iron-withholding capacity of transferrin, we considered the possibility that norepinephrine can overcome this effect by supplying transferrin-bound iron for growth. Incubation with concentrations of norepinephrine that stimulated bacterial growth in serum-SAPI medium resulted in loss of bound iron from iron-saturated transferrin, as indicated by the appearance of monoferric and apo- isoforms upon electrophoresis in denaturing gels. Norepinephrine also caused the loss of iron from lactoferrin. The pharmacologically inactive metabolite norepinephrine 3-O-sulfate, by contrast, did not result in iron loss from transferrin or lactoferrin and did not stimulate bacterial growth in serum-SAPI medium. Norepinephrine formed stable complexes with transferrin, lactoferrin, and serum albumin. Norepinephrine-transferrin and norepinephrine-lactoferrin complexes, but not norepinephrine-apotransferrin or norepinephrine-albumin complexes, stimulated bacterial growth in serum-SAPI medium in the absence of additional norepinephrine. Norepinephrine-stimulated growth in medium containing (55)Fe complexed with transferrin or lactoferrin resulted in uptake of radioactivity by bacterial cells. Moreover, norepinephrine-stimulated growth in medium containing [(3)H]norepinephrine indicated concomitant uptake of norepinephrine. In each case, addition of excess iron did not affect growth but significantly reduced levels of radioactivity ((55)Fe or (3)H) associated with bacterial cells. A role for catecholamine-mediated iron supply in the pathophysiology of infectious diseases is proposed.

Growth stimulation of intestinal commensal Escherichia coli by catecholamines: a possible contributory factor in trauma-induced sepsis.

Trauma is well recognized to result in the immediate and sustained release of stress-related neurochemicals such as the catecholamine norepinephrine. Past work has shown that in addition to their ability to function as neurotransmitters, catecholamines can also directly stimulate the growth of a number of pathogenic bacteria. The development of trauma-associated sepsis has often been linked to the ability of otherwise normal commensal bacteria to invade and penetrate the gut mucosal barrier. Therefore, the aim of our study was to examine whether catecholamines could also stimulate the growth of commensal Escherichia coli strains of the type present in the intestinal tract at the time of a traumatic event. Herein we report that the growth of a range of non-pathogenic isolates of E. coli of human and environmental origin was significantly increased in the presence of catecholamines. A primary mechanism by which catecholamines increase bacterial growth was shown to be iron removal from lactoferrin and transferrin and subsequent acquisition by bacteria. The 3,4-dihydroxybenzoyl (catechol) structure of the catecholamines was further demonstrated to be critical to iron acquisition. The synthetic catecholamine inotropes dobutamine and isoprenaline, as well as norepinephrine metabolites that retained the catechol structure were also active, whereas norepinephrine metabolites in which the catechol moiety had been modified were not. A role for catecholamine-mediated bacterial iron supply in the pathophysiology of gut-derived sepsis due to trauma is proposed.

Stimulation of Staphylococcus epidermidis growth and biofilm formation by catecholamine inotropes

BACKGROUND Bacterial colonisation of indwelling medical devices by coagulase-negative staphylococci is a prevalent risk in intensive-care units. Factors determining biofilm formation and progression to catheter- related infection are incompletely understood. We postulated that administration of inotropic agents via indwelling intravenous catheters may stimulate growth and formation of biofilms by Staphylococcus epidermidis. METHODS Inocula representing physiologically relevant infecting doses of S epidermidis were incubated in a minimum medium supplemented with fresh human plasma in the presence or absence of pharmacological concentrations of norepinephrine or dobutamine. Biofilm formation on polystyrene and medical-grade silicone was examined. After incubation, cultures were assessed for growth and formation of biofilms by colony counting and scanning electronmicroscopy. The production of exopolysaccharide, a major constituent of S epidermidis biofilms, was also assessed by use of immunofluorescence microscopy. FINDINGS Incubation of S epidermidis with catecholamine inotropes in the presence of human plasma resulted in a significant increase in growth compared with control on both polystyrene and silicone surfaces, with pronounced increases in biofilm formation as visualised by scanning electronmicroscopy. Experiments with transferrin labelled with radioactive iron showed the ability of catecholamine inotropes to facilitate acquisition of iron by S epidermidis. Immunofluorescence microscopy revealed extensive exopolysaccharide production associated with S epidermidis biofilms. INTERPRETATION The ability of catecholamine inotropic drugs to stimulate bacterial proliferation and biofilm formation may be an aetiological factor in the development of intravascular catheter colonisation and catheter-related infection. The removal of iron from transferrin for subsequent use by S epidermidis is a possible mechanism by which catecholamine inotropes stimulate bacterial growth as biofilms.

Elucidation of the Mechanism by Which Catecholamine Stress Hormones Liberate Iron from the Innate Immune Defense Proteins Transferrin and Lactoferrin

The ability of catecholamine stress hormones and inotropes to stimulate the growth of infectious bacteria is now well established. A major element of the growth induction process has been shown to involve the catecholamines binding to the high-affinity ferric-iron-binding proteins transferrin (Tf) and lactoferrin, which then enables bacterial acquisition of normally inaccessible sequestered host iron. The nature of the mechanism(s) by which the stress hormones perturb iron binding of these key innate immune defense proteins has not been fully elucidated. The present study employed electron paramagnetic resonance spectroscopy and chemical iron-binding analyses to demonstrate that catecholamine stress hormones form direct complexes with the ferric iron within transferrin and lactoferrin. Moreover, these complexes were shown to result in the reduction of Fe(III) to Fe(II) and the loss of protein-complexed iron. The use of bacterial ferric iron uptake mutants further showed that both the Fe(II) and Fe(III) released from the Tf could be directly used as bacterial nutrient sources. We also analyzed the transferrin-catecholamine interactions in human serum and found that therapeutically relevant concentrations of stress hormones and inotropes could directly affect the iron binding of serum-transferrin so that the normally highly bacteriostatic tissue fluid became significantly more supportive of the growth of bacteria. The relevance of these catecholamine-transferrin/lactoferrin interactions to the infectious disease process is considered.

Involvement of enterobactin in norepinephrine-mediated iron supply from transferrin to enterohaemorrhagic Escherichia coli

Exposure of bacteria to members of the stress-associated family of catecholamine hormones, principally norepinephrine, has been demonstrated to increase both growth and production of virulence-related factors. Mutation of genes for enterobactin synthesis and uptake revealed an absolute requirement for enterobactin in norepinephrine-stimulated growth of Escherichia coli O157:H7. The autoinducer produced by norepinephrine-stimulated E. coli could not substitute for enterobactin. We also demonstrate that norepinephrine promotes iron shuttling between transferrin molecules, thereby enabling the bacterial siderophore enterobactin to more readily acquire iron for growth. These results suggest one of the possible mechanisms by which the hormonal output of stress may affect enterohaemorrhagic E. coli pathogenicity.

A random six-phase switch regulates pneumococcal virulence via global epigenetic changes

Streptococcus pneumoniae (the pneumococcus) is the world’s foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or ‘phases’) that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.

Blockade of catecholamine-induced growth by adrenergic and dopaminergic receptor antagonists in Escherichia coli O157:H7, Salmonella enterica and Yersinia enterocolitica

BackgroundThe ability of catecholamines to stimulate bacterial growth was first demonstrated just over a decade ago. Little is still known however, concerning the nature of the putative bacterial adrenergic and/or dopaminergic receptor(s) to which catecholamines (norepinephrine, epinephrine and dopamine) may bind and exert their effects, or even whether the binding properties of such a receptor are similar between different species.ResultsUse of specific catecholamine receptor antagonists revealed that only α, and not β, adrenergic antagonists were capable of blocking norepinephrine and epinephrine-induced growth, while antagonism of dopamine-mediated growth was achieved with the use of a dopaminergic antagonist. Both adrenergic and dopaminergic antagonists were highly specific in their mechanism of action, which did not involve blockade of catecholamine-facilitated iron-acquisition. Use of radiolabeled norepinephrine suggested that the adrenergic antagonists could be acting by inhibiting catecholamine uptake.ConclusionThe present data demonstrates that the ability of a specific pathogen to respond to a particular hormone is dependent upon the host anatomical region in which the pathogen causes disease as well as the neuroanatomical specificity to which production of the particular hormone is restricted; and that both are anatomically coincidental to each other. As such, the present report suggests that pathogens with a high degree of exclusivity to the gastrointestinal tract have evolved response systems to neuroendocrine hormones such as norepinephrine and dopamine, but not epinephrine, which are found with the enteric nervous system.

Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China

BackgroundWe are profoundly ignorant about the diversity of viruses that infect the domain Archaea. Less than 100 have been identified and described and very few of these have had their genomic sequences determined. Here we report the genomic sequence of a previously undescribed archaeal virus.ResultsHaloarchaeal strains with 16S rRNA gene sequences 98% identical to Halorubrum saccharovorum were isolated from a hypersaline lake in Inner Mongolia. Two lytic viruses infecting these were isolated from the lake water. The BJ1 virus is described in this paper. It has an icosahedral head and tail morphology and most likely a linear double stranded DNA genome exhibiting terminal redundancy. Its genome sequence has 42,271 base pairs with a GC content of ~65 mol%. The genome of BJ1 is predicted to encode 70 ORFs, including one for a tRNA. Fifty of the seventy ORFs had no identity to data base entries; twenty showed sequence identity matches to archaeal viruses and to haloarchaea. ORFs possibly coding for an origin of replication complex, integrase, helicase and structural capsid proteins were identified. Evidence for viral integration was obtained.ConclusionThe virus described here has a very low sequence identity to any previously described virus. Fifty of the seventy ORFs could not be annotated in any way based on amino acid identities with sequences already present in the databases. Determining functions for ORFs such as these is probably easier using a simple virus as a model system.