E Coli: Pathogenic Strains and Regulators

E. coli Commensal

Escherichia coli (E. coli) is a Gram-negative, facultative anaerobic, rod-shaped bacterium of the genus Escherichia (Tenaillon et al. 2010).. It is commonly found in the lower intestine of warm-blooded organisms (Secher T 2016). E. coli is an extremely diverse bacterial species which forms part of the gut microbiome, a term which describes the ecological community of commensal, symbiotic and pathogenic microorganisms found in the intestine (Bull M J et al., 2014). Throughout life, the population of E. coli usually settles around 107–108 colony-forming units per gram of faeces (Secher T 2016). The E. coli population of the intestines usually includes a set of durable core strains, as well as temporary transients that vary with health, nutrition, infection and antibiotic exposure (Blount 2015). E. coli grows in the thin mucous layer that lines the gut where it competes with many other microorganisms for nutrients. This results in it being a non-fastidious organism, with a wide-ranging diet (Blount 2015). E. coli is the primary aerobic microorganism in the gastrointestinal tract (Tenaillon et al. 2010). As E. coli is a facultative anaerobe it is able to optimise growth based on the availability of oxygen. It is able to utilise both anaerobic and microaerobic respiration in order to survive in the low oxygen environment in the intestine (Tenaillon O 2010). Although E. coli competes with other microorganisms for resources such as surface area and nutrition, it is suggested to also share a mutualistic relationship with some. It aids anaerobic commensals in the gut by consuming any oxygen that may enter the gut, helping the maintain an anaerobic environment. In exchange, it is believed that E. coli may benefit from the breakdown of mucosal polysaccharides and dietary fibres by obligate anaerobes in the intestines (Tenaillon O 2010). E. coli is one of the first commensal microorganisms that the human infant gains exposure to. This is thought to occur via exposure to maternal faecal microbiota during childbirth. Interestingly, this exposure appears to be reducing in industrialised countries due to more stringent hospital hygiene standards and an increase in the number of caesarean sections being performed (Nowrouzian et al. 2003). Although the relationship between E. coli and the host is often considered to be commensal in which one party notably benefits and the other is neither helped nor harmed, there is some evidence that E. coli provides some succour to the host in the form of the production of vitamins K and B12, as well as the competitive exclusion of pathogens from the gut (Katouli 2010) (Hudault, Guignot and Servin 2001). In return, the human intestinal tract provides E. coli with a steady supply of carbon and energy sources, a comfortable environment with a moderate pH and temperature as well as protection against certain stresses and transport and dissemination facilities.

E. coli Pathogenic

However, E. coli is not entirely harmless, and some strains comprise several major foodborne pathogens. E. coli is an organism that frequently crosses the line between commensalism and pathogenicity (Leimbach, Hacker and Dobrindt 2013). This is due in part to its highly flexible genome. E. coli carries between 4.5 and 5.5 million base pairs of DNA but fewer than half of all genes encoded are conserved among all members of this species (Figler and Dudley 2016). Sequencing information estimates that the core genome of E. coli comprises less than 20% of the more than 16,000 genes in the E. coli pan-genome (Blount 2015). As E. coli is able to acquire genetic variation through horizontal gene transfer, it is enabled to adapt to particular niches, improve its metabolic capacity and assimilate virulence factors (Blount 2015). This gives rise to the numerous pathotypes of E. coli which pose a substantial threat to both human health and the economy. Per year it is estimated that E. coli infections cause 2 million deaths in humans (Russo and Johnson 2003). E. coli is conventionally serotyped based on three types of antigens; somatic (O), capsular (K) and flagellar (H) (Figler and Dudley 2016). E. coli O157 and O104 are among the most widespread foodborne pathogens.

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There are five major foodborne E. coli pathotypes. These are Enteroaggregative E. coli, (EAEC), Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterotoxigenic E. coli (ETEC) and Enterohemorrhagic E. coli (EHEC), (Yang et al. 2017). Pathogenic E. coli infection usually causes severe diarrhoea. Other symptoms include abdominal pain, nausea, emesis and fever (Yang et al. 2017). Strains such as EHEC and EAEC can cause haemolytic uremic syndrome a condition characterised by thrombocytopenia, microangiopathic haemolytic anaemia and acute renal failure (Naylor et al. 2003). E. coli can also cause extraintestinal diseases such as urinary tract infections, neonatal meningitis and sepsis (Poolman and Wacker 2016).

Pathogenic E. coli have a number of virulence mechanisms available to them. The most significant of these is known as the locus of enterocyte effacement (LEE) and is comprised of a cluster of virulence genes on the chromosomal pathogenicity island (PAI) (Yang et al. 2017). This locus encodes a type III secretion system. The type III secretion system transports virulence factors to the host epithelial cells. The pathogenic mechanism of E. coli is characterised by attachment and effacement of the host epithelium (Gaytan et al. 2016).

In order to affect this pathogenicity in the human, however, E. coli has a long journey of survival that involves the utilisation of a vast number of attributes, all of which are controlled by its highly active genome. The first barrier to infection in the human is survival outside of the human.

E. coli Environmental

One of the factors which make certain E. coli strains such important and formidable pathogens is the ability to survive in extraneous environments. As a commensal intestinal lodger, E. coli by design must regularly enter the outside environment in faecal waste discharged by humans and animals. The extensive dispersal of E. coli in the environment is exacerbated through human activity in the form of fertiliser made from animal wastes, wastewater from abattoirs and factory farms and effluent from water treatment plants and sewage schemes (Ishii and Sadowsky 2008). Although it was previously believed that E. coli did not survive well outside of the host, recent studies have refuted this and have shown that it can persist for long periods of independent of its host and may indeed become part of the indigenous flora of its new environment, a process called naturalisation (Jang et al. 2017). However, the external environment is much less stable than the mammalian host and there is huge variation in factors such as nutrition, temperature, oxygen, moisture, pH, and/or the surrounding microbial community (Blount 2015). E. coli has been able to acquire certain features which enable it to survive in such unforgiving environments. One such adaptation is the development of persister variants, which are metabolically inactive dormant versions of the E. coli strain (Shah et al. 2006). Although most E. coli strains are non-pathogenic, there do exist strains with the potential to cause significant harm to the human host. Many of these are acquired from environmental sources, either through the ingestion of unsanitary water, colonised vegetables or contaminated meat. E. coli is known to colonize leafy green vegetables such as spinach and lettuce, causing food poisoning outbreaks such as the outbreak of E. coli O157:H7 in 2006 linked with spinach and the outbreak of STEC O104 in 2011 associated with fenugreek seeds. The most recent outbreak which occurred in late 2018 was of STEC O157:H7 and was linked to romaine lettuce. Comment by Siobhan Ladden: https://www.cdc.gov/ecoli/2018/o157h7-11-18/index.html

Pathogenic E. coli colonising the human from an outside source must first overcome barriers mounted both explicitly and ambiguously by the human host. Before it reaches the intestine, where it thrives and proliferates it must first survive the passage through the upper and mid gastrointestinal tract.

During its passage to the intestinal tract, E. coli must first pass through extremely acidic conditions in the stomach. In order to do so intact, it must survive an extremely low pH of between 1.5 and 3.0.(Kanjee and Houry 2013). Imperative to E. coli’s survival in the stomach is its outer membrane. This is the first barrier the acid must breach in order to cause injury to the organism. The membrane of E. coli is highly adaptive and can respond to acid stress in a number of ways. E. coli can change the composition its membranes in order to prevent the entry of damaging acid. E. coli achieves this by increasing the concentration of cyclopropane fatty acids and reducing the concentration of unsaturated lipids. Additionally, the entry of acid may be decreased by obstruction of the outer membrane porins (OMPs) through the binding of polyphosphate to the OMPs. In the periplasm, the chaperone proteins HdeA and HdeB protect substrate proteins from aggregation and upon return to a neutral pH aid in substrate refolding (Ding et al. 2015). In the cytoplasm, the Hsp31 chaperone binds to and stabilizes unfolded intermediates until the stress is alleviated and then allows the proteins to refold either naturally or through ATP-dependent chaperone systems (Ding et al. 2015). Additionally, an amino acid decarboxylase system helps maintain pH homeostasis in the cytoplasm. This consists of a cytoplasmic decarboxylase, which converts its substrate into a related amine and an antiporter, which exchanges the imported amino acid for the cytoplasmic amine produced (Kern et al. 2007). Protection of DNA under acid stress is handled by the DNA-binding Dps (DNA-binding protein from starved cells) protein which binds to and protects DNA (Ding et al. 2015).

The survival of intestinal bacteria such as E. coli is reliant on its ability to survive not only the acidic environment of the stomach but also the high concentration of up to 30mM of bile salts in the human intestinal tract. It has been speculated that the pathogenicity of such enteric bacteria is dependent on its ability to survive and grow in the presence of bile salts. Bile is a fluid manufactured in the liver and stored in the gallbladder. In humans, primary bile acids consist of cholate and chenodeoxycholate. Later, the acids are conjugated to either glycine or taurine via amide bonds for secretion (Thanassi, Cheng and Nikaido 1997). Bile acids function in the emulsion of lipids to aid digestion. As well as in digestion, bile salts aid in the body’s defence against microbial pathogens. This is illustrated by the fact that the small intestine, which maintains high concentrations of bile acids, typically contains very scarce bacterial populations (Merritt and Donaldson 2009). Unconjugated bile salts can traverse both the outer and inner membranes of Gram-negative bacteria and accrue in the cell cytoplasm. Here, bile salts may destroy bacteria through a number of mechanisms, namely the disruption of cell membrane integrity, oxidative stress, DNA damage, upregulation of RNA secondary structure formation, and denaturation of cellular proteins. Central to the resistance of E. coli to bile are two important multidrug efflux systems AcrAB-TolC and EmrAB-TolC (Wang et al. 2017). Efflux pumps such as these are mechanisms which appear to remove toxic bile salts from the cytoplasm after they have penetrated the cell membrane. Other contributors to bile resistance include the protein YdhE of the multidrug and toxic compound extrusion (MATE) family, and the YdgEF small multidrug resistance (SMR) protein (Paul et al. 2014). A 2014 study proposed that a small multidrug resistance transporter called MdtM promotes persistence of E. coli in bile salts through the catalysis of secondary active transport of bile salts out of the cell cytoplasm. A functional cooperation between this proposed system and the AcrAB-TolC system discussed above results in an enhanced resistance of E. coli to bile (Paul et al. 2014)

E. coli Transcriptional Regulators

The initial step in gene expression occurs when particular segment of DNA is copied into RNA in order to be translated into a protein sequence and is known as transcription. Transcriptional regulators help to control the expression of certain genes involved in this process. Regulators can work by either activating or repressing single or operonic genes. Transcription regulators allow the RNA polymerase access to promotors which allows for response to environmental changes. This can happen by positive or negative regulation (Flores-Bautista et al. 2018). It is estimated that less than a tenth of all genes in E. coli function as transcription regulators by directly binding to DNA, though other proteins may indirectly participate in regulation of transcription (Perez-Rueda and Collado-Vides 2000).

E. coli has a large number of regulatory genes, due to its ability to adapt to a wide-ranging group of environments. This is important as it needs to be able to survive both inside the mammalian gut and in the extraneous environment. This helps to shape it as a significant pathogen, as it can survive the journey from one mammal to another, via soil and in turn food. It must be able to survive under a diverse range of environments with a huge range in temperature, pH and osmolality.

The most common type of transcriptional regulator in the prokaryotes are the LsyR type transcriptional regulators (LTTRs)(Maddocks and Oyston 2008). They were first identified by Henikoff et al in the 1980s and are believed to include over 40,000 members (Knapp and Hu 2010). As the LTTR family comprises such a large membership, it is no surprise that the genes it regulates encompass a vast number of functions. Some of these include genes involved in virulence, metabolism, transport, detoxification and motility. LTTRs are also highly involved in the biosynthesis of amino acid pathways (Perez-Rueda and Collado-Vides 2000). In E. coli some well-characterised LTTRs include ArgP which regulates arginine transport (Nandineni and Gowrishankar 2004), CynR which regulates cyanate detoxification, (Sung and Fuchs 1992), CysB which regulates Cysteine biosynthesis (van der Ploeg et al. 1997), LrhA which functions in motility and chemotaxis (Lehnen et al. 2002), LysR which regulates lysine biosynthesis (Stragier and Patte 1983), OxyR, involved in the oxidative stress response to H2O2 (Farr and Kogoma 1991) and QseA involved in quorum sensing (Sperandio, Torres and Kaper 2002).

LTTRs are highly structurally conserved and most comprise of about 330 amino acids. They contain a co-factor binding domain at the C terminus and a helix-turn-helix (HTH) motif at the N terminus. These domains afford a means of binding to DNA. The LTTRs make up a unique group of HTH-containing transcriptional regulators, as the HTH is situated 20-90 amino acids from the N terminus irrespective of whether it is activating or repressing transcription. By contrast, the HTH is situated at the C terminus in transcriptional activators and at the N terminus in transcriptional repressors (Maddocks and Oyston 2008). For this reason, LTTRs are known as dual regulators (Maddocks and Oyston 2008).

E. coli Y genes

E. coli is one of the most well-characterised microorganisms, due to its central role as a model organism in microbiology and molecular biology (Blount 2015). However, the function of many of its genes remains unknown. Less than 67% of E. coli’s protein-encoding genes appear with a recognised function in the HAMAP (High-quality Automated and Manual Annotation of Proteins) database (Pedruzzi et al. 2015). A 2018 study reported that 1563 of 4653 unique E. coli genes lack direct experimental evidence of function (Ghatak et al. 2019). A total of 131 of these have absolutely no evidence of function. An additional 304 of these genes (6.6%) are pseudogenes or phantom genes ((Flores-Bautista et al. 2018, Ghatak et al. 2019). Traditionally, genes which lack an annotated function have been referred to as “y-genes”. The lack of experimentally characterised function of so many genes is in part due to the recent explosion in protein sequencing technology and bioinformatics. The increase in biological data has led to a huge backlog of sequenced genes for which the function has not yet been elucidated experimentally. Many genes have hypothetical functions, derived by comparing sequences and structures between proteins with experimentally characterised functions and proteins with only theoretical functions (Flores-Bautista et al. 2018).

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