The gut microbiota encompasses all the microorganisms which colonise the gut. There is in excess of four hundred bacterial species contained in the gut (Gorbach, 1996) Gram-negative anaerobes including Bacteroides, Prevotella, Porphyromonas and Fusobacterium are linked to a protective role of the human microbiota (Salyers and Shoemaker, 2009). Moreover, gram-negative anaerobes are consummate opportunists, and one example of this opportunism is the ability to resist the damaging effects and toxicity of bile.
Bile salts are bio-surfactants which perform two main physiological functions in the body. Firstly, they play a vital role in both the digestion and the absorption of nutrients. Secondly, bile salts facilitate the excretion of numerous waste products from the blood. Additionally, the bile-mediated solubilisation of fat-soluble nutrients allows for transport to the small intestine (Maldonado-Valderrama et al., 2011).
Bile acids can also prevent bacterial overgrowth in the small gut. The Farnesoid X Receptor (FXR) is a steroid/thyroid hormone receptor, activated by bile acids such as cholate and chenodeoxycholate, which regulates several proteins with well-established antimicrobial activity. Angiogenin, for example, is involved in the acute phase response to infection. Inagaki et al. demonstrated that FXR knockout mice had increased quantities of bacteria in the ileum (Inagaki et al., 2006)
Bile salts can be toxic to unadapted microorganisms. The lipophilic steroid ring of bile acids targets the bacterial cell membrane and can disturb the lipid packaging and dissipate the proton motive force (Kurdi et al., 2006). Passive diffusion of unconjugated bile acids, which act as weak acids results in the cytoplasm becoming acidified which can result in cell death Bile toxicity can also precipitate changes in sugar metabolism and protein folding (Ruiz et al., 2013).
Increasing bile concentrations along the gastrointestinal tract is coupled with increased proportions of gram-positive bacteria (Ridlon et al., 2014) Thus far, research has predominantly focused on the mechanisms underpinning bile resistance in gram-positive bacteria. Counteracting oxidative and acid stresses, the use of efflux systems and bile modification via bile salt hydrolases are among the mechanisms which have been studied in bacterial groups such as Bifidobacterium and Lactobacillus (Ruiz et al., 2013). In this study, we will focus on the resistance of gram-negative bacteria to bile, a less explored area.
Mechanisms of bile resistance
The PhoPQ system is utilised by some bacteria to sense/identify a bile-rich environment. PhoPQ is a two-component system composed of the PhoQ protein which performs its function as a sensor for extracytoplasmatic Mg2+ and Ca2+ which subsequently controls the activity of PhoP, the response regulator. Bacteria utilise the PhoPQ system to determine its subcellular location, thus enabling the immediate activation of factors and pathways to ensure survival in the adverse conditions (Monsieurs et al., 2005). In a study carried out by Van Delkinburgh et al. a mutant PhoP– Salmonella typhimurium strain proved to be threefold more bile sensitive than a wild-type strain (van Velkinburgh and Gunn, 1999).
Bile resistance is dependent on the integrity of the bacterial outer membrane. The functions of the Tol-Pal system and Dam methylase are critical in maintaining membrane stability. The Tol-Pal system has been studied in a variety of gram-negative bacteria such as Escherichia Coli, Haemophilus ducreyi, S. enterica and Vibrio cholerae (Dubuisson et al., 2005). It is composed of five proteins which can connect the outer membrane to the peptidoglycan and inner membrane layers (Gerding et al., 2007). TolQRA is involved in activation of cps facilitating capsule synthesis in order to respond to perceived membrane damage (Prouty et al., 2002). Mutations to any of the Tol-Pal genes induces disruption in membrane stability and integrity thus rendering the bacterium more sensitive to bile salts (Dubuisson et al., 2005). Moreover, Dam methylase, which functions to methylate adenosine residues that are embedded in 5’-GATC-3’ sites, can alter protein binding. Pucciarelli illustrated that a lack of Dam methylase altered the association of envelope proteins to peptidoglycan. The study also demonstrated that S. enterica Typhimurium strains with Dam methylase were resistant to 1% deoxycholate while the bacterial strains devoid of Dam methylase were sensitive. (Pucciarelli et al., 2002)
Deconjugation via bile salt hydrolases is a mechanism by which some enteric bacteria can modify bile acids. It is believed to supply cellular carbon, nitrogen and sulphur for the bacteria. Taurine is also produced which can be utilised as an energy source by some microbes. Deconjugation involves the enzymatic hydrolysis of the C-24 N-acyl amide bond which links bile acids to their amino acid conjugates (Ridlon et al., 2006).
Although bile salt hydrolases have been studied mostly in gram-positive bacteria, Kawamoto et al. identified a BSH from Bacteroides vulgatus and named it chenodeoxycholyltaurine hydrolase. This BSH illustrated specificity for taurine conjugates (Kawamoto et al., 1989).
Several enteric species such as Bacteroides and E. coli can perform epimerisation and oxidation of hydroxy groups at positions C3, C7 and C12 of bile salts generating iso-bile salts (Urdaneta and Casadesús, 2017). This process functions to detoxify the bile acid for the bacterium. The epimerisation of the 7-alpha-hydroxy group of a bile acid, for example, acts to decrease the toxicity of chenodeoxycholic acid for gut microbiota (Ridlon et al., 2016).
Repair of bile-induced damage
There are many strategies employed to repair the damage induced by bile. One example of such damage is the DNA lesions induced. The SOS response system is an inducible network composed of over 40 genes involved in mechanisms to tolerate DNA damage. Once activated, this system can rescue cells in the cases of severe DNA damage, such as that caused by oxidative stress. Rodriguez- Beltran et al. studied the implications of one of these genes in bile tolerance in E. coli. dinF encodes a polypeptide sequence with a high degree of homology to MATE (multidrug and toxic compound extrusion) membrane proteins. Expression of dinF was found to increase the minimum inhibitory concentration (MIC) of bile salts. Competition assays were employed to compare wild-type E. coli strains against strains with mutated dinF. Fitness tests presented a 50% difference between the mutant and the WT in 4% bile salts.
It was also shown that cells lacking DinF had a higher susceptibility to hydrogen peroxide-mediated killing and a significantly decreased mutation rate (Rodriguez-Beltran et al., 2012).
Prieto et al. investigated the repair of bile=induced DNA oxidative damage in S. enterica with regard to other mechanisms of repairing DNA oxidative damage induced by bile. Exonuclease III and Endonuclease IV are needed for base excision repair in the correction of oxidative damage. In this study, mutants devoid of exonuclease III and endonuclease IV were found to be bile sensitive further highlighting the requirement for base excision repair in S. enterica’s resistance to bile (Prieto et al., 2006)
S. enterica also utilises homologous recombination to correct the oxidative damage. S.enterica employs a recombination repair pathway, RecBCD pathway. Double-strand breaks result in the activation of this pathway (Merritt and Donaldson, 2009) The induction of the SOS response, which aids survival in a bile rich environment, blocks DNA replication, emphasising the requirement for homologous recombination with pathways such as RecBCD. (Urdaneta and Casadesús, 2017
A recent study by Gourley et al. demonstrated the role of the AddAB DNA repair system in bile resistance in C. jejuni. Although expression of the addA and addB genes was not shown to be upregulated in the presence of deoxycholate, there was constitutive expression of these genes in the presence of the bile salts. As aforementioned RecBCD is utilised by E. coli for recombination repair. Gourley demonstrated that upon transformation of addAand addB with an E. coli strain without recBCD genes, AddAB performed the same function as RecBCD in repairing double-stranded breaks. The contribution of AddAB to bile tolerance in C.jejuni was directly demonstrated when an addAB– mutant was transformed with wild-type addAB genes, restoring the deoxycholate resistance(Gourley et al., 2017)
Link between bile tolerance and antibiotic resistance
Efflux pumps can play a key role in exporting bile from host bile-tolerant bacteria. These pumps, specifically multi-drug resistance (MDR) efflux pumps can prevent the entry or facilitate the export of antibiotics (Piddock, 2006).
In some cases, MDR pumps present in pathogenic bacteria and can aid the establishment of persistent infection and complicate the choice of appropriate antimicrobial therapy. Campylobacter jejuni, which is the most prevalent causative agent of bacterial gastroenteritis in first world countries, possesses a Resistance-Nodulation-cell Division (RND) family efflux pump termed CmeABC. Ciprofloxacin and erythromycin which are both commonly prescribed if C.jejuni infection is diagnosed, are also substrates of the CmeABC pump. Lin et al. demonstrated that overexpression of cmeB alone confers resistance to these two antibiotics along with ampicillin, chloramphenicol and tetracycline.(Lin et al., 2002)
Bile is also a substrate for the MDR AcrAB-TolC. Both S.enterica typhimurium and E.coli possess this pump. The transporter AcrAB has been comprehensively studied and has been shown to export chloramphenicol, tetracyclines, macrolides, B- lactams and fluoroquinolones (Blanco et al., 2016)
Prouty et al. demonstrated that marRAB operon induces both bile resistance and antibiotic resistance in S. enterica typhimurium. Its activity is regulated in a concentration-dependent manner by bile. (Prouty et al., 2004) When activated by bile, this operon can confer increased resistance to tetracycline, ampicillin and chloramphenicol (Kunonga et al., 2000)
Vibrio parahaemolyticus is a principal causative agent of food poisoning. As this bacterium is ingested, it comes into contact with several toxic compounds including bile. Matsuo characterised several multi-drug efflux transporters belonging to the RND family (Matsuo et al., 2013) When one of these transporters VmeAB was cloned into E.coli, there were significant increases in MIC values in a variety of antibiotics. For example, there was a 60-fold increase in MIC for novobiocin and a 4-fold increase for erythromycin. The MIC for sodium deoxycholate was also at least 30 times higher with the vmeAB mutant also, indicating the critical role the transporter plays in bile resistance. (Matsuo et al., 2007)
Acquiring more information about MDR efflux pumps and their mechanism of action could be advantageous for the treatment of gastrointestinal infections. Phenylalanine-arginine β-naphthylamide (PAβN) is currently in clinical use in combination with fluoroquinolones. It acts as a competitive inhibitor of the antibiotic, minimising the expulsion of the antibiotic from the cell. This facilitates the antibiotic to rise in intracellular concentration and act unimpededly within the bacterium. (Tsai et al., 2012)
Bile resistance and increased virulence
Numerous enteric pathogens can not only tolerate the presence of bile but can utilise it as a signal to regulate virulence gene expression. This enhanced virulence can facilitate both colonisation and maintenance of infection in the human gastrointestinal tract (Sistrunk et al., 2016) For example, following bile salt exposure Shigella flexneri, had increased adherence to colonic epithelium. Faherty et al. focused specifically on outer surface proteins E1(OSP E1) and E2. Upon induction by bile salts, these two proteins become localised to the outer membrane of the bacterium thus improving adhesion to colonic epithelial cells. This study also evaluated protein secretion by a plasmid-encoded Type III secretion system possessed by S. flexneri. It was shown that the level of secretion was increased in the presence of bile salts (Faherty et al., 2012)
Campylobacter jejuni synthesises and secretes proteins to ensure effective invasion of host epithelial cells. These proteins are referred to as Campylobacter invasion antigens or Cia proteins (Malik-Kale et al., 2008) Rivera- Amill deduced that the presence of physiological concentrations of bile was a necessary stimulus for the production of these proteins. Insertional disruption of one gene encoding a Cia protein namely ciaB renders the C. jejuni non-invasive (Rivera-Amill et al., 2001) This highlights the importance of these proteins, and in turn, the role bile plays in the virulence of C. jejuni.
Bacteroides fragilis is the most common anaerobic bacterial species isolated from intestinal tract infections. The impact of bile salts on B. fragilis cells and their behaviour was analysed in a study by Pumbwe. On exposure to 0.15% non-conjugated bile salts, the cells produced many appendages similar to fimbria which projected out from the surface of the cell. This appendage enabled adherence of the bacterium to the substrate and to neighbouring cells. It was deduced that these fimbriae are likely to exhibit haemagglutinin function, the activity of a sialidase and are also likely to carry endotoxin (Pumbwe et al., 2007)
Deoxycholate is a secondary bile acid produced from primary/free bile salts via reductive 7-alpha-dehydroxylation (Philipp, 2011) Payne evaluated the possible role of deoxycholate as an aetiological agent of colon cancer. In this study, the results indicated that deoxycholate acts as an inducer of the autophagic pathway via an oxidative nitrosative mechanism (Payne et al., 2009)
Biliary tract disease is predominantly associated with bacterial infection. E. coli and Klebsiella are among two of the most common causes of hospital-acquired cholangitis. Colonisation and infection by enteric bacteria frequently follow the blockage of the bile duct. As a part of a study of E. coli and choledocholithiasis bile resistance of 100 patient samples was quantified. Every E. coli strain survived in 1% and 3% deoxycholate (Razaghi et al., 2017) It is evident in the case of choledocholithiasis that bile tolerance acts as a vital virulence factor.
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