Chapter Five - The function, biogenesis and regulation of the electron transport chains in Campylobacter jejuni: New insights into the bioenergetics of a major food-borne pathogen

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Abstract

Campylobacter jejuni is a zoonotic Epsilonproteobacterium that grows in the gastrointestinal tract of birds and mammals, and is the most frequent cause of food-borne bacterial gastroenteritis worldwide. As an oxygen-sensitive microaerophile, C. jejuni has to survive high environmental oxygen tensions, adapt to oxygen limitation in the host intestine and resist host oxidative attack. Despite its small genome size, C. jejuni is a versatile and metabolically active pathogen, with a complex and highly branched set of respiratory chains allowing the use of a wide range of electron donors and alternative electron acceptors in addition to oxygen, including fumarate, nitrate, nitrite, tetrathionate and N- or S-oxides. Several novel enzymes participate in these electron transport chains, including a tungsten containing formate dehydrogenase, a Complex I that uses flavodoxin and not NADH, a periplasmic facing fumarate reductase and a cytochrome c tetrathionate reductase. This review presents an updated description of the composition and bioenergetics of these various respiratory chains as they are currently understood, including recent work that gives new insights into energy conservation during electron transport to various alternative electron acceptors. The regulation of synthesis and assembly of the electron transport chains is also discussed. A deeper appreciation of the unique features of the respiratory systems of C. jejuni may be helpful in informing strategies to control this important pathogen.

Introduction

Campylobacteriosis is the commonest food-borne bacterial zoonosis in many countries around the world, with laboratory confirmed cases in the UK alone running at ∼60,000 in 2016 (Public Health England, 2018) but with the true community incidence estimated to be about ten times this level. The result is a huge public health and economic burden. The major causative agent is C. jejuni with fewer but still significant numbers of cases caused by Campylobacter coli. C. jejuni is divided into two subspecies: C. jejuni subsp. jejuni, and C. jejuni subsp. doylei, which are often distinguished phenotypically by the inability of the latter to reduce nitrate to nitrite (Miller, Parker, Heath, & Lastovica, 2007). Campylobacters are most commonly associated with a range of wild and domesticated bird species, where they form part of the intestinal microbiota. Undercooked chicken is the major source of human campylobacteriosis in most countries; in the UK for example, 60–80% of cases are attributable to this source (Sheppard et al., 2009). However, Campylobacters also colonize other farm animals and can be isolated from the environment (e.g. water and soil). The population structure is complex, with both host/source-specific and generalist strains capable of colonizing a variety of niches. In humans, C. jejuni is an intestinal mucosal pathogen that causes an acute bloody diarrhea, severe abdominal cramps and fever, with ∼0.1% of infections leading to transient paralysis (and sometimes death) due to inflammatory neuropathies such as Guillain-Barré syndrome and Miller-Fisher syndrome (Jacobs, van Belkum, & Endtz, 2008).

In chickens, the highest counts (up to ∼109 CFU g1) are found in the caeca where the bacteria can persist for long periods. The caeca is emptied daily and ingestion of excreted caecal material from infected birds is a major mechanism of transmission within flocks. In addition, antimicrobial resistance is a major problem in Campylobacters with >50% of human chicken and other livestock isolates being ciprofloxacin resistant (Sproston, Wimalarathna, & Sheppard, 2018). Accordingly, antibiotic resistant C. jejuni has been added to the WHO list of priority organisms for developing new antimicrobial agents. As there are currently no licensed vaccines or colonisation-resistant chickens available, control strategies have focused on reducing Campylobacter levels in poultry (e.g. by phage treatment, dietary interventions) or contamination on carcasses (e.g. by chemical, physical or phage treatment) with variable success and consumer acceptability. It is clear that new insights into C. jejuni biology are needed if control measures are to be put in place that more effectively reduce chicken colonization and/or food-chain contamination.

C. jejuni is a microaerophilic bacterium, which implies that although requiring oxygen for growth, it is unable to grow at normal atmospheric oxygen tensions (Krieg & Hoffman, 1986). This interesting physiological characteristic is arguably the key, defining feature of its biology and is an adaptation for growth at low oxygen concentrations in the host. Indeed, C. jejuni preferentially colonizes the mucus layer and the intestinal crypts close to the epithelium, where the oxygen concentration will be higher than in the intestinal lumen. Most strains of C. jejuni grow optimally at 3%–10% (v/v) oxygen, with growth inhibited under either atmospheric oxygen levels or strict anaerobiosis. A possible explanation for the latter is the requirement of low amounts of oxygen for deoxyribonucleotide (and thus DNA) synthesis, which is catalyzed by a single “aerobic-type” class I ribonucleotide reductase in C. jejuni (NrdAB-type RNR; Sellars, Hall, & Kelly, 2002).

Microaerophily might result from a deficiency in oxidative stress defences. Non-specific electron transfer from the respiratory chain to oxygen occurs in all oxygen-dependent bacteria (Cabiscol, Tamarit, & Ros, 2000). The stepwise one-electron reduction of O2 results in the formation of reactive oxygen species (ROS) such as the superoxide radical (O2), hydrogen peroxide (H2O2) and the hydroxyl radical (HO.), which damage cellular targets including DNA, proteins and membranes. ROS must be constantly removed to allow continued growth. ROS production also occurs in the host immune system in response to bacterial infection. Interestingly, C. jejuni strains express a variety of ROS-detoxifying enzymes (Flint, Stintzi, & Saraiva, 2016) including the superoxide dismutase SodB (Cj0169; Pesci, Cottle, & Pickett, 1994), the alkyl hydroxide reductase AhpC (Cj0334; Baillon, van Vliet, Ketley, Constantinidou, & Penn, 1999), the catalase KatA (Cj1385; Day, Sajecki, Pitts, & Joens, 2000), the thiol peroxidases Tpx (Cj0779; Atack, Harvey, Jones, & Kelly, 2008), the bacterioferritin comigratory protein Bcp (Cj0271; Atack et al., 2008), two cytochrome c peroxidases (Cj0020c and Cj0358; Bingham-Ramos & Hendrixson, 2008), and the methionine sulfoxide reductases MsrA and MsrB (Cj0637 and Cj1112; Atack & Kelly, 2008). However, comparisons of superoxide production in aerobes like Escherichia coli with anaerobic bacteria like Bacteroides, do indicate a higher level of production of ROS under oxygen stress in the latter bacteria, possibly due to lower levels of expression of Sod (Imlay, 2018). This may also apply to microaerophiles like C. jejuni, but has been little investigated.

Another reason for the microaerophilic nature of C. jejuni seems to be the possession of several inherently ROS and oxygen-sensitive metabolic enzyme systems that are essential for growth. The evidence for this is most clear in the case of the citric-acid cycle. The available genome sequences indicate that C. jejuni strains possess all of the usual enzymes that are necessary for operating a complete oxidative cycle (Parkhill et al., 2000). However, a key distinctive feature is that the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are absent, but are replaced by two related oxidoreductases normally found in anaerobic bacteria. The pyruvate: acceptor oxidoreductase (Por; encoded by cj1476c) is responsible for the oxidative decarboxylation of pyruvate to acetyl-CoA and the 2-oxoglutarate: acceptor oxidoreductase (Oor; encoded by oorDABC, cj0535 to cj0538) catalyses the analogous conversion of 2-oxoglutarate to succinyl-CoA. These are both crucial reactions in central carbon metabolism; Por seems to be an essential enzyme for viability based on an inability to isolate mutants in the cj1476c gene, most likely because this is the only mechanism to synthesize acetyl-CoA (Kendall et al., 2014, Weerakoon and Olson, 2008). Both Por and Oor contain iron-sulphur (Fe-S) clusters that are very sensitive to inactivation by ROS; in intact cells of C. jejuni exposed to atmospheric oxygen levels, their activity has been shown to decline rapidly (Kendall et al., 2014). Interestingly, mutant studies suggest that the hemerythrin proteins HerA (Cj0241c) and HerB (Cj1224) may help to partially protect Por and Oor in some way from oxygen damage, as their activities are lower in the cognate mutants, but once damaged through exposure to atmospheric oxygen concentrations, repair of Por and Oor activity seems to be inefficient (Kendall et al., 2014). The electron acceptor for Oor and probably Por is the sole flavodoxin encoded in the genome, FldA (Kendall et al., 2014, Weerakoon and Olson, 2008). The use of an easily autooxidisable flavodoxin as an electron carrier may also contribute to an inability to grow at high ambient oxygen levels. This not only impacts respiratory electron transport, but also other metabolic processes requiring reduced flavodoxin. For example, FldA is essential as an electron donor in the methyl erythritol phosphate (MEP) pathway of isoprenoid biosynthesis that is present in this bacterium (Frank & Groll, 2017); isoprenoids are necessary for building peptidoglycan and also the respiratory quinones. Thus, we suggest that a potential for higher ROS production during growth with oxygen, along with ROS labile enzyme targets and a reliance on flavodoxin as an essential cytoplasmic electron carrier are major factors that combine to produce the microaerophilic growth phenotype of C. jejuni.

Section snippets

Carbon and electron sources for C. jejuni growth

Until relatively recently, there has been a consensus view that Campylobacter isolates are unable to catabolize sugars, especially glucose, due to the lack of glucokinase (Glk) and phosphofructokinase (PfkA) of the classical Embden-Meyerhof-Parnas (EMPs) glycolysis pathway (Parkhill et al., 2000, Velayudhan and Kelly, 2002). However, it was shown that l-fucose can be utilized as carbon and energy source in about 30%–50% of C. jejuni strains (Muraoka and Zhang, 2011, Stahl et al., 2011). More

Overview

C. jejuni strains can carry out substrate-level phosphorylation but are unable to grow by a purely fermentative metabolism, therefore growth must occur by electron-transport linked oxidative phosphorylation. Bacterial electron transport chains are enormously diverse in composition and function, but there are several crucial factors that govern their roles; (i) the degree of branching at both dehydrogenase and reductase ends of the chain determines the diversity of electron donors and acceptors

Overview

In the past decade, advances in transcriptomic technology and availability has generated a wealth of information on the regulatory responses of C. jejuni. Recent transcriptomic studies on growth phase transitions, colonization, exposure to host extracts, phage infection, nutrient availability, exposure to host signaling molecules and antibiotics, oxygen availability and others, all show statistically significant alterations in the expression of an often overlapping set of metabolism and

Acknowledgments

We wish to acknowledge the contributions of current and previous members of the Kelly laboratory, especially Dr. Edward Guccione, Dr. Andrew Hitchcock and Dr. Yang-Wei Liu, who have worked on several projects directly related to electron transport in C. jejuni. We also acknowledge the contributions of our collaborators in the UK and internationally. Work in the author's laboratory has been supported by grants and studentships from the UK Biotechnology and Biological Sciences Research Council

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