METABOLIC AND PHYSIOLOGICAL DETERMINANTS IN LISTERIA
MONOCYTOGENES ANAEROBIC VIRULENCE REGULATION
Dissertation
Submitted to
The College of Arts and Sciences of the
UNIVERSITY OF DAYTON
In Partial Fulfillment of the Requirements for
The Degree of
Doctor of Philosophy in Biology
By
Nathan Christopher Wallace
Dayton, OH
December 2018
METABOLIC AND PHYSIOLOGICAL DETERMINANTS IN LISTERIA
MONOCYTOGENES ANAEROBIC VIRULENCE REGULATION
Name: Wallace, Nathan Christopher
APPROVED BY:
Yvonne Sun, Ph.D. Faculty Advisor
Amit Singh, Ph.D. Committee Member
Donald Comfort, Ph.D. Committee Member
Jayne Robinson, Ph.D. Committee Member
Pothitos Pitychoutis, Ph.D. Committee Member
ii ABSTRACT
METABOLIC AND PHYSIOLOGICAL DETERMINANTS IN LISTERIA
MONOCYTOGENES ANAEROBIC VIRULENCE REGULATION
Name: Wallace, Nathan C. University of Dayton
Adviser: Dr. Yvonne Sun
In order for enteric pathogens to be successful in causing their infection they must overcome various environmental factors including: low pH, decreased oxygen, high osmolarity, nutrient competition, and host immune system etc. One such pathogen, Listeria monocytogenes (L. monocytogenes), is well known for being extremely adaptable and avoids host immune defenses by causing its infection intracellularly. This unique infection route makes L. monocytogenes an attractive pathogen for gaining further understanding host immune responses to intracellular pathogens. However, the majority of studies involving L. monocytogenes take place in the presence of oxygen, which completely omits the anaerobic phase of gastrointestinal infection. The main goal of this study is to understand the effects of anaerobic growth on L. monocytogenes pathogenesis.
Chapter I provides a brief introduction including background information on L. monocytogenes discovery, lifestyle, outbreaks, and pathogenesis.
iii Following the introduction in Chapter II, I give a review on the role of oxygen in the pathogenesis of various other relevant enteric pathogens. Moving into
Chapter III, I look at key morphological differences between aerobic and anaerobic growth conditions and explores how exogenous supplementation of key intermediates of the tricarboxylic acid cycle (TCA) affects subsequent pathogenesis. This transitions into Chapter IV, where I explore the role of respiratory activity in priming and sustaining intracellular pathogenesis. And finally, Chapter V evaluates the importance of menaquinone biosynthesis in L. monocytogenes growth and subsequent pathogenesis. Together these data support a regulatory role for metabolic activity in the success of L. monocytogenes infection.
iv ACKNOWLEDGMENTS
I express my deepest gratitude to my mentor, Dr. Yvonne Sun. You took a chance on me back in 2014, and I am eternally grateful for the opportunity you gave me to continue my education. I am appreciative of your ability to push me and realize my potential as a scientist. Thank you so very much. Thanks also go to my graduate advisory committee: Dr. Amit Singh, Dr. Jayne Robinson, Dr.
Pothitos Pitychoutis, and Dr. Don Comfort. I appreciate you all meeting with me every semester and always providing insightful comments and suggestions for future experiments.
To my older brother Ryan, thanks for always being a role model for me growing up. You don’t understand how much I appreciate your willingness to provide everything from advice on a particular experiment or just listening when I was having a bad day. Thanks for being an awesome big brother and an even better father to my adorable niece Emilia. I have always wanted to be like my older brother, and now we are now a house of learned doctors.
To my parents, Brian and Karen, I am deeply thankful for the undying support you have both provided me in the pursuit of my education. Ever since I
v was in grade school, you instilled the value of education in me. I came to understand this value on my own in obtaining this degree, and I am grateful for the constant reassurance, especially when I was unsure if I could do this. This dissertation would not have been possible if it weren’t for you two, thank you.
To my sisters Morgan and Madison, thanks for providing me with constant encouragement during the course of my Ph.D. Even if you didn’t know it, our short conversations or simple exchange of texts helped pick me up when I was down. I appreciate all of the support, especially in the form of tasty treats. I also have to give a very special thanks to Shane Childs for helping me keep my car together for the length of my studies, the Mazdarati will live on long past when we put her to rest.
I wouldn’t have made it this far without the help of so many of my lab mates. To everyone who was a part of the Sun lab both past and present thanks for doing everything including: helping me with an experiment, bringing in a tasty treat, or providing some comic relief on a stressful day. I want to specifically thank Ashley Zani and Eric Newton. Ashley, you were the first undergraduate student I worked with during my Ph.D. and you will always hold a special place in my heart. I am so proud of you and so happy to have been a part of your scientific career. I have no doubts that you will be a wonderful scientific writer someday. Eric, you were the second undergraduate student who worked with me. We developed a true friendship over the course of your time in the lab, and I
vi am very grateful for your willingness to help with my experiments. “Lab pops” will live on forever thanks to you.
Thanks to all of the support staff in the University of Dayton Department of
Biology. Thanks to Grover Allen for helping with any technical problems and teaching me so very much about fixing things. Cathy Wolfe for keeping me always on track for graduation. Thanks to Rita McGinn and Jan Bertke for being the best office staff. I always knew you always would be willing to answer my questions or just have a nice chat.
vii TABLE OF CONTENTS
ABSTRACT ...... iii
ACKNOWLEDGMENTS ...... v
LIST OF TABLES ...... xii
LIST OF FIGURES ...... xiii
LIST OF ABBREVIATIONS AND NOTATIONS ...... xv
CHAPTER I BACKGROUND ...... 1
Identification/Nomenclature ...... 1
Environmental Niches ...... 2
L. monocytogenes Outbreaks ...... 3
L. monocytogenes infection ...... 4
Pathogenesis...... 6
Role of Oxygen in Virulence Regulation ...... 8
Thesis Overview ...... 9
References ...... 10
CHAPTER II THE IMPACT OF OXYGEN ON BACTERIAL ENTERIC
PATHOGENS ...... 17
viii Abstract ...... 17
Keywords ...... 18
Enteric pathogens exposed to varying oxygen levels ...... 18
Enteric pathogens responding to varying oxygen levels ...... 22
Bacillus cereus ...... 22
Campylobacter jejuni ...... 25
Enterohaemorrhagic E. coli ...... 27
Helicobacter pylori...... 29
Listeria monocytogenes ...... 31
Salmonella enterica serotype Typhimurium ...... 32
Shigella flexneri ...... 34
Vibrio cholerae ...... 37
Yersinia enterocolitica ...... 38
Conclusion ...... 39
References ...... 41
CHAPTER III METABOLIC DETERMINANTS IN LISTERIA
MONOCYTOGENES ANAEROBIC LISTERIOLYSIN O PRODUCTION...... 72
Abstract ...... 72
Introduction ...... 73
Materials and Methods:...... 75
ix Results ...... 81
Discussion ...... 90
References ...... 96
CHAPTER IV STIMULATING RESPIRATORY ACTIVITY PRIMES
ANAEROBICALLY GROWN LISTERIA MONOCYTOGENES FOR
INTRACELLULAR INFECTION ...... 102
Abstract ...... 102
Introduction ...... 103
Results ...... 106
Discussion ...... 117
Materials and Methods...... 122
References ...... 129
CHAPTER V EVALUATING THE IMPORTANCE OF MENAQUINONE
BIOSYNTHESIS IN LISTERIA MONOCYTOGENES VIRULENCE
REGULATION ...... 133
Abstract ...... 133
Introduction ...... 134
Results ...... 135
Materials and Methods...... 140
Discussion ...... 143
x References ...... 144
Supplementary Figures ...... 146
CHAPTER VI CONCLUDING REMARKS ...... 147
APPENDIX I The Effect of Oxygen on Hly Transcript ...... 149
Methods: ...... 150
APPENDIX II Evaluation of Redox Homeostasis In Listeria monocytogenes .. 154
Methods ...... 155
xi LIST OF TABLES
Table 3.1: Characterizations of L. monocytogenes strain 10403s in vitro growth...... 83
Table A1.1 Primer design used for qRT-PCR analysis ...... 149
xii LIST OF FIGURES
Figure 1.1 Schematic of L. monocytogenes intracellular infection ...... 8
Figure 3.1 Anaerobically grown L. monocytogenes exhibits decreased maximal growth in vitro and morphological differences under TEM ...... 82
Figure 3.2 Anaerobic growth of L. monocytogenes leads to increased initial intracellular CFU but decreased intracellular growth and actin co-localization ... 85
Figure 3.3 Anaerobically grown L. monocytogenes secretes less LLO ...... 87
Figure 3.4 Buffering media or exogenous supplementation of lactate or acetoin does not alleviate the reduced LLO production under anaerobic conditions relative to aerobic conditions ...... 88
Figure 3.5 Supplementation of intermediates involved in central carbon metabolism alters carbon metabolism and increases supernatant LLO activity of anaerobically grown L. monocytogenes ...... 90
Figure 4.1. Fumarate enhances L. monocytogenes anaerobic respiratory activity...... 108
Figure 4.2. Fumarate-treated L. monocytogenes exhibited enhanced transition to aerobic in vitro and intracellular growth ...... 110
Figure 4.3. Inhibition of aerobic respiratory activity with CCCP phenocopies anaerobically grown L. monocytogenes ...... 112
xiii Figure 4.4. Modulations of respiratory activity alter production of LLO and actin co-localization ...... 115
Figure 4.5. Prior or concurrent fumarate treatments enhance cell-cell spread . 117
Figure 5.1: Anaerobic growth increase with fumarate supplementation abolished in the ∆MenB strain possibly due to manipulation of the fate of pyruvate ...... 137
Figure 5.2: MenB is critical for aerobic LLO production and intracellular fitness ...... 139
Supplemental Figure 5.1: Screen of respiratory mutants on the basis of growth, acetoin production and hemolytic activity ...... 146
Figure A1.1: Anaerobically grown L. monocytogenes shows increased hly transcript activity and stability but does not result in increased LLO protein abundance ...... 150
Figure A2.1 Anaerobically grown L. monocytogenes exhibit increased oxidative stress and increased susceptibility to killing by H2O2 due to lack of catalase activity...... 155
xiv LIST OF ABBREVIATIONS AND NOTATIONS
L. monocytogenes Listeria monocytogenes
TCA Cycle Tricarboxylic acid cycle
TCA Trichloroacetic acid
LLO Listeriolysin O
GABA Gamma aminobutyric acid
PrfA Positive regulatory factor A
InlA,B Internalin A and B
PlcA,B Phospholipase C A and B
LAP Listeria adhesion protein
TMAO Trimethylamine N-oxide
DMSO Dimethyl sulfoxide
SigB Alternative sigma factor B
DHNA 1,4-dihydroxy-2-napthoate
LB Lysogeny broth
BHI Brain heart infusion
IPTG Isopropyl thiogalactopyranoside
DTT Dithiothreitol
xv CHAPTER I
BACKGROUND
Identification/Nomenclature
Listeria monocytogenes (L. monocytogenes) is a Gram-positive microorganism capable of causing disease in humans and animals. In history, L. monocytogenes was first characterized as “Bacillus monocytogenes” by G. D.
Murray, R. A. Webb, and M. B. R. Swann in 1924 when the bacterium was isolated from rabbits and guinea pigs who all perished from similar lesions
(Murray, Webb, and Swann 1926). The first case of a human infected with L. monocytogenes was not reported until 1929 in Denmark, but there may have been isolated cultures of L. monocytogenes from meningitis patients as early as
1921 (Dumont, J. and Cotuni, L. 1921). The current genus, Listeria, was not coined until 1939 by J.H. Harvey Pirie at the Third International Congress for
Microbiologists, held in New York City (J.H. Harvey Pirie 1940). As of now, there are several other species of the Listeria genus including: L. welshimeri, L. ivanovii, L. innocua, L. seeligeri, and L. grayi. However only L. monocytogenes and L. ivanovii are pathogenic. Listeria monocytogenes (L. monocytogenes) is
1 now divided into 13 serotypes based on their somatic (O) and flagellar (H) antigens, which are as follows: 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, 5,
7 (Seeliger and Höhne 1979). Clinically speaking, there has been higher prevalence of serotype 4b isolated from human listeriosis patients. For example, one study demonstrated that in a population of over 1300 infected individuals
64% of the isolated L. monocytogenes were of the 4b serotype, followed by 1/2a and 1/2b with 15% and 10%, respectively (McLauchlin 1990). This differs from cultures isolated from food, which are typically from the 1/2a serotype. In one
Belgian study a prevalence of L. monocytogenes isolated from food stuffs was heavily in favor of the 1/2a serotype (54%) followed by 4b (20%) (Gilot, Genicot, and André 1996). Additional characterization of L. monocytogenes separates into
3 different lineages (Lineage 1, 2, and 3) based on differences in the nucleotide sequences of the virulence factors hly, actA, and inlA (Zhang et al. 2003;
Rasmussen et al. 1995). Serotypes 1/2b, 3b, 3c, 4b, 4d, and 4e fall into lineage
1; 1/2a, 1/2c and 3a are in lineage 2; and lineage 3 contains 4a and 4c. Further analysis supported a higher incidence of lineage 1 L. monocytogenes isolated from clinics when compared to lineages 2 and 3 (Zhang et al. 2003).
Environmental Niches
L. monocytogenes is a naturally occurring organism found in the soil
(Welshimer 1960) and water (Lyautey et al. 2007). It is considered to be a saprophyte (Gray and Killinger 1966), which is an organism that lives off of dead
2 or decaying organic matter. As a saprophyte, L. monocytogenes encounters various other microbes, fungi, and protists with which it must compete and persist
(Hilbi et al. 2007). Several studies have looked at the spatial and temporal prevalence of L. monocytogenes found in the soil. Prevalence of L. monocytogenes was much higher (~30%) in samples from uncultivated fields or meadows compared to a much lower (~8%) in samples from cultivated fields
(Dowe et al. 1997). In one study, during the summer months the prevalence of L. monocytogenes is much higher in natural environments, and simultaneously at its lowest in urban environments (Sauders et al. 2012). However, in another study the prevalence of L. monocytogenes was much higher in vegetable farms in 2 out of 3 years (Strawn et al. 2013), which points to variability of L. monocytogenes in terms of environmental prevalence. When L. monocytogenes ceases life as a saprophyte and enters the realm of human food it becomes a major public health concern.
L. monocytogenes Outbreaks
L. monocytogenes infections were considered to be a laboratory rarity from its discovery until around 1970, when the incidence of foodborne contamination steadily increased through the 1980’s (Fleming et al. 1985; Büla,
Bille, and Glauser 1995; Schlech 2000). One of the deadliest outbreaks occurred in 1985 in Los Angeles County, California. Over 142 cases of Listeriosis were reported, and of those individuals affected, 48 died as a result; 20 of which were
3 unborn fetuses (Linnan et al. 1988). The source of the contamination was L. monocytogenes-contaminated milk that was used to make Mexican-style soft cheese (queso fresco). The cause of the contamination was due to the improper pasteurization of the milk prior to the cheese being made. This is one the largest cases of food-poisoning related deaths reported in the United States. Another such example of a large-scale L. monocytogenes contamination occurred in
2011. In this case, a multi-state L. monocytogenes outbreak occurred in contaminated cantaloupe. Over the course of 3 months, a total of 146 cases of listeriosis were reported, of which 30 individuals died and one pregnant mother miscarried. Upon further investigation, the source of the contamination was traced to Jensen Farms in Colorado. Multiple surfaces in the processing facility were contaminated with L. monocytogenes, and due to the poor design, proper sanitization of the facility was difficult (Centers for Disease Control and
Prevention 2011).
L. monocytogenes infection
L. monocytogenes infection can lead to various disease outcomes such as: invasive listeriosis, non-invasive gastroenteritis, and in some rare cases eczematous skin infection. Listeriosis as a disease is defined as an invasive L. monocytogenes infection, which typically infects sterile areas in the body such as: liver (Wing and Gregory 2002), cerebral spinal fluid (Cone et al. 2003), spleen
(Aoshi et al. 2009), blood (Bhat et al. 2013), and in some rare cases the heart
4 (Summa and Walker 2010). Although typically a rare infection (World Health
Organization and Nations 2004), Listeriosis has a high mortality rate when compared to other foodborne pathogens (Scallan et al. 2011b) with mortality rates ranging from 14-30% (Hernandez-Milian and Payeras-Cifre 2014). The majority of the population that develop invasive listeriosis infection are immunocompromised. The major risk groups include: pregnant women, the elderly, infants, and anyone on immunosuppressants (Buchanan et al. 2017).
Generally, L. monocytogenes is susceptible to most first choice antibiotics such as: penicillin, ampicillin, and aminoglycosides (Swaminathan and Gerner-Smidt
2007). However, in some cases even with early treatment L. monocytogenes can still cause bacteremia and patient death. In fact, one such study showed that even adequately timed antibiotic treatment of affected individuals still had a 15% mortality rate.(Thønnings et al. 2016) .
Most healthy individuals will not develop invasive listeriosis infection, but may instead experience acute, self-limiting febrile gastroenteritis. It is believed that this non-invasive form of L. monocytogenes infection is much more common than is reported due to the nature of the disease’s symptoms. Common symptoms include: fever, diarrhea, nausea, headache, and musculoskeletal pain.
Typically, this occurs when the patient has ingested large quantities of L. monocytogenes and symptoms will generally appear in a matter of 24 hours and last about 2 days (Ooi and Lorber 2005). In very rare cases, L. monocytogenes may also cause a cutaneous skin infection that is typically identified by the presence of nonpainful, localized, skin lesions. These are typically as a result of
5 a veterinarian coming into direct contact with infected animals. Specifically, most reported cases are during bovine calf delivery or abortion, and there have also been few reports of cutaneous infection as a result of direct contact with soil where L. monocytogenes was present (Godshall, Suh, and Lorber 2013).
Pathogenesis
In order for L. monocytogenes to cause invasive listeriosis it must enter host cells through facilitated endocytosis or phagocytosis. Invasion of non- phagocytic cells is a unique characteristic and L. monocytogenes expresses two main virulence factors for this process to occur: Internalin A and B (InlA and InlB)
(Gaillard et al. 1991). InlA interacts with the host cell surface protein e-cadherin
(Mengaud et al. 1996) and InlB binds to the met receptor tyrosine kinase (Shen et al. 2000). Successful binding initiates a signaling cascade that promotes the internalization of L. monocytogenes into the host cell by a vacuole. Escape from the vacuole is mediated by the secretion of the pore-forming toxin listeriolysin O
(LLO) (Cossart et al. 1989) and two phospholipases C (PlcA and PlcB)
(Leimeister-Wächter, Domann, and Chakraborty 1991). When inside the cytosol,
L. monocytogenes expresses another virulence factor, actin assembly-inducing protein (ActA), which polymerizes host actin filaments to one bacterial cell pole and through the polymerization process propels itself into the host cell membrane
(Portnoy, Auerbuch, and Glomski 2002; Vázquez-Boland et al. 2001). This
6 propulsion leads to the formation of a protrusion into neighboring cells and the escape process repeats.
The only known virulence regulator in L. monocytogenes is the transcription factor positive regulatory factor A (PrfA), which is similar in structure and function to other cyclic AMP receptor proteins (CRP) (Chakraborty et al.
1992; Lampidis et al. 1994; Menguad et al. 1991; Sheehan et al. 1995). All known L. monocytogenes virulence factors are under the direct control of PrfA by binding to its site upstream of each virulence gene locus in an area known as the
PrfA box (Luo et al. 2004; Milohanic et al. 2003; Vega et al. 2004). PrfA is regulated by carbon sources such as glucose and cellobiose, which decrease virulence factor production (Milenbachs et al. 1997). Alternatively, carbon sources such as glucose-1-phosphate increase virulence factor production (Ripio et al. 1997). This is an example of the importance of environmental factors such as carbon source on virulence regulation in L. monocytogenes.
7
Listeria Phagosome
Disrupted Phagosome InlA Actin propelled Listeria InlB
LLO PlcA/B
LLO PlcA/B
Figure 1.1 Schematic of L. monocytogenes intracellular infection
Role of Oxygen in Virulence Regulation
One other such example of the importance of environmental factors on virulence regulation is the presence or absence of oxygen. The majority of research performed on L. monocytogenes virulence regulation has the bacteria cultured aerobically, however this fails to address the anaerobic phase of infection that occurs in the intestines. As an enteric pathogen, L. monocytogenes is exposed to decreasing concentrations of oxygen as it is transmitted through the gastrointestinal tract. The environment of the gastrointestinal tract has
8 oxygen concentrations ranging from less than 1mmHg in the cecum (Albenberg et al. 2014b) to 100mmHg (Pittman 2011) in the arteries of the submucosal tissue. Therefore, it is important to consider what role these changes in oxygen levels may have on virulence regulation.
There is limited evidence for the role of anaerobic conditions in L. monocytogenes virulence regulation. Several studies have shown increased invasion of anaerobically grown L. monocytogenes in cultured cell lines (James and Keevil 1999; Müsken et al. 2008; Schüller and Phillips 2010). Specifically, under anaerobic conditions the Listeria Adhesion Protein (LAP) is upregulated and promotes adhesion to cultured intestinal epithelial cells and is necessary for in vivo infection in mice (Burkholder et al. 2009). It was later determined that increased LAP secretion under anaerobic conditions promotes the expression of the LAP receptor-Hsp60 (Burkholder and Bhunia 2010). Further studies are required to have a more in-depth understanding of the effects of oxygen on L. monocytogenes infection outcome.
Thesis Overview
This thesis utilized various molecular, biochemical, and tissue culture techniques to evaluate anaerobic virulence regulation in Listeria monocytogenes.
In chapter II we provide an in-depth review on the known information regarding the role of oxygen in virulence regulation of various common enteric pathogens.
Then in chapter III we specifically look at L. monocytogenes and evaluate the
9 role of TCA cycle intermediates in the production of a key virulence factor, LLO.
This research showed that exogenous supplementation of key intermediates of the TCA cycle had the potential to enhance the production of LLO. In chapter IV we evaluate the role of respiratory activity in L. monocytogenes infection.
Supplementation of exogenous fumarate showed the potential to enhance intracellular growth, particularly that of L. monocytogenes that was cultured anaerobically. In chapter V we looked to the role of menaquinone biosynthesis in virulence regulation as a means of better establishing a connection between metabolic activity and pathogenesis.
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16 CHAPTER II
THE IMPACT OF OXYGEN ON BACTERIAL ENTERIC PATHOGENS
This work was originally published in the peer-reviewed scientific journal
Advances in Applied Microbiology in 2016 with the following citation: Wallace, N.,
A. Zani, E. Abrams, and Y. Sun. 2016. The Impact of Oxygen on Bacterial
Enteric Pathogens. Advances in Applied Microbiology 95: 179–204.
Abstract
Bacterial enteric pathogens are responsible for a tremendous amount of foodborne illnesses every year through consumption of contaminated food products. During their transit from contaminated food sources to the host gastrointestinal tract, these pathogens are exposed and must adapt to fluctuating oxygen levels in order to successfully colonize the host and cause diseases.
However, the majority of enteric infection research has been conducted under aerobic conditions. To raise awareness of the importance in understanding the impact of oxygen, or a lack of oxygen, on enteric pathogenesis, we describe in this review the metabolic and physiological responses of nine bacterial enteric pathogens while exposed to environments with different oxygen levels. We
17 further discuss the effects of oxygen levels on virulence regulation to establish potential connections between metabolic adaptations and bacterial pathogenesis.
While not providing an exhaustive list of all bacterial pathogens, we highlight key differences and similarities among nine facultative anaerobic and microaerobic pathogens in this review to argue for a more in-depth understanding of the diverse impact oxygen levels have on enteric pathogenesis.
Keywords
Bacillus cereus, Campylobacter jejuni, Enterohaemorrhagic E. coli, Helicobacter pylori, Listeria monocytogenes, Salmonella enterica serotype Typhimurium,
Shigella flexneri, Vibrio cholerae, Yersinia enterocolitica, anaerobiosis, virulence regulation, anaerobic respiration
Enteric pathogens exposed to varying oxygen levels
Foodborne illnesses result in high medical and societal burden at a global level (Kirk et al. 2015). Bacterial pathogens are among the top leading causes for foodborne diseases (Scallan et al. 2011a). In United States, Salmonella enterica,
Campylobacter, and Listeria monocytogenes together are responsible for over
50% of annual cost of illness related to foodborne infections (Hoffmann, Batz, and Morris 2012). Understanding the incidence rate and cost has helped establishing and prioritizing appropriate strategies to reduce foodborne diseases.
18 For example, optimization in food packaging and storage conditions can be an effective approach to reduce or eliminate bacterial contamination and subsequently prevent foodborne infections.
Modified atmosphere packaging (MAP) is an extensively studied and investigated method that aims to reduce microbial growth and maintain freshness of food products to extend shelf life by reducing oxygen levels in food packaging
(Kader, Zagory, and Kerbel 1989; Lee et al. 2015). MAP works by replacing air with altered concentrations of oxygen, CO2, or nitrogen to extend the life and quality of the food product and simultaneously reduce microbial growth.
However, while the overall microbial growth is reduced with decreased oxygen levels, many foodborne bacterial pathogens are anaerobes, facultative anaerobes, or microaerophiles and can survive and even proliferate under suboxic conditions (Al-Qadiri et al. 2015). The ability for pathogens to persist under suboxic conditions raises two important questions. First, it is not clear if suboxic conditions contribute to the enrichment of pathogens in food products by suppressing the growth of non-pathogenic bacteria while allowing the growth of pathogenic bacteria. Second, because the majority of bacterial pathogenesis research is conducted under the optimal growth conditions for the pathogen, it is not clear how exposure to the potentially stressful suboxic conditions by the pathogen may impact subsequent infectivity. Therefore, it is beneficial to obtain a better understanding of how foodborne pathogens respond to suboxic conditions to develop and implement strategies to prevent foodborne infections with MAP.
19
In addition to environmental exposure of changing oxygen levels, enteric pathogens also face a dynamic gastrointestinal (GI) tract with fluctuating oxygen concentrations. Under normal, healthy conditions, there is a natural oxygen gradient in the GI tract with a decreasing trend in oxygen tension from stomach to large intestines (Sheridan, Lowndes, and Young 1990; He et al. 1999; Cooper,
Sherry, and Thorpe 1995). There is also a transverse gradient in the lower gastrointestinal tract where the lumen is devoid of oxygen, which is depleted by the endogenous microbes, while the mucosal surface is relatively enriched with oxygen (Albenberg et al. 2014a; Karhausen et al. 2004; Marteyn et al. 2010), which is supplied by the underlying vasculature. These two intersecting oxygen gradients are further complicated by temporal fluctuations. For example, as flood flow to the intestines increases after meal, the supply of oxygen to the intestinal mucosa likely increases as well. Moreover, during inflammation and other enteric disease states, mucosal oxygenation level significantly decreases, often leading to induction of hypoxia responses (Jantsch and Schödel 2015; Shehade,
Oldenhove, and Moser 2014; Colgan and Taylor 2010; Campbell et al. 2014;
Karhausen et al. 2004; Cooper, Sherry, and Thorpe 1995). These scenarios paint a complex and dynamic picture of oxygen levels in the GI environment during pathogen transit through the GI tract and further support a need to understand how enteric pathogens respond to fluctuating oxygen levels.
20 The ability for enteric pathogens to sense and respond to fluctuating oxygen levels is critical in surviving environments with changing oxygen levels and depends on various metabolic and physiological adaptations. This global scale of adaptations to fluctuating oxygen levels is choreographed by a multitude of transcriptional regulators. For example, Fnr and ArcAB are commonly found in enteric bacterial pathogens and represent two best-studied transcriptional regulators heavily involved in adaptations to fluctuating oxygen levels. Fnr is a cytosolic protein with an iron-sulfur cluster cofactor as the oxygen-sensing domain and a DNA-binding domain to regulate a variety of genes involved in anaerobic processes (Körner, Sofia, and Zumft 2003; Spiro 1994; Spiro and
Guest 1990; Unden and Bongaerts 1997). ArcAB is a two-component signal transduction system in which the sensor kinase ArcB responds to the redox state of the cell (Bekker et al. 2010; Georgellis, Kwon, and Lin 2001; Malpica et al.
2004), and the response regulator ArcA modifies the transcription of a large number of genes to adapt to the changing redox states (Evans et al. 2011;
Gunsalus and Park 1994). Many more transcriptional regulators, often working with Fnr or ArcAB, are also involved in the response to fluctuating oxygen levels.
Together, these regulators lay the foundation for enteric pathogens to sense the changing oxygen levels and respond in both metabolic adaptations and virulence regulation.
21
Enteric pathogens responding to varying oxygen levels
Most research on enteric pathogenesis is conducted under normal atmospheric conditions. Nevertheless, a significant body of literature has provided key insights into the impact of suboxic conditions on enteric pathogens.
In this review, in order to provide a concise and focused comparison between different enteric pathogens, we purposely avoid discussions on oxidative stresses, which can take place regardless of oxygen concentrations, and choose to focus on work where oxygen levels were intentionally modulated during experimental procedures. Moreover, we also limit the discussions on facultative anaerobes and microaerophiles to provide a better comparison among organisms with wider range of oxygen tolerance for growth. Therefore, although not an exhaustive list of enteric pathogens and their responses, the following sections represent our sincere effort to highlight key similarities and differences.
Bacillus cereus
Bacillus cereus is a facultative anaerobe and a spore-forming enteric pathogen. B. cereus cause two distinct types of gastrointestinal diseases, the emetic and diarrheal syndromes (Bottone 2010). The emetic syndrome is caused by the emetic toxin cereulide found in the food products and therefore exhibits a short incubation period. In contrast, the diarrheal syndrome has a longer
22 incubation period and is caused by vegetative B. cereus in the intestines producing cytotoxins—hemolysin BL (Hbl), non-hemolytic enterotoxin (Nhe), and cytotoxin K (CytK) (Arnesen, Fagerlund, and Granum 2008).
The expression of these toxins are highly regulated by metabolism and environmental conditions (Arnesen, Fagerlund, and Granum 2008; van der Voort et al. 2008; Han, Sullivan, and Wilson 2015; Hayrapetyan et al. 2015). Anaerobic growth, in particular, has been consistently shown to result in increased toxin production (Duport et al. 2004; Zigha et al. 2006; Zigha et al. 2007a; Clair et al.
2010; van der Voort and Abee 2009; Gilois et al. 2007). B. cereus toxin production is regulated by redox-sensitive regulators, such as OhrRA, a redox- sensitive system involved in the expression of Hbl and Nhe (Clair et al. 2013) and
ResDE, a redox-sensitive two component signal transduction system involved in anaerobic induction of Hbl and Nhe production (Duport et al. 2006; Esbelin et al.
2009; Laouami et al. 2014). Mutations in resDE, without inhibiting anaerobic growth, lead to compromised anaerobic Hbl and Nhe production (Duport et al.
2006). The role of the ResDE system in transcriptional regulation of hbl and nhe was demonstrated by the response regulator ResD binding directly to the toxin gene promoters (Esbelin et al. 2009). Interestingly, ResD was also shown to bind to promoter sequence of plcR as well as fnr. PlcR, or phospholipase C regulator, is a transcriptional regulator activated by quorum sensing (Slamti and Lereclus
2002; Declerck et al. 2007) and is involved in the activation of toxin production
(Agaisse et al. 1999; Gohar et al. 2002; Gohar et al. 2008). Fnr, which is up- regulated in strain F4430/73 but not in strain ATCC 14579 under anaerobic
23 conditions, also contributes to Nhe and Hbl production (Esbelin et al. 2008; Zigha et al. 2007a; Messaoudi et al. 2010). Moreover, Fnr binds directly to the promoter sequences of the toxin genes, nhe and hbl, as well as regulator genes plcR and resDE (Esbelin et al. 2008). Evidence is also available that suggests Fnr directly interact with PlcR and ResD for DNA binding (Esbelin et al. 2009; Esbelin,
Jouanneau, and Duport 2012). These studies demonstrate a complex regulatory network where toxin production can be controlled in response to a variety of signaling pathways and their respective signals.
Exposure to anaerobic conditions impacts many aspects of B. cereus growth. For example, during anaerobic growth, glycolytic genes are up-regulated while tricarboxylic acid cycle genes show no change in the expression level (van der Voort and Abee 2009), indicating changes in carbon metabolism. Moreover, anaerobic growth results in modified membrane fatty acid composition (de Sarrau et al. 2012) and reduced spore formation (Abbas et al. 2014). However, spores formed under anaerobic conditions are more resistant to wet heat and extreme pH (Abbas et al. 2014). Although B. cereus can grow anaerobically through fermentation as well as anaerobic respiration of multiple alternative electron acceptors (Rosenfeld et al. 2005), anaerobic growth through glucose fermentation or fumarate respiration, but not nitrate respiration, induces toxin production (Zigha et al. 2007a), a phenotype indicative for additional regulatory signals beyond simple aerobic versus anaerobic switch. Therefore, the exact role
24 of oxygen in the hierarchy and connection among metabolism and virulence regulation is not clear and required further investigations.
Campylobacter jejuni
Campylobacter jejuni is a Gram-negative microaerophilic bacterium and one of the major causes for diarrheal illnesses. A variety of animal hosts and epithelial cell lines have been adapted to determine the mechanisms by which C. jejuni causes intestinal illnesses (Backert et al. 2013) and have shown that while
C. jejuni mainly replicates in the extracellular space, its intracellular persistence can be detected in vivo (Babakhani, Bradley, and Joens 1993; van Spreeuwel et al. 1985). The growth preferences of C. jejuni play an important role in transmission and pathogenesis. Although C. jejuni optimally grows between 37-
42°C and fails to grow at temperature below 30°C, it remains metabolic active and can survive at temperatures as low as 4°C for extended periods of time
(Penner 1988; Hazeleger et al. 1998). Its ability to survive through stressful conditions (Murphy, Carroll, and Jordan 2006) contributes to its persistence through antimicrobial controls during food processing and storage. Once inside the host, as an obligate microaerophile, C. jejuni has been found in regions of the
GI tract with reduced oxygen tension such as the mucosal surface (Lee et al.
1986; Beery, Hugdahl, and Doyle 1988).
25 To support the growth under microaerobic conditions, C. jejuni employs a highly branched respiratory chain capable of using a variety of electron donors and acceptors (Hofreuter 2014; Sellars, Hall, and Kelly 2002). Under microaerobic conditions (5% CO2 and 10-12% O2 in N2 balance), C. jejuni is able to grow using fumarate, nitrate, nitrite, DMSO, and TMAO as electron acceptors
(Weingarten, Grimes, and Olson 2008; Sellars, Hall, and Kelly 2002).
Interestingly, in the presence of formate, C. jejuni is also capable of growing using nitrate, DMSO, and TMAO as electron acceptors under anaerobic conditions (5% CO2 and 10% H2 in N2 balance) (Weingarten, Grimes, and Olson
2008). Fumarate can also support growth under oxygen-limited conditions (0.3%
O2) (van der Stel et al. 2015). The preferential utilization for different electron acceptors is established through the RacRS two component signal transduction system (van der Stel et al. 2015; Brás et al. 1999; Apel et al. 2012). The sensor kinase RacS has a predicted periplasmic sensing domain while the response regulator RacR suppresses fumarate reduction in the presence of more favorable electron acceptors, nitrate and DMSO, by directly suppressing the transcription of
AspA, an enzyme that synthesizes fumarate from aspartate (van der Stel et al.
2015; Brás et al. 1999; Apel et al. 2012).
Mutants deficient in these respiratory pathways, or the RacR/RacS system, exhibited a significantly reduced adhesion and invasion in cultured cells
(Kassem et al. 2012; Kassem et al. 2014; Apel et al. 2012) and decreased bacterial burden in animals (Hitchcock et al. 2010; Weingarten, Grimes, and
26 Olson 2008). Transcriptional profiling experiments using day-of-hatch chicken
(Taveirne et al. 2013; Woodall et al. 2005) and rabbit ileal loop (Stintzi et al.
2005) models also support the role of respiration during C. jejuni colonization.
Genes encoding components of the electron transport chain and the terminal reductases were found up-regulated in vivo compared to in vitro growth conditions (Woodall et al. 2005; Taveirne et al. 2013; Stintzi et al. 2005). These studies strongly indicate a low oxygen environment during C. jejuni colonization and the ability for C. jejuni to maintain respiratory activity in response to reduced oxygen levels.
Enterohaemorrhagic E. coli
Enterohaemorrhagic E. coli (EHEC) is a Gram-negative, facultative anaerobe capable of causing haemorrhagic colitis. EHEC pathogenesis involves production of shiga-like toxins (Stx) as well as a type III secretion system (T3SS) and its effector proteins. These virulence factors are encoded by genes in a pathogenicity island, locus of enterocyte effacement (LEE), which is regulated by a variety of regulators in response to various environmental conditions
(Rosenshine, Ruschkowski, and Finlay 1996; Kenny et al. 1997; Connolly, Finlay, and Roe 2015).
Both Stx production and T3SS secretion are modulated by oxygen levels.
In vitro, Stx production was enhanced under oxygen-limited or anaerobic
27 conditions in chemostat cultures (James and Keevil 1999a). In a vertical diffusion chamber model, translocation of Stx2 was enhanced during infections of polarized human colon epithelial T84 cells when the apical side was maintained under microaerobic conditions compared to aerobic conditions (Tran et al.
2014). Similarly, production of T3SS components, EspA and EspB, as well as the effector protein Tir (translocated intimin receptor) were increased during microaerobic infections in polarized T84 cells in the apical chamber (Schüller and
Phillips 2010a). These studies indicate that both prior exposure to reduced oxygen levels by bacteria as well as infections under reduced oxygen levels result in increased toxin production and T3SS functions. Anaerobic growth, specifically during respiration of nitrate or TMAO, but not fumarate or DMSO, also induced the production of EspB and Tir by EHEC strain Sakai (Ando et al.
2007). The critical role for respiration during infections is further supported by the observation that mutants lacking respiratory activities were out-competed by wild type bacteria during co-infections of Streptomycin-treated mice (Jones et al.
2007).
In oxygen-limiting or anaerobic conditions, EHEC typically exhibits enhanced adhesion to host cells. EHEC strain P2822 grown under oxygen- limiting or anaerobic conditions in chemostat exhibited increased adhesion to
HEp-2 cells (James and Keevil 1999a). EHEC strain TUV 93-0 also exhibited increased adhesion to polarized T84 cells cultured in a vertical diffusion chamber under microaerobic (1-2% atmospheric oxygen level) apical chamber (Schüller
28 and Phillips 2010a). Anaerobic growth of several non-motile EHEC isolates
(O157:NM strains) also exhibited enhanced adhesion to HCT-8 cells (Müsken et al. 2008a). However, the expression of the E. coli common pilus, which contributes to host cell adherence, was strongly expressed in response to 5%
CO2 but not anaerobicity (Rendón et al. 2007). Moreover, enhanced toxin production by growth in simulated ileal and colonic media was only observed under aerobic but not microaerobic conditions (Polzin et al. 2013). These data suggest an inherent sensitivity for the oxygen-responsive network underlying virulence regulation to be modulated by additional environmental factors.
Helicobacter pylori
Helicobacter pylori is typically considered a microaerophilic organism and requires reduced oxygen levels for cultivation. When oxygen levels increase or decrease from the optimal microaerobic conditions, mutation frequency increases
(Park et al. 2004) and susceptibility to redox active drug metronidazole changes
(Gerrits et al. 2004; Martínez-Júlvez et al. 2012). However, this microaerophilic notion has been challenged. Comparison of transcriptional profiles of H. pylori under microaerobic (5% O2) or aerobic (20% O2) conditions show a notable up- regulation of genes in protein synthesis and nucleic acid metabolism in addition to redox processes that are induced likely to mediate oxidative stress under aerobic conditions (Kaakoush et al. 2009). Moreover, growth under atmospheric oxygen levels by H. pylori is possible at high cell density or in the presence of
29 elevated CO2 (Park, Ko, and Lee 2011; Bury-Moné et al. 2006). The reason behind the CO2 requirement for aerobic growth is not clear but, considering CO2 as a product formed from H. pylori neutralization of stomach acid with urease, these studies highlight a potential role for CO2 during pathogenesis.
Under conditions with oxygen levels either above or below the optimal microaerobic concentrations, H. pylori goes through a morphological transformation from bacillary cells to U-shaped and finally coccoid forms (Cellini,
Allocati, Di Campli, et al. 1994; Kusters et al. 1997; Tominaga et al. 1999; Donelli et al. 1998; Park et al. 2004). The coccoid H. pylori contains dramatic modifications in cell envelope (Costa et al. 1999; Shimomura et al. 2004) and likely maintains low levels of metabolic activities (Sörberg et al. 1996; Kusters et al. 1997; Mizoguchi et al. 1998). The exact role of the coccoid form in H. pylori pathogenesis has been difficult to establish. While the coccoid forms of H. pylori do not colonize gnotobiotic piglets (Eaton et al. 1995), they have been found in infected human gastrectomy specimens (Chan et al. 1994) and stomach mucosa of infected BALB/c mice (Cellini, Allocati, Angelucci, et al. 1994). Serological analyses identified coccoid-specific antigens in infected patients (Vijayakumari et al. 1995; Benaissa et al. 1996). During cell culture infections, coccoid forms of H. pylori have also been reported to exhibit adherence to a gastric carcinoma cell line (KATOIII) (Vijayakumari et al. 1995). However, cellular adhesion by coccoid forms of H. pylori is reduced compared to bacillary bacteria, resulting in reduced
NF-κB activation in HEK293T cells and IL-8 production in infected gastric
30 epithelial cells (Cole et al. 1997). Therefore, it is proposed that the coccoid form contributes to H. pylori subversion of immune surveillance during colonization and infection (Cellini 2014).
Listeria monocytogenes
Listeria monocytogenes is a Gram-positive bacterium capable of growing in the cytosol of a variety of mammalian cells. The intracellular life cycle of L. monocytogenes relies first on the ability of L. monocytogenes to gain access to the host cell cytosol through bacterial surface and secreted virulence factors.
Secondly, L. monocytogenes is capable of invading neighboring cells through actin-based motility, thus maintaining its intracellular residence without being exposed to extracellular immune surveillance. At an organismal level, L. monocytogenes survival in the gastrointestinal tract relies on its ability to resist acid (Smith, Liu, and Paoli 2013), bile salts (Begley et al. 2005; Dussurget et al.
2002; White et al. 2015; van der Veen and Abee 2011; Quillin, Schwartz, and
Leber 2011; Zhang et al. 2011; Dowd et al. 2011; Watson et al. 2009; Kim et al.
2006; Sleator et al. 2005), and likely other environmental stresses (Gahan and
Hill 2014; Lungu, Ricke, and Johnson 2009).
As a facultative anaerobe, L. monocytogenes is capable of growing under anaerobic conditions through mixed acid fermentation (Pine et al. 1989; Romick,
Fleming, and McFeeters 1996a). In addition to metabolic modifications (Müller-
31 Herbst et al. 2014a; Nilsson et al. 2013), anaerobic exposure also induces acid tolerance and bile salt resistance (Sewell, Allen, and Phillips 2015; White et al.
2015), that likely contribute to L. monocytogenes surviving and colonizing the intestines. Moreover, several studies have noted that anaerobically grown L. monocytogenes exhibits enhanced adherence and invasion phenotype. First, transcriptional studies have shown that, internalins, which are surface proteins involved in adherence to host cells, were up-regulated in suboxic conditions
(Toledo-Arana et al. 2009; Müller-Herbst et al. 2014a; Jydegaard-Axelsen et al.
2004). Second, in both cell culture and animal infection models, anaerobically grown L. monocytogenes exhibited an increased adhesion and invasion phenotype (Burkholder et al. 2009a; Andersen et al. 2007). At least one factor,
Listeria adhesion protein (LAP), is up-regulated under anaerobic condition and is required for the anaerobic induction of adhesion of cultured Caco-2 and HCT-8 cells (Burkholder et al. 2009a). However, the genetic network for anaerobic virulence regulation remains to be determined.
Salmonella enterica serotype Typhimurium
Salmonella enterica serotype Typhimurium (S. Typhimurium) is a common foodborne pathogen that can cause gastroenteritis. S. Typhimurium pathogenesis involves two bacterial type III secretion systems that facilitate S.
Typhimurium invasion of the intestinal epithelium and survival in phagocytes (van der Heijden and Finlay 2012; Schlumberger and Hardt 2006). Several studies
32 have shown that S. Typhimurium adherence and invasion are induced under oxygen-limiting conditions (Lee and Falkow 1990; Ernst, Dombroski, and Merrick
1990; Schiemann and Shope 1991; Singh, Khullar, and Ganguly 2000). With as short as 3 hours of static incubation without agitation under aerobic conditions, the invasion of S. Typhimurium in Madin-Darby Canine Kidney Epithelial (MDCK) cells was increased (Lee and Falkow 1990). S. Typhimurium grown aerobically with agitation or 20% O2 also exhibits the lowest invasion phenotype compared to those grown without agitation or with 0 or 1% O2 (Lee and Falkow 1990). With
HEp-2 cells, in contrast, aerobically or anaerobically grown S. Typhimurium exhibited similar invasion rates when infection was carried out under aerobic conditions. However, if infections were carried out under anaerobic conditions, anaerobically grown S. Typhimurium exhibited a stronger invasion phenotype
(Ernst, Dombroski, and Merrick 1990). Similar anaerobic induction of invasion was also reported using human Henle intestine 407 epithelial cells, mouse peritoneal cells (Schiemann and Shope 1991), spleen macrophages, and immobilized mucins (Singh, Khullar, and Ganguly 2000). In a rabbit ileal loop infection model, anaerobically grown S. Typhimurium also resulted in increased fluid accumulation compared with aerobically grown S. Typhimurium (Singh,
Khullar, and Ganguly 2000).
By carefully curating and comparing 15 completed S. enterica genomes,
Nuccio et al. identified that the genomes of gastrointestinal S. enterica pathogens are enriched with genes involved in anaerobic metabolism that can contribute to
33 intestinal colonization (Nuccio and Bäumler 2014). Two transcriptional regulators have been described to facilitate S. Typhimurium metabolic adaptations to anaerobic conditions. The global regulator Fnr controls the expression of over
36% of the coding sequences in response to anaerobic conditions (Fink et al.
2007), suppressing genes involved in aerobic metabolism while activating genes involved in anaerobic metabolism, flagella synthesis, and ethanolamine utilization
(Fink et al. 2007; Wei and Miller 1999). Interestingly, although Fnr is a negative regulator for the expression of hilA (Golubeva et al. 2012), which encodes the main transcriptional activator for Salmonella pathogenicity island 1 (SPI1), the fnr mutant is more attenuated in mice compared to wildtype bacteria (Fink et al.
2007). In contrast, ArcA controls the expression of 44% of the coding sequences under anaerobic conditions, including suppression of some virulence genes
(Evans et al. 2011). However, the arcA mutant, despite exhibiting a reduced growth rate under anaerobic conditions, has no virulence defect in intraperitoneal or oral infections in mice (Evans et al. 2011). These studies demonstrate the complexity in anaerobic responses and highlight the importance of metabolic and physiological adaptation to anaerobicity for S. Typhimurium to establish infections.
Shigella flexneri
Shigella flexneri is a Gram-negative enteric pathogen and together with other 3 other Shigella species, S. dysenteriae, S. sonnei, and S. boydii, is
34 responsible for about half a million cases of shigellosis in the United States each year (Scallan et al. 2011a). The shigellosis disease is manifested with 5-7 days of diarrhea, fever, and abdominal pain and is a result of the intestinal tissue destruction and inflammatory responses by Shigella infections (Sansonetti 2006).
S. flexneri is capable of invading and disseminating the intestinal epithelium
(Agaisse 2016; Bonnet and Tran Van Nhieu 2016; Yang et al. 2015; Valencia-
Gallardo, Carayol, and Tran Van Nhieu 2015) and replicates rapidly inside the host cell cytosol (Kentner et al. 2014). One of the virulence factors required for S. flexneri virulence is the type 3 secretion system (T3SS), encoded on a large virulence plasmid (Sansonetti, Kopecko, and Formal 1982; Le Gall et al. 2005).
The effector proteins secreted by T3SS contribute heavily to S. flexneri pathogenesis (Ogawa et al. 2008; Phalipon and Sansonetti 2007; Campbell-
Valois and Pontier 2016; Picking and Picking 2016; Ashida, Mimuro, and
Sasakawa 2015). The expression of T3SS and its effector proteins is induced at temperature higher than 32°C and the secretion is activated upon contact with host cells (Campbell-Valois and Pontier 2016). A seminal report further demonstrated a critical role for oxygen in the T3SS construction and activation in
S. flexneri (Marteyn et al. 2010). Specifically, after growth at a permissive temperature of 37°C, the length of the T3SS needle on S. flexneri was significantly longer under anaerobic conditions than aerobic conditions in an Fnr- dependent manner (Marteyn et al. 2010). As a result, exposure to the anaerobic lumen was hypothesized as a priming signal for S. flexneri for subsequent aerobic encountering of host cells (Marteyn et al. 2010).
35
The ability to respire oxygen is important for S. flexneri virulence. Mutants deficient in cytochrome bd oxidase are compromised in cell culture and animal models of infections (Way et al. 1999). In the absence of oxygen, S. flexneri up- regulates genes encoding processes contributing to anaerobic growth, such as fermentation, anaerobic respiration, and acid responses (Vergara-Irigaray et al.
2014). As expected, genes encoding components in aerobic processes, such as the tricarboxylic acid cycle, are down-regulated (Vergara-Irigaray et al. 2014).
The presence or absence of oxygen also plays a key role in the expression of iron acquisition systems (Carpenter and Payne 2014; Wei and Murphy 2016).
The genome of S. flexneri contains at least 4 different iron acquisition systems—
2 ferrous transporters, Feo and Sit, and 2 ferric transporters, aerobactin (Iuc/IutA) and Fec (Wyckoff, Boulette, and Payne 2009). Two of the iron acquisition systems, encoded by iuc/iutA and sit genes, are expressed under aerobic conditions (Vergara-Irigaray et al. 2014; Boulette and Payne 2007). This is likely a result of an ArcA-dependent suppression under anaerobic conditions (Boulette and Payne 2007). In contrast, the feo system is expressed under anaerobic conditions, in an Fnr-dependent manner (Boulette and Payne 2007). These studies provide an important insight into the connection between metabolism and physiology with S. flexneri pathogenesis.
36
Vibrio cholerae
Vibrio cholerae is a Gram-negative, facultative anaerobe and the causative agent for cholera, an acute diarrheal disease that can be fatal from severe dehydration. Vibrio species are able to grow anaerobically through mixed acid fermentation (Nobechi 1925; Unger, Rahman, and Demoss 1961) as well as respiration (Shi et al. 2006). Since 1977, it has been reported that anaerobically grown V. cholerae at 37°C exhibited a pleomorphic phenotype and increased cholera toxin production (Cobaxin et al. 2014; Fernandes, Clark, and Smith 1977;
Fernandes and Smith 1977). More recently, it was demonstrated that anaerobic growth using trimethylamine N-oxide (TMAO), not DMSO or fumarate, specifically induced anaerobic cholera toxin production likely through activation of stringent response (Lee et al. 2012; Oh et al. 2014; Oh et al. 2015). Using an infant mouse infection model, wildtype V. cholerae outcompeted mutant incapable of TMAO respiration (Oh et al. 2014) and while mutants either lacking or overproducing the intracellular stringent response signal ppGpp were attenuated (Oh et al. 2015).
V. cholerae from human stool samples not only outcompeted in vitro- grown bacteria in an infant mouse infection model (Merrell et al. 2002) but also exhibited increased transcript levels of genes involved in anaerobic growth
(Merrell et al. 2002; Bina et al. 2003). Using a rabbit ileal loop model to further
37 investigate V. cholerae in vivo transcriptional responses, higher expressions of genes involved in anaerobic metabolism were also identified (Xu, Dziejman, and
Mekalanos 2003). Multiple factors involved in colonization are also induced under anaerobic conditions (Marrero et al. 2009). Together, these studies strongly supported an anaerobic phase during V. cholerae infections. One of the known regulators involved in V. cholerae anaerobic responses is the transcription factor
AphB, which works with AphA and activates V. cholerae virulence regulatory cascade (Kovacikova and Skorupski 1999). AphB mainly responds to low pH and anaerobic conditions (Kovacikova, Lin, and Skorupski 2010; Taylor et al. 2012) and the activity of AphB is reversibly modulated by the presence and absence of oxygen through a cysteine residue (C235 in El Tor C6706) (Liu et al. 2011), which contributes to AphB dimerization under oxygen limiting conditions (Fan et al. 2014; Liu et al. 2011). In addition to AphAB, ArcA has also be found to play a role in the overall expression of ToxT, a key transcriptional activator for V. cholerae virulence genes (Sengupta, Paul, and Chowdhury 2003).
Yersinia enterocolitica
Yersinia enterocolitica is a Gram-negative pathogen typically transmitted through consumption of contaminated animal products. In the absence of oxygen, Y. enterocolitica is a mix acid fermenter and likely exhibits reduced TCA cycle activity based on a global transcriptional profiling study using a related organism, Y. intermedia (ATCC29909) (Babujee et al. 2013). Y. enterocolitica
38 virulence regulation responds strongly to a variety of environmental conditions
(Straley and Perry 1995; Chen, Thompson, and Francis 2016). Limited evidence also demonstrates a complex pathogenic response to different oxygen levels.
Expression of Ail, an adhesion protein, is down regulated under static growth at
37°C compared to aerobic, agitated growth (Pederson and Pierson 1995). Similar sub-oxic growth conditions also resulted in reduced invasion of Chinese hamster ovary cells and decreased resistance to serum killing (Pederson and Pierson
1995). In contrast, the expression of the invasin gene, inv, was not affected by anaerobic growth at 37°C. Moreover, expression of YplA, a phospholipase involved in pathogenesis, exhibited an increasing trend over growth conditions with increasing aeration (Schmiel, Young, and Miller 2000). These contrasting virulence gene responses indicate a complex behavior during Yersinia transit through the GI tract with fluctuating oxygen levels that remain to be determined.
Conclusion
It is clear that there is not a unifying response to fluctuating oxygen levels for all enteric pathogens. Within our limited selection of organisms, some, such as EHEC, L. monocytogenes, and S. Typhimurium, clearly exhibit enhanced adhesion or invasion of host cells, while others do not. Different electron acceptors for anaerobic respiration also exhibit different effects on virulence regulation. Even within the same organism, V. cholerae, despite multiple studies agree with the anaerobic induction of cholera toxin production, one study reports
39 the opposite observation where cholera toxin production is higher in aerobic but not anaerobic conditions (Krishnan et al. 2004). The variety of responses justify more in-depth investigations into potential cross-talks between oxygen-sensitive regulatory networks with pathways responsive to other environmental stimuli.
Moreover, the potential cross-talk strongly argues for a second and anaerobic look at the established virulence regulon of many well-studied transcription factors to reveal the connections between virulence regulation and pathogen physiology.
It is also important to consider the role of oxygen gradient and temporal exposure in enteric pathogenesis. In vitro growth in batch cultures does not realistically represent the oxygen gradient and temporal exposure experienced by enteric pathogens in vivo. Using chemostat for in vitro cultures, which can generate distinctive results from batch cultures (James and Keevil 1999a), may provide confirmation of batch culture findings and generate novel insight into the role of a dynamic system with systemic fluctuations in oxygen levels and temporal exposures. Similarly, cell culture infection models commonly used to determine the impact of oxygen levels on host-pathogen interactions should also incorporate oxygen gradient, similarly to a vertical diffusion chamber, across a polarized epithelium to better mimic the impact of oxygen gradient on both host cells and the pathogens.
In summary, enteric pathogens have complex and specific responses to fluctuating oxygen levels. Future research will enhance our understanding in
40 pathogen response during food processing and storage and gastrointestinal transit and colonization. These pieces of knowledge together will greatly improve our ability to control pathogen transmission and further reduce the medical and societal burden of foodborne infections.
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71 CHAPTER III
METABOLIC DETERMINANTS IN LISTERIA MONOCYTOGENES ANAEROBIC
LISTERIOLYSIN O PRODUCTION
This work was originally published in the peer-reviewed scientific journal Archives of Microbiology in 2017 with the following citation: Wallace, N., Newton, E.,
Abrams, E. et al. Arch Microbiol (2017) 199: 827. https://doi.org/10.1007/s00203-
017-1355-4
Abstract
Listeria monocytogenes is a human pathogen and a facultative anaerobe.
To better understand how anaerobic growth affects L. monocytogenes pathogenesis, we first showed that anaerobic growth led to decreased growth and changes in surface morphology. Moreover, compared to aerobically grown bacteria, anaerobically grown L. monocytogenes established higher level of invasion but decreased intracellular growth and actin polymerization in cultured cells. The production of listeriolysin O (LLO) was significantly lower in anaerobic cultures—a phenotype observed in wildtype and isogenic mutants lacking transcriptional regulators SigB or CodY or harboring a constitutively active PrfA.
72 To explore potential regulatory mechanisms, we established that addition of central carbon metabolism intermediates, such as acetate, citrate, fumarate, pyruvate, lactate, and succinate, led to an increase in LLO activity in the anaerobic culture supernatant. These results highlight the regulatory role of central carbon metabolism in L. monocytogenes pathogenesis under anaerobic conditions.
Introduction
Listeria monocytogenes is a foodborne pathogen and a leading cause of death from foodborne illnesses. While immuno-competent individuals may develop mild gastroenteritis after ingestion of large amounts of L. monocytogenes, immuno-compromised individuals have a higher risk of developing systemic infections. These infections can cause more severe symptoms and lead to fatal outcomes despite early antibiotic treatments.
Therefore, there is a need to better understand L. monocytogenes behavior during transmission to develop effective strategies to prevent infections. Upon ingestion, L. monocytogenes transits through the gastrointestinal tract and must adapt to host lumenal conditions in order to establish infections. However, despite the fact that the intestinal lumen is characterized by varying degrees of oxygenation, most of our understanding of L. monocytogenes pathogenesis is based on research conducted under aerobic conditions. The extent and the
73 mechanism by which anaerobic exposure impacts L. monocytogenes pathogenesis is unclear.
As a facultative anaerobe, L. monocytogenes can grow under strict anaerobic conditions with altered carbon metabolism. Chemical analyses have shown that in the presence of oxygen, L. monocytogenes incompletely oxidizes glucose to acetate, lactate, and acetoin. In the absence of oxygen, L. monocytogenes produces lactate as its major fermentation product along with ethanol, formate, and carbon dioxide (Romick, Fleming, and McFeeters 1996;
Pine et al. 1989; Jydegaard-Axelsen et al. 2004; Romick and Fleming 1998).
Moreover, transcriptional analyses using L. monocytogenes strain EGD showed a decreased transcript level for genes encoding pyruvate dehydrogenase and those involved in acetoin synthesis under anaerobic conditions (Müller-Herbst et al. 2014a) (Müller-Herbst et al. 2014). Genes encoding phosphotransferases systems also exhibited differential transcript levels in response to suboxic conditions (Toledo-Arana et al. 2009). Together these studies suggest that oxygen levels play a key role in regulating carbon metabolism in L. monocytogenes. However, it is not clear whether or how these metabolic adaptations influence L. monocytogenes pathogenesis under anaerobic conditions.
L. monocytogenes is an intracellular pathogen capable of growing and spreading between the cytosol of mammalian host cells. Its ability to invade non- phagocytic cells contributes to invasion of intestinal epithelium and subsequent systemic infections. Available evidence suggests that anaerobic growth results in
74 an enhanced invasion phenotype (Burkholder et al. 2009; Bo Andersen et al.
2007). However, the subsequent intracellular growth in the aerobic host cytosol is not known. Moreover, the signals mediating the anaerobic effects on L. monocytogenes infection have not been established. In this study, to provide a better understanding of L. monocytogenes behavior under anaerobic conditions, we investigated how anaerobic growth and the associated signals from anaerobic metabolism affect L. monocytogenes pathogenesis
Materials and Methods:
Bacterial strains and culture conditions
Culture of the wild-type and isogenic mutants of L. monocytogenes strain
10403s were grown from colonies on a freshly streaked BHI plate (<1 week old) at 37°C. Mutants used in this study include those with clean deletion in sigB
(∆sigB) and codY (∆codY) and one with a constitutively active PrfA (PrfA*)
(Bruno and Freitag 2010). All cultures were grown in filter-sterilized brain heart diffusion (BHI) media (Lot 4176589) to ensure consistency. Aerobic cultures were grown with agitation at 250 RPM to ensure adequate oxygen diffusion. Anaerobic cultures were grown in a temperature-controlled incubator inside an anaerobic chamber (Coy Laboratory, Type A) with a nitrogenous atmosphere containing
2.5% hydrogen. Optical density (OD) was measured in an optically clear 96-well plate at 600 nm with a volume of 200 µL per well using a 96-well plate reader
75 (Biotek Synergy4). Supplements included sodium acetate (Fisher Scientific
BP334-500), sodium fumarate (Acros Organics AC21553-1000), sodium succinate (Acros Organics AC20874-5000), sodium citrate (Fisher Scientific
S279-500), acetoin (Acros Organics AC 41195-100), sodium pyruvate (Alfa
Aesar A11148), and lithium lactate (Acros Organics 413331000). All supplements were prepared as 1 M stock solutions in deionized water, filter-sterilized, and added directly to the media to the desired concentration before inoculation.
Measurement of lactate, acetoin, and ethanol concentrations
Supernatant lactate was measured using a commercially available enzymatic kit following the manufacturer’s suggested protocol (Fisher 50-489-
257). The Voges-Proskauer test (Nicholson 2008) was adapted to quantify acetoin production in the supernatant of overnight L. monocytogenes cultures.
Supernatant or standard (100 µL) was placed into a sterile micro-centrifuge tube followed by additions of 70 µL of .5% creatine monohydrate (Sigma C3630-
100G), 100 µL of 1-Napthol (Sigma N1000-10G), and 100 µL of 40% KOH
(Chempure 831-704) in 95% EtOH. Samples were centrifuged between each addition, and incubated at room temperature for 15 minutes after the final addition. After incubation 200 µL of each sample was placed into a flat bottom
96-well plate and the absorbance was read at 560 nm. A standard curve was constructed to calculate the concentration of acetoin in culture supernatant
76 samples. Ethanol percentage was measured using a commercially available enzymatic kit following manufacturer’s suggested protocol (Fisher 50-489-254).
Transmission electron microscopy
Overnight aerobic and anaerobic cultures of L. monocytogenes were visualized using transmission electron microscopy. Bacterial cultures (3 mL) were spun down to collect pellets, which were first fixed using 2 mL of a 2% paraformaldehyde (Alfa Aesar 30525-89-4) and 2% glutaraldehyde (Alfa Aesar
111-30-8) in phosphate buffer solution for 24 hours at 4°C. Following fixation, cells were washed 3 times for 10 minutes in phosphate buffer. Washed cells were then post fixed using a 2% solution of OsO4 in phosphate buffer for 24 hours at 4°C. Following post fixation cells were stained with a 2% lead citrate in phosphate buffer solution at 4°C for 24 hours. After staining the cells were treated to a series of dehydrations in ethanol (30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, 100%) each for 10 minutes. The dehydrated cells were then embedded in API-PON 812 epoxy resin monomer (SPI-CHEM 90529-77-4) and dried for 24 hours at 70°C in an oven. The dried samples were sectioned using an ultra-microtome with a diamond blade to 100 nm sections. The sections were then embedded on lacy carbon grids and read using a Hitachi H-7600
Transmission Electron Microscope at 120kv. Measurements of cell envelope thickness were made using GNU Image Manipulation Program (GIMP).
77 Cell culture infection
The murine peritoneal macrophages RAW 264.7 (ATCC TIB-71), Caco-2 colorectal adenocarcinoma cells (ATCC HTB-37), and LS174T mucin-secreting colorectal adenocarcinoma cells (ATCC CL-188) were grown in DMEM media
(Thermo Scientific SH30285.01) supplemented with 10% (v/v) heat inactivated fetal bovine serum (JRScientific REF 4365-500, Lot N056-6), HEPES (10 mM), and glutamine (2 mM) in a 37°C incubator with a 5% CO2 atmosphere. Prior to infections, cells were seeded in a 24-well tissue culture plate and grown for 14-18 hours. Overnight cultures of L. monocytogenes were used for infections at an
MOI of 10. Bacteria diluted in cell culture medium were added to each well (500
µL) and incubated for 30 minutes. Following incubation media was aspirated and cells were washed twice with sterile DPBS. Fresh media (1 mL per well) containing 10 µg/mL gentamicin stock was added to each well. To enumerate intracellular bacteria, cell culture media was aspirated off and sterile 0.1% (v/v) triton X-100 was added to each well (200 µL per well) to lyse host cells. Lysates were diluted and spread on LB plates. Colonies on plates were counted using an automatic colony counter (Synbiosis aCOLyte 3) after 24-48 hours of incubation in a 37°C incubator.
78 Immunofluorescence microscopy
RAW264.7 macrophages were plated onto sterile coverslips (18 by 18 mm) inside 6-well plates at 1 million cells per well in the afternoon prior to infections. Overnight L. monocytogenes cultures were washed twice and diluted in cell culture media for infection at an MOI of 10. At 2 hpi, coverslips were fixed in paraformaldehyde (3.7% in PBS) overnight at 4°C. For immunofluorescence microscopy, each coverslip was washed with TBS-T (25 mM Tris-HCl, 150 mM
NaCl, 0.1% Triton X-100) and blocked with TBS-T with 1% bovine serum albumin
(BSA). Anti-Listeria serum (1:500 in TBS-T with 1% BSA; Thermo Scientific PA1-
30487) was added onto each coverslip and incubated at room temperature for one hour. Each coverslip was washed in 5 ml of TBS-T prior to incubation with secondary antibodies: phalloidin-iFluor 594 (1: 400, abcam ab176757) and
AlexaFluor 488–goat anti-rabbit antibody (1:400, abcam ab150077) in TBS-T with 1% BSA. One hundred intracellular bacteria per experimental replicate were scored for the presence or absence of actin clouds.
Hemolytic assays
Hemolytic assays were performed using overnight culture supernatant samples. Each sample was incubated at room temperature with 0.1 M DTT (5
µL) for 15 minutes. A positive control (0.4% triton X-100) and a negative control
(blank BHI media) were included for each experiment. After incubation, samples
79 were serially diluted using hemolysis buffer containing: dibasic sodium phosphate
(35 mM) and sodium chloride (125 mM) brought to pH 5.5 with acetic acid.
Defibrinated sheep’s blood (Hemostat Laboratories DSB050) was diluted to a hematocrit of 2% and then added to each sample for a final hematocrit of 1%.
Samples were incubated at 37°C for 30 minutes. After incubation, all samples were spun down at 2000 RPM for 5 minutes to pellet intact blood cells.
Supernatant lysate (120 µL) was transferred to a flat bottom 96-well plate for OD measurement at 541 nm as an indicator for LLO activity. Hemolytic unit was calculated as the inverse of the dilution factor at which half complete lysis occurred and subsequently normalized with original culture OD measured at absorbance at 600 nm. Samples that did not produce lysis at a level more than half of complete lysis were designated as “Below Detection” for their hemolytic units. Supernatant samples from anaerobic cultures typically generate activities at or slightly above “Below Detection” levels.
SDS-PAGE, silver staining, and immunoblotting
Samples from overnight cultures of L. monocytogenes were used for SDS-
PAGE and western blotting. Cultures were normalized by optical density (600 nm) using BHI media and centrifuged to separate supernatant and bacterial cell pellets. Supernatant samples were precipitated with 1% trichloroacetic acid at
4°C for 1 hour. Following precipitation, a cold acetone wash was performed. Both the pellet and supernatant samples were resuspended in 12 µL of 2x sample
80 buffer and heated at 95°C for 5 min. The samples were then separated via SDS-
PAGE (8% acrylamide in the separating gel). Following SDS-PAGE, gels were either subjected to silver staining (Thermo Scientific 24612) following manufacturer’s protocol or proteins in gel were transferred to a PVDF membrane for subsequent immunoblotting using anti-LLO rabbit antibody (1:10,000, abcam ab43018) followed by goat-anti-rabbit HRP antibody (1:10,000, abcam ab6721).
Bands were visualized using chemilluminescent substrate (BIO-RAD 170-5060) and captured with x-ray films (WorldWide Medical Products 41101002).
Results
Characterization of anaerobic growth by Listeria monocytogenes strain
10403s
Current knowledge of anaerobic metabolism in L. monocytogenes is built from research using different laboratory strains (Pine et al. 1989; Romick,
Fleming, and McFeeters 1996; Müller-Herbst et al. 2014). Strain 10403s is widely used as a model organism, but its anaerobic metabolism has not been investigated. Therefore, we first monitored in vitro growth of strain 10403s in the presence or absence of oxygen in standard BHI medium. As expected for a facultative anaerobe, static growth in the absence of oxygen resulted in a lower maximal optical density compared to agitated aerobic growth (Fig. 1A).
Compared to aerobic growth, anaerobic growth of strain 10403s resulted in lower
81 pH, higher concentrations of ethanol and lactic acid, and no detectable levels of
acetoin (Table 1). Using TEM to visualize strain 10403s also highlighted a
morphological difference between aerobically and anaerobically grown cells (Fig.
1B, C). Anaerobically grown strain 10403s exhibited a notably increased space
between cytoplasm and the outer edge of the cells.
Figure 1. Anaerobically grown L. monocytogenes exhibits decreased maximal growth in vitro and morphological differences under TEM. AFigure Growth 3.1 cur vAnaerobicallyes of L. monoc ygrowntogene L.s s monocytogenestrain 10403s grown exhibits in BHI ar edecreased plotted on amaximal linear Y- axis to show the decreased magrowthximal O Din o vitrover 8 andh of gmorphologicalrowth. Averages differences of triplicates aunderre plo tTEMted w.i tAh eGrowthrror bar scurves repre sofen L.tin g the standard deviation and monocytogenesstatistics were pe strainrforme 10403sd using agrown two-ta inile BHId stu areden plottedt’s t test onwi tha slinearignific Ya-naxist diff etore shownces ithendic ated by asterisks (***p<.001). Adecreasederobically ( maximalB) or ana eODrob overically 8(C h) gofro growth.wn L. m oAveragesnocytoge nofe striplicates were visu arealiz eplottedd with T withEM. Serrorpac e between bars representing the standard deviation and statistics were performed using a two-tailed cytoplasm and outer edge of cells (n=10) was measured and shown under their respective images as averages±standard student’s t test with significant differences indicated by asterisks (***p < 0.001). Aerobically deviation (B) or anaerobically (C) grown L. monocytogenes were visualized with TEM. Space between cytoplasm and outer edge of cells (n = 10) was measured and shown under their respective images as averages ± standard deviation
82 Table 3.1: Characterizations of L. monocytogenes strain 10403s in vitro growth. Values shown are averages of triplicate standard deviation a p values were calculated between aerobic and anaerobic samples using a two-tailed student’s t test
Culture Culture pH [Lactate] [Acetoin] [Ethanol] pH (Buffered (mM) (mM) (%) Figure 3.2 Anaerobic(BHI) growth BHI)of L. monocytogenes leads to increased initial intracellular CFU but decreased intracellular growth and actin co-localizationTable 3.1: Characterizations of L. monocytogenes Aerobic 5.41 6.57 0.01 0 1.37 0.22 strain 10403s in vitro growth. Values shown are0.14 averages of triplicate standard deviation 0.51 0.000 Anaerobic 4.67 6.51 0.03 1.75 0 1.43 a p values were calculated0.12 between aerobic and anaerobic0.31 samples using a two0.002-tailed student’sP value ta test 0.002 0.48 0.009
Figure 3.2 Anaerobic growth of L. monocytogenes leads to increased initial intracellular CFU but decreased intracellular growth and actin co-localization. Cell Effectsculture infections of anaerobic were performed exposure with humanon cell colonic culture epithelial infections cell lines, Caco-2 (A) and LS174T (B), and with murine peritoneal macrophages, RAW264.7 (C, D). All infections were performed with MOI of 10 using aerobically or anaerobically grown L. monocytogenes. Approximately 100 L. monocytogenes cells were counted for actin co- localization per infection condition at 2 hpi. Averages of triplicates are plotted with error bars representingTo determine standard the deviation impact andof anaerobic statistics were growth performed on L. using monocytogenes a two-tailed student’s t test with significant differences indicated by asterisks (*p < 0.05, **p < 0.01, infections,***p < 0.001) we infected murine macrophages (RAW264.7) and human colonic epithelial cells (Caco-2 and LS174T) with overnight L. monocytogenes grown underFigure aerobic3.3 Anaerobically or anaerobic grown conditions. L. monocytogenes At 1 hpi, secretes there wasless LLOa significantlyFigure 3.2 higher Anaerobic growth of L. monocytogenes leads to increased initial intracellular CFU but decreased intracellular growth and actin co-localizationTable 3.1: intracellularCharacterizations CFU of in L both. monocytogenes Caco-2 (Fig. 2A) and LS174T (Fig. 2B) cells infected strain 10403s in vitro growth. withValues anaerobically shown are averages grown of L. triplicate monocytogenes standard deviation compared to those infected with aerobicaa p values llywere grown calculated bacteria. between We aerobic also investigatedand anaerobic thesamples impact using of a anaerobic two-tailed growth student’s t test on infection stages beyond the initial invasion by monitoring intracellular growth of aerobically or anaerobically grown L. monocytogenes in RAW264.7 macrophages.Figure 3.2 Anaerobic While growth there of was L. monocytogenes a higher intracellular leads to number increased of initial bacteria in intracellular CFU but decreased intracellular growth and actin co-localizationTable 3.1: Characterizations of L. monocytogenes macrophages strain 10403s in infected vitro growth with. anaerobically grown bacteria at 1 hpi, intracellular Values shown are averages of triplicate standard deviation
a p values were calculated between aerobic and83 anaerobic samples using a two-tailed student’s t test growth by anaerobically grown L. monocytogenes was significantly reduced in later time points post infection (Fig. 2C). Because intracellular growth relies on L. monocytogenes escape from phagosomes into the cytosol, we enumerated the proportion of cytosolic bacteria by measuring actin co-localization at 2 hpi inside macrophages. L. monocytogenes grown under anaerobic conditions exhibited significantly compromised actin co-localization compared to those grown under aerobic conditions (Fig. 2D). These data suggest that anaerobic growth has a strong effect on the outcome of infections. Moreover, because all infections were performed under aerobic conditions, the observed differences between aerobically and anaerobically grown bacteria suggest that anaerobic exposure may have a long-term impact on subsequent interactions with host cells under aerobic conditions.
84
FigureFigur e3.2 2. AnaerobicAnaerobic growthgrowth o off L L.. m monocytogenesonocytogenes le aleadsds to itonc increasedreased init iinitialal intra intracellularcellular CFU but CFUdec butrea sdecreaseded intracel lintracellularular growth a ngrowthd actin cando-lo actincaliza cotio-nlocalization. Cell culture. Cellinfe cculturetions w infectionsere perfor mwereed w ith performedhuman c owithlon ichuman epithe coloniclial cell epitheliallines, Ca ccello-2 lines, (A) a nCacod LS-127 (4AT) (andB), a LS174Tnd with m(Bu),r iandne p witherito murineneal peritonealmacroph macrophages,ages, RAW26 4RAW264.7.7 (C, D). A (lC,l in Dfe).c tAllion infectionss were pe rwereform eperformedd with MO Iwith of 1MOI0 us ofin g10 a eusingrobic ally or aerobically or anaerobically grown L. monocytogenes. Approximately 100 L. monocytogenes cells wereana countederobical lfory g ractinown Lco. m-localizationonocytoge pernes .infection Approxi mconditionately 10 at0 L 2. mhpi.on Averagesocytogene ofs ctriplicatesells were arecou nted for plottedactin cwitho-lo errorcaliz abarstion representingper infection cstandardondition deviationat 2 hpi. A andvera statisticsges of tr iwereplica tperformedes are plot tusinged wi tah twoerro-r bars tailedrepr estudent’ssenting st tatestnd awithrd d significanteviation a ndifferencesd statistics windicatedere perf obyrm asterisksed using (* ap tw < 0.05,o-taile **d spt u< 0.01,dent’s t test with ***spig < 0.001)nificant d ifferences indicated by asterisks (*p<0.001)
Figure 3.3 Anaerobically grown L. monocytogenes secretes less LLOFigure 3.2 Anaerobic Effectsgrowth of L.anaerobic monocytogenes growth leads on to LLO increased production initial intracellular CFU but decreased intracellular growth and actin co-localization. Cell culture infections were performed with human colonic epithelial cell lines, Caco-2 (A) and LS174T (B), and with murine peritoneal macrophages, RAW264.7 (C, D). All infections were performed with MOI of 10 using aerobically or anaerobically grown L. monocytogenes. Approximately 100 L. monocytogenes cells were countedLLO for actinis a secretedco-localization hemolysin per infection and condition its pore at-form 2 hpi.ing Averages activity of contributes triplicates are to plotted L. with error bars representing standard deviation and statistics were performed using a two-tailed monocytogenesstudent’s t test with escape significant from differences phagosomes indicated to by the asterisks cytosol. (*p Therefore,< 0.05, **p < 0.01, based on ***p < 0.001)
85 Figure 3.3 Anaerobically grown L. monocytogenes secretes less LLO. A LLO activity is decreased in anaerobic culture supernatant compared to aerobic culture supernatant of wildtype strain 10403s and isogenic mutants. Averages of triplicates are plotted with error bars representing standard deviation and statistics were performed using a two-tailed student’s t test the infection phenotypes, we hypothesized that anaerobic growth, in contrast to enhancing invasion (Fig. 2A and 2B) (Burkholder et al. 2009; Bo Andersen et al.
2007), resulted in decreased LLO production. We tested supernatant samples from overnight aerobic or anaerobic cultures for LLO activities through hemolytic assays and found little to no detectable hemolytic activity in the anaerobic culture supernatant (Fig. 3A). Using immunoblotting and silver staining, it was clear that while anaerobic growth did not alter the overall protein abundance in the supernatant (Fig. 3B bottom), it resulted in a clear decrease in LLO abundance
(Fig. 3B top). Because LLO production can be regulated by multiple transcription factors PrfA, SigB, and CodY (de las Heras et al. 2011; Rauch et al. 2005; Lobel et al. 2015) we tested isogenic mutants lacking known transcriptional regulators
SigB (∆sigB) or CodY (∆codY) or harboring a constitutively active virulence master regulator PrfA (PrfA*) for their LLO production in response to anaerobic growth. While the PrfA* mutant exhibited higher levels of LLO production, all 3 mutants tested, similarly to wild type, produced significantly lower levels of LLO under anaerobic conditions compared to aerobic conditions (Fig. 3D). These results highlighted that LLO production is under strong regulation by the presence or absence of oxygen. Moreover, this anaerobic suppression of LLO production is not directly mediated by known virulence regulators PrfA, SigB, and
CodY.
86 A B SUP LYS * 160 rLLO ∆hly A AN A AN ** 3000
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Figure 3.3 Anaerobically grown L. monocytogenes secretes less LLO. A LLO activity is decreased in anaerobic culture supernatant compared to aerobic culture supernatant of wildtype strain 10403s and isogenic mutants. Averages of triplicates are plotted with error bars representing standard deviation and statistics were performed using a two-tailed student’s t test with significant differences indicated by asterisks (*p<0.05 ** p<.01). B top Abundance of LLO is lower in anaerobic (“AN”) culture supernatant (“SUP”) compared to aerobic (“A”) culture supernatant. Lysate (“LYS”) of samples shows similar total protein levels. B bottom Silver stain was used as a loading control and shows similar total protein levels between aerobic and anaerobic samples. Recombinant LLO (“rLLO”) was used as a positive control and supernatant from mutant lacking the hly gene (∆hly) was used as a negative control
Figure 3.4 Buffering media or exogenous supplementation of lactate or acetoin does not alleviate the reduced LLO production under anaerobic conditions relative Effectsto aerobic of conditionsFiguremetabolic signals 3.3 Anaerobicallyon anaerobic grown LLO L. production monocytogenes secretes less LLO. A LLO activity is decreased in anaerobic culture supernatant compared to aerobic culture supernatant of wildtype strain 10403s and isogenic mutants. Averages of triplicates are plotted with error bars representing standard deviation and statistics were performed using a two-tailed student’s t test with significant differences indicated by asterisksTo (*p<0.05 identify ** factors p<.01). contributingB top Abundance to regulation of LLO is lower of LLO in anaerobic production (“AN”) in culture response supernatant (“SUP”) compared to aerobic (“A”) culture supernatant. Lysate (“LYS”) of tosamples the presence shows similar or absence total protein of oxygen,levels. B bottomwe investigated Silver stain thewas roleused ofas physiological a loading control and shows similar total protein levels between aerobic and anaerobic samples. Recombinant LLO (“rLLO”) was used as a positive control and supernatant from mutant andlacking metabolic the hly gene signals (∆hly) differentially was used as agenerated negative control during aerobic or anaerobic growth. We first considered the role of lactic acid, a fermentation acid produced Figure 3.4 Buffering media or exogenous supplementation of lactate or acetoin fromdoes pyruvate not alleviate during the reducedL. monocytogenes LLO production anaerobic under anaerobic growth, conditionsin regulation relative of LLO to aerobic conditions. A Compared to aerobic cultures, LLO activity in supernatant of anaerobic cultures in BHI or BHI buffered with MOPS (pH 7.0) was significantly lower. B production.Lactate supplementation The signal enhancedfrom lactic culture acid supernatant could be twoLLO foldactivity— thein aerobically acidification and of the anaerobically grown L. monocytogenes. C Acetoin supplementation did not enhance LLO mediumactivity in or aerobically the organic or anaerobically acid itself. grown To test L. monocytogenes. the role of medium Averages acidification, of triplicates we are plotted with error bars representing standard deviation and statistics were performed measuredusing a two LLO-tailed activity student’s in t thetest withsupernatant significant ofdifferences cultures indicated grown inby bufferedasterisks medium (*p<.05 **p<.01)
87 Figure 3.5 Supplementation of intermediates involved in central carbon metabolism alters carbon metabolism and increases supernatant LLO activity of anaerobically grown L. monocytogenesFigure 3.4 Buffering media or exogenous supplementation of lactate or acetoin does not alleviate the reduced LLO to prevent medium acidification with or without oxygen. In MOPS-buffered medium (pH 7.0), while there was no significant difference in pH between aerobic and anaerobic cultures (Table 1), LLO activity was significantly lower in anaerobic culture supernatant than that in aerobic culture supernatant (Fig. 4A).
Exogenous supplementation of lactate (2 mM) resulted in increased LLO activity in both aerobic and anaerobic culture supernatant but didn’t alleviate the relatively lower levels of anaerobic LLO production. (Fig. 4B). In contrast, exogenous supplementation of the aerobic metabolite, acetoin, did not affect LLO activity in aerobic or anaerobic cultures (Fig. 4C). These results suggest that while acetoin and lactate are both metabolite products of pyruvate, only lactate supplementation influenced anaerobic LLO production.
Figure 3.4 Buffering media or exogenous supplementation of lactate or acetoin does not alleviate the reduced LLO production under anaerobic conditions relative to aerobic conditions. A Compared to aerobic cultures, LLO activity in supernatant of anaerobic cultures in BHI or BHI buffered with MOPS (pH 7.0) was significantly lower. B Lactate supplementation enhanced culture supernatant LLO activity in aerobically and anaerobically grown L. monocytogenes. C Acetoin supplementation did not enhance LLO activity in aerobically or anaerobically grown L. monocytogenes. Averages of triplicates are plotted with error bars representing standard deviation and statistics were performed using
88
Effects of central carbon metabolites on LLO production
Lactate production is catalyzed by a reversible enzyme, lactate dehydrogenase, from pyruvate—a metabolite that connects to multiple carbon metabolic pathways in L. monocytogenes (Fig. 5A). Therefore, the effect of lactate on anaerobic LLO production is likely mediated by signals generated through pyruvate metabolism. When pyruvate was supplemented in the culture medium, we observed a dramatic increase in both aerobic and anaerobic LLO production (Fig. 5B). The pyruvate supplementation also resulted in an increase in acetoin production under both aerobic and anaerobic conditions (Fig. 5C), a phenotype suggesting exogenous pyruvate was taken up and metabolized.
Because pyruvate is also metabolized to generate acetyl-coA for tricarboxylic acid (TCA) cycle, we tested the effects of TCA intermediates on anaerobic LLO production. If increase in the carbon flux through pyruvate was important in enhancing anaerobic LLO production, then supplementation of downstream metabolites in the TCA cycle should exhibit similar anaerobic enhancement of
LLO production. Indeed, supplementations of acetate, citrate, succinate, and fumarate all resulted in higher levels of anaerobic LLO production (Fig. 5D).
These data highlighted a potential role for central carbon metabolites in influencing LLO production in the absence of oxygen.
89 Figure 3.5 Supplementation of intermediates involved in central carbon metabolism alters carbon metabolism and increases supernatant LLO activity of anaerobically grown L. monocytogenes. A Simplified schematic shows three possible fates of pyruvate in L. monocytogenes central carbon metabolism. B Exogenous supplementation of pyruvate enhanced LLO activity in both aerobic and anaerobic culture supernatant. C Exogenous pyruvate supplementation increased acetoin concentrations in both aerobic and anaerobically grown L. monocytogenes. D Supplementation of intermediates of the TCA cycle (50mM) enhanced anaerobic LLO activity. Averages of triplicates are plotted with error bars representing standard deviation and statistics were performed using a two-tailed student’s t test with significant differences indicated by asterisks (*p<.05 ***p<0.001)
Discussion
As an enteric pathogen, L. monocytogenes encounters fluctuating levels of oxygen from the aerobic oral cavity to the anaerobic intestinal lumen. As a result, metabolic adaptations to anaerobic conditions are an inevitable process
90 during intestinal phase of infections. Here we show that anaerobic growth resulted in major changes in carbon metabolism characterized by the lack of acetoin production and the increased production of lactate and ethanol. Ethanol concentrations for aerobic cultures may be underestimated because of the loss through culture agitation during aerobic growth. Curiously, anaerobic growth led to different morphologies under TEM. It is not clear if the differences in morphology are a result of specific structural differences or a result of different responses to TEM sample preparation processes. Both scenarios suggest surface modifications in anaerobically grown L. monocytogenes that can potentially lead to changes in stress resistance during transit through the anaerobic lumen during intestinal phase of infections.
Anaerobic growth also resulted in significant changes in subsequent interactions with host cells under aerobic conditions. Anaerobically grown L. monocytogenes exhibited a significant increase in cell invasion but a significant decrease in actin co-localization and intracellular growth compared to aerobically grown bacteria. These results suggest that while anaerobic growth results in enhanced internalization into host cells, likely as a result of the increased expressions of internalins (Toledo-Arana et al. 2009) and LAP (Burkholder et al.
2009), it does not provide advantages in subsequent intracellular growth.
Because L. monocytogenes entry into the host cytosol mainly relies on the activity of LLO (Hamon et al. 2012), the lack of actin co-localization phenotype can be partially attributed to the reduced LLO production exhibited by anaerobically grown bacteria. Alternatively, it is also possible that anaerobically
91 grown L. monocytogenes have compromised intracellular expression of ActA, which facilitates actin polymerization as a means for bacterial motility and cell- cell spread. ActA is typically expressed by intracellular L. monocytogenes.
However, the role of physiological or metabolic states of L. monocytogenes prior to entering host cells in intracellular ActA expression is not clear. Given the role of L. monocytogenes dissemination in lethal infections, knowledge of how extracellular conditions influence subsequent intracellular behavior can be used to develop strategies to restrict L. monocytogenes infections in the intestines without spreading to peripheral organs.
To begin investigating the regulatory mechanism, we first tested the anaerobic LLO production in isogenic mutants either lacking known transcription regulators (∆sigB and ∆codY) or harboring constitutively active regulator (PrfA*).
In all the mutants tested, hemolytic activities in anaerobic culture supernatant were significantly lower than those in aerobic culture supernatant. These results suggest that these known transcriptional regulators are not directly involved in the anaerobic suppression of LLO production. L. monocytogenes genome contains 15 putative members in the Crp/Fnr protein family (Glaser et al. 2001), which is known for their ability to detect and respond to environmental signals such as fluctuating oxygen levels (Körner, Sofia, and Zumft 2003). Although mutations in each of these genes did not result in compromised growth in reduced oxygen conditions (Uhlich, Wonderling, and Luchansky 2006), these regulators may still play a direct or indirect role in detecting oxygen levels and modulating virulence gene expressions. In addition to the Crp/Fnr protein family,
92 L. monocytogenes has 15 histidine kinases and 16 response regulators with demonstrated functions in fitness and pathogenesis (Williams et al. 2005; Larsen et al. 2006; Vivant et al. 2014; Gottschalk et al. 2008; Kallipolitis et al. 2003;
Dons et al. 2004; Flanary et al. 1999; Brøndsted et al. 2003; Collins et al. 2012;
Cotter, Guinane, and Hill 2002; Nielsen et al. 2012; Kallipolitis and Ingmer 2001;
Pöntinen et al. 2015). However, it’s not clear how the signal transduction system is involved in L. monocytogenes anaerobic adaptations. Future investigations into their activities under anaerobic conditions can dramatically enrich our current understanding of L. monocytogenes anaerobic virulence regulation.
To further explore potential signals involved in the regulation of anaerobic
LLO production, we first tested the effects of lactic acid, the main product of L. monocytogenes anaerobic metabolism, on anaerobic LLO production. We considered lactic acid as two separate signals, medium acidification and the organic acid itself, and found that the lower LLO production under anaerobic conditions compared to aerobic conditions cannot be explained by medium acidification or lactate. While lactate supplementation does not influence the potential suppression of anaerobic LLO production compared to aerobic LLO production, it enhances anaerobic LLO production compared to no lactate anaerobic control. This led us to consider anaerobic carbon metabolism as part of the signaling pathway leading to decreased anaerobic LLO production. Lactate is typically produced by L. monocytogenes from pyruvate through a reversible enzyme, lactate dehydrogenase. Therefore, the exogenous supplementation of lactate may potentially be converted back to pyruvate, which can then enter
93 multiple carbon metabolic pathways. In contrast, the lack of effect from acetoin suggests that the acetoin production is a non-reversible pathway or that the expression of pathway enzymes are suppressed under anaerobic conditions. To directly confirm the role of pyruvate, we tested and demonstrated the positive effects of exogenous pyruvate on LLO and acetoin production. The dramatic effects of pyruvate observed in our study suggest that LLO production is sensitive to modulation by signals generated through pyruvate metabolism.
The TCA cycle is one of the main metabolic pathways utilizing pyruvate as the main carbon substrate. L. monocytogenes has an incomplete TCA cycle (Fig.
5A), lacking 2-oxoglutarate dehydrogenase, succinyl-CoA synthetase, and succinic dehydrogenase (Glaser et al. 2001; Trivett and Meyer 1971). Although an incomplete TCA cycle is not an uncommon genotype in bacteria (Huynen,
Dandekar, and Bork 1999), its presence often demands additional means for bacteria to generate TCA intermediates to support anabolic pathways. L. monocytogenes is capable of generating oxaloacetate from pyruvate by pyruvate carboxylase (Schär et al. 2010) and succinate from gamma-aminobutyrate
(GABA) by the glutamate decarboxylase system coupled with the GABA shunt under acid stress conditions (Feehily, O’Byrne, and Karatzas 2013; Cotter,
Gahan, and Hill 2001). As a result, the carbon flux of TCA cycle in L. monocytogenes might not be unidirectional and might change under different physiological conditions. In E. coli and Bacillus subtilis, TCA cycle is known to be suppressed under anaerobic conditions (Spencer and Guest 1987; Nakano,
Zuber, and Sonenshein 1998; Gray, Wimpenny, and Mossman 1966) and by
94 catabolite repression (Gosset et al. 2004; Nakano, Zuber, and Sonenshein
1998). While catabolite repression has been associated with L. monocytogenes virulence regulation (Gilbreth, Benson, and Hutkins 2004), which is known to respond to the presence of fermentable carbohydrates (Behari and Youngman
1998), the anaerobic TCA cycle activities have not been investigated in detail. If
TCA cycle activity is reduced in L. monocytogenes under anaerobic conditions similarly to E. coli and B. subtilis, our results showing the positive effects of TCA cycle intermediates on anaerobic LLO production suggest a connection between the reduced TCA cycle activity and the decreased anaerobic LLO production.
All TCA cycle intermediates, when supplemented exogenously, resulted in an increase in anaerobic LLO production. Curiously, only citrate supplementation led to a significantly decreased aerobic LLO production compared to no supplementation control. Citrate has a multifaceted role in bacterial metabolism and physiology. As an intermediate metabolite in the TCA cycle, it serves as a feedback molecule that binds to the catabolite control protein C (CcpC) and suppresses the transcription of the first two genes in the TCA cycle—citrate synthase (citZ) and aconitase (citB) (Kim, Mittal, and Sonenshein 2006a; Mittal et al. 2013). However, when the intracellular level of citrate is artificially high, as established with citB mutation, citrate-bound CcpC acts as a transcriptional activator for citB
. Therefore, the relationship between citrate levels and CcpC activities is not linear. It is possible that the opposing effects of exogenous citrate on aerobic or
95 anaerobic LLO production reflect the different intracellular citrate levels achieved by exogenous citrate supplementations and the corresponding citrate synthase and aconitase activities under aerobic or anaerobic conditions.
In summary, our study highlights a critical role of anaerobic exposure in L. monocytogenes infections. L. monocytogenes grown anaerobically exhibit higher levels of internalization into host cells but compromised actin polymerization and intracellular growth, both of which might be attributed to the decreased LLO production. To better understand the mechanism underlying the anaerobic regulation of LLO production, our study suggests TCA cycle metabolites as positive signaling molecules for anaerobic LLO production. With anaerobic exposure a necessary step during infections, results from our study help strengthen current knowledge on L. monocytogenes adaptations and responses under anaerobic conditions.
Conflict of Interest
The authors declare that they have no conflict of interest.
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101 CHAPTER IV
STIMULATING RESPIRATORY ACTIVITY PRIMES ANAEROBICALLY GROWN
LISTERIA MONOCYTOGENES FOR INTRACELLULAR INFECTION
Abstract
Listeria monocytogenes is a Gram-positive, enteric pathogen and the causative agent of listeriosis. During transition through the gastrointestinal tract,
L. monocytogenes routinely encounters suboxic conditions. However, how the exposure to the low oxygen environment affects subsequent pathogenesis is not completely understood. Our lab previously reported that anaerobically grown L. monocytogenes exhibited an intracellular growth defect in macrophages even though the infections took place under aerobic conditions. This phenotype suggests that prior growth conditions have a prolonged effect on the outcome of subsequent intracellular infection. In this study, we investigated further the mechanisms that contribute to the compromised intracellular growth after anaerobic exposure. We hypothesized that the lack of respiratory activity under anaerobic conditions prevented anaerobically grown L. monocytogenes to establish subsequent intracellular growth under aerobic conditions. Therefore, we stimulated respiratory activity in anaerobically grown L. monocytogenes by
102 supplementing exogenous fumarate and assessed subsequent intracellular pathogenesis. We found that fumarate supplementation significantly increased the respiratory activity of anaerobically grown L. monocytogenes and rescued the subsequent intracellular growth defect, likely through promoting the production of listeriolysin O, phagosomal escape, and cell-cell spread. This study highlights the importance of anaerobic exposure in the intestinal lumen in the outcome of subsequent intracellular infections.
Keywords: Listeria monocytogenes, anaerobic respiration, fumarate, intracellular infection
Introduction
Listeria monocytogenes (L. monocytogenes) is a Gram-positive, facultative anaerobe found in a variety of environmental habitats (Vivant,
Garmyn, and Piveteau 2013; Linke et al. 2014). When L. monocytogenes enters an animal host through ingestion and establishes infections as an intracellular pathogen, it can cause diseases with high mortality rate in immunocompromised individuals. For example, within the 1998-2008 decade, confirmed L. monocytogenes outbreaks led to a total of 38 deaths out of 359 illnesses
(Cartwright et al. 2013). Moreover, it was estimated that L. monocytogenes infections were responsible for an annual cost burden of $2.8 billion in the United
States (USDA ERS - Cost Estimates of Foodborne Illnesses n.d.). The high
103 mortality rate and the cost burden associated with L. monocytogenes infections together render L. monocytogenes a critical foodborne pathogen that needs to be under routine surveillance in the food industry. However, L. monocytogenes exhibits a unique ability to survive and grow in food preservation processes that are generally considered antimicrobial, such as high osmotic stress, low pH, and refrigeration temperatures (Dykes and Moorhead 2000; Walker, Archer, and
Banks 1990; Angelidis and Smith 2003). Therefore, to effectively prevent L. monocytogenes contamination in food products and protect high-risk individuals from L. monocytogenes exposure, it is necessary to consider and understand how L. monocytogenes responds and adapts to different environmental conditions.
Being a facultative anaerobe, L. monocytogenes is able to survive and proliferate in conditions of low to no oxygen. As L. monocytogenes traverses the human gastrointestinal tract during infection, it encounters varying concentrations of oxygen (Fisher et al. 2013; Albenberg et al. 2014). How this exposure and potential adaptation to microaerobic to anaerobic conditions impact subsequent infections is not completely understood. L. monocytogenes cultured under strict anaerobic conditions have an initial invasion advantage compared to those cultured under aerobic condition, a phenotype mediated through the upregulating the Listeria adhesion protein (LAP) under anaerobic (Burkholder and Bhunia
2010; Burkholder et al. 2009). We have also observed this initial invasion advantage in anaerobically grown L. monocytogenes during infections of different cell lines. However, we reported that over a period of 8 hours, prior anaerobic
104 exposure actually led to an intracellular growth defect (Wallace et al. 2017). This observation suggests a sustained effect of anaerobic growth on subsequent aerobic intracellular infections.
L. monocytogenes adaptation to anaerobic conditions includes shifts in carbon metabolism. In defined medium using glucose as the sole carbon source, aerobic growth results in the production lactate, acetate, and acetoin, whereas anaerobic growth results in the production of lactate with minor accumulation of acetate, formate, and ethanol (Romick, Fleming, and McFeeters 1996). In rich
BHI medium, acetoin production was observed under aerobic but not anaerobic conditions (Wallace et al. 2017). Although L. monocytogenes is primarily a lactic acid fermenter, it contains genes coding for a fumarate reductase. The gene lmo0355, which encodes a subunit in fumarate reductase in strain EGDe, is upregulated under anaerobic conditions and allows L. monocytogenes to show an enhanced anaerobic growth in the presence of fumarate (Müller-Herbst et al.
2014). Therefore, it is likely that L. monocytogenes is capable of carrying out anaerobic respiration using fumarate as the terminal electron acceptor.
To further understand the impact of anaerobic adaptation on L. monocytogenes pathogenesis, we tested whether the defective intracellular growth after anaerobic exposure was caused by anaerobically grown L. monocytogenes having reduced respiratory activity and thus a delay in aerobic growth. We utilized fumarate as an alternative electron acceptor to stimulate respiratory activity in anaerobically grown L. monocytogenes and assessed the effects of fumarate on subsequent infections. We reported here that stimulating
105 anaerobic respiratory activity with fumarate significantly enhanced subsequent intracellular growth. Conversely, reducing respiratory activity in aerobically grown
L. monocytogenes by adding the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) caused aerobic L. monocytogenes to display phenotypes similar to anaerobically grown L. monocytogenes. Therefore, our findings indicate that the state of respiratory activity in L. monocytogenes prior to infections is a strong indicator for subsequent success in pathogenesis.
Results
If the lack of respiratory activity contributes to the intracellular growth defect in anaerobically grown L. monocytogenes, stimulating anaerobic respiratory activity using an alternative electron acceptor such as fumarate should rescue the intracellular growth defect. To confirm that supplementation of fumarate can indeed stimulate respiratory activity in L. monocytogenes under anaerobic conditions, we performed multiple in vitro assays investigating the physiological responses to fumarate. During anaerobic growth in BHI at 37°C, supplementation of 50 mM fumarate resulted in a significant increase in overnight culture optical density (OD), compared to no fumarate controls (Figure 1a). This anaerobic, fumarate-induced increase in optical density is accompanied by a significant increase in culture pH (Figure 1b) and acetoin level (Figure 1c), a significantly higher tetrazolium reduction activity (Figure 1d), and a significantly higher intracellular ATP levels (Figure 1e). In contrast, supplementation of
106 fumarate under aerobic conditions did not significantly alter culture OD, pH, acetoin level, or tetrazolium reduction activity (Figures 1a-d) but resulted in a significantly decreased intracellular ATP levels (Figure 1e) compared to aerobic, no fumarate controls. These data collectively show that exogenous supplementation of fumarate can significantly alter the anaerobic physiology and metabolism of L. monocytogenes, likely reducing fermentative activities but increasing respiratory activities.
107
Figure 4.1. Fumarate enhances L. monocytogenes anaerobic respiratory activity. L. monocytogenes was grown overnight (16-20 hours) aerobically or anaerobically in BHI at 37°C with or without supplementation of fumarate (50 mM). Culture optical density was measured at 600 nm (a). The pH (b) and acetoin concentration (c) were determined in culture supernatant. Reduction of tetrazolium salt (d) was determined using washed bacterial pellets. Relative intracellular ATP levels (e) was quantified using a luciferase-based assay. Average of triplicates were plotted with error bars representing standard deviation. Data represent at least three independent experiments. Significant differences (*, p<0.05; **, 0.001 Next, we investigated whether the anaerobic, fumarate-stimulated L. monocytogenes exhibits a faster growth transition from anaerobic to aerobic growth by first analyzing in vitro growth in BHI at 37°C. Anaerobically grown L. monocytogenes exhibited a decreased growth phenotype after transitioning into 108 aerobic conditions (Figure 2a). In comparison, anaerobically grown, fumarate- stimulated L. monocytogenes exhibited a faster adaptation to aerobic growth (Figure 2a). Similar phenotypes were also observed during transitions into aerobic, intracellular growth. Overnight L. monocytogenes cultures were used to infect RAW264.7 macrophages for 30 minutes at a multiplicity of infection (MOI) of 10 and assayed for intracellular growth. The extent of intracellular growth was similar in macrophages infected with aerobically grown L. monocytogenes with or without prior fumarate stimulation (Figure 2b). However, in macrophages infected with anaerobically grown L. monocytogenes, the intracellular growth was greatly enhanced by exposing bacteria to fumarate prior to infection (Figure 2b). These in vitro and intracellular growth characteristics support our hypothesis that, compared to anaerobically grown L. monocytogenes, anaerobic, fumarate- stimulated L. monocytogenes exhibits an increased respiratory activity and a faster anaerobic to aerobic growth transition in vitro and inside host cells. 109 Figure 4.2. Fumarate-treated L. monocytogenes exhibited enhanced transition to aerobic in vitro and intracellular growth. Overnight cultures grown under aerobic or anaerobic conditions were used to inoculate fresh BHI media and grown under aerobic conditions (a) or to infect RAW264.7 macrophages for 30 minutes at an MOI of 10 (b). Averages of triplicates were plotted with error bars representing standard deviation. Data represent three independent experiments. Significant differences (*, p<0.05; **, 0.001 If respiratory activity prior to infection is key in subsequent intracellular success, compromising respiratory activity in aerobically grown L. monocytogenes should, in theory, result in reduced pathogenesis. To test this hypothesis, we grew L. monocytogenes in BHI at 37°C in the presence of CCCP to reduce its respiratory activity and assessed the impact on L. monocytogenes and L. monocytogenes-host interactions. We first established a working concentration of 1 µM CCCP to reduce aerobic growth to a level similar to anaerobic growth without complete growth inhibition. Compared to no 110 supplement, both aerobic and anaerobic growth with 1 µM CCCP was significantly reduced (Figure 3a). However, compared to no supplement, CCCP treatment resulted in significantly reduced culture pH and tetrazolium reduction activity under aerobic but not anaerobic conditions (Figure 3b, c). We then grew L. monocytogenes with or without CCCP treatment to infect RAW264.7 macrophages and assessed the impact of prior CCCP treatment in subsequent intracellular growth. At one hour post infection (hpi), there was a significantly higher intracellular CFU in macrophages infected with the aerobically grown, CCCP-treated L. monocytogenes than those infected with aerobically grown, untreated controls. However, there was no difference in intracellular growth over an eight-hour period for bacteria grown with or without CCCP (data not shown). These results suggest that compromising respiratory activity in aerobically grown L. monocytogenes prior to infection leads to early but not sustained defects in intracellular growth. 111 Figure 4.3. Inhibition of aerobic respiratory activity with CCCP phenocopies anaerobically grown L. monocytogenes. L. monocytogenes was grown at 37°C in BHI with or without 1 µM CCCP. Culture optical density was measured at 600 nm (a). pH was measured in culture supernatant (b) while reduction of tetrazolium salts was measured in washed bacterial pellets (c). Bacteria from overnight cultures were used to infect RAW264.7 macrophages for 30 minutes at an MOI of 10 where intracellular colony forming units (CFU) was determined and compared to the CFU in inoculum (d). Averages of triplicates were plotted with error bars representing standard deviation. Data represent three independent experiments. Significant differences (**, 0.001 To establish a successful intracellular life cycle, L. monocytogenes needs to escape the phagosome in order to grow in the host cytoplasm. This phagosomal escape is mediated by the secreted virulence factor, listeriolysin O (LLO). Therefore, to determine whether the intracellular fate of fumarate- stimulated or CCCP-treated L. monocytogenes was attributed to different levels 112 of LLO production, we first investigated the effects of fumarate and CCCP on LLO production in vitro. The activity of the secreted LLO was significantly higher in aerobically or anaerobically grown L. monocytogenes with fumarate supplementation (Figure 4a). Overnight cultures of L. monocytogenes treated with 1 µM CCCP decreased secreted LLO activity in aerobic but not anaerobic cultures when normalized to culture optical density. (Figure 4b). To confirm supernatant LLO activity is representative of protein abundance, we quantified secreted LLO protein abundance using immunoblotting (Figure 4c, left) followed by pixel density analysis (Figure 4c, right). Compared to no fumarate controls, the abundance of LLO protein was significantly increased, under both aerobic and anaerobic conditions, in cultures stimulated with fumarate. However, CCCP treatment led to no significant difference in LLO abundance compared to no CCCP controls. These results suggest that while in general the LLO protein activities correlate with protein abundance, a similar correlation was not observed in aerobic, CCCP-treated bacteria. We further investigated whether the effects of fumarate and CCCP on in vitro LLO production would result in an altered phenotype during infections by assaying bacterial actin co-localization as a proxy for phagosomal escape. We found that fumarate supplementation in anaerobically grown L. monocytogenes resulted in a significantly increased level of actin co-localization at 2 hpi compared to no fumarate controls (Figure 4d), a phenotype consistent with the LLO activity result. However, fumarate supplementation in aerobically grown L. monocytogenes resulted in a significantly reduced level of actin co-localization 113 compared to untreated samples (Figure 4d). Additionally, while CCCP treatment in anaerobically grown L. monocytogenes did not significantly alter actin co- localization, CCCP treatment in aerobically grown L. monocytogenes led to a significant decrease in the actin co-localization (Figure 4e). These results suggest that respiratory activity in L. monocytogenes prior to infection may play a role in both vacuolar escape as well as subsequent actin polymerization. 114 Figure 4.4. Modulations of respiratory activity alter production of LLO and actin co- localization. L. monocytogenes was grown overnight at 37°C in BHI. Culture supernatant was used to quantify LLO activity using a hemolytic assay (a, b). Averages of triplicates were plotted with error bars representing standard deviation. Data represent three independent experiments. LLO abundance in culture supernatant was determined using immunoblotting (c, left) followed by pixel density analysis (c, right). Blot shown is representative of six independent experiments. (rLLO, recombinant LLO control; NS, no supplement; SB, sample buffer; FUM, fumarate-treated L. monocytogenes; CCCP, CCCP-treated L. monocytogenes.) Averages of pixel density from six blots were plotted with error bars representing standard error of the mean (n=6). RAW264.7 macrophages were infected with overnight L. monocytogenes for 30 minutes at a MOI of 10. Actin colocalization was determined at 2 hours post infection (hpi). BD, or below detection, indicates lack of colocalization. Significant differences (*, p<0.05; **, 0.001 115 To further understand the sustained effects of L. monocytogenes respiratory activity prior to host cell entry on long-term infections, we performed L2 plaque assays with bacteria grown aerobically or anaerobically with or without furmarate or CCCP treatments. With the diameter of the resulting plaques, measured at 3 days post infection, as a proxy for cell-to-cell spread, fumarate supplementation in L. monocytogenes grown under either aerobic or anaerobic conditions resulted in significantly larger plaques (Figure 5a). In contrast, CCCP treatment in aerobically but not anaerobically grown L. monocytogenes resulted in significantly decrease in plaque size (Figure 5a). It is important to note that during the 3 days of infection, neither fumarate nor CCCP was added to the cell culture media. Therefore, the differences in plaque sizes reflect a long-lasting impact of prior fumarate or CCCP exposure on subsequent infections. To investigate the effects of concurrent fumarate exposure on cell-cell spread, we compared plaque sizes from infections where fumarate was supplemented only in the growth of L. monocytogenes inoculum or in both the growth of L. monocytogenes inoculum and during infection. For aerobically grown L. monocytogenes, fumarate supplementation, either for the growth of inoculum or during infections, resulted in a significantly increased plaque sizes (Figure 5b). For anaerobically grown L. monocytogenes, the presence of fumarate during infection resulted in complete clearing with undeterminable plaque size. These results confirm that respiratory activities prior to infections can impact subsequent infection outcomes with observable effects for as long as 3 days post 116 infections. Moreover, the presence of fumarate during infection greatly enhanced infections, particularly for anaerobically grown L. monocytogenes. Figure 4.5. Prior or concurrent fumarate treatments enhance cell-cell spread. Overnight cultures of L. monocytogenes were washed and used to infect fibroblast monolayer cells for 1 hour. After 72 hours of incubation, plaque sizes were measured by neutral red staining. Diameters of at least 30 plaques were measured. (a) Fumarate (50 mM) or CCCP (1 µM) was supplemented only in the growth of the inoculum. (b) Compared to no fumarate control (NS_NS), fumarate was added only to growth of the L. monocytogenes inoculum (FUM_NS) or to both the growth of L. monocytogenes and during the 72 hours of incubation (FUM_FUM). Fumarate supplementation during 72 hours of incubation in cells infected with anaerobically grown, fumarate treated L. monocytogenes resulted in complete lysis where plaque sizes were not determinable (CL, Complete Lysis) Discussion L. monocytogenes is a facultative anaerobe and is exposed to suboxic to anoxic environments during its transmission into and colonization of a host. How this exposure to fluctuating levels of oxygen affects subsequent infections is not completely understood. We and others have shown that anaerobic growth prior to infection resulted in a significant increase in invasion (Burkholder and Bhunia 2010; Burkholder et al. 2009; Wallace et al. 2017). However, we noted that this invasion advantage of anaerobically grown L. monocytogenes did not extend into 117 long-term intracellular growth. In fact, L. monocytogenes that was grown anaerobically exhibited a significantly compromised intracellular growth compared to those grown aerobically prior to infection (Wallace et al. 2017). It’s important to note that these cell culture-based infections lasted 8 hours under aerobic incubation. Therefore, the defect observed at 8 (Figure 2b) and 72 hours post infection (Figure 5a) suggests that prior adaptation to anaerobic conditions severely compromises the ability of L. monocytogenes to establish a productive, intracellular infection under aerobic conditions. In this study, we reported that the lack of respiratory activity in anaerobically grown L. monocytogenes contributes the subsequent intracellular growth defect. Using fumarate as an alternative electron acceptor, we were able to stimulate respiratory activity in anaerobically grown L. monocytogenes and assess its subsequent infection outcomes. As predicted, fumarate supplementation prior to infections resulted in a significantly enhanced intracellular growth that could be attributed to increased LLO production, phagosomal escape, and cell-cell spread. Conversely, we reduced respiratory activity in aerobically grown L. monocytogenes using the proton ionophore, CCCP, and were able to compromise LLO activity, phagosomal escape, and cell- cell spread. Because fumarate or CCCP treatments took place during the growth of L. monocytogenes inoculum, not during infections, the results highlight the respiratory activity in L. monocytogenes prior to infection as a key determinant in infection outcome. 118 The choice for fumarate in our study was based mainly on the predicted ability of L. monocytogenes to utilize fumarate as an alternative electron acceptor in the absence of oxygen (Orsi et al. 2015; Trivett and Meyer 1971). However, the extent by which fumarate is involved in L. monocytogenes growth and pathogenesis goes beyond anaerobic respiration. Supplementation of fumarate as an intermediate in the tricarboxylic acid (TCA) cycle, as reported here (Figure 4a-c) and elsewhere (Wallace et al. 2017), led to a significant increase in L. monocytogenes LLO production under both aerobic and anaerobic conditions. Therefore, it is possible that the increase in LLO production in anaerobic, fumarate-treated L. monocytogenes was a result of both stimulating TCA cycle and anaerobic respiratory activities. Interestingly, while LLO production was increased in both aerobically and anaerobically grown L. monocytogenes by fumarate treatment, actin colocalization was only increased in macrophages infected by fumarate-treated, anaerobically grown L. monocytogenes. In macrophages infected by aerobically grown L. monocytogenes, prior fumarate treatment resulted in a decreased level of actin colocalization. First, in vitro LLO production is not the absolute predictor for phagosomal escape, especially when LLO production was measured using LLO accumulated in vitro over 16-20 hours of overnight culturing. Second, it is possible that while fumarate supplementation enhances LLO production in both aerobically and anaerobically grown L. monocytogenes, its effect on subsequent intracellular ActA production is not equivalent between aerobic or anaerobically grown L. monocytogenes. Given that aerobically grown, fumarate-treated bacteria exhibited no defects in 119 intracellular growth compared to aerobically grown, no fumarate controls (Figure 2b), it is likely that the decrease in actin colocalization is not a result of defects in phagosomal escape but in ActA production. How respiratory activity prior to infections affects subsequent intracellular ActA production is not known and is under current investigation. The enhancing effect of exogenous fumarate on L. monocytogenes pathogenesis also raises concerns over the use of fumarate as a food preservative. United States Food and Drug Administration recommends fumaric acid and salts of fumaric acid as safe to be used in food for direct human consumption “at a level not in excess of the amount reasonably required to accomplish the intended effect” (CFR - Code of Federal Regulations Title 21 n.d.). As a result, fumarate and its acid form, fumaric acid, have been tested as an antimicrobial additive in a variety of food products. For example, addition of fumaric acid (1%) to the surface of lean beef inoculated with L. monocytogenes, followed by 5 seconds of incubation at 55°C, resulted in a 1-log reduction in CFU (Podolak et al. 1996). Fumaric acid (0.25%) also caused a complete inhibition of planktonic L. monocytogenes (Tango, Mansur, and Oh 2015). Therefore, co-occurrence of fumarate and suboxic packaging might create an environment where L. monocytogenes can persist and develop an increased capability for subsequent intracellular growth. Based on the enhanced cell-cell spread in the presence of fumarate (Figure 5b), the potential presence of fumarate inside host cytosol might also increase the severity of L. monocytogenes infections. Curiously, when fumarate 120 was supplemented both to the growth of inoculum and to the infected cells during incubation, the resulted enhancement in plaque size was much more pronounced in cells infected with anaerobically grown L. monocytogenes compared to those infected with aerobically grown L. monocytogenes (Figure 5b). This observation suggests that the presence of fumarate during infections might contribute to host response to aerobically or anaerobically grown L. monocytogenes. Therefore, we consider possible scenarios for intracellular L. monocytogenes to be exposed to cytosolic fumarate. Although fumarate is typically generated and consumed as part of the TCA cycle pathway located inside mitochondria, a cytosolic isoform of fumarate hydratase (FH), which catalyzes the hydration of fumarate to malate, has been described. In fact, individuals with FH deficiency exhibit severe neurological and developmental diseases (Fumarase Deficiency | Genetic and Rare Diseases Information Center (GARD) – an NCATS Program n.d.). Moreover, the oncogenic effects of fumarate accumulated in FH-deficient cells have helped introduce fumarate as a potential oncometabolite (Yang et al. 2012). Elevated levels of fumarate in FH-deficient cells also lead to succination of glutathione, resulting in increased production of reactive oxygen species (Sullivan et al. 2013), and defects in respiratory chain activities (Tyrakis et al. 2017). Whether exogenous fumarate supplementation to fibroblasts in our experimental design leads to an increase in cytosolic fumarate remains to be determined. However, our study highlighted that host-derived fumarate might greatly alter L. monocytogenes-host interactions. 121 Finally, the role of anaerobic respiration in modulating bacterial pathogenesis has been demonstrated in other enteric pathogens. For example, tetrathionate respiration provides Salmonella enterica serovar Typhimurium with advantage to colonize the inflamed intestines (Thiennimitr et al. 2011; Winter et al. 2010). Cholera toxin production by Vibrio cholerae is also enhanced by anaerobic respiration (Lee et al. 2012; Oh et al. 2016). Recently, it was demonstrated that the ability of L. monocytogenes to perform extracellular electron transfer conferred a clear fitness advantage in vivo (Light et al. 2018). When enteric bacterial pathogens transit through the suboxic host intestinal lumen, adaptive strategies to maintain redox homeostasis can directly modulate virulence regulation and offer a competitive advantage amidst the endogenous fermentative organisms. Moreover, based on findings from this study, these anaerobic adaptations can sustain long into aerobic environment and alter infection outcomes. Understanding the extent of the anaerobic adaptations in the pathogens will greatly enrich our ability to provide protection from infections. Materials and Methods Bacterial strains and culture conditions L. monocytogenes strain 10403s was grown from isolated colonies on freshly streaked brain–heart infusion (BHI) plates (<1 week) at 37 °C. All cultures were grown in filter-sterilized BHI media (Lot 4176589) to ensure consistency. 122 Aerobic cultures were grown with agitation at 250 RPM to ensure adequate oxygen diffusion. Anaerobic cultures were grown in a temperature-controlled incubator inside an anaerobic chamber (Coy Laboratory, Type A) with a nitrogenous atmosphere containing 2.5% hydrogen. Optical density (OD) was measured in an optically clear 96-well plate at 600 nm with a volume of 200 µL per well using a 96-well plate reader (Biotek Synergy4). Sodium fumarate (Acros Organics) was prepared as 1M stock solution in deionized water, filter-sterilized, and added directly to the media at the desired concentration. Intracellular Growth Curves Murine peritoneal macrophages RAW264.7 (ATCC TIB-71) were grown in DMEM (Thermo Scientific) supplemented with 10% (v/v) heat inactivated fetal bovine serum (JRScientific), HEPES (10 mM), and glutamine (2 mM) in a 37 °C incubator with a 5% CO2 atmosphere. Prior to infections, cells were seeded in a 24-well tissue culture plate and grown for 14–18 h. Overnight cultures of L. monocytogenes were used for infections at a MOI of 10. Bacteria diluted in cell culture medium were added to each well (500 µL) and incubated for 30 min. Following incubation, media were aspirated, and cells were washed twice with sterile DPBS. Fresh media (1 mL per well) containing 10 µg/mL gentamicin stock was added to each well. To enumerate intracellular bacteria, cell culture media were aspirated off and sterile 0.1% (v/v) Triton X-100 (Fisher BP151-100) was added to each well (200 µL per well) to lyse host cells. Lysates were diluted and 123 spread on LB plates. Colonies on plates were counted using an automatic colony counter (Synbiosis aCOLyte 3) after 24–48 h of incubation in a 37 °C incubator. Tetrazolium Reduction Assay Overnight cultures of L. monocytogenes were washed with PBS and normalized by OD600nm to a final volume of 1 mL. Tetrazolium Salt (MTT) working solution (50 µL, 0.5 mg/mL) was added to 50 µL of bacterial sample or blank control and incubated at 37°C for 1 hour in a 96-well flat-bottomed plate. Following incubation, 100 µL of DMSO was added to each sample and the plate was placed in a 37°C incubator shaking for 15 minutes. Following incubation, samples were read at 540 nm to quantify the level of MTT reduction. Measurement of Supernatant Acetoin The Voges–Proskauer test was adapted to quantify acetoin production in the supernatant of overnight L. monocytogenes cultures. Supernatant or acetoin standards (100 µL) were placed into a sterile micro-centrifuge tube followed by additions of 70 µL of 0.5% creatine monohydrate in water (Sigma), 100 µL of 5% 1-Napthol in water (Sigma), and 100 µL of 40% KOH (Chempure) in 95% EtOH. Samples were incubated at room temperature for 15 min and the absorbance was read at 560 nm. A standard curve was constructed to calculate the concentration of acetoin in culture supernatant samples. 124 Hemolytic Assay Hemolytic assays were performed using supernatant from overnight cultures to measure the activity of secreted listeriolysin O (LLO). Each sample (100 µL) was incubated at room temperature with DTT (5 µL, 0.1M) for 15 min. A positive control (0.1% Triton X-100) and a negative control (blank BHI media) were included for each experiment. After incubation, samples were serially diluted using hemolysis buffer containing: dibasic sodium phosphate (35 mM) and sodium chloride (125 mM) brought to pH 5.5 with acetic acid. Defibrinated sheep’s blood (Hemostat Laboratories) was diluted to a hematocrit of 2% and then added to each sample for a final hematocrit of 1%. Samples were incubated at 37 °C for 30 min. After incubation, all samples were spun down at 2000 RPM for 5 min to pellet intact blood cells. Supernatant lysate (120 µL) was transferred to a flat bottom 96-well plate for OD measurement at 541 nm as an indicator of LLO activity. Hemolytic unit was calculated as the inverse of the dilution factor at which half complete lysis using 0.4% Triton X-100 occurred and subsequently normalized with original culture OD measured at absorbance at 600 nm. Samples that did not produce lysis at a level more than half of complete lysis were designated as “Below Detection” for their hemolytic units. Supernatant samples from anaerobic cultures typically generate activities at or slightly above “Below Detection” levels. 125 LLO Immunoblotting SDS-PAGE and Immunoblotting Analysis: Overnight cultures of L. monocytogenes were used to perform SDS-PAGE and Immunoblotting Analysis. Cultures were normalized to OD (600nm) using BHI media. Pellet samples were frozen (-20°) and supernatant samples were precipitated with 10% trichloroacetic acid at 4° for 1 hour. Following supernatant protein precipitation, a cold acetone wash was performed. Both the pellet and supernatant samples were resuspended in 12µL of 2x sample buffer and heated at 95°C for 5 min after which it was separated by SDS-PAGE. Following separation immunoblots were performed using anti-LLO rabbit antibody (abcam® ab43018) followed by goat- anti-rabbit HRP antibody (abcam® ab6721). Actin Co-localization by Immunofluorescence Microscopy RAW264.7 macrophages were plated onto sterile coverslips (18 by 18 mm) inside 6-well plates at 1 million cells per well one day prior to infections. L. monocytogenes overnight cultures were washed twice and diluted in cell culture media for infection at a MOI of 10. At 2 hours post infection (hpi), coverslips were fixed in paraformaldehyde (3.7% in PBS) overnight at 4°C. For immunofluorescence microscopy, each coverslip was washed with TBS-T (25 126 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100) and blocked with TBS-T with 1% bovine serum albumin (BSA). Anti-L. monocytogenes serum (1:500 in TBS-T with 1% BSA; Thermo Scientific PA1-30487) was added onto each coverslip and incubated at room temperature overnight. Each coverslip was washed in 5 ml of TBS-T prior to incubation with secondary antibodies, phalloidin-iFluor 594 (1:400, abcam ab176757) and AlexaFluor 488-goat anti-rabbit antibody (1:400, abcam ab150077), in TBS-T with 1% BSA. One hundred intracellular bacteria per experimental replicate were scored visually for the presence or absence of actin clouds. Quantification of Intracellular ATP Intracellular ATP levels were quantified using a Molecular Probes™ ATP determination kit. Samples were prepared according to manufacturer’s suggested protocol. Overnight cultures of L. monocytogenes were washed and resuspended with PBS to a final concentration of 108 CFU/ml. Washed bacterial cells were incubated in a dry heating block at 100°C for 5 minutes to release intracellular ATP. Samples were then centrifuged briefly at 10,000 RPM to pellet any intact cells. Supernatant from each sample (10 µL) was placed in a black 96- well plate with 90 µL of reaction master mix (double deionized H2O reaction buffer, DTT, D-Luciferin, and firefly luciferase as described in manufacturer’s protocol). Bioluminescence was measured by a BioTek Synergy 4 plate reader. 127 L2 Fibroblast plaque assay L2 cells were seeded in 6-well plates and allowed to form a monolayer for 72 hours. On day of infection, 1 mL of overnight L. monocytogenes cultures were washed and resuspended in 100 µL of DPBS. Monolayers were washed three times with DPBS and were infected with 1 mL per well of fresh DMEM containing 6 µL of L. monocytogenes suspension. At 1 hpi, the cells were covered with a 3 mL mixture of DMEM containing 10 µg/mL gentamicin and 0.7% agarose. Plates were incubated at 37°C in a 5% CO2 incubator for 72 hours. Following incubation, wells were stained with neutral red for one hour followed by PBS washes. Plaque diameters were measured using the ruler function in GIMP software. Author Contributions: Conceptualization, N.W. and Y.S.; Methodology, All; Validation, All; Formal Analysis, N.W. and Y.S.; Investigation, All; Resources, Y.S.; Writing- Original Draft Preparation, N.W. and Y.S.; Writing-Review & Editing, N.W. and Y.S.; Visualization, All; Supervision, Y.S.; Project Administration, Y.S.; Funding Acquisition, Y.S. 128 Funding: This research was funded by American Heart Association (16GRNT27260219). E.R. and N.W. were supported by the University of Dayton Graduate Student Summer Fellowship. Y.S. was supported by the University of Dayton Research Council, Hanley Sustainability Institute, and Department of Biology. Acknowledgments: N/A Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. 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Frontiers in Oncology 2: 85. 132 CHAPTER V EVALUATING THE IMPORTANCE OF MENAQUINONE BIOSYNTHESIS IN LISTERIA MONOCYTOGENES VIRULENCE REGULATION Abstract Listeria monocytogenes (L. monocytogenes) is a facultative intracellular pathogen and causative agent of the foodborne disease, listeriosis. L. monocytogenes is ubiquitous and must persist and grow in environments with varying levels of oxygen ranging from the soil to the human intestine. However, most of our understanding of L. monocytogenes pathogenesis comes from work done in the presence of oxygen, completely leaving out the anaerobic phase of infection. In this study we evaluated the importance of (menB), a gene involved in menaquinone biosynthesis, L. monocytogenes pathogenesis. Additionally, our previous work has shown that the supplementation of exogenous fumarate can stimulate respiratory activity and enhance anaerobic pathogenesis, and this study shows that MenB may be involved in the effect of fumarate on pathogenesis of both aerobic and anaerobically grown L. monocytogenes. 133 Introduction Listeria monocytogenes (L. monocytogenes) is a Gram-positive foodborne pathogen capable of causing the invasive disease, listeriosis. As a facultative pathogen, L. monocytogenes must be able to adapt and respond to various oxygen conditions, especially when transmitting through the gastrointestinal tract (Albenberg et al. 2014). However, the majority of research on L. monocytogenes takes advantage of standard aerobic culturing techniques, which creates a knowledge gap in the importance of anaerobic conditions during L. monocytogenes infection. Limited research points to the increased adherence and initial infection from anaerobically cultured L. monocytogenes compared to aerobic (Burkholder and Bhunia 2010; Burkholder et al. 2009; Wallace et al. 2017). However, the actual molecular underpinnings of this differential anaerobic response have yet to be determined. Our lab previously explored the importance of central carbon metabolism and respiratory activity as potential points for virulence regulation to take place, and this research aims to further explore the importance of respiratory activity by evaluating the importance of genes involved in electron transport. The electron transport chain (ETC) consists of a series of oxidation reactions which move electrons through various intermediates with the ultimate goal of generating a proton gradient to drive the production of ATP through ATP synthase. This process has been linked to successful production of virulence 134 factors in other pathogens. Staphylococcus aureus mutants who lack a functional electron transport chain are incapable of producing hemolysins, eukocidins, and the antioxidant staphyloxanthin. However, stimulation of ETC activity in those mutants led to restoration of virulence factor production. Additionally, stimulation of Enterococcus faecalis ETC activity led to restoration of catalase activity (Painter et al. 2017). Although eukaryotic, Aspergillus fumigates mutants with impaired ETC displayed attenuated virulence in a mouse model of infection (Grahl et al. 2012). In order to evaluate the importance of the ETC in L. monocytogenes pathogenesis, we used mutants lacking key components of the ETC and evaluated their subsequent growth and pathogenesis. Our data show that one component of menaquinone biosynthesis, MenB, is important in growth and pathogenesis. Results Intermediate in menaquinone biosynthesis is important in L. monocytogenes response to fumarate As a means of further understanding the importance of respiratory activity in L. monocytogenes virulence regulation, overnight cultures of various mutants lacking components of the electron transport chain were grown in either presence or absence of oxygen as well as exogenous fumarate (Supplementary Figure 1). Previous work in our lab successfully demonstrated that 135 supplementation of fumarate to anaerobic, overnight cultures led to increased respiratory activity and culture optical density. However, overnight growth of the ∆MenB mutant led to significantly decreased aerobic growth both with and without fumarate (Figure 1A) and the anaerobically grown L. monocytogenes showed no increase in growth when fumarate supplementation (Figure 1B). As a means of understanding if the changes in overnight growth in the ∆MenB strain were because of media acidification, we measured the culture pH. Under both aerobic and anaerobic conditions there was no significant change in pH between no supplement WT and ∆MenB, and fumarate supplementation led to an increase in pH in both strains with ∆MenB having a slightly smaller increase (Figure 1 C,D). To ascertain if the ∆MenB growth defect was associated with a change in central carbon metabolism we measured the production of acetoin. When cultured aerobically, the ∆MenB strain produced very little acetoin independent of fumarate supplementation (Figure 1E). Anaerobically grown ∆MenB did not show the characteristic increase in acetoin production when supplemented with fumarate shown in WT (Figure 1F). These results suggest that MenB may be mediating L. monocytogenes response to exogenous fumarate supplementation potentially through modulation of central carbon metabolism. 136 A Aerobic B Anaerobic No Supplement 50mM Fumarate No Supplement 50mM Fumarate 1.2 NS 0.8 * 0.7 1 NS 0.6 m 0.8 m n n 0.5 0 0 0 0.6 0 0.4 6 NS 6 D 0.4 D 0.3 O O 0.2 0.2 0.1 0 0 WT ∆menB WT ∆MenB C Aerobic D Anaerobic 7.00 6.00 *** *** *** 6.00 * 5.00 5.00 4.00 4.00 H H 3.00 p 3.00 p 2.00 2.00 1.00 1.00 0.00 0.00 WT ∆MenB WT ∆MenB E Aerobic F Anaerobic 3 NS 1.6 *** 2.5 1.4 1.2 2 / ] / m ] m n 1 n i n n i 0 o 0 t o 0 t 1.5 0 e 0.8 6 e 6 c c D a D [ a [ O 0.6 O 1 0.4 0.5 NS 0.2 NS 0 0 WT ∆menB WT ∆menB FFigureigure 1: 5.An1:a eAnaerobicrobic growt hgrowth increas increasee with fum witharate fumaratesupplemen supplementationtation abolished in tabolishedhe ∆MenB in sthetrain ∆MenB possibl ystrain due to possibly manipula tdueion o tof t hmanipulatione fate of pyruv aofte .the A,B fate) Cul tofure pyruvate OD600nm. wA,B)as ta Cultureken in both WT and ∆MenB strain grown with or without oxygen and fumarate. C,D) Overnight culture OD600nm was taken in both WT and ∆MenB strain grown with or without oxygen and supernatant was taken for pH measurement. E,F) Supernatant from overnight cultures was taken to qfumarate.uantify ace toC,D)in pr oOvernightduction. Re culturepresent asupernatanttive of three in wasdepe takenndent e forxpe pHrim emeasurement.nts done in triplic E,F).ate ar e pRepresentativelotted with error b aofrs threerepres independententing standar dexperiments deviation and done statis tiincs triplicatewere perf oarerme plottedd using awi twtho -error tabarsiled s representingtudent’s t-test w standardith signific deviationant differen andces instatisticsdicated b ywere aster iperformedsks (*p<.05 *using** p<. 0a0 1two NS-=tailedNot Sstudent’significant ) t-test with significant differences indicated by asterisks (*p<.05 *** p<.001 NS=Not Significant ) Figure 5.2: MenB is critical for aerobic LLO production and intracellular fitnessFigure 5.1: Anaerobic growth increase with fumarate supplementation abolished in the ∆MenB strain possibly due to manipulation of the fate of pyruvate. A,B) Culture OD600nm was taken in both WT and ∆MenB strain grown with or without oxygen and fumarate. C,D) Overnight culture137 supernatant was taken for pH measurement. E,F). Representative of three independent experiments done in triplicate are plotted with error bars representing standard deviation and statistics were performed using a two-tailed student’s t-test with significant differences indicated by asterisks (*p<.05 *** p<.001 NS=Not Significant ) MenB is important for aerobic LLO production and intracellular growth Previous work in our lab demonstrated that supplementation of fumarate to aerobically and anaerobically grown L. monocytogenes led to an increase in the production of the pore-forming toxin, listeriolysin O (LLO). Because we saw marked changes in ∆MenB response to fumarate in regard to growth and acetoin production we wanted to see if there were similar differences in LLO production and subsequent intracellular growth. Interestingly, deletion of MenB led to a bifurcated response in aerobic and anaerobic conditions. Aerobically grown ∆MenB showed undetectable production of LLO independent of fumarate supplementation (Figure 2A), whereas anaerobically cultured ∆MenB showed increased LLO production when compared to anaerobic WT and fumarate enhanced LLO production in both WT and ∆MenB (Figure 2B). This response could indicate that there are different signals regulating MenB involvement in the production of LLO under different oxygen conditions. Previously we identified increased intracellular fitness of aerobically cultured L. monocytogenes when compared to anaerobically cultured L. monocytogenes. To test if the importance of MenB during intracellular growth we infected RAW264.7 macrophages with the ∆MenB strain and performed an 8-hour intracellular growth curve. Interestingly, deletion of MenB led to decreased intracellular fitness of aerobically grown L. monocytogenes compared to anaerobically grown (Figure 2C). This is another example of a bifurcated response with the ∆MenB strain between different oxygen conditions, which may mean that MenB is far more important in aerobic 138 virulence regulation compared to anaerobic virulence regulation. A Aerobic B Anaerobic No Supplement 50mM Fumarate No Supplement 50mM Fumarate 400 *** 40 t t n n *** e 350 e 35 m m e e l l y 300 y 30 t p t p i i p p v v i i u u t t c c S 250 S 25 A A o o c c i i N N t t 200 20 y y T T l l o o W W 150 15 m m c c i i e e b b H H o 100 o 10 r r e e A 50 A 5 % % ( BD BD ( BD 0 0 WT ∆MenB WT ∆MenB C Aerobic ∆MenB Anaerobic ∆MenB Aerobic ∆MenB+50mM Fumarate Anaerobic ∆MenB+50mM Fumarate 60 50 *** U F 40 C ) r t a u l p u l n 30 l i e % c ( a r 20 t n I 10 NS 0 0 1 2 3 4 5 6 7 8 Time (h) Figure 2: MenB is critical for aerobic LLO production and intracellular fitness. A,B) Supernatant from overnight cultures of both WT and ∆MenB grown both aerobically and anaerobically with and without Figurefuma r5.at2:e w MenBas take nis a ncriticald a hem oforlyti caerobic assay wa LLOs perf oproductionrmed looking fandor the intracellular production of L LfitnessO. C) ∆M. Aen,B)B Supernatantgrown both fromaerob overnightically, anae rculturesobically, a nofd wbothith o rWT with andout fu ∆MenBmarate w grownas infec bothted in taerobicallyo RAW 264.7 and anaerobicallymacrophage withs and and a su bwithoutsequent ifumaratentracellula rwas grow takenth curv eand was a p ehemolyticrformed. Re assaypresen twasative performedof three lookingindep forend theent eproductionxperiments d ofon eLLO. in trip C)lica ∆teMenB are plo growntted with both error baerobically,ars represen tanaerobically,ing standard dev iandation and withs taortis withouttics were fumarate performed wasusing infected a two-tail eintod stu RAWdent’s t264.7-test w imacrophagesth significant diff eandrenc eas subsequentindicated by asterisks (*** p<.001 NS=Not Significant BD=Below Detection limit of the assay ) intracellular growth curve was performed. Representative of three independent experiments done in triplicate are plotted with error bars representing standard deviation and statistics were performed using a two-tailed student’s t-test with significant differences indicated by asterisks (*** p<.001 NS=Not Significant BD=Below Detection limit of the assay ) Supplemental Figure 5.1: Screen of respiratory mutants on the basis of growth, acetoin production and hemolytic activityFigure 5.2: MenB is critical for aerobic LLO production and intracellular fitness. A,B) Supernatant from overnight cultures of both WT and ∆MenB grown both aerobically and anaerobically with and without fumarate was taken and a hemolytic assay was performed looking for the production of LLO. C) ∆MenB grown both aerobically, anaerobically, and with or without fumarate was infected into RAW 264.7 macrophages and a subsequent139 intracellular growth curve was performed. Representative of three independent experiments done in triplicate are plotted with error bars representing standard deviation and statistics were performed using a two- tailed student’s t-test with significant differences indicated by asterisks (*** p<.001 NS=Not Significant BD=Below Detection limit of the assay ) Materials and Methods Bacterial strains and culture conditions L. monocytogenes strain 10403s was grown from isolated colonies on freshly streaked brain–heart infusion (BHI) plates (<1 week) at 37 °C. All cultures were grown in filter-sterilized BHI media to ensure consistency. Aerobic cultures were grown with agitation at 250 RPM to ensure adequate oxygen diffusion. Anaerobic cultures were grown in a temperature-controlled incubator inside an anaerobic chamber (Coy Laboratory, Type A) with a nitrogenous atmosphere containing 2.5% hydrogen. Optical density (OD) was measured in an optically clear 96-well plate at 600 nm with a volume of 200 µL per well using a 96-well plate reader (Biotek Synergy4). Sodium fumarate (Acros Organics) was prepared as 1M stock solution in deionized water, filter-sterilized, and added directly to the media at the desired concentration before inoculation. Hemolytic assays Hemolytic assays were performed using supernatant from overnight cultures to measure the activity of listeriolysin O (LLO). Each sample was incubated at room temperature with 0.1 M DTT (5 µL) for 15 min. A positive control (0.4% triton X-100) and a negative control (blank BHI media) were 140 included for each experiment. After incubation, samples were serially diluted using hemolysis buffer containing: dibasic sodium phosphate (35 mM) and sodium chloride (125 mM) brought to pH 5.5 with acetic acid. Defibrinated sheep’s blood (Hemostat Laboratories DSB050) was diluted to a hematocrit of 2% and then added to each sample for a final hematocrit of 1%. Samples were incubated at 37 °C for 30 min. After incubation, all samples were spun down at 2000 RPM for 5 min to pellet intact blood cells. Supernatant lysate (120 µL) was transferred to a flat bottom 96-well plate for OD measurement at 541 nm as an indicator of LLO activity. Hemolytic unit was calculated as the inverse of the dilution factor at which half complete lysis using 0.1% Triton X-100 occurred and subsequently normalized with original culture OD measured at absorbance at 600 nm. Samples that did not produce lysis at a level more than half of complete lysis were designated as “Below Detection” for their hemolytic units. Supernatant samples from anaerobic cultures typically generate activities at or slightly above “Below Detection” levels. Acetoin Production The Voges-Proskauer test (Nicholson 2008) was adapted to quantify acetoin production in the supernatant of overnight L. monocytogenes cultures. Supernatant or standard (100 µL) was placed into a sterile micro-centrifuge tube followed by additions of 70 µL of .5% creatine monohydrate (Sigma C3630- 100G), 100 µL of 1-Napthol (Sigma N1000-10G), and 100 µL of 40% KOH 141 (Chempure 831-704) in 95% EtOH. Samples were centrifuged between each addition and incubated at room temperature for 15 minutes after the final addition. After incubation 200 µL of each sample was placed into a flat bottom 96-well plate and the absorbance was read at 560 nm. A standard curve was constructed to calculate the concentration of acetoin in culture supernatant samples. Intracellular Growth Curves Murine peritoneal macrophages RAW264.7 (ATCC TIB-71) were grown in DMEM (Thermo Scientific) supplemented with 10% (v/v) heat inactivated fetal bovine serum (JRScientific), HEPES (10 mM), and glutamine (2 mM) in a 37 °C incubator with a 5% CO2 atmosphere. Prior to infections, cells were seeded in a 24-well tissue culture plate and grown for 14–18 h. Overnight cultures of L. monocytogenes were used for infections at a MOI of 10. Bacteria diluted in cell culture medium were added to each well (500 µL) and incubated for 30 min. Following incubation, media were aspirated, and cells were washed twice with sterile DPBS. Fresh media (1 mL per well) containing 10 µg/mL gentamicin stock was added to each well. To enumerate intracellular bacteria, cell culture media were aspirated off and sterile 0.1% (v/v) triton X-100 (Fisher BP151-100) was added to each well (200 µL per well) to lyse host cells. Lysates were diluted and spread on LB plates. Colonies on plates were counted using an automatic colony counter (Synbiosis aCOLyte 3) after 24–48 h of incubation in a 37 °C incubator. 142 Discussion In this study we set out to better understand the importance of the ETC in L. monocytogenes virulence regulation both with and without oxygen. After screening many mutants for growth, acetoin production, and hemolytic activity (Supplementary Figure 1), we chose to specifically highlight the ∆MenB mutant due to its strong bifurcated response to both oxygen and exogenous fumarate. Overnight growth of the ∆MenB mutant was significantly attenuated in aerobic but not anaerobic growth, but both oxygen conditions showed no significant response to exogenous fumarate supplementation. These differences were not due to pH, but there was a significant effect on the production of acetoin both aerobically and anaerobically. MenB deletion led to no acetoin production aerobically and anaerobically grown L. monocytogenes supplemented with fumarate did not display enhanced acetoin production. Furthermore, deletion of MenB led to attenuated LLO production and intracellular growth of aerobically cultured L. monocytogenes. Thus, we demonstrated the importance of menaquinone biosynthesis in L. monocytogenes growth and pathogenesis. MenB is an enzyme in the shikimate biosynthesis pathway that catalyzes the conversion of O-succinlybenzoyl-CoA (OSB-CoA) to 1,4-dihydroxy-2- napthoate (DNHA), which is necessary for cytosolic survival and the lack of it leads to increased bacteriolysis (Chen et al. 2017). This is not unique to L. 143 monocytogenes, Staphylococcus aureus menaquinone biosynthesis is necessary for survival and resistance to haem toxicity (Wakeman et al. 2012). We believe it is possible that the decreased intracellular growth of aerobically cultured L. monocytogenes may be due to increased bacteriolysis. Therefore, it is clear that there are downstream effects of shikamate biosynthesis beyond just the production of menaquinones. Due to the complete ablation of acetoin production in the aerobically cultured MenB mutant we believe this indicates that MenB may be involved in central carbon metabolism. Further investigation into the role of MenB in L. monocytogenes virulence regulation is warranted as a means of better understanding its importance during infection. Acknowledgments N. Wallace is supported in part by the University of Dayton office for Graduate Academic Affairs through the Graduate Student Summer Fellowship Program, the University of Dayton College of Arts and Sciences, and the Department of Biology. A special thanks goes to Dr. John-Demian Sauer for providing us with the respiratory mutants. References Albenberg, Lindsey, Tatiana V. Esipova, Colleen P. Judge, et al. 2014 Correlation Between Intraluminal Oxygen Gradient and Radial Partitioning of Intestinal Microbiota. Gastroenterology 147(5): 1055–1063.e8. 144 Burkholder, Kristin M., and Arun K. Bhunia 2010 Listeria Monocytogenes Uses Listeria Adhesion Protein (LAP) to Promote Bacterial Transepithelial Translocation and Induces Expression of LAP Receptor Hsp60. Infection and Immunity 78(12): 5062–5073. Burkholder, Kristin M., Kwang-Pyo Kim, Krishna K. Mishra, et al. 2009 Expression of LAP, a SecA2-Dependent Secretory Protein, Is Induced under Anaerobic Environment. Microbes and Infection 11(10): 859–867. Chen, Grischa Y., Courtney E. McDougal, Marc A. D’Antonio, Jonathan L. Portman, and John-Demian Sauer 2017 A Genetic Screen Reveals That Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria Monocytogenes Cytosolic Survival. mBio 8(2): e00119-17. Grahl, Nora, Taisa Magnani Dinamarco, Sven D. Willger, Gustavo H. Goldman, and Robert A. Cramer 2012 Aspergillus Fumigatus Mitochondrial Electron Transport Chain Mediates Oxidative Stress Homeostasis, Hypoxia Responses and Fungal Pathogenesis. Molecular Microbiology 84(2): 383–399. Nicholson, Wayne L. 2008 The Bacillus Subtilis ydjL (bdhA) Gene Encodes Acetoin Reductase/2,3- Butanediol Dehydrogenase. Applied and Environmental Microbiology 74(22): 6832–6838. Painter, Kimberley L., Alex Hall, Kam Pou Ha, and Andrew M. Edwards 2017 The Electron Transport Chain Sensitizes Staphylococcus Aureus and Enterococcus Faecalis to the Oxidative Burst. Infection and Immunity 85(12). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695107/. Wakeman, Catherine A., Neal D. Hammer, Devin L. Stauff, et al. 2012 Menaquinone Biosynthesis Potentiates Haem Toxicity in Staphylococcus Aureus. Molecular Microbiology 86(6): 1376–1392. Wallace, Nathan, Eric Newton, Elizabeth Abrams, Ashley Zani, and Yvonne Sun 2017 Metabolic Determinants in Listeria Monocytogenes Anaerobic Listeriolysin O Production. Archives of Microbiology: 1–11. 145 Supplementary Figure A Aerobic B Anaerobic No Supplement 50mM Fumarate 1.2 No Supplement 50mM Fumarate 1 0.9 0.8 m 0.8 0.7 n m 0 0.6 n 0 0.6 0 6 0.5 0 D 0.4 6 0.4 O D 0.3 0.2 O 0.2 0 0.1 0 S A B H B A A N n n p A x x e e t d o o S A B H B A A m m a y q q N n n tp A x x ∆ ∆ ∆ c ∆ ∆ e e a d o o ∆ + m m ∆ y q q ∆ ∆ c ∆ ∆ B ∆ + A B yd A c C D yd ∆ c Aerobic ∆ 9 Anaerobic 7 8 6 7 / ] m / ] n m 6 n 5 i n n 0 i o 5 t 0 0 o 4 t e 0 6 4 e c 6 3 D c A 3 D [ A O [ 2 O 2 1 1 0 0 T A B H B A B T A B H B A B n n p A x A n n p A x A W e e t d o W e e t d o a y q yd a y q yd m m ∆ c ∆ c m m ∆ c ∆ c E ∆ ∆ ∆ ∆ F ∆ ∆ ∆ ∆ + + A A x Anaerobic x o o q q Aerobic ∆ 200 ∆ ) t 600 n 180 ) t e n 160 m e 500 y t e 140 i l m y v t p i i e 400 120 t l v p c i p t u 100 A p c 300 S c u 80 A i o t S c y i 60 200 N l t o y o c l 40 i N o m 100 b 20 T e o m r H W e 0 0 e H A % ( T A B H B A B T A B H B A B T x n n p A x A W n n tp A o A W e e t d o d e e a d q d a W y y m m ∆ y q y m m ∆ c ∆ c ∆ ∆ c ∆ c ∆ ∆ ∆ ∆ ∆ ∆ % + ( + A A x x o o q q ∆ ∆ Supplmentary Figure 1: Screen of respiratory mutants on the basis of growth, acetoin production and hemolytic activity. A,B) Culture OD600nm was taken in both WT mutant strains grown with or without oxygen aSupplementalnd fumarate. C,D )Figure Superna 5.ta1:nt frScreenom overn ofigh respiratoryt cultures was tmutantsaken to qu aonnti fythe ace basistoin pro ofdu cgrowth,tion. E,F) Sacetoinupernata nproductiont samples we rande tak ehemolyticn and assay eactivityd for LLO. pA,B)rodu cCulturetion usin gOD600nm as hemolyt iwasc ass atakeny. Rep inre sbothentat ive of thWTree mutantindepen dstrainsent exp egrownriments with done or in twithoutriplicate aoxygenre plotte dand with fumarate. error bars r eC,D)prese Supernatantnting standard dfromeviat ion. overnight cultures was taken to quantify acetoin production. E,F) Supernatant samples were taken and assayed for LLO production using as hemolytic assay. Representative of three independent experiments done in triplicate are plotted with error bars representing standard deviation. Table A1.1 Primer design used for qRT-PCR analysisSupplemental Figure 5.1: Screen of respiratory mutants on the basis of growth, acetoin production and hemolytic activity. A,B) Culture OD600nm was taken in both WT mutant strains grown with or without oxygen and fumarate. C,D) Supernatant from overnight cultures was taken to quantify acetoin production. E,F) Supernatant146 samples were taken and assayed for LLO production using as hemolytic assay. Representative of three independent experiments done in triplicate are plotted with error bars representing standard deviation. CHAPTER VI CONCLUDING REMARKS The aim of my dissertation research was to gain a better understanding of the effect of anaerobic conditions on Listeria monocytogenes virulence regulation. I approached understanding this effect by measuring the effect of strict anaerobic culture on the production of a key virulence factor in L. monocytogenes, LLO, intracellular growth using various cultured cell lines, and various other biochemical and molecular techniques. Due to the inherent difference in metabolic function between aerobic and anaerobic growth of L. monocytogenes, I wanted to establish a connection between metabolic activity and subsequent pathogenesis. I showed that exogenous supplementation of TCA cycle intermediates to anaerobically grown L. monocytogenes led to an increase in the production of LLO. We also identified an intracellular growth defect for anaerobically cultured L. monocytogenes during subsequent infection. We hypothesized that decreased respiratory activity in anaerobically cultured L. monocytogenes was responsible for the intracellular growth defect. We stimulated respiratory activity in anaerobically cultured L. monocytogenes by supplying exogenous fumarate, an alternative electron acceptor. The stimulation 147 of respiratory activity alleviated the intracellular growth defect, highlighting the importance of respiratory activity in subsequent intracellular infection. Based on these findings we then evaluated the importance of several genes associated with menaquinone biosynthesis and electron transport. We identified the necessity of MenB in the success of L. monocytogenes intracellular growth when cultured aerobically but not anaerobically. This finding further supports the importance of respiratory activity in the success of L. monocytogenes infection. This dissertation provides the framework for further exploration into the connection between metabolic activity and pathogenesis for L. monocytogenes. 148 APPENDIX I The Effect of Oxygen on Hly Transcript Rationale: The aim of these experiments was to characterize the effects of both oxygen conditions as well as fumarate supplementation on the transcriptional activity and stability of hly-the gene, which codes for LLO. Additionally, a mutant L. monocytogenes containing an IPTG inducible promoter was used to control the transcription oh hly. This mutant was used as a means of understanding if fumarate supplementation effects LLO production through hly transcription. Table A1.1 Primer design used for qRT-PCR analysis Gene F Primer R primer Tablehly A1.1 Primer5' - C CdesignG GAA C usedTT ACC for AC TqRT TGT- PCRGAA - analysis3' 5' - ACT ATA TTT CGG ATA AAG CGT GGT - 3' gyrA 5' - CCC AAC TGC TGG GAT GAT TAT GGG A - 3' 5' - GGC AAT ACG TTC AAC TAG GCG TGC - 3' Table A1.1 Primer design used for qRT-PCR analysis Table A1.1 Primer design used for qRT-PCR analysis Table A1.1 Primer design used for qRT-PCR analysis Table A1.1 Primer design used for qRT-PCR analysis Table A1.1 Primer design used for qRT-PCR149 analysis Table A1.1 Primer design used for qRT-PCR analysis Figure A1.1: Anaerobically grown L. monocytogenes shows increased hly transcript activity and stability but does not result in increased LLO protein abundance. A) Overnight cultures of a hly-gus reporter strain Listeria were grown aerobically, anaerobically, with, or without fumarate and subsequent fluorescence was quantified. B) RNA was isolated and purified from overnight cultures of Listeria grown aerobically or anaerobically with or without fumarate. It was then reverse transcribed, and the resulting cDNA was used for qPCR. C) Hemolytic activity was measured from overnight cultures of Listeria grown aerobically, anaerobically, with, or without fumarate in both WT and iLLO strains. D) Western blot showing LLO protein abundance for Listeria grown aerobically, anaerobically, with, or without fumarate. (NS= No Supplement, FUM= 50mM Fumarate, iLLO= inducible LLO promoter strain, +O2 = aerobic, -O2= anaerobic. Averages of triplicates are plotted with error bars representing standard deviation and statistics were performed using a two-tailed student’s t test with significant differences indicated by asterisks (*p<0.05 ** p<.01 *** p<.001) Figure A1.1: Anaerobically grown L. monocytogenes shows increased hly Methods:transcript activity and stability but does not result in increased LLO protein abundance. A) Overnight cultures of a hly-gus reporter strain Listeria were grown aerobically, anaerobically, with, or without fumarate and subsequent fluorescence was quantified. B) RNA was isolated and purified from overnight cultures of Listeria grown aerobically or anaerobically with or without fumarate. It was then reverse transcribed, RNAand theExtract resultingion cDNA and wasPurification used for qPCR. C) Hemolytic activity was measured from overnight cultures of Listeria grown aerobically, anaerobically, with, or without fumarate in both WT and iLLO strains. D) Western blot showing LLO protein abundance for Listeria grown aerobically, anaerobically, with, or without fumarate. (NS= No RNASupplement, extraction FUM= was 50mM performed Fumarate, using iLLO= amresco® inducible LLO RiboZol™ promoter strain, (N580 +O-30ML)2 = reagent aerobic, -O2= anaerobic. Averages of triplicates are plotted with error bars representing standard deviation and statistics were performed using a two-tailed student’s t test with accordingsignificant differencesto manufacturer’s indicated byprotocol. asterisks Bacterial (*p<0.05 ** cells p<.01 were *** p<.001) homogenized with a Figure A1.1: Anaerobically grown L. monocytogenes150 shows increased hly transcript activity and stability but does not result in increased LLO protein abundance. A) Overnight cultures of a hly-gus reporter strain Listeria were grown aerobically, anaerobically, with, or without fumarate and subsequent fluorescence was BeadBug® microtube homogenizer using lysing matrix B (MP Biomedicals 6911- 100) 4x each sample for 40 seconds at maximum speed. Samples were kept on ice between runs 2 and 3 for 40 seconds. The RNA was then purified further including an on-column DNA digestion using an RNeasy® kit (Qiagen Cat. No. 74106) according to the manufacturer’s protocol. The final product was eluted with RNase free water and the concentration was measured using a NanoDrop spectrophotometer. cDNA synthesis and qPCR cDNA was synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase (OPTIZYME™ Fisher BP81045) according to the manufacturer’s protocol using 1µg of RNA and 1µM of reverse primer. A no RT control was included for each sample where the M-MLV was replaced with RNase free water. qPCRs were set up using Power SYBR® Green PCR master mix (applied biosystems® ref. no. 4367659) with 1µg of cDNA as a template and primer sets with a final concentration of 1µM. Reactions (40 cycles of 95° for 15s and 60° for 60s) were performed in an Applied biosystems StepOne Plus™ and analyzed using StepOne software v2.3. The transcript levels of gyrA a gene coding for the DNA gyrase subunit A were used as a normalizer. 151 MUG Assay The L. monocytogenes reporter strain (Phly-gus-neo) was generously provided by Dr. Nancy Freitag at University of Illinois College of Medicine at Chicago to establish transcriptional responses of hly to propionate. The reporter strain was grown on LB plates with neomycin sulfate (1 µg/mL). Colonies were selected and used to inoculate into BHI with or without propionate for growth overnight. Optical density (OD) of the overnight cultures was measured for normalization. The bacteria (1 mL) were harvested by centrifugation at 4 ◦C, washed twice with PBS, and resuspended in 100 µL of PBS with 1% Triton-X100. Bacterial cells were then lysed using a sonicator for three 30-s cycles. Samples were put on ice between each cycle. The lysate samples were centrifuged at 10,000 rpm for 5 min at 4 ◦C, and the resulting 100 µL of supernatant was collected into a 96-well plate. In the dark, 20 µL of 4-Methylumbelliferyl-β-D-glucuronide solution (1.8 mg/mL MUG; AAB21190MD, Fisher Scientific) was added to each well, and the plate was incubated at 37 ◦C. After 10 min, 10 µL of 0.2 M sodium carbonate was added as a stop solution in the dark. Fluorescence was measured at 365 nm excitation wavelength and 400 nm emission wavelength using a 96-well plate reader (Synergy4, Biotek). 152 LLO Immunoblotting SDS-PAGE and Immunoblotting Analysis: Overnight cultures of L. monocytogenes were used to perform SDS-PAGE and Immunoblotting Analysis. Cultures were normalized to OD (600nm) using BHI media. Pellet samples were frozen (-20°) and supernatant samples were precipitated with 10% trichloroacetic acid at 4° for 1 hour. Following supernatant protein precipitation, a cold acetone wash was performed. Both the pellet and supernatant samples were resuspended in 12µL of 2x sample buffer and heated at 95°C for 5 min after which it was separated by SDS-PAGE. Following separation immunoblots were performed using anti-LLO rabbit antibody (abcam® ab43018) followed by goat- anti-rabbit HRP antibody (abcam® ab6721). Acknowledgments: I am thankful to Dr. Nancy Freitag for sharing phly-GUS and Dr. Mary O’Riordan for the iLLO strains of L. monocytogenes. I am also grateful to Erica Rinehart for performing the studies using the phly-GUS mutant. 153 APPENDIX II Evaluation of Redox Homeostasis in Listeria monocytogenes Rationale: The aim of these experiments was to characterize the differences in redox balance between L. monocytogenes grown aerobically or anaerobically. Redox balance measurements indicated that anaerobically grown L. monocytogenes was experiencing oxidative stress. We discovered that anaerobically grown L. monocytogenes displayed no catalase activity and this led to a subsequent increase in killing when exposed to hydrogen peroxide. 154 Figure A2.1 Anaerobically grown L. monocytogenes exhibit increased oxidative stress and increased susceptibility to killing by H2O2 due to lack of catalase activity. A) Overnight cultures of Listeria were grown in the presence and absence of oxygen and the ratio of NAD:NADH was calculated. B) Catalase activity was measured by measuring he height of bubbles resulting from the addition of hydrogen peroxide to Listeria was measured. C) Listeria was challenged with .4% hydrogen peroxide for 30 minutes and the surviving bacteria were calculated as a percent survival compared to the starting concentration. D) Image showing the formations of bubbles with the addition of hydeogen peroxide to Listeria cultures grown under different conditions. NS= No Supplement, FUM= 50mM Fumarate,+O2 = aerobic, -O2= anaerobic. Averages of triplicates are plotted with error bars representing standard deviation and statistics were performed using a two-tailed student’s t test with significant differences indicated by asterisks (*p<0.05 , *** p<.001) Me thods Figure A2.1 Anaerobically grown L. monocytogenes exhibit increased oxidative stress and increased susceptibility to killing by H2O2 due to lack of catalase activity. A) Overnight cultures of Listeria were grown in the presence and absence of oxygen and NAD/NADHthe ratio of NAD:NADH Quantification was calculated. B) Catalase activity was measured by measuring he height of bubbles resulting from the addition of hydrogen peroxide to Listeria was measured. C) Listeria was challenged with .4% hydrogen peroxide for 30 minutes and the surviving bacteria were calculated as a percent survival compared to the starting concentration. D) Image showing the formations of bubbles with the addition of hydeogen peroxide to Listeria The concentration of NAD and NADH was determined by using an cultures grown under different conditions. NS= No Supplement, FUM= 50mM Fumarate,+O2 = aerobic, -O2= anaerobic. Averages of triplicates are plotted with error bars representing EnzyChrom™standard deviation NAD/NADH and statistics Assay were performedKit from Bioassayusing a two -Systemstailed student’ accordings t test with to the significant differences indicated by asterisks (*p<0.05 , *** p<.001) manufacturer’s suggested protocol. In brief: bacterial samples were Figure A2.1 Anaerobically grown L. monocytogenes155 exhibit increased oxidative stress and increased susceptibility to killing by H2O2 due to lack of catalase activity. A) Overnight cultures of Listeria were grown in the presence and absence of oxygen and the ratio of NAD:NADH was calculated. B) Catalase activity was measured by measuring he height of bubbles resulting from the addition of hydrogen peroxide to Listeria was measured. homogenized and assayed for [NAD] and [NADH] separately, and the ratio was determined by dividing [NAD] by [NADH]. Catalase Activity Assay Overnight samples of L. monocytogenes were normalized and subsequently concentrated to 109 CFU/mL. 100µL of the culture suspension was added to 13x100mM glass tubes in addition to 100µL of 30% hydrogen peroxide and 100µL of 1% Triton X-100 detergent. Each sample was thoroughly mixed and the corresponding height of bubble formation was measured to the nearest mm with a ruler. Survival Assay Overnight samples of L. monocytogenes were pelleted in microcentrifuge tubes by spinning at 10,000rpm for 3 minutes. The pellets were washed and resuspended in PBS with calcium and magnesium. All samples were diluted and plated on LB agar plates for time point zero. Undiluted samples were incubated with .4% hydrogen peroxide for 30min at 37°C. Following incubation samples were diluted and plated on LB agar plates for time point 30min. Plates were incubated at 37°C for 48 hours and colonies were enumerated. The percentage survival was calculated as the number of bacteria present at 30min divided by the number of bacteria at time point zero. 156