Transcriptomic Analysis of Listeria monocytogenes in Response to Bile Under Aerobic and Anaerobic Conditions.

Listeria monocytogenes is a gram-positive facultative anaerobic bacterium that causes the foodborne illness listeriosis. The pathogenesis of this bacterium depends on its survival in anaerobic, acidic, and bile conditions encountered throughout the gastrointestinal (GI) tract. This transcriptomics study was conducted to analyze the differences in transcript levels produced under conditions mimicking the GI tract. Changes in transcript levels were analyzed using RNA isolated from L. monocytogenes strain F2365 at both aerobic and anaerobic conditions, upon exposure to 0 and 1% bile at acidic and neutral pH. Transcripts corresponding to genes responsible for pathogenesis, cell wall associated proteins, DNA repair, transcription factors, and stress responses had variations in levels under the conditions tested. Upon exposure to anaerobiosis in acidic conditions, there were variations in the transcript levels for the virulence factors internalins, listeriolysin O, etc., as well as many histidine sensory kinases. These data indicate that the response to anaerobiosis differentially influences the transcription of several genes related to the survival of L. monocytogenes under acidic and bile conditions. Though further research is needed to decipher the role of oxygen in pathogenesis of L. monocytogenes, these data provide comprehensive information on how this pathogen responds to the GI tract.

Introduction

Listeria monocytogenes is a gram-positive foodborne pathogen that is responsible for the disease listeriosis (Scallan et al., 2011). Pregnant women, infants, elderly, and immunocompromised individuals are more susceptible to listeriosis, with meningitis, septicemia, and spontaneous abortions being possible manifestations of the disease (Thigpen et al., 2011). Being a foodborne pathogen, this bacterium must be able to respond to the stressors encountered following ingestion of contaminated food. Low pH, bile, and hypoxic/anoxic environments are some of the key stressors that are encountered by L. monocytogenes within the gastrointestinal (GI) tract (Davis et al., 1996).

Low pH of the stomach is one of the initial stressors encountered by L. monocytogenes upon ingestion (White et al., 2015). The low pH of the gastric secretion is a roadblock to invasion by the bacteria. Listeria’s acid response involves the SOS response, LisRK (a two-component regulatory system that regulates listerial osmotolerance), components of sigma B regulon, ATPase proton pump, and enzymatic systems that regulate internal hydrogen ion concentration (Sleator and Hill, 2005). A transcriptomic study that was performed on Listeria grown in the presence of organic acids revealed an increase in the transcript levels of sigma B and prfA regulated genes, which included internalins, phospholipases, and other virulence genes. This previous study also indicated an up-regulation of oxidative stress defenses, DNA repair, intermediary metabolism, cell wall modification, and cofactor and fatty acid biosynthesis (Tessema et al., 2012). A proteomic study performed on Listeria grown in the presence of organic salts demonstrated an up-regulation of oxidoreductases and lipoproteins. Upon exposure to hydrochloric acid, it was also observed that proteins involved in respiration (enzyme dehydrogenases and reductases), osmolyte transport, protein folding and repair, general stress resistance, flagella synthesis and metabolism were expressed in the response to the acidic conditions (Bowman et al., 2012).

Listeria is also exposed to bile within the GI tract (White et al., 2015). Bile is synthesized by the liver and stored in the gall bladder. It is released into the duodenum during digestion (Monte et al., 2009). The bile acids are the antibacterial component of bile; bile acids induce damage to the cell wall and DNA (Coleman et al., 1979; Bernstein et al., 1999; Prieto et al., 2004, 2006). Within the gall bladder, bile is found at a nearly neutral pH (7.5), while in the duodenum it is more acidic (pH 5.5) (White et al., 2015). Bile is more bactericidal at acidic pH than at a neutral pH, as indicated in a study that showed a decrease in survival in bile under pH 5.5 in comparison to a pH of 7.5 (Dowd et al., 2011). Many studies have been conducted to determine the global response of L. monocytogenes to bile encountered within the GI tract. For instance, the transcription factor brtA, which senses cholic acid and regulates efflux pumps (MdrM and MdrT) is involved in bile tolerance (Quillin et al., 2011). Bile salt hydrolases neutralize conjugated bile acids, thereby providing protection against the bactericidal properties of bile (Dowd et al., 2011). The bilE gene is also involved in detoxifying bile acids (Dowd et al., 2011).

In addition to changes in pH and bile, L. monocytogenes is also exposed to changes in oxygen concentrations. The duodenum is considered microaerophilic in nature, while the gall bladder is anaerobic (Zheng et al., 2015). Oxygen availability has been found to influence bile resistance. A proteomics study performed under anaerobic conditions in the presence of bile observed notable alterations in cell wall associated proteins, DNA repair proteins and oxidative stress response proteins. Under anaerobic conditions the Listeria adhesion protein has been observed to have a significant role in intestinal infection (Burkholder et al., 2009). Additionally, oxygen deprivation has been found to affect the survival of L. monocytogenes in vitro (Payne et al., 2013; Wright et al., 2016), as well as in cell cultures, guinea pigs (Bo Andersen et al., 2007), and gerbils (Harris et al., 2019). These studies highlight the importance of oxygen in regulation of virulence. However, it is not known what the transcriptomic response of L. monocytogenes is to conditions that mimic the GI tract under physiologically relevant anaerobic conditions. Therefore, the goal of this study was to determine the impact of oxygen on the transcriptomic response of L. monocytogenes to bile in conditions that mimic the duodenum (pH 5.5) and the gall bladder (pH 7.5).

Results

Survival of L. monocytogenes in Conditions Mimicking Gastrointestinal Tract

Listeria monocytogenes exhibits slightly slower growth rates under anaerobic conditions (Figures 1A vs. 1B). Bile also impacted the viability of L. monocytogenes strain F2365 differently under anaerobic conditions. Under neutral pH, bile did not have a significant impact on survival of L. monocytogenes strain F2365 under either aerobic (Figure 1A) or anaerobic conditions (Figure 1B).

FIGURE 1

Impact of oxygen on the survival of L. monocytogenes in conditions mimicking the gall bladder. F2365 was exposed to either aerobic (A) or anaerobic (B) conditions with 0% bile (black bars) or 1% bile (gray bars) at a pH of 7.5 and survival was measured by viable plate counts over 7 h. Data represent averages of three independent replicates. Error bars represent standard deviation from biological replicates.

At acidic pH in the presence of bile, which mimics the exposure to bile in the duodenum, the percentage of L. monocytogenes that survived significantly declined (Figure 2A; p < 0.05). This further demonstrates the increase in toxicity exhibited by bile when in acidic conditions. Survival also declined under anaerobic conditions in comparison to time 0 h (Figure 2B, p < 0.05). However, the decrease in viability was not as severe under anaerobic conditions (Figure 2B) in comparison to aerobic conditions (Figure 2A; p < 0.05). This indicates that anaerobic conditions improve the survival of L. monocytogenes to the toxic effects of bile.

FIGURE 2

Impact of oxygen on the survival of L. monocytogenes in conditions mimicking the duodenum. F2365 was exposed to either aerobic (A) or anaerobic conditions (B) with 0% bile (black bars) or 1% bile (gray bars) at a pH of 5.5 and survival was measured by viable plate counts over 7 h. Data represent averages of three independent replicates. Error bars represent standard deviation from biological replicates. * Indicates p < 0.05 in comparison to time 0.

Overall Changes in Transcript Levels in Response to Conditions Mimicking the Gastrointestinal Tract

As significant alterations in survival were observed following 1 h of bile exposure under acidic conditions, this time point was selected to compare the impact that oxygen had on the transcriptome. Table 1 shows the overall changes in transcripts detected. Under anaerobic conditions, a total of 190 transcripts in media at pH 7.5 and 268 at pH of 5.5 were identified to be differentially expressed in comparison to aerobic conditions. In the presence of bile and absence of oxygen, 304 and 434 transcripts were differentially produced at pH 7.5 and 5.5, respectively. Under anaerobic conditions, upon exposure to bile, variations in the transcript levels of 200 genes were identified at pH 7.5 and 419 at pH 5.5. For all conditions tested, there were globally more transcripts identified to be up-regulated than down-regulated, except for acidic bile conditions under anaerobic growth.

TABLE 1

Total changes in transcript levels following exposure to bile at pH of 7.5 or 5.5 under either aerobic or anaerobic conditions.

	Aerobic vs. Anaerobic	Bile Aerobic vs. Bile Anaerobic	Anaerobic vs. Bile Anaerobic
pH 7.5	Total = 190	Total = 304	Total = 200
	Up = 125 Down = 65	Up = 207 Down = 97	Up = 131 Down = 69
pH 5.5	Total = 268	Total = 434	Total = 419
	Up = 147 Down = 121	Up = 213 Down = 221	Up = 264 Down = 155

Changes in Transcript Levels in Response to Anaerobic Conditions

Transcripts representative of five genes were found to be increased in expression levels under exposure to anaerobic conditions regardless of whether the cultivation was conducted under either neutral or acidic pH (Table 2 and Supplementary Figure 1). These included genes involved in membrane transport, protein folding, and stress response. Of these transcripts the amino acid transporter (LMOf2365_2333) had nearly a 9-fold increase in levels at neutral pH in comparison to acidic pH. Transcripts representative of the dnaJ (LMOf2365_1491) and dnaK (LMOf2365_1492) genes, which encode for molecular chaperones and have roles in phagocytosis and protein homeostasis, were also increased under anaerobic conditions at both pH conditions tested. The transcript representative of the cadA (LMOf2365_0672) gene, which encodes for a heavy metal translocating P-type ATPase and is a component of the CadAC efflux cassette, was also increased 6.1-fold at pH 7.5 and 3.8 at pH 5.5 under oxygen depleted conditions (Table 2).

TABLE 2

Transcript levels increased in response to anaerobiosis at pH 7.5 or 5.5.

Gene ID	Gene product	Transcript fold changes
	Membrane transport	pH 7.5	pH 5.5
cadA LMOf2365_0672	Cadmium translocating P-type ATPase	6.1	3.8
LMOf2365_2333	Amino acid antiporter	137.2	15.0
Protein folding
dnaK LMOf2365_1492	Chaperone protein	5.3	8.6
dnaJ LMOf2365_1491	Chaperone protein	7.2	4.8
Stress response
gadG LMOf2365_2405	Glutamate decarboxylase gamma	11.2	3.2

The transcript levels of 18 genes were decreased under anaerobic conditions regardless of the pH condition tested (Table 3 and Supplementary Figure 2). Out of these eighteen transcripts, six were representative of uncharacterized hypothetical proteins; all of these had lower transcript levels under neutral conditions in comparison to acidic conditions. This could suggest that these hypothetical genes are regulated similarly. The remaining transcripts identified encoded for stress response, membrane associated protein, and metabolism protein (Table 3).

TABLE 3

Transcript levels decreased in response to anaerobiosis at pH 7.5 or 5.5.

Gene ID	Gene product	Transcript fold changes
		pH 7.5	pH 5.5
Membrane transport
LMOf2365_2554	Sensor histidine kinase	−4.46	−12.1
Metabolism
acpP LMOf2365_1834 LMOf2365_0511	Acyl carrier protein Heme oxygenase (staphylobilin-producing)	−13.6	−5.3
gcvT LMOf2365_1365	Glycine cleavage system T protein	−8.9	−4.7
LMOf2365_0585	Phosphoglycerate mutase family protein	−7.7	−3.6
Stress response
LMOf2365_0544	Universal stress protein family	−5.9	−5.8
Hypothetical proteins
LMOf2365_0964	Conserved hypothetical protein	−13.7	−5.9
LMOf2365_0511	Conserved hypothetical protein	−13.6	−5.3
LMOf2365_1087	Conserved hypothetical protein	−12.1	−3.9
LMOf2365_0808	Conserved hypothetical protein	−11.2	−3.1
LMOf2365_1179	Hypothetical protein	−8.3	−3.7
LMOf2365_2288	Conserved hypothetical protein	−6.3	−5.8

Changes in Transcript Levels in Response to Anaerobic Acidic Conditions

In acidic conditions, transcript levels of 140 genes were increased (Table 4 and Supplementary Figure 1) and 104 were decreased under anaerobiosis (Table 5). Analyzing these transcripts up-regulated in response to acidic conditions under anaerobiosis revealed that several biological pathways related to pathogenesis, stress response, membrane associated proteins, transcription factors and DNA repair mechanisms influenced the survival of L. monocytogenes (Table 4). Transcripts representative of genes involved in metabolism, transcription factor and pathogenesis were down-regulated (Table 5). Certain transcripts encoding for glycolytic enzymes increased under acidic anaerobic conditions as well (Table 4). These included the glyceraldehyde-3-phosphate dehydrogenase (5.4-fold increase), phosphoglycerate mutase (4.7-fold increase), and pyruvate kinase (6.7-fold increase).

TABLE 4

Transcript levels increased for select genes in response to anaerobiosis at pH 5.5.

Gene ID	Gene product	Transcript fold changes
Metabolism
hemL LMOf2365_1574	Glutamate-1-semialdehyde-2,1-aminomutase	3.1
nrdD LMOf2365_0299	Anaerobic ribonucleoside-triphosphate reductase	3.1
LMOf2365_1386	Phosphate acetyl/butyryltransferase family protein	3.1
panD LMOf2365_1929	Aspartate 1-decarboxylase	3.1
LMOf2365_0434	Polysaccharide deacetylase family protein	3.1
pepQ LMOf2365_1600	Proline dipeptidase	3.1
ldh-2 LMOf2365_1553	L-lactate dehydrogenase	3.2
LMOf2365_2670	N-acetylmuramoyl-L-alanine amidase, family 4	3.3
LMOf2365_1275	Hydrolase, alpha/beta fold family	3.3
LMOf2365_0372	Transcriptional regulator, DeoR family	3.4
LMOf2365_2200	Putative lactoylglutathione lyase	3.4
LMOf2365_0846	Pyruvate flavodoxin/ferredoxin oxidoreductase	3.4
LMOf2365_0277	Glycosyl hydrolase, family 1	3.7
asnB LMOf2365_1687	Asparagine synthase (glutamine-hydrolyzing)	3.8
pfl-1 LMOf2365_1425	Formate acetyltransferase	3.8
LMOf2365_2673	Orn/Lys/Arg decarboxylase	3.9
LMOf2365_0330	Threonine aldolase family protein	4.1
mvaS LMOf2365_1434	Hydroxymethylglutaryl-CoA synthase	4.2
LMOf2365_1633	Putative glutamyl-aminopeptidase	4.3
LMOf2365_1642	Dipeptidase	4.3
LMOf2365_0603	Glycosyl hydrolase, family 1	4.4
LMOf2365_0550	Glycosyl hydrolase, family 4	4.6
pnp LMOf2365_134	Polyribonucleotide nucleotidyltransferase	4.6
Gpm LMOf2365_2238	Phosphoglycerate mutase	4.7
LMOf2365_1226	Putative peptidase	5.2
LMOf2365_2528	Putative fructose-bisphosphate aldolase	5.3
gap LMOf2365_2432	Glyceraldehyde-3-phosphate dehydrogenase, type I	5.4
LMOf2365_1083	Inositol monophosphatase family protein	5.5
LMOf2365_2199	Metallo-beta-lactamase family protein	5.6
LMOf2365_1400	Putative acylphosphatase	5.7
LMOf2365_1299	4-hydroxybenzoyl-CoA thioesterase family protein	6.2
Pyk LMOf2365_1592	Pyruvate kinase	6.7
ldh-1 LMOf2365_0221	L-lactate dehydrogenase	7.5
pflA LMOf2365_1426	Pyruvate formate-lyase activating enzyme	7.6
galU LMOf2365_1099	UTP-glucose-1-phosphate uridylyltransferase	7.7
LMOf2365_0582	CBS domain protein	8.5
LMOf2365_2144	Nitroreductase family protein	9.3
LMOf2365_0802	Putative acyl-carrier protein phosphodiesterase	9.4
ald LMOf2365_1601	Alanine dehydrogenase	11.9
manA LMOf2365_2143	Mannose-6-phosphate isomerase, class I	13.6
LMOf2365_1608	Putative inorganic polyphosphate/ATP-NAD kinase	13.6
LMOf2365_2308	Aminopeptidase C	13.9
pfl-2 LMOf2365_1946	Formate acetyltransferase	40.3
murI LMOf2365_1246	Glutamate racemase	68
Transcription factors
LMOf2365_2140	Transcriptional regulator, DeoR family	3.1
argR LMOf2365_1384	Arginine repressor	3.2
LMOf2365_1526	DNA-binding response regulator	4.1
LMOf2365_1907	Iron-dependent repressor family protein	4.3
LMOf2365_0755	Transcriptional regulator, PadR family	4.6
LMOf2365_0480	Putative transcriptional regulator	4.8
LMOf2365_1986	Transcriptional regulator, Fur family	4.8
LMOf2365_0814	Transcriptional regulator, MarR family	7.8
LMOf2365_1707	Peroxide operon transcriptional regulator	8.6
Pathogenesis
LMOf2365_1812	Internalin family protein	5.4
hly LMOf2365_0213	Listeriolysin O	10.2
Motility
LMOf2365_1723	Methyl-accepting chemotaxis protein	4.4
DNA repair
topA LMOf2365_1293	DNA topoisomerase I	3.3
nth LMOf2365_1923	Endonuclease III	3.5
exoA LMOf2365_1807	Exodeoxyribonuclease	4.2
LMOf2365_1643	MutT/nudix family protein	4.4
ung-2 LMOf2365_1236	Uracil-DNA glycosylase	5.3
Stress response
LMOf2365_1997	Putative tellurite resistance protein	3.1
LMOf2365_0783	Glyoxalase family protein	3.4
LMOf2365_0963	Peroxide resistance protein Dpr	3.5
LMOf2365_2735	General stress protein 26	5.1
LMOf2365_1121	Glyoxalase family protein	5.2
Protein folding
groEL LMOf2365_2099	Chaperone protein GroEL	4.0
atpB LMOf2365_2508	ATP synthase F0, A subunit	4.1

TABLE 5

Transcript levels decreased for select genes in response to anaerobiosis at pH 5.5.

Gene ID	Gene product	Transcript fold changes
Metabolism
pheA LMOf2365_1555	Prephenate dehydratase	−18.8
LMOf2365_2263	Putative arsenate reductase	−14.8
LMOf2365_1556	GTP-binding protein, GTP1/OBG family	−13.4
LMOf2365_0148	Ser/Thr protein phosphatase family protein	−13.2
LMOf2365_2831	Sucrose phosphorylase	−9.3
LMOf2365_0128	Lipase	−8.9
cah LMOf2365_0827	Carbonic anhydrase	−8.9
LMOf2365_2647	Galactitol PTS system EIIA component	−8.5
tkt-3 LMOf2365_2640	Transketolase	−6.2
arcA LMOf2365_0052	Arginine deiminase	−6.1
LMOf2365_2643	Alcohol dehydrogenase, zinc-dependent	−5.7
qoxA LMOf2365_0016	Cytochrome aa3-600 menaquinol oxidase subunit II, Oxidative phosphorylation	−5.5
gabD LMOf2365_0935	Succinate-semialdehyde dehydrogenase	−5.4
LMOf2365_2364	Ferredoxin/flavodoxin—NADP+ reductase	−5.3
LMOf2365_0209	UDP-N-acetylglucosamine pyrophosphorylase	−4.9
guaB LMOf2365_2746	Inosine-5′-monophosphate dehydrogenase	−4.3
LMOf2365_0566	Putative N-carbamoyl-L-amino acid amidohydrolase	−4.1
ctaB LMOf2365_2088	Heme o synthase	−4.1
prs-1 LMOf2365_0210	Ribose-phosphate pyrophosphokinase	−3.9
LMOf2365_1048	Metallo-beta-lactamase family protein	−3.6
LMOf2365_2576	Acetamidase/formamidase family protein	−3.4
LMOf2365_2824	Glycosyl transferase, family 65	−3.0
Transcription Factors
ada, LMOf2365_0093	AraC family transcriptional regulator	−9.4
LMOf2365_0127	Transcriptional regulator, AraC family	−7.2
purr LMOf2365_0203	Pur operon transcriptional repressor	−4.3
LMOf2365_1683	Phosphosugar-binding transcriptional regulator, RpiR family	−4.2
LMOf2365_0023	Transcriptional regulator, GntR family	−4.0
LMOf2365_2467	Phosphate transport system protein PhoU	−4.0
LMOf2365_2017	LacI family transcriptional regulator	−3.3
LMOf2365_2224	ArsC family protein, regulatory protein spx	−3.3
LMOf2365_1010	Transcriptional regulator, MarR family	−3.1
Membrane Transport
LMOf2365_1428	MFS transporter, ACDE family, multidrug resistance protein	−7.9
LMOf2365_2542	Peptide/nickel transport system substrate-binding protein; bacterial extracellular solute-binding protein, family 5	−7.7
LMOf2365_2575	Putative Mg2+ transporter-C (MgtC) family protein	−5.4
LMOf2365_0759	Methyl-accepting chemotaxis protein	−4.2
LMOf2365_0267	Sugar ABC transporter, sugar-binding protein	−4.0
LMOf2365_0167	Peptide/nickel transport system substrate-binding protein	−3.9
LMOf2365_2351	Multicomponent Na+ :H+ antiporter subunit A	−3.3
LMOf2365_0876	Sugar ABC transporter, sugar-binding protein	−3.1
LMOf2365_2732	ATP-binding cassette, subfamily B, bacterial AbcA/BmrA	−3.1
Pathogenesis
LMOf2365_0128	Lipase	−8.9
inlE LMOf2365_0283	Internalin E	−6.7
LMOf2365_2467	Phosphate transport system protein PhoU	−4.0

Changes in Transcript Levels in Response to Bile Under Anaerobic Conditions

Transcripts representative of 53 genes were found to be up-regulated in response to exposure to bile under anaerobic conditions (Table 6 and Supplementary Figure 3). Transcripts encoding for transcription regulators of virulence, antibiotic resistance, metabolism, and membrane associated proteins were also observed to increase in their levels of expression (Table 6). Transcripts representative of nine genes were down-regulated under anaerobic conditions in presence of bile at both pH 7.5 and 5.5 (Table 7 and Supplementary Figure 4). Fold changes of the transcript levels of genes associated with metabolism, translation, pathogenesis, and transcription were down-regulated (Table 7).

TABLE 6

Transcript levels increased for select genes in response to anaerobiosis at pH of 7.5 and 5.5.

Gene ID	Gene product	Transcript fold changes
	Transcription factors	pH 7.5	pH 5.5
LMOf2365_0641	Transcriptional regulator, MarR family	6.5	13.7
prfA LMOf2365_0211	Listeriolysin regulatory protein	11.5	3.7
LMOf2365_1986	Fur family transcriptional regulator, ferric uptake regulator	12.7	18.8
glnR LMOf2365_1316	Transcriptional repressor GlnR	13.6	13.9
Metabolism
LMOf2365_2358	Thioesterase family protein	4.2	6.4
LMOf2365_0884	ATP-dependent RNA helicase DeaD	4.4	3.1
LMOf2365_1433	Acetyl-CoA acetyltransferase	4.5	6.6
LMOf2365_1729	Deoxynucleoside kinase family protein	4.6	10.5
LMOf2365_1660	Muramoyltetrapeptide carboxypeptidase	5.1	4.4
cysK LMOf2365_0234	Cysteine synthase A	6.1	6.2
LMOf2365_1038	Putative PTS system, glucose-specific, IIA component	6.3	4.4
LMOf2365_2371	NifU family protein	6.9	27.1
Cah LMOf2365_0827	Carbonic anhydrase	7.1	7.2
LMOf2365_1419	Acetyltransferase, GNAT family	7.3	3.7
trxB LMOf2365_2451	Selenocompound metabolism	8.7	5.0
glnA LMOf2365_1317	Glutamine synthetase, type I	9.9	3.3
LMOf2365_2364	Pyridine nucleotide-disulfide oxidoreductase family protein	10.1	5.1
LMOf2365_0861	Putative endoribonuclease L-PSP	10.6	4.2
LMOf2365_0391	Messenger RNA biogenesis	10.7	7.8
divIVA LMOf2365_2045	Cell division protein DivIVA	14.1	5.1
LMOf2365_0997	Acetyltransferase, GNAT family	14.5	7.1
alsS LMOf2365_2030	Acetolactate synthase	16.5	20.8
LMOf2365_0640	Flavodoxin-like fold domain protein	35.9	37.4
Membrane transport
LMOf2365_0761	Putative membrane protein	4.0	6.0
LMOf2365_2229	Oligopeptide ABC transporter, oligopeptide-binding protein	4.3	3.6
LMOf2365_1443	Transporter, NRAMP family	5.7	6.3
LMOf2365_0168	Zinc ABC transporter, zinc-binding protein	6.9	52.5
LMOf2365_1435	Putative transporter	8.2	7.4
LMOf2365_1012	Membrane protein, TerC family	9.6	257.7
LMOf2365_2330	Putative membrane protein	18.9	46.3

TABLE 7

Transcript levels decreased for select genes in response to anaerobiosis at pH 7.5 and 5.5.

Gene ID	Gene product	Transcript fold changes
		pH 7.5	pH 5.5
Metabolism
adhE LMOf2365_1656	Acetaldehyde dehydrogenase/alcohol dehydrogenase	−48.1	−71.2
LMOf2365_0250	Serine O-acetyltransferase	−5.8	−4.4
murE LMOf2365_2070	UDP-N-acetylmuramoyl-L-alanyl-D-glutamate–2,6-diaminopimelate ligase	−5.7	−4.5
Translation
LMOf2365_2879	tRNA-Glu	−25.3	−4.8
LMOf2365_2913	tRNA Leu	−11.5	−4.1
hly LMOf2365_0213	Listeriolysin O	−70.0	−3.7
Transcription factors
LMOf2365_2205	Sigma-54 dependent transcriptional regulator	−10.7	−5.5

Changes in Transcript Levels in Response to Bile Under Acidic and Anaerobic Conditions

Transcript levels of 210 genes were up-regulated in response to bile at acidic pH under anaerobic conditions (Table 8 and Supplementary Figure 3). Transcripts encoding for transcription factors, metabolism, replication and repair, cell signaling, protein folding, and pathogenesis were also found to be up-regulated. Additionally, transcripts representing 146 genes were down-regulated under anaerobic conditions with acidic bile (Table 9 and Supplementary Figure 4), with these being primarily associated with metabolism, membrane transport, replication and repair, pathogenesis, and transcription factors.

TABLE 8

Transcript levels increased for select genes in response to bile in anaerobic conditions at pH 5.5.

Gene ID	Gene product	Transcript levels
Metabolism
LMOf2365_0638	Rhodanese-like domain protein	3.4
LMOf2365_0686	Serine/threonine protein phosphatase family protein	4.1
mvaS LMOf2365_1434	Hydroxymethylglutaryl-CoA synthase	4.8
LMOf2365_1406	Putative pyrroline-5-carboxylate reductase	38
Pathogenesis
inlE LMOf2365_0283	Internalin E	3.6
LMOf2365_0508	Putative antigen	4.4
LMOf2365_2725	CBS domain protein	5.2
hlY-III LMOf2365_1893	Hemolysin III	6.2
LMOf2365_0726	Flagellin	29.2
LMOf2365_1503	DNA-binding protein, ComEA family	130.5
Cell Signaling
LMOf2365_0626	Cyclic nucleotide-binding protein	6.8
Protein Folding
LMOf2365_1018	ATP-dependent Clp protease, ATP-binding subunitE	3.9
clpP LMOf2365_2441	ATP-dependent Clp protease, protease subunit	5.2
trx-1 LMOf2365_1242	Thioredoxin	6.2
clpP-1 LMOf2365_1146	ATP-dependent Clp protease, proteolytic subunit P	25.0
Membrane Transport
LMOf2365_0153	Oligopeptide ABC transporter	3.0
LMOf2365_0288	Putative transporter	3.1
LMOf2365_2265	CBS domain protein	3.1
LMOf2365_0295	Competence protein ComEC/Rec2-related protein	3.3
LMOf2365_1088	Cell division protein, FtsW/RodA/SpoVE family	3.3
LMOf2365_1219	Putative membrane protein	3.4
acsA LMOf2365_2700	Acetyl-coenzyme A synthetase	3.6
LMOf2365_2554	Sensor histidine kinase	3.7
LMOf2365_2835	Major facilitator family transporter	3.7
LMOf2365_2647	PTS system, IIA component	3.8
zurM-2 LMOf2365_1465	Zinc ABC transporter, permease protein	4.0
LMOf2365_0622	Formate/nitrite transporter family protein	4.0
LMOf2365_1002	Drug resistance transporter, EmrB/QacA family	4.7
LMOf2365_0930	Putative membrane protein	5.0
LMOf2365_0967	Putative transporter	5.1
LMOf2365_0810	Putative membrane protein	5.6
LMOf2365_1721	Cation efflux family protein	6.4
LMOf2365_0588	Magnesium transporter, CorA family	6.5
LMOf2365_0701	ABC transporter, ATP-binding protein	7.1
lmrB-2 LMOf2365_2560	Lincomycin resistance protein LmrB	7.3
LMOf2365_1695	Putative laminin-binding surface protein	8.2
LMOf2365_2119	MATE efflux family protein	8.5
LMOf2365_2222	CoiA-like family protein	10.6
LMOf2365_0570	ABC transporter, substrate-binding protein	12.0
LMOf2365_0812	RarD protein	13.6
LMOf2365_0941	ABC transporter, ATP-binding protein	18.1
LMOf2365_1011	MATE efflux family protein	19.1
LMOf2365_0167	Bacterial extracellular solute-binding protein	20.4
LMOf2365_1502	Zinc-binding, ComEB family protein	21.8
LMOf2365_1428	Major facilitator family transporter	25.6
LMOf2365_1000	ABC transporter, ATP-binding protein	46.6
LMOf2365_0034	Putative membrane protein	60.2
Replication and Repair
LMOf2365_0196	Deoxyribonuclease, TatD family	3.1
LMOf2365_1533	ATPase, AAA family domain protein	3.3
LMOf2365_1998	Putative DNA-damage-inducible protein P	4.2
LMOf2365_0949	Putative DNA-3-methyladenine glycosylase	4.7
rnhA LMOf2365_1909	Ribonuclease HI	4.9
LMOf2365_2784	Replication and repair	5.9
dbpA LMOf2365_1260	ATP-dependent RNA helicase DbpA	8.4
recA LMOf2365_1417	Recombination protein RecA	9.2
LMOf2365_0863	Excinuclease ABC subunit C domain protein	11.4
LMOf2365_2339	MutT/nudix family protein	11.6
LMOf2365_0849	Putative transposase OrfA, IS3 family	12.7
dnaG LMOf2365_1474	DNA primase	18.7
Transcription Factors
LMOf2365_1427	Transcriptional regulator, PadR family	3.3
LMOf2365_1515	Transcription elongation factor GreA	3.4
nusG LMOf2365_0258	Transcription antitermination factor NusG	3.4
LMOf2365_2467	Phosphate transport system protein PhoU	3.4
LMOf2365_2223	MecA family protein	3.6
LMOf2365_0023	Transcriptional regulator, GntR family	3.6
LMOf2365_0576	Putative DNA-binding transcriptional regulator	3.6
LMOf2365_2337	Transcriptional regulator, DeoR family	3.7
ctsR LMOf2365_0241	Transcriptional regulator CtsR	3.8
LMOf2365_0119	Transcriptional regulator, ArsR family	4.0
LMOf2365_0446	Transcriptional regulator, LysR family	4.0
LMOf2365_2017	Transcriptional regulator, LacI family	4.1
LMOf2365_2841	Transcriptional regulator, AraC family	4.4
LMOf2365_1051	Transcriptional regulator, LacI family	4.4
LMOf2365_0906	Conserved hypothetical protein	4.8
LMOf2365_0794	ROK family protein	5.1
LMOf2365_2466	Transcriptional regulator, ArsR family	5.8
LMOf2365_2669	Transcriptional regulator, TetR family	5.8
LMOf2365_0266	Transcriptional regulator, DegA family	6.1
LMOf2365_0665	Rrf2 family protein	6.5
LMOf2365_0841	Transcriptional regulator, MerR family	7.7
LMOf2365_0394	Transcriptional regulator, DeoR family	9.5
LMOf2365_1894	DeoR family transcriptional regulator, catabolite repression regulator	11.5
LMOf2365_2224	ArsC family protein	11.7
LMOf2365_0940	PRD/PTS system IIA 2 domain protein	12.4
LMOf2365_2322	LysR family transcriptional regulator, regulator of the ytmI operon	13.1
LMOf2365_0435	DNA-binding protein	14.2
LMOf2365_2799	DNA-binding protein	14.7
Gene ID	Gene product	Transcript levels
LMOf2365_1010	Transcriptional regulator, MarR family	18.4
LMOf2365_2233	Transcriptional regulator, MarR family	19.1
LMOf2365_0755	Transcriptional regulator, PadR family	19.5
LMOf2365_0387	GntR family transcriptional regulator	25.7
LMOf2365_0326	DNA-binding protein	41.2

TABLE 9

Transcript levels decreased for select genes in response to bile in anaerobic conditions at pH 5.5.

Gene ID	Gene name	Transcript levels
Metabolism
LMOf2365_2610	Putative lipoprotein	−29.9
LMOf2365_0802	FMN-dependent NADH-azoreductase	−21.6
LMOf2365_1226	Putative peptidase	−18.2
LMOf2365_0565	6-phospho-beta-glucosidase	−18.2
pflA LMOf2365_1426	Pyruvate formate lyase activating enzyme	−11.1
LMOf2365_1975	Riboflavin transporter	−10.2
pyrH LMOf2365_1330	Uridylate kinase	−8.7
LMOf2365_1597	Bifunctional oligoribonuclease and PAP phosphatase NrnA	−8.5
LMOf2365_0277	Glycosyl hydrolase, family 1	−8.5
LMOf2365_0776	Hydrolase, alpha/beta fold family	−8.2
pfl-2 LMOf2365_1946	Formate C-acetyltransferase	−8.2
rplS LMOf2365_1814	Large subunit ribosomal protein L19	−7.7
pepQ LMOf2365_1600	Proline dipeptidase	−7.6
cadA LMOf2365_0672	Zn2+/Cd2+-exporting ATPase	−7.6
LMOf2365_2666	Cell division protein, FtsW/RodA/SpoVE family	−7.3
LMOf2365_0021	Glycosyl hydrolase, family 1	−6.9
LMOf2365_2146	Hydrogen peroxide-dependent heme synthase	−6.5
glmS LMOf2365_0762	Glutamine—fructose-6-phosphate transaminase	−6.3
LMOf2365_1093	N-acetylmuramoyl-L-alanine amidase	−6.3
LMOf2365_0057	Accessory gene regulator B	−5.9
LMOf2365_1386	Phosphate butyryltransferase	−5.7
thiI LMOf2365_1614	tRNA uracil 4-sulfurtransferase	−5.7
galU LMOf2365_1099	UTP–glucose-1-phosphate uridylyltransferase	−5.6
LMOf2365_1702	Methionine synthase/methylenetetrahydrofolate reductase (NADPH)	−5.6
LMOf2365_2609	FAD:protein FMN transferase	−5.6
eno LMOf2365_2428	Enolase	−5.5
LMOf2365_2670	N-acetylmuramoyl-L-alanine amidase, family 4	−5.3
fabI LMOf2365_0990	Enoyl-[acyl-carrier-protein] reductase I	−5.2
LMOf2365_1880	Copper chaperone; heavy metal binding protein	−5.1
LMOf2365_2711	PhnB protein	−5.1
LMOf2365_2673	Orn/Lys/Arg decarboxylase	−5.1
LMOf2365_1368	Rhodanese-like domain protein	−5.0
LMOf2365_2510	UDP-N-acetylglucosamine 2-epimerase	−4.8
mraY LMOf2365_2069	Phospho-N-acetylmuramoyl-pentapeptide-transferase	−4.7
purA LMOf2365_0065	Adenylosuccinate synthase	−4.7
ald LMOf2365_1601	Alanine dehydrogenase	−4.7
plcA LMOf2365_0212	1-phosphatidylinositol phosphodiesterase	−4.6
menE LMOf2365_1696	O-succinylbenzoate–CoA ligase	−4.6
murC LMOf2365_1627	UDP-N-acetylmuramate–alanine ligase	−4.5
LMOf2365_2743	Hydrolase, CocE/NonD family	−4.4
gpmA LMOf2365_2429	2,3-bisphosphoglycerate-independent phosphoglycerate mutase	−4.4
LMOf2365_0434	Peptidoglycan-N-acetylglucosamine deacetylase	−4.1
tmk LMOf2365_2672	Thymidylate kinase	−4.1
LMOf2365_1643	8-oxo-dGTP diphosphatase	−4.1
LMOf2365_2133	Pyridoxal 5′-phosphate synthase pdxS subunit	−3.9
pyk LMOf2365_1592	Pyruvate kinase	−3.9
alaS LMOf2365_1523	Alanyl-tRNA synthetase	−3.9
fhs LMOf2365_1906	Formate–tetrahydrofolate ligase	−3.9
LMOf2365_1033	N-acetyldiaminopimelate deacetylase	−3.8
LMOf2365_0872	D-alanine-D-alanine ligase	−3.8
LMOf2365_0987	Putative GTP pyrophosphokinase	−3.8
LMOf2365_1299	Acyl-CoA thioester hydrolase	−3.8
LMOf2365_1512	Peptidase, M3 family	−3.7
pfl-1 LMOf2365_1425	Formate C-acetyltransferase	−3.7
LMOf2365_2144	Nitroreductase family protein	−3.6
folA LMOf2365_1903	Dihydrofolate reductase	−3.6
LMOf2365_1371	Xaa-Pro aminopeptidase	−3.6
upp LMOf2365_2511	Uracil phosphoribosyltransferase	−3.5
uppS LMOf2365_133	Undecaprenyl diphosphate synthase	−3.5
LMOf2365_0239	Dihydrouridine synthase family protein	−3.5
LMOf2365_1633	Putative glutamyl-aminopeptidase	−3.4
LMOf2365_1476	[pyruvate, water dikinase]-phosphate phosphotransferase	−3.4
LMOf2365_0293	Acetyltransferase, GNAT family	−3.4
LMOf2365_1691	L-lactate dehydrogenase	−3.3
LMOf2365_0101	Oxidoreductase, aldo/keto reductase family	−−3.3
LMOf2365_1644	ADP-dependent NAD(P)H-hydrate dehydratase	−3.3
LMOf2365_0846	Pyruvate-ferredoxin/flavodoxin oxidoreductase	−3.3
LMOf2365_1915	Carboxypeptidase Taq	−3.3
hemE LMOf2365_2245	Uroporphyrinogen decarboxylase	−3.3
nrdD LMOf2365_0299	Ribonucleoside-triphosphate reductase	−3.3
sdhB LMOf2365_1841	L-serine dehydratase	−3.3
LMOf2365_2207	Oxidoreductase, short-chain dehydrogenase/reductase family	−3.2
LMOf2365_2514	L-threonylcarbamoyladenylate synthase	−3.2
pepT LMOf2365_1805	Tripeptide aminopeptidase	−3.1
LMOf2365_1048	Ribonuclease J	−3.1
mpl LMOf2365_0214	Zinc metalloproteinase	−3.1
LMOf2365_0488	Undecaprenyl diphosphate synthase	−3.1
LMOf2365_2308	Bleomycin hydrolase	−3.1
manA LMOf2365_2143	Mannose-6-phosphate isomerase, class I	−3.0
ftsX LMOf2365_2479	Cell division ABC transporter, permease protein FtsX	−3.0
gap LMOf2365_2432	Glyceraldehyde 3-phosphate dehydrogenase	−3.0
Pathogenesis
plcB LMOf2365_0216	Phospholipase C	−10.0
LMOf2365_1812	Internalin family protein	−6.1
Replication and repair
dnaE LMOf2365_1596	DNA polymerase III subunit alpha	−4.9
LMOf2365_1628	DNA segregation ATPase FtsK/SpoIIIE, S-DNA-T family	−4.3
ligA LMOf2365_1783	DNA ligase, NAD-dependent	−3.2
recG LMOf2365_1839	ATP-dependent DNA helicase RecG	−3.0
Transcription factor
LMOf2365_2335	Transcriptional regulator, RofA family	−8.6
argR LMOf2365_1384	Arginine repressor	−4.4
LMOf2365_2715	Transcriptional regulator, MerR family	−3.4
LMOf2365_2780	DNA-binding protein	−3.2
Membrane transport
LMOf2365_2388	D-methionine transport system substrate-binding protein	−9.1
LMOf2365_0606	Putative membrane protein	−8.4
Ffh LMOf2365_1828	Signal recognition particle subunit SRP54	−7.3
LMOf2365_2553	Putative ABC transport system permease protein	−6.3
ptsI LMOf2365_1024	Phosphoenolpyruvate-protein phosphotransferase	−5.9
LMOf2365_0803	D-serine/D-alanine/glycine transporter	−5.8
agrC LMOf2365_0059	Two-component system, LytTR family, sensor histidine kinase AgrC	−5.1
LMOf2365_0673	Putative membrane protein	−4.4
cydD LMOf2365_2695	ATP-binding cassette, subfamily C, bacterial CydC	−4.3
LMOf2365_1034	Moderate conductance mechanosensitive channel	−4.3
prf1 LMOf2365_2516	Peptide chain release factor 1	−4.2
ldh-1 LMOf2365_0221	L-lactate dehydrogenase	−4.2
LMOf2365_2148	ABC transporter, permease protein	−4.0
LMOf2365_1450	ABC transporter, ATP-binding protein	−3.8
LMOf2365_1994	ABC-2 type transport system ATP-binding protein	−3.8
LMOf2365_1264	Putative transporter	−3.3
LMOf2365_2323	Monovalent cation/hydrogen antiporter	−3.2
LMOf2365_0845	Na/Pi-cotransporter family protein	−3.2
LMOf2365_1091	Teichoic acid transport system permease protein	−3.1
LMOf2365_2844	YidC/Oxa1 family membrane protein insertase	−3.0
LMOf2365_0317	Putative membrane protein	−3.0
Translation
tsf LMOf2365_1678	Elongation factor Ts	−11.4
rpsB LMOf2365_1679	Small subunit ribosomal protein S2	−5.4
valS LMOf2365_1573	Valyl-tRNA synthetase	−4.1
gatB MOf2365_1779	Aspartyl-tRNA(Asn)/glutamyl-tRNA(Gln) amidotransferase subunit B	−4.1
efp LMOf2365_1372	Translation elongation factor P	−3.4
thrS LMOf2365_1580	Threonyl-tRNA synthetase	−3.1
infA LMOf2365_2583	Translation initiation factor IF-1	−3.1

Discussion

Anaerobiosis Improves Survival of L. monocytogenes in Conditions Mimicking the Gastrointestinal Tract

Survival of L. monocytogenes strain F2365 was analyzed under conditions mimicking the GI tract. This strain was chosen as it is a serotype 4b strain, which represents the serotype of a large portion of outbreak strains. F2365 was isolated from one of the deadliest outbreaks of L. monocytogenes (Linnan et al., 1988). F2365 has been sequenced (Nelson et al., 2004) and has been extensively studied for genomic analyses (Chatterjee et al., 2006; Liu and Ream, 2008; Payne et al., 2013), making it an ideal strain to analyze transcriptomic responses.

Bile is made in the liver, stored in the gall bladder, and released to the duodenum upon ingestion. The environment in the gall bladder is anaerobic and neutral pH, while the duodenum is acidic and microaerophilic (Zheng et al., 2015). The alterations in oxygen availability within the GI tract are essential to developing the redox relationship between microbes and host (He et al., 1999; Espey, 2013). Therefore, we tested how oxygen influenced the survival of L. monocytogenes under either acidic (mimicking the duodenum) or neutral (mimicking the gall bladder) bile conditions.

Since variations in transcript levels were observed due to alterations in oxygen availability, we wanted to determine which genes were commonly expressed under anaerobiosis. Transcript levels of five genes were found to be up-regulated under exposure to anaerobic conditions regardless of whether the cultivation was conducted under either neutral or acidic pH (Table 2), though there were differential expressions between the two conditions. Transcripts common to both conditions included two membrane transporters LMOf2365_2333 and cadA (LMOf2365_0672), two chaperones, and the stress response related gene gadG (LMOf2365_2405). CadA has been previously shown to be involved in formation of biofilms at 25°C by L. monocytogenes (Parsons et al., 2017). CadA also has been implicated in having roles in virulence and pathogenesis (Parsons et al., 2017). Therefore, it is possible that CadA is involved in stress response mechanisms related to anaerobic survival and that the formation of biofilms may be a critical component to survival. Previous studies have also shown that various stressors (i.e., heat shock, nutrient limitation, acidic condition, etc.) cause an increase in the expression of chaperones (Wright et al., 2016). Indeed, the data showed an increase in the transcript levels of two chaperones (dnaK and dnaJ) under anaerobic conditions at both pH 7.5 and 5.5. Therefore, it is possible that L. monocytogenes uses molecular chaperones to combat anaerobic stress, which in turn assists with phagocytosis. The gadG encodes for an amino acid antiporter that is part of the glutamate decarboxylase system, which is a defense mechanism up-regulated by L. monocytogenes under acid stress and anaerobiosis. This system alleviates the acidification of the cytoplasm by consuming a proton (Cotter et al., 2001; Jydegaard-Axelsen et al., 2004; Paudyal et al., 2020). The fact that this transcript was up-regulated in response to anaerobic conditions suggests that there may be overlapping functions of the GAD system in both acid resistance and anaerobiosis. The transcript level of the LMOf2365_2333 gene was increased by nearly 9-fold in comparison to acidic pH. There is a possibility that this amino acid anti-transporter may function with gadG in response to bile. This should be further explored in future studies.

Transcript levels of eighteen genes were down-regulated under anaerobic conditions regardless of the pH, including histidine kinase, metabolic genes, a universal stress response gene, and genes coding for hypothetical proteins. As histidine kinases are involved in two-component systems, it is possible that suppression of this sensor is responsible for the response to oxygen availability. One of the metabolic genes, the phosphoglycerate mutase, has been shown in Bacillus subtilis to be responsible for the control of the two-component system required for sensing and responding to aerobic and anaerobic respiration (Nakano et al., 1999). The fact that the transcript level of this gene was down-regulated suggests that the accumulation of the product 1,3-bisphosphoglycerate, which is the intermediate in the reaction catalyzed by phosphoglycerate mutase, might impact the regulation of the histidine kinase LMOf2365_2554. The impact of this precursor on regulation of two-component systems needs to be explored in further detail. The transcript level of the gene acpP was also down-regulated. This gene product is involved in biosynthesis of fatty acids as a lipid transporter. This gene has been found to be differentially regulated under anaerobic conditions in many other bacteria, including Escherichia coli and Neisseria gonorrhoeae (Isabella and Clark, 2011). This indicates that the regulation of the fatty acid synthesis is necessary for the adaptability to anaerobiosis.

Differential Transcript Levels in Response to Anaerobic Acidic Conditions

An increase in the transcript levels of nrdD (LMOf2365_0299), which is an anaerobic ribonucleoside-triphosphate reductase that catalyzes the synthesis of dNTPs required for DNA replication, was observed under anaerobic conditions at acidic pH. NrdD is an essential enzyme required by L. monocytogenes and other GI pathogens, such as E. coli, to survive under anaerobic conditions (Garriga et al., 1996; Ofer et al., 2011). Since our study showed acidic conditions influence the up-regulation of this gene under anaerobic conditions, there is a possibility that this enzyme is involved in growth under acidic conditions. This may be required to stabilize the redox potential of the cell under acidic conditions. Ribonucleotide reductases have been explored as potential biomedical targets for bacterial infections (Torrents, 2014). Since the ribonucleotide reductase was up-regulated under anaerobic acidic conditions, it will be necessary for future studies to analyze the activity of antibacterial compounds under these conditions to effectively target the protein expressed.

Transcript levels of genes coding for a glycosyl hydrolases, which are involved in hydrolyzing the glycosidic linkages in sugars, were also up-regulated. Certain glycosyl hydrolases have been previously identified as virulence factors in gram positive pathogenic bacteria, including Streptococcus pneumoniae (Niu et al., 2013). Glycosyl hydrolase PssZ has been observed to degrade extracellular polymeric substance, thereby disrupting biofilm formation by L. monocytogenes (Wu et al., 2019). L. monocytogenes, which is an intracellular bacterium, may synthesize glycosyl hydrolases upon exposure to acidic pH under anaerobic conditions, which thereby hinders formation of biofilms and facilitates the bacterium’s entry into the host cells.

One of the virulence factors of L. monocytogenes is metalloproteases. Few such proteases were identified to have an increase in transcript levels at pH 5.5 in anaerobic conditions, including the aminopeptidase (LMOf2365_2308) (Table 4). It has been shown that the bacterial burden of L. monocytogenes EGDe strain in host cells decreased significantly when the aminopeptidase T of family M29 was deleted (Cheng et al., 2015). Thus, at anaerobic conditions under acidic pH, aminopeptidases may be up-regulated and function as virulence factors.

GalU (LMOf2365_1099), UTP-glucose-1-phosphate uridyltransferase, which catalyzes cell wall teichoic acid glycosylation, had an increase in transcript levels under anaerobic conditions at pH 5.5 (Table 4; Kuenemann et al., 2018). In silico design of GalU inhibitors attenuated virulence of L. monocytogenes, proving GalU to be an instrumental part in virulence pathways (Kuenemann et al., 2018). Various transcription factors were up-regulated under anaerobic conditions at pH 5.5 (Table 4), including the fur regulator that controls virulence of various pathogenic bacteria. We also observed that transcripts coding for virulence genes, such as listeriolysin O and internalin family proteins, were also up-regulated under these conditions. The transcript level of a methyl accepting chemotaxis protein was also increased. In L. monocytogenes chemotaxis genes cheA and cheY have been shown to facilitate to adhesion and thereby invasion into the host epithelial cells. As L. monocytogenes is an intracellular pathogen, it may be possible that along with the CheA and CheY system, it is using the methyl accepting chemotaxis proteins to attach to epithelial cells under anaerobic conditions at pH 5.5 (Dons et al., 2004).

Internalins A and B are required by L. monocytogenes for facilitating entry inside host cells. Transcript levels for genes encoding internalin proteins were found to be up-regulated under the acidic environment in absence of oxygen. Interestingly, the transcript level of inlE (LMOf2365_0283), which is a gene coding for the secreted protein Internalin E, was decreased. Internalins A and B are involved in adhesion and invasion by Listeria, but Internalin E is not involved in invasion (Dramsi et al., 1997). This indicates anaerobiosis influences the invasive potential of L. monocytogenes. The impact of anaerobiosis on invasion has been shown in vitro and in vivo, but the exact mechanism of such interplay has not been well characterized (Bo Andersen et al., 2007; Harris et al., 2019).

Differential Transcript Levels in Response to Bile Under Anaerobic Conditions

Previous studies have shown that following ingestion of L. monocytogenes into host systems, the prfA regulon is up-regulated (Scortti et al., 2007). prfA, the positive regulatory factor A, is a transcription factor that regulates major virulence factors of L. monocytogenes. prfA regulates listeriolysin O, phospholipase C and metalloproteases, all of which were up-regulated in anaerobiosis in presence of bile (Table 6). Following bile exposure, the transcript levels of the virulence regulator prfA were decreased (Boonmee et al., 2019); however these data show that under anaerobic conditions in presence of bile, prfA is up-regulated independent of pH. We have also observed that L. monocytogenes survives bile better under anaerobic conditions (Figure 2).

Previous transcriptomics studies in L. monocytogenes 10403S (Boonmee et al., 2019) have found that following exposure to bile, the house keeping sigma factor σA has a significant role in survival. marR [multiple antibiotic resistance regulator (LMOf2365_0641)] is a transcriptional regulator that was up-regulated in response to bile in anaerobic conditions regardless of the pH tested (Table 6). In pathogens such as Salmonella and Staphylococcus, marR homologs slyA and sarZ regulate virulence gene expression. marR homologs have also been found to regulate genes involved in stress response, degradation or efflux of harmful substances and metabolic pathways (Grove, 2013). Bile exposure under anaerobic environments may trigger the up-regulation of marR to export bile out of the bacterial cell, thereby contributing to the bile resistance of L. monocytogenes along with other factors. The role of marR in bile resistance needs to be further explored.

Glutamine synthetase catalyzes the condensation of ammonia and glutamate to form glutamine. The transcript level of the glutamine synthetase repressor, glnR (LMOf2365_1316) was increased following exposure to bile in anaerobic conditions. It is a central nitrogen metabolism regulator which is activated in presence of glutamine. When glutamine is in excess, GlnR represses the synthesis of glutamine synthetase (Kaspar et al., 2014). Another probable transcriptional regulator (tnrA or codY) represses glutamine synthetase and its activation have been found to be essential in replication Listeria intracellularly (Kaspar et al., 2014). Interestingly glutamine synthetase was also up-regulated under the same conditions, which indicates the possibility of a feedback loop.

Metalloenzyme carbonic acid catalyzes hydration of carbon dioxide into bicarbonate and proton (Supuran, 2016). The infection cycle of Legionella has similarities with that of L. monocytogenes, such as invasion and escaping the phagosome. Legionella has been shown to evade the destruction by maintaining neutral pH (Supuran, 2016). One of the enzymes involved in regulating the pH is carbonic anhydrase; the transcript level of carbonic anhydrase increased under anaerobic conditions in the presence of bile in L. monocytogenes (Table 6). This could indicate that environmental conditions mimicking parts of intestine can contribute to Listeria’s pathogenic potential. Interestingly, the transcript level of this gene was down-regulated under acidic conditions (Table 5), suggesting that the influence of bile is important to the expression of this gene.

Transcript levels representative of an uncharacterized membrane protein LMOf2365_1012 that belongs to the TerC family was up-regulated following exposure to bile in anaerobic conditions (Table 6). In B. subtilis, TerC has been found to confer manganese resistance (Paruthiyil et al., 2020). In Streptococcus, manganese homeostasis is linked to oxidative stress as well as virulence (Turner et al., 2015). It is possible that TerC is linked with manganese homeostasis and therefore virulence in the presence of bile under anaerobic conditions. Transcripts coding for several other membrane transporters were also increased in their levels under the anaerobic environment in response to bile. The zinc ABC transporter has been shown to have a role in virulence of L. monocytogenes in a mouse infection model (Corbett et al., 2012). Thus, bile exposure in absence of oxygen probably impacts uptake of zinc by the bacteria thereby impacting the virulence. NRAMP, which functions as a metal ion transporter on membranes, was up-regulated (Nevo and Nelson, 2006).

The transcript level of the oligopeptide ABC transporter, which is an oligopeptide binding protein that helps the bacteria survive intracellularly, was increased (Slamti and Lereclus, 2019). It is the substrate binding component or receptor of an ABC type oligopeptide transport system that binds extracellular peptides, relays it to the membrane component of the system and inside the bacterial cell afterward. Gram positive bacteria such as Listeria, Streptococcus, and Enterococcus, use peptides to sense and respond to environmental changes. The gene oppA, which encodes for an oligopeptide binding protein, has been found to be required for invasion (Borezee et al., 2000). Thus, the oligopeptide ABC transporter observed in our study could be responsible for intracellular survival of bacteria in presence of bile under anaerobic conditions.

Interestingly, there was a decrease in the transcript levels of hly (LMOf2365_0213), which encodes for listeriolysin O, at both pH 7.5 and 5.5 following exposure to bile under anaerobic conditions. This was different than what was observed under anaerobiosis at pH 5.5 alone, as hly (LMOf2365_0213) was up-regulated in these conditions (Table 4). This suggests that bile has an important role in regulating the invasiveness of L. monocytogenes. This correlates well with previous studies that have shown that L. monocytogenes remains extracellular in the gall bladder, which has high concentrations of bile (Hardy et al., 2004; Dowd et al., 2011).

Differential Transcript Levels in Response to Bile Under Acidic and Anaerobic Conditions

There was an increase in transcript levels for the myosin cross reactive antigen (McrA) (LMOF2365_0508; Table 8). Although its function in L. monocytogenes is yet unknown, in Streptococcus pyogenes McrA is a fatty acid double bond hydratase that adds water to double bonds of fatty acids. Upon deletion of this gene, decreased oleic acid resistance and reduced adherence and internalization in the host cell was observed in S. pyogenes (Volkov et al., 2010). Conditions encountered within the duodenum may directly or indirectly contribute to up-regulation of mcrA, which may regulate the pathogen’s resistance to bile.

Internalin E and hemolysin III are both virulence factors responsible for internalization and invasion for L. monocytogenes. Both had an increase in transcript levels, indicating that bile exposure at acidic and anaerobic conditions, which mimics the duodenum, is conducive to the pathogenesis of the bacteria.

The transcript level of the LPXTG-motif cell wall anchor domain (LMOF2365_1144) was also up-regulated. In the L. monocytogenes EGDe strain, it has been shown that a LPXTG protein encoded by the Listeria mucin binding invasion A gene, or lmiA, has roles in promoting bacterial adhesion and entry into the host cell (Mariscotti et al., 2014). MucBP domain present in LPXTG was observed to bind to mucin. Thus, up-regulation of LPXTG gene under conditions mimicking the duodenum indicates that these conditions may facilitate invasion of host cells by the bacteria.

The level of transcripts representing flagellin also increased. It has been shown that flagellin helps in motility soon after ingestion in vivo (O’Neil and Marquis, 2006) and invasion (Dons et al., 2004). A previous study has also observed up-regulation of motility under exposure to bile at pH 5.5 (Guariglia-Oropeza et al., 2018). The fact that expression increased in conditions that would be encountered soon after ingestion suggests that the flagellin are important for the motility of the bacteria to the location in the GI tract where they will invade the intestinal lining.

The transcript level of the histidine kinase LMOf2365_2554 was also up-regulated under conditions mimicking the duodenum. Histidine kinase is the signal receiver a two-component regulatory system. Its counterpart in the system is the response regulator (Chang and Stewart, 1998; Stock et al., 2000; West and Stock, 2001; Krell et al., 2010). Response regulators in L. monocytogenes have been proven to have roles in virulence and pathogenesis. Sensor histidine kinase, ChiS, regulates the chitin utilization pathway required by Vibrio cholerae, which is needed to survive in aquatic environments. Chourashi et al. (2016) observed that ChiS has an important role in adherence and intracellular survival of V. cholerae in HT-29 cell cultures. They also showed that the sensor histidine kinase ChiS was activated in the presence of intestinal mucin (Chourashi et al., 2016). In the case of L. monocytogenes, it could be possible that the conditions in the duodenum are favorable for activation of the sensor histidine kinase, which could in turn relay information that would result in the activation of transcription factors responsible for adhesion and invasion.

Transcript levels representative of replication and repair genes were also up-regulated. In L. monocytogenes strain EGDe, RecA has been shown to have roles in bile and acid resistance, as well as in adhesion and invasion to Caco-2 cell cultures (van der Veen and Abee, 2011). Our data indicate that in the pathogenic strain F2365, RecA has the similar role of bile and acid resistance. In our study, we have also found that under anaerobic conditions (along with bile and acidic) the transcript level of recA changed, indicating absence of oxygen may have impact on activation of RecA.

The transcript level for a gene encoding for the transcriptional regulator padR was up-regulated (Table 8). In L. monocytogenes EGDe, LftR, which is a PadR like transcriptional regulator, has been shown to influence invasion of human host cells (Kaval et al., 2015). It is already known that Listeria uses internalin proteins for adhering and internalizing into the cell. Kaval et al. (2015) found that LftR, which is an uncharacterized protein, is required for invasion.

Transcript level of the gene encoding for ctsR, (LMOf2365_0241) a class III stress gene repressor that negatively regulates clp, was up-regulated under these conditions (Table 8). CtsR has been shown to be required for virulence in mice. PrfA which regulates many virulence genes of L. monocytogenes has been shown to down-regulate ClpC production (Karatzas et al., 2003). Although Karatzas et al. (2003) could not find any relationship between clp and prfA, there is still a possibility that there is a connection between the regulation of Clp by CtsR under anaerobic conditions in exposure to bile at acidic pH (Cui et al., 2018).

The transcript level of the transcription elongation factor greA (LMOf2365_1515) also increased under anaerobic conditions with acidic bile. GreA has been found to have roles in affecting functions of virulence gene expression in the pathogen Francisella tularensis subsp. Novicida (Cui et al., 2018). In F. tularensis, GreA was found to be required for invasion and intracellular growth of bacteria. Cui et al. (2018) also observed suppression of virulence of the greA mutant in mouse model. Transcriptomics analysis of the greA mutant revealed down-regulation of various genes responsible for virulence. Thus, with respect to our work, conditions in the duodenum are favorable for induction of the transcription elongation factor greA, which may in turn regulate genes responsible for invasion and multiplication of L. monocytogenes.

This study indicates that not only one stressor, but combinations of different stressors impact the transcription of various virulence genes. Transcriptomic and phenotypic studies in absence of these genes under mimicking physiological condition could give us an insight into this mechanism. A better understanding of how these biological processes help the survival of L. monocytogenes will lead us to understand how the physiological conditions contribute to the pathogenesis.

Materials and Methods

Bacterial Strain and Culture Conditions

Listeria monocytogenes str. 4b F2365 was used for this study. Overnight cultures of L. monocytogenes str. 4b F2365 were grown at 37°C aerobically in Brain Heart Infusion (BHI) media at pH 7.5. Next day, inoculum (1:100) from the overnight culture was used to grow the cells to mid exponential phase in fresh BHI media (OD600 = 0.3 to 0.5) under either aerobic or anaerobic conditions in 5 mL aliquots. Anaerobic culture conditions were obtained using an incubator shaker set at 37°C inside a Coy Anaerobic Chamber with a gas mixture of 95% N2 and 5% H2 (Coy Laboratory Products, United States). Cells were then pelleted at 8000 × g at 23°C and resuspended in fresh BHI at a pH of either 7.5 or 5.5; pH was adjusted with either HCl or NaOH. For bile treated cells, mid exponential phase cells were resuspended in BHI at a pH of either 7.5 or 5.5 supplemented with 1% porcine bile extract (Sigma Aldrich, United States). Cells were then grown under either aerobic or anaerobic conditions at 37°C. This study had eight different conditions that mimicked parts of the GI tract. The conditions tested were: (1) aerobic at pH 5.5; (2) anaerobic at pH 5.5; (3) aerobic at pH 7.5; (4) anaerobic at pH 7.5; (5) aerobic at pH 5.5 with 1% porcine bile; (6) anaerobic at pH 5.5 with 1% porcine bile; (7) aerobic at pH 7.5 with 1% porcine bile; and (8) anaerobic at pH 7.5 with 1% porcine bile. For each time point during a 7 h incubation period, aliquots were serially diluted in phosphate buffered saline (PBS) and plated onto BHI agar plates. Plates were incubated overnight at 37°C prior to enumeration. Three independent replicates were performed in parallel for each individual condition tested.

RNA Extraction, Library Preparation and RNA Sequencing

To isolate the RNA for analysis of the transcript level expression, cells were collected after 1 h of incubation in the eight culture conditions described above. Three biological replicates were assayed. Briefly, 5 mL of culture was pelleted by centrifugation at 8,000 × g for 5 min at room temperature. Cell pellets were then treated with RNA Protect Bacterial Reagent (Qiagen, Germany). Total RNA was isolated using the RNeasy® Mini Kit (Qiagen, Germany) per manufacturer’s instructions. The extracted RNA was quantitated using Qubit 3 Fluorometer (Invitrogen, United States) using the Qubit RNA BR assay kit (Thermo Fisher, United States). Extracted samples with values of A260/280 ∼ 2.0 were selected for sequencing. Illumina HiSeqTM 2000 paired-end 50 bp sequencer (PE50) was used. Ribosomal RNA was reduced with Epicentre RiboMinus kit (Illumina, United States) coupled with Directional RNA-Seq library prep with TruSeq indexes (Illumina, United States) per manufacturer’s instructions.

Data Analysis

Differences in survival were determined using a student’s t-test (Prism 8). Tophat-2.0.8.b (Trapnell et al., 2009) was used to align the RNA-Seq data to the reference genome, AE017262.2 L. monocytogenes str. 4b F2365. Transcript level calculation and FPKM normalization were performed using Cufflinks-2.1.1 (Trapnell et al., 2010). FPKM filtering cutoff of 1.0 was maintained to determine expressed transcripts. Differential transcript levels of the genes were determined using Cuffdiff (Trapnell et al., 2013). Differential transcript levels which had a greater than 3-fold expression and were statistically significant (p < 0.01 and q < 0.01) were subjected to Gene Ontology (GO) enrichment analysis using Blast2GO (Conesa et al., 2005). In this software, the up- and down-regulated transcripts were selected, and BLAST was performed against the L. monocytogenes nucleotide database in NCBI. The BLAST results were then mapped and annotated.

Data Availability Statement

SRA IDs of the submitted data: SRR13859772, SRR13859774, and SRR13859773: F2365 pH 5.5 Aerobic, SRR13859144, SRR13859143, and SRR13859142: F2365 pH 5.5 Anaerobic, SRR13859527, SRR13859526, and SRR13859525: F2365 pH 5.5+ Bile Anaerobic, SRR13859600, SRR13859599, and SRR13859598: F2365 pH 7.5+ Bile aerobic, SRR13858938, SRR13858937, and SRR13858936: F2365 pH 7.5+ Bile Anaerobic, SRR13858765, SRR13858767, and SRR13858766: F2365 pH 7.5 Anaerobic, SRR13853432, SRR13853433, and SRR13853431: F2365 pH 5.5+ Bile Aerobic, SRR13849951, SRR13849952, and SRR13849950: F2365 pH 7.5 aerobic.

Author Contributions

JD: conceptualization, supervision, and project administration. MA, MD, JD, GS, and DC: methodology. GS and DC: software. GS, DC, and JD: validation and visualization. DC and JD: investigation and writing–review and editing. MA and GS: resources. DC: data curation and writing–original draft preparation. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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