The DNA glycosylase, Adlp Affects Flagellar Motility, Biofilm Formation, Virulence, and Stress 1 Response in Listeria monocytogenes 2

3

Ting Zhang, Dongryeoul Bae, and Chinling Wang* 4

Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University 5

6

Running title: The discovery of a novel virulence factor, adlp, in Listeria monocytogenes 7

*Corresponding author 8

Mailing address: P.O. Box 6100, Department of Basic Sciences, College of Veterinary Medicine, 9

Mississippi State University, Mississippi State, MS 39762. 10

Phone: (662) 325 1683 11

Fax: (662) 325 1031 12

E-mail: cw57@msstate.edu 13

Keywords: DNA glycosylase, flagellar motility, virulence, stress response, Listeria 14

monocytogenes 15

Zhang’s current address: Department of Microbiology and Immunobiology, Harvard Medical 16

School, Boston, MA 021153 17

Bae’s current address: Division of Microbiology, National Center for Toxicological Research, 18

U.S. Food and Drug Administration, Jefferson, Arkansas 720791 19

AEM Accepted Manuscript Posted Online 17 June 2016Appl. Environ. Microbiol. doi:10.1128/AEM.00719-16Copyright © 2016 Zhang et al.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Abstract 20

The temperature-dependent alteration of flagellar motility gene expression is critical for the 21

food-borne pathogen, Listeria monocytogenes, to respond to a changing environment. In this 22

study, a genetic determinant (lmof2365_0220) encoding a putative protein that is structurally 23

similar to the Bacillus cereus alkylbase DNA glycosylase (AlkD) was identified. This 24

determinant was involved in the transcriptional repression of flagellar motility genes and was 25

named adlp (AlkD-like Protein, Adlp). Deletion of adlp activated the expression of flagellar 26

motility genes at 37°C and disrupted the temperature-dependent inhibition of L. monocytogenes 27

motility. The adlp null strains demonstrated decreased survivability in murine macrophage-like 28

RAW264.7 cells and less virulence in mice. Furthermore, the deletion of adlp significantly 29

decreased biofilm formation and impaired the survivability of bacteria under several stress 30

conditions, including the presence of a DNA alkylation compound (methyl methanesulfonate), 31

an oxidative agent (H2O2), and aminoglycoside antibiotics. Our findings strongly suggest that 32

adlp may encode a bi-functional protein that transcriptionally represses the expression of 33

flagellar motility genes and influences stress responses through its DNA glycosylase activity. 34

35

Importance 36

We discovered a novel protein named AlkD-like Protein (Adlp). This protein affected flagellar 37

motility, biofilm formation and virulence. Our data suggest that adlp may be a bi-functional 38

protein that represses flagellar motility genes and influences stress responses through its DNA 39

glycosylase activity. 40

41

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Introduction 42

Listeria monocytogenes is a gram-positive, food-borne intracellular pathogen that causes severe 43

illnesses in neonates, pregnant women, and immunocompromised individuals (1, 2). The switch 44

between the pathogenic stage and saprophytic stage of this opportunistic bacterium is 45

accompanied with altered transcription pattern (3), which is orchestrated by its regulatory 46

systems, including global transcription regulators (4–9), two-component systems (10–13), and 47

emerging non-coding RNAs (14–17). 48

49

Early studies reveal that the motility of L. monocytogenes is temperature dependent; it is non-50

motile at high temperatures (37°C) and highly motile at low temperatures (28°C and below). The 51

motility of bacteria is correlated with its temperature-dependent expression of flagella (18, 19), 52

as well as flagellar motility genes (3, 20). The expression of flagellar motility genes in L. 53

monocytogenes is under sophisticated regulation. MogR, a transcriptional repressor, inhibits the 54

transcription of flagellar motility genes at high temperatures (37°C and above) by binding to the 55

promoter regions of these genes (20, 21). At low temperatures, GmaR, which is a 56

glycosyltransferase that is capable of beta-O-GlcNAcylating its substrates (flagellin), works as 57

an anti-repressor by forming a stable complex with MogR (22), thus resulting in the non-58

hierarchical expression of flagellar motility genes (21). GmaR transcription is under the 59

regulation of DegU (22), which is an orphan response regulator with a dispensable receiver 60

domain that activates flagellar motility expression independent of its phosphorylation state (23–61

26). Remarkably, GmaR itself can work as a thermometer to regulate flagellar motility by 62

switching the binding affinity to MogR at different temperatures (27). In addition, sigB can 63

influence motility through a sigB-controlled mogR promoter (3). 64

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65

Recently, a novel alkylbase DNA glycosylase (AlkD) was discovered in Bacillus (B.) cereus and 66

has been classified as a new family of DNA glycosylases, based on its unique DNA-binding 67

protein architecture (tandem α-helical repeats, known as HEAT-like repeats). It has been shown 68

to play a central role in N3- and N7-alkylated base repair (28–31). The objective of this study 69

was to understand the function of AlkD-like gene in L. monocytogenes and determine its 70

physiological significance with respect to biofilm formation, mobility and stress response. 71

Materials and methods 72

Bacterial strains and culture conditions 73

Listeria strains that were used in this study are listed in Supplementary table 1. L. 74

monocytogenes strains were cultured in BHI broth (Difco Laboratories, Detroit, MI) at 37°C. E. 75

coli DH5α were cultured in Luria-Bertani (Difco) broth. To make the deletion mutant, 76

transformed L. monocytogenes were selected on a BHI agar plate supplemented with 77

erythromycin (5 μg/ml) or tetracycline (10 μg/ml) when necessary. RAW 264.7 murine 78

macrophage cells were cultured in RPMI 1640 medium (Invitrogen, Gland Island, NY) 79

supplemented with 10% fetal bovine serum (Invitrogen) and 50 U/ml penicillin/streptomycin. All 80

cells were cultured at 37°C in a 5% CO2 incubator. 81

For the growth curve of L. monocytogenes, three strains (F2365∆adlp, wild-type, and 82

∆adlp::pMAD_adlp) of L. monocytogenes were cultured in BHI broth and shaken at 180 rpm for 83

16 hours at 37°C. The cultures were then diluted 1000 times, and 50 µl diluted bacteria were re-84

inoculated into 5 ml fresh BHI broth. Bacteria were incubated at 37°C in the shaker, and 85

bacterial numbers were counted at the specified time points by plating. 86

87

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Deletion mutagenesis 88

A temperature-sensitive shuttle plasmid (pMAD) was used to generate the deletion mutant (35). 89

Using the genomic DNA of the L. monocytogenes F2365 or EGD strain as the template, the 90

upstream and downstream regions flanking the target gene were amplified using PCR with 91

primers (Supplementary Table 2). The PCR products were digested with the designated 92

restriction enzymes (Supplementary Table 2) and then cloned in tandem into a pMAD_tet or 93

pMAD_cat plasmid. The recombinant plasmid was introduced into L. monocytogenes by 94

electroporation at 2.5 KV, 200 Ω, and 25 µF. The deletion of target genes was conducted by 95

allelic exchange. Transformed bacteria were incubated at 43°C for 2 days for the first integration, 96

and colonies were inoculated into antibiotic-free BHI media at 30°C and passaged three times (1 97

day per passage) for the second recombination. Deletion mutants were selected on BHI agar 98

plates supplemented with erythromycin (5 μg/ml) or tetracycline (10 μg/ml) when necessary. 99

RNA extraction and real-time quantitative PCR 100

For real-time quantitative PCR analysis, overnight cultures of bacterial strains were diluted in 101

fresh media and collected at the early exponential growth phase (OD600nm 0.1–0.2) and late 102

exponential growth phase (OD600nm =~0.8). Bacteria were pelleted, resuspended in TRIZOL 103

(Invitrogen), and disrupted in a lysing matrix B tube (MP Biomedicals, Solon, Ohio) by shaking 104

in the Disruptor Genie (Scientific Industries, Inc., Bohemia, NY) for 5 min. Total RNA was 105

extracted and treated with the RNase-free DNase kit (Qiagen, Valencia, CA) to remove genomic 106

DNA. RNA was further purified using the RNeasy mini kit (Qiagen), according to the 107

manufacturer’s instructions. Purified RNA (2 µg) was reverse transcribed into cDNA using a 108

reverse transcription kit (Applied Biosystems, Foster City, CA). The concentration of cDNA was 109

measured with the Nanodrop ND1000 spectrophotometer (Nanodrop Technologies, Wilmington, 110

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DE), and cDNA (approximately 100 ng) was used as the template for a 25-µl reaction system 111

using the primers listed in Supplementary table 2. Quantitative real-time quantitative PCR was 112

performed using the SYBR Green Master kit under the following conditions: 95°C for 10 min 113

and 40 cycles involving 95°C for 30 seconds and 58°C for 60 seconds on the Mx3005PTM Real 114

Time PCR system (Stratagene, La Jolla, CA). The internal control was rlpM, and the ΔΔCt 115

method was used to calculate relative mRNA levels. 116

Motility assay 117

The motility of all strains was tested on a 0.3% agar plate with 1% tryptone and 0.25% NaCl at 118

30°C and 37°C. Toothpicks were used to inoculate BHI broth-grown bacteria to the agar plate, 119

and colony sizes were measured after 24 hours and 48 hours of incubation. 120

Biofilm formation assay 121

Bacteria that were cultured overnight (100 µl) were inoculated into 0.9 ml fresh BHI medium in 122

24-well plastic cell culture plates (Corning, NY) (~107 cells/well). After 24 hours of incubation 123

at 37°C, the unattached bacteria were removed, and fresh medium was added. After further 124

incubations of 48 hours (total 72 hours), the wells were gently washed twice with phosphate-125

buffered saline (PBS) and then stained with 0.5% crystal violet in 10% methanol. A similar 126

incubation protocol was used to analyze biofilm formation via confocal microscopy. After 72 127

hours of incubation, the biofilm was fixed with 3.7% formaldehyde, washed gently twice with 128

PBS, and stained with filmTracer (Invitrogen) according to the manufacturer’s protocol. Z-axial 129

serial collection was performed using the Zeiss laser scanning confocal microscope (LCSM, Carl 130

Zeiss, Germany) at 2.0 µm per interval for 15 scans (Z-axis thickness: 30 µm). 131

MMS assays 132

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L. monocytogenes cells at the mid-exponential growth phase were harvested by centrifugation 133

and washed three times with PBS. Approximately 106 cells were incubated with the indicated 134

concentrations of MMS (Sigma-Aldrich Co., St. Louis, MO) for 1 hour at room temperature. The 135

numbers of recovered bacteria were counted by serial plating. We calculated the percentages of 136

surviving bacteria by comparing the numbers of bacteria (colony-forming units [cfu]) recovered 137

from the MMS solution to numbers of bacteria (cfu) recovered from the PBS solution. 138

H2O2 sensitivity assay 139

L. monocytogenes cells were prepared, as described above. Bacteria were incubated with the 140

indicated concentrations of H2O2 (Sigma-Aldrich Co) for 10 minutes at room temperature, and 141

then Micrococcus lysodeikticus catalase (1,000 U/ml) (Sigma-Aldrich Co.) was added to 142

inactivate the remaining H2O2. We calculated the percentages of surviving bacteria by comparing 143

the numbers of bacteria (cfu) recovered from the H2O2 solution with the numbers of bacteria 144

(cfu) recovered from the PBS solution. 145

Antibiotic survival assay 146

Bacterial cells (107) from overnight cultures were inoculated into BHI broth supplemented with 147

the indicated concentrations of antibiotics. After 4 hours of incubation in a 37°C shaker, bacterial 148

numbers were counted by plating. For the time-kill curve, bacteria were incubated in BHI broth 149

supplemented with 0.5 µg/ml gentamicin and 1 µg/ml ciprofloxacin in a 37°C shaker for the 150

indicated time points. Bacterial numbers were counted by serial plating. We calculated the 151

percentage of surviving bacteria by comparing the numbers of bacteria (cfu) recovered after 152

antibiotic exposure to the numbers of bacteria (cfu) at time zero. 153

Macrophage survival assay 154

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Murine macrophage RAW 264.7 cells were seeded onto 24-well plates and incubated at 37°C in 155

a 5% CO2 incubator. L. monocytogenes strains were grown in BHI until they reached the mid-log 156

phase, after which they were collected by centrifugation. The bacterial pellet was washed twice 157

with PBS and resuspended in RPMI 1640 medium. RAW 264.7 cells were infected with bacteria 158

at a multiplicity of infection of 10:1 for 1 hour. After 1 hour of infection, cells were incubated 159

with fresh medium supplemented with 100 μg/ml gentamicin to kill extracellular bacteria. Cells 160

were washed with PBS three times at the indicated time points and then lysed with 0.2% Triton 161

X-100. The amount of intracellular bacteria was quantified in cell lysates by serial dilution and 162

plating. 163

Infection of mice 164

Four groups (4 mice per cage) of 10- to 12-week-old female BALB/c mice were injected 165

intravenously with L. monocytogenes F2365 wild-type, F2365∆adlp, EGD wild-type, or 166

EGD∆adlp strains at a dose of 6×103 viable bacteria. All mice were euthanized at 72 hours post-167

infection. The spleens and livers were collected and homogenized in PBS. All samples were 168

serially diluted and plated on BHI agar, after which they were incubated at 37°C for 48 hours. 169

The colony-forming units were recorded. 170

171

172

Results 173

The adlp locus flanks Listeria pathogenicity island-1 (LIPI-1) 174

According to the complete genome sequence data that are available for the L. monocytogenes 175

F2365 strain, the adlp locus, which was previously designated “vclA”, is located in the 176

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downstream flanking region of the virulence cluster locus (vcl, also known as LIPI-1). The adlp 177

locus is evolutionarily conserved among six species in the Listeria genus and is located between 178

the housekeeping genes, vclB and ldh-1 (lactate dehydrogenase) (Figure 1A). Along with vclB 179

and ldh-1, adlp directly flanks LIPI-1 on the downstream side. 180

181

Adlp is structurally similar to AlkD in B. cereus and has DNA-binding properties 182

The conserved-domain search tool from the National Center for Biotechnology Information 183

indicated that Listeria Adlp contains a putative AlkD-like domain (residues 20–216, Figure 1B), 184

which is a newly identified protein architecture in the family of DNA glycosylases. We used the 185

SwissModel server (32) to predict the protein structure of Adlp. The B. cereus hypothetical 186

protein (protein ID: 1T06 A) was used as a template. A structural homology search with the 187

DALI server (33) indicated that Adlp is structurally similar to AlkD (an alkylbase DNA 188

glycosylase in B. cereus [Protein ID: 3BVS]), AlkF (a branched DNA-binding DNA glycosylase 189

[Protein ID: 3ZBO]), and the B. cereus hypothetical protein (Protein ID: 1T06 A). 190

191

A cartoon representation of the Listeria Adlp homology model and the published B. cereus AlkD 192

structure is shown in Figure 1C. Adlp possesses a solenoid superhelical structure consisting of 13 193

alpha helices, which is similar to AlkD in its overall structure and protein fold. We found minor 194

differences in the C-terminus regions. AlkD has a tail of HEAT repeats, whereas Adlp has a long 195

loop linking α12 and α13 (Figure 1C). This loop is considered to be a unique feature of the 196

AlkF/G subfamily (34). In Adlp, except for α2, the 12 α-helices form the HEAT-like motifs: six 197

pairs of antiparallel α-α repeats in tandem (Figure 1 C, D). A concave area on the surface of Adlp 198

is formed by α3, α5, α7, α9, α11, and α13. Similar to AlkD, the helices that form the concave 199

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surface are rich in Lysine and Arginine residues (Figure 1D). The Delphi-calculated surface 200

electrostatic potential showed an extremely electropositive concave Adlp surface (Figure 1E), 201

which is a typical feature for accommodating DNA. 202

203

Deletion of adlp disrupted the temperature-dependent motility of L. monocytogenes 204

To study the role of Adlp, we constructed adlp deletion mutants in the L. monocytogenes F2365 205

and EGD strains (Supplementary Table 1) by using a pMAD shuttle-vector system (35). To 206

ensure that the deletion of adlp did not cause growth defects, we compared the growth kinetics of 207

the ∆adlp strain, the F2365 parental strain, and the ∆adlp::pMAD_adlp strain by standard plate 208

counting. We observed no significant differences in the growth rates of all three strains 209

(Supplementary Figure 1), which suggests that the deletion of adlp does not affect bacterial 210

growth. 211

The motility of L. monocytogenes on soft agar is temperature dependent. We analyzed the 212

motility of wild-type and ∆adlp strains on 0.3% agar with 1% tryptone and 0.25% NaCl. 213

Consistent with previous studies, EGD and F2365 wild-type strains were motile on soft agar 214

when grown at 30°C but were non-motile when grown at 37°C (Figure 2A). Surprisingly, the 215

F2365∆adlp strain and EGD∆adlp strain maintained their motility at 37°C (Figure 2A), 216

suggesting that the deletion of adlp disrupted the temperature-dependent inhibition of motility at 217

37°C. 218

Deletion of adlp activated the expression of flagellar motility genes 219

Based on our structural predictions and surface electrostatic potential calculations, Adlp had 220

typical DNA-binding properties. Thus, we speculated that Adlp may play a role in transcriptional 221

regulation. To address this hypothesis, real-time quantitative polymerase chain reaction (PCR) 222

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analysis was performed to investigate the transcription levels of flagellar motility genes. RNA 223

was isolated from strains that were harvested from the early exponential growth phase (OD600nm 224

0.1–0.2) and late exponential growth phase (OD600nm ~0.8) at 37°C. Remarkably, the 225

transcription levels of flagellar motility genes in the F2365∆adlp strain were significantly higher 226

than those in its parental strain at both early and late exponential phases (Figure 2B) at 37°C. 227

Notably, similar results were observed in the EGD∆adlp strain (Figure 2C). These results suggest 228

that the deletion of aldp activates the expression of flagellar motility genes at 37°C. 229

Deletion of adlp impaired biofilm formation 230

Due to the affected motility and altered expression levels of flagellar motility genes, we 231

compared biofilm formation between the F2365∆adlp strain and its parental strain. At three days 232

after incubation, the F2365∆adlp strain demonstrated significantly impaired biofilm formation on 233

plastic cell culture plates, as compared with its parental strain (Figure 3A). This result was 234

further confirmed using confocal microscopy (Figure 3B). The thickness of the biofilm that was 235

formed by the F2365∆adlp strain was significantly less than that of the F2365 wild-type strain 236

(Figure 3B). These data demonstrate that the inactivation of adlp affected the biofilm formation 237

of the L. monocytogenes F2365 strain. 238

Deletion of adlp impaired survival in macrophages and virulence in mice 239

To assess the role of adlp in macrophage survival, we further evaluated the survivability of the 240

three strains (F2365 wild-type, F2365∆adlp, and F2365∆adlp::pMAD_adlp) in RAW 264.7 cells. 241

The ∆adlp strain exhibited decreased survival at all observed time points (Figure 4A). In 242

particular, the number of intracellular bacteria in the ∆adlp strain was significantly lower than 243

that in the parental strain at 12 and 16 hours after infection (Figure 4A). 244

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To assess the role of Adlp in the infection process, we tested the virulence of these two mutants 245

(F2365∆adlp and EGD∆adlp) and their parental strains in BALB/c mice. The mice were injected 246

intravascularly (i.v.) with 6×103 viable bacteria per strain. The bacterial numbers of the 247

F2365∆adlp and EGD∆adlp strains were significantly lower than those of their parental strains 248

in the liver and spleen at 72 hours post-infection (Figure 4B, 4C). Interestingly, the deletion of 249

adlp on the F2365 strain resulted in about 2 log lower CFU in liver of infected mice comparing 250

to F2365 wild type, whereas 1 log difference was observed on EGD strain (Figure 4B, 4C). 251

The F2365∆adlp strain was more sensitive to methyl methanesulfonate (MMS) and H2O2 252

Due to the structural similarity between AlkD and Adlp, we wanted to address whether adlp 253

deletion affects the sensitivity of L. monocytogenes to DNA-alkylating compounds. We tested 254

the survivability of L. monocytogenes in solutions with different concentrations of the alkylating 255

reagent, MMS, for 1 hour. As predicted, the ∆adlp strain had a significantly lower recovery rate 256

after treatment with 20 mM and 40 mM MMS compared with wild-type and 257

F2365∆adlp::pMAD_adlp strains (Figure 5A). In contrast, we found no significant differences in 258

recovery rates after treatments with MMS solutions at low (10 mM) or high (60 mM) 259

concentrations (Figure 5A). The low and high concentrations represented a tolerable dose and 260

lethal dose for the L. monocytogenes F2365 strain, respectively. These data suggest that Adlp 261

plays a role in MMS resistance in L. monocytogenes. 262

To assess the antioxidant effect of Adlp, we compared the dose-dependent and time-dependent 263

sensitivities of the three L. monocytogenes F2365 strains to the oxidative reagent, H2O2 (Figure 264

5B, Supplementary figure 2). After 10 minutes of exposure to H2O2, the ∆adlp strain had 265

approximately 20–30% decreased survivability compared with the parental and 266

F2365∆adlp::pMAD_adlp strains (Figure 5B). Indeed, we observed a difference in recovery rate 267

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after exposure to a low dose (0.5 mM) of H2O2 (Figure 5B). Surprisingly, increasing the 268

concentration of H2O2 (up to 30 mM) did not have much effect on the overall survival rates of 269

the three strains (Figure 5B). We also investigated the survivability of the three strains in 10 mM 270

H2O2 at multiple time points. The ∆adlp strain was more sensitive to H2O2 after 1 hour of 271

exposure (Supplementary Figure 2). 272

Deletion of adlp impaired aminoglycoside resistance 273

To examine the role of Adlp in antibiotic resistance, we tested the survivability of the three 274

strains in the presence of three categories of antibiotics: β-lactam (ampicillin), aminoglycoside 275

(gentamicin), and quinolone (ciprofloxacin). We exposed the strains to 0.5, 0.75, or 1 μg/ml 276

gentamicin for 4 hours. The survival rate of the ∆adlp strain was approximately one order of 277

magnitude lower than those of the parental and ∆adlp::pMAD_adlp strains in 0.5 and 0.75 μg/ml 278

gentamicin (Figure S4-A). We also performed a time-kill study using 0.5 μg/ml gentamicin. 279

Starting from 2 hours of exposure, the ∆adlp strain had a significantly lower recovery rate than 280

the parental and ∆adlp::pMAD_adlp strains (Figure 5C). After 4 hours of exposure, we observed 281

a difference of one order of magnitude in the recovery rates between the ∆adlp and parental 282

strains (Figure 5C). In contrast to the wild-type and ∆adlp::pMAD_adlp strains, a survival defect 283

was observed in the ∆adlp strain after 4 hours of shaking at 37°C and subsequent overnight 284

incubations at room temperature in brain-heart infusion (BHI) broth with 0.5 μg/ml gentamicin 285

(Figure 5D). We observed similar results with streptomycin, which is another drug in the 286

aminoglycoside category (Figure S3). 287

We also tested the survivability of the three L. monocytogenes strains in the presence of a 288

quinolone (ciprofloxacin) and β-lactam (ampicillin). No significant differences in recovery rates 289

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were observed between the ∆adlp strain and the other two strains following treatments with 290

ampicillin and ciprofloxacin (Figure S4B, C). 291

Discussion 292

In this study, we performed real-time quantitative PCR analysis and found that deletion of adlp 293

activated the expression of flagellar motility genes at 37°C in both F2365 and EGD strains. This 294

suggests that Adlp may be involved in the transcriptional repression of flagellar motility genes at 295

37°C. Adlp is evolutionary conserved and is found in all species of Listeria. Therefore, the role 296

of Adlp on flagellar motility regulation might also be evolutionary conserved within different 297

species of Listeria. In contrast to other types of bacteria, in which flagellar motility genes are 298

hierarchically regulated (36), inactivation of adlp results in evenly elevated expression levels of 299

flagellar motility genes in L. monocytogenes. This indicates that flagellar motility genes in L. 300

monocytogenes are non-hierarchically controlled and is consistent with findings from previous 301

studies (3, 21). The initial speculation of the regulatory function of Adlp was from its predicted 302

DNA-binding property. Unlike the MogR repressor, which binds to AT-rich sequences of 303

flagella promoters by its helix-turn-helix motif and inhibits the expression of flagellar motility 304

genes at 37°C (21, 37), Aldp possesses 13 alpha helices and forms a solenoid structure with an 305

extremely electropositive concave. It is highly possible for Adlp to directly bind to the promoter 306

regions of flagellar motility genes to block the initiation of transcription. This hypothesis is 307

supported by the biochemical property of its structurally related protein, AlkF, in B. cereus, 308

which has a unique long loop that forms a β sheet and allows AlkF to bind to branded DNA, 309

such as holiday junctions (34). Furthermore, solenoid structures exhibit high numbers of HEAT 310

motifs and are able to serve as a scaffold for protein-protein interactions (38). Therefore, Adlp 311

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may also work as an adaptor protein that facilitates other regulatory machinery to regulate the 312

expression of flagellar motility genes. 313

The expression of flagellar motility genes is controlled at physiological temperatures (37°C) 314

(19), because flagellin is a major target that is recognized by Toll-like receptor-5 and Nod-like 315

receptor NLRC4 (Ipaf) to trigger the innate immune response (39–42). Indeed, down-regulation 316

of flagellin production is used by L. monocytogenes as a strategy to avoid activation of the 317

NLRC4 inflammasome (43). In our study, deletion of aldp in the F2365 and EGD strains 318

significantly attenuated virulence in the murine infection model, as compared with their wild-319

type strains. We suggested that disrupted regulation in flagellar genes is one of the reasons 320

(probably an important one) that causes attenuation in mice. However, it is total possible that 321

other factors (such as stress responses) that affected by deletion of adlp are responsible for the 322

attenuation in mice. Further study such as comparing the transcription profiles of adlp deletion 323

mutant to wild type (by RNA-seq) would reveal more information other than flagellar 324

regulation”.Similar results have been observed on flagellin-hyperexpressed strains, such as mogR 325

and flgK mutant strains (20, 44). The activation of caspase-1 through NLRC4 by uncontrolled 326

flagellin expression might also be responsible for the reduced survival of the F2365∆adlp strain 327

in RAW264.7 cells, as similar observations on the survival of mogR and flgK mutant strains in 328

macrophages have been documented (20, 44). 329

Our results show that adlp inactivation constitutively activated the expression of flagellar 330

motility genes at 37°C and severely affected biofilm formation. This suggests that regulated 331

flagella-based motility is required for efficient biofilm formation. Indeed, for motile bacteria like 332

L. monocytogenes, a transition process that allows the bacteria to switch from a motile phase to a 333

non-motile phase is a prerequisite for the efficient formation of biofilm (45). Within the biofilm 334

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of Bacillus subtilis, flagellar motility is tightly inhibited by a molecular clutch, EpsE (46, 47). In 335

addition to regulated flagellar activity, type IV pili-based motility has been shown to substitute 336

flagella-based motility in the biofilm of Pseudomonas putida (48). Interestingly, the deletion of 337

DegU and FlaA decreases biofilm formation in L. monocytogenes (49, 50), indicating that 338

flagella are required for biofilm formation. One possible explanation for this is that flagella are 339

required for the initial attachment stage, because non-motile bacteria are less attachable than 340

wild-type bacteria (51, 52). In conjunction with previous findings, our data indicate that the 341

stage-dependent regulation of flagellar activity is required for successful biofilm formation, 342

which involves a short stage of activation for the initial attachment and a long-term inhibitory 343

stage during biofilm formation. 344

The deletion of adlp altered the stress response of the L. monocytogenes F2365 strain to multiple 345

stimuli. Thus, we speculate that Adlp might be working as a functional DNA glycosylase, as the 346

base-excision repair pathway plays a central role in counteracting DNA base lesions (53, 54). 347

Surprisingly, recent data showed that AlkF and AlkG, which are structurally similar to Adlp in B. 348

cereus, do not exhibit DNA glycosylase activity to alkylated and oxidized bases, although a 349

weak reduction in resistance to MMS and NAL is also observed in AlkF- and AlkG-deletion 350

mutants (34). In our study, the deletion of adlp in L. monocytogenes mildly decreased the 351

resistance of bacteria to H2O2 and MMS (20–30% difference in recovery rate compared with 352

wild-type), indicating that Adlp might play a functional role in clearing alkylated and oxidative 353

bases in L. monocytogenes. One explanation for the minor role of Adlp in helping L. 354

monocytogenes to counteract oxidative stress is that other glycosylases might be involved. These 355

bases include MutM, MutY, and Nth, which are responsible for clearing DNA lesions that are 356

caused by reactive oxygen species. Examples of DNA lesions are the highly prevalent 7,8-357

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dihydro-8-oxo-2’-deoxyguanosine (8-oxogaunine or GO) (55) and the 5,6-358

dihydroxydihydrothymine (thymine glycol), which is a lethal lesion that blocks DNA replication 359

and RNA synthesis in bacteria (56, 57). Thus, double- or triple-deletion mutants are necessary to 360

clearly reveal the role of glycosylases in oxidative stress resistance. Indeed, functional 361

redundancies and substrate overlaps are found among DNA glycosylases. A recent report on the 362

pathogen Neisseria meningitidis showed that a network of glycosylases is involved in the repair 363

of oxidatively damaged bases (58). Interestingly, a time-dependent, “two-mode” killing pattern 364

was observed in the time-dependent H2O2 killing experiment, such that the adlp mutant strain 365

had two sensitive time zones (at 10 minutes and after 60 minutes) and a resistance intermediate 366

time zone (around 30 minutes) to H2O2 killing. This “two-mode” killing by H2O2 was also 367

observed in E.coli in a dose-dependent manner (59) and might be another indication that a 368

network of glycosylases exists in L. monocytogenes. 369

In summary, we found a putative protein in L. monocytogenes with a similar overall structure of 370

AlkD that is encoded by a genetic determinant, aldp (Lmof2365_0220). Deletion of adlp 371

activated the expression of flagellar motility genes at 37°C and disrupted the temperature-372

dependent motility of L. monocytogenes. In addition, adlp null mutants showed attenuated 373

virulence in mice, decreased ability to form biofilm, and less resistance to several stresses. . 374

These findings suggest that adlp might encode a bi-functional protein that represses flagellar 375

motility gene expression and influences stress response. Further studies are needed to clarify the 376

protein function of Adlp. 377

378

Acknowledgments 379

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This work was supported by grants from the USDA/ARS (#58-6402-7-230) and the Mississippi 380

Agriculture and Forestry Experiment Station. 381

382

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Figure legends 559

560

Figure 1: Listeria (L.) monocytogenes Adlp is structurally similar to Bacillus (B.) cereus 561

AlkD. (A) Genomic organization of the adlp locus in the L. monocytogenes F2365 strain. The 562

map was generated from the complete genome sequence of the L. monocytogenes F2365 strain. 563

Adlp is located between the housekeeping genes, ldh-1 and vclB, and flanks the downstream 564

region of Listeria pathogenicity island 1. (B) Domain architecture of Adlp. An AlkD-like domain 565

is located from aa 20 to aa 216 (grey box) (C) Predicated three-dimensional structure of Adlp 566

and published structure of B. cereus AlkD (Protein ID: 3BVS). Alpha helices are marked in 567

order, and six pairs of anti-parallel HEAT-like repeats are shown with six different colors. (D) 568

Structure-based sequence alignment of HEAT motifs in Adlp. Positively charged residues that 569

contribute to the electropositive concave surface are highlighted in red. Aromatic residues are in 570

bold, and acidic residues are in blue. A glutamine residue that has a potent role in catalytic 571

activity is marked in orange. Conserved residues that maintain the structure of alpha helices are 572

shown in green. (E) Solvent-accessible protein surface with the same orientation of the models in 573

(c) is colored by electrostatic potential (red, negative; blue, positive; −5 to 5 kBT). Potentials 574

were calculated with Delphi (60), and images were demonstrated with PyMOL 575

(http://pymol.org/) and Chimera (http://www.cgl.ucsf.edu/chimera/). 576

577

Figure 2: Deletion of adlp disrupted temperature-dependent motility and activated the 578

expression of flagellar motility genes. (A) The motility of the strains (EGD WT, F2365 WT, 579

EGD∆adlp, and F2365∆adlp) at 30°C and 37°C was tested using 0.3% agar plates with 1% 580

tryptone and 0.25% NaCl at 24 hours and 48 hours. (B, C) F2365 wild-type and F2365∆adlp 581

strains were harvested at the early exponential growth phase (OD600nm 0.1–0.2) and late 582

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exponential growth phase (OD600nm ~0.8). EGD wild-type and EGD∆adlp strains were harvested 583

at early exponential growth phases. Transcript levels of target genes in the F2365 (A) and EGD 584

strains (B) were evaluated using real-time quantitative PCR. The levels of target gene transcripts 585

in the F2365∆adlp and EGD∆adlp strains were normalized with the housekeeping gene, rplM, 586

and then compared to their paternal strains. Data are the mean and standard deviation (SD) of 587

results from two independent experiments with triplicates in each experiment (n=6). 588

Figure 3: Deletion of adlp impaired the biofilm formation ability of the F2365 strain. A. The 589

biofilm formation abilities of the F2365 wild-type and ∆adlp strains were assessed on plastic cell 590

culture plates (A) and 8-well glass chambers (B). (A) The biofilm was stained with crystal violet 591

and 10% methanol. (B) FilmTracer (Invitrogen) was used to stain the biofilm, and 30 µm of 592

thickness on the Z-axis was determined by Z-axis serial scanning (Z-stack 2 -axis s on the Zeiss 593

confocal microscope. Z-axis panels were constructed to determine the thickness of biofilm. 594

Figure 4: Adlp is involved in bacterial survival in RAW 264.7 and is required for virulence 595

of L. monocytogenes in mice. (A) Three strains (F2365 wild-type, ∆adlp strain, and 596

complementary strain) of bacteria were incubated with RAW 264.7 cells at a 10:1 multiplicity of 597

infection and allowed to replicate for the indicated time points. Survivability was calculated as 598

the ratio of recovered bacteria to inoculated bacteria. Data are the mean and standard error of the 599

mean (SEM) of three independent experiments. (N=3; *p<0.05, **p<0.01, ANOVA) (B, C) Ten- 600

to 12-week-old BALB/c mice were infected intravenously with 6×103 L. monocytogenes F2365 601

wild-type, EGD wild-type, F2365∆adlp, and EGD∆adlp strains. At 72 hours post-infection, 602

bacterial counts in the spleen (B) and liver (C) were calculated. Data are results from two 603

independent experiments (N=2; n=8). Mann-Whitney tests were used to calculate the p value. 604

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605

Figure 5: Role of Adlp in the stress response. (A) Adlp is associated with resistance to DNA-606

alkylating compounds. Three strains (F2365 wild-type, ∆adlp, , and ∆adlp::pMAD_adlp) of L. 607

monocytogenes were incubated with 10, 20, 40, and 60 mM of MMS or PBS at room 608

temperature for 1 hour, after which survival rates were measured. The survival percentages were 609

calculated by comparing the recovered bacteria (colony-forming units [cfu]) from MMS with the 610

recovered bacteria (cfu) from PBS. Data are the mean and SEM of at least three independent 611

experiments. (N≥3; *p<0.05, **p<0.01, ANOVA) (B) Three strains of bacteria were treated with 612

the indicated concentrations of H2O2 or PBS at room temperature for 10 minutes, and the 613

survival rates were measured. Survival percentages were calculated by comparing the recovered 614

bacteria (cfu) from H2O2 to the recovered bacteria (cfu) from PBS. Data are the mean and SEM 615

of three independent experiments. (N=3; *p<0.05, **p<0.01, ANOVA) (C, D): Contribution of 616

Adlp to bactericidal antibiotic resistance. (C) Three strains of bacteria were exposed to 0.5 µg/ml 617

gentamicin for the indicated time points, and the survival rates were measured. Data are the 618

mean and SEM of three independent experiments. (N=3; *p<0.05, **p<0.01, ANOVA) (D) 619

Three strains of bacteria were exposed to 0.5 µg/ml of gentamicin, shaken at 37°C for 4 hours, 620

and then left at room temperature for 16 hours. The image is representative of two independent 621

experiments. 622

623

624

625

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